OA13295A - Compounds specific to adenosine A1, A2a, and A3 receptor and uses thereof. - Google Patents

Abstract

This invention pertains to compounds which specifically inhibit the adenosine A1, A2A, and A3 receptors and the use of these compounds to treat a disease associated with A1, A2A, and A3 adenosine receptors in a subjects, comprising administering to the subject a therapeutically effective amount of the compounds.

Description

1 013295

This application is a continuation-in-part and. daimspriority of U.S. Serial Nos. 09/728,316, filed December 1,2000, 09/728,607, filed December 1, 2000, and 09/728,616, filed December 1, 2000, each of which is hereby incorporatedby reference in its entirety.

Throughout this application, reference is made to compoundsthat specifically bind to I) adenosine A3 receptors (such asinter alia, pages 4-76, 130-175, and 257-287), ii) adenosineA2a receptors (such as inter alia, pages 176-201, and pages288-293), and adenosine A3 receptors (such as inter alia,pages 202-256 and 294-300).

Backcrround of the Invention

Adenosine is an ubiquitous modulator of numerous physiological activities, particularly within thecardiovascular and nervous Systems. The effects of adenosineappear to be mediated by spécifie cell surface receptorproteins. Adenosine modulâtes diverse physiologicalfunctions including induction of sédation, vasodilation,suppression of cardiac rate and contractility, inhibition ofplatelet aggregability, stimulation of gluconeogenesis andinhibition of lipolysis. In addition to its effects onadenylate cyclase, adenosine has been shown to open potassiumchannels, reduce flux through calcium channels, and inhibitor stimulate phosphoinositide turnover through receptor-mediated mechanisms (See for example, C.E. Muller and B.Stein "Adenosine Receptor Antagonists: Structures andPotential Therapeutic Applications," Current PharmaceuticalDesign, 2:50.1 (1996) and C.E. Muller "A-^-Adenosine ReceptorAntagonists," Exp. Opin. Ther. Patents 7(5):419 (1997)).

Adenosine receptors belong to the superfamily of purine 2 013295 receptors which are currently subdivided into P-j_ (adenosine)and P2 (ATP, ADP, and other nucléotides) receptors. Fourreceptor subtypes for the nucleoside adenosine hâve beencloned so far from various species including humans. Tworeceptor subtypes (Ax and A2a) exhibit affinity for adenosinein the nanomolar range while two other known subtypes A2b andA3 are low-affinity receptors, with affinity for adenosine inthe low-micromolar range. A 1 and A3 adenosine receptoractivation can lead to an inhibition of adenylate cyclaseactivity, while A2a and A2k activation causes a stimulationof adenylate cyclase. A few A3 antagoniste hâve been developed for the treatment ofcognitive disease, rénal failure, and cardiac arrhyfchmias.It has been suggested that A2a antagonists may be bénéficiaifor patients suffering from Morbus Parkinson (Parkinson'sdisease). Particularly in view of the potential for localdelivery, adenosine receptor antagonists may be valuable fortreatment of allergie inflammation and asthma. Availableinformation (for example, Nyce & Metzger "DNA antisenseTherapy for Asthma in an Animal Model" Nature (1997) 385:721-5)indicates that in this pathophysiologic context, A xantagonists may block contraction of smooth muscle underlyingrespiratory epithelia, while A2£ or A3 receptor antagonistsmay block mast cell degranulation, mitigating the release ofhistamine and other inflammatory mediators. A 2b receptorshâve been discovered throughout the gastrointestinal tract,especially in the colon and the intestinal epithelia. It hasbeen suggested that A2b receptors médiate cAMP response(Strohmeier et al., J. Bio. Chem. (1995) 270:2387-94).

Adenosine receptors hâve also been shown to exist on theretinas of various mammalian species including bovine,porcine, monkey, rat, guinea pig, mouse, rabbit and human(See, Blazynski et al., Discrète Distributions of AdenosineReceptors in Mammalian Retina, Journal of Neurochemistry,volume 54, pages 648-655 (1990); Woods et al.,Characterization of Adenosine A1-Receptor Binding Sites in 013295

Bovine Retinal Membranes, Experimental Eye Research, volume53,. pages 325-331 (1991) ; and Braas et al., Endogenousadenosine and adenosine receptors localized to ganglion cellsof the retina, Proceedings of the National Academy ofScience, volume 84, pages 3906-3910 (1987)). Recently,Williams reported the observation of adenosine transportsites in a cultured human retinal cell line (Williams et al.,Nucleoside Transport Sites in a Cultured Human Retinal CellLine Established By SV-40 T Antigen Gene, Current EyeResearch, volume 13, pages 109-118 (1994)).

Compounds which regulate the uptake of adenosine uptake hâvepreviously been suggested as potential therapeutic agents forthe treatment of retinal and optic nerve head damage. InU.S. Patent No. 5,780,450 to Shade, Shade discusses the useof adenosine uptake inhibitors for treating eye disorders.Shade does not disclose the use of spécifie A 3 receptorinhibitors. The entire contents of U.S. Patent No. 5,780,450are hereby incorporated herein by reference.

Additional adenosine receptor antagonists are needed aspharmacological tools and are of considérable interest asdrugs for the above-referenced disease States and/orconditions. 4 013295

Suiranarv of the Invention

The présent invention is based on compounds which selectivelybind to adenosine Ax receptor, thereby treating a diseaseassociated with Ai adenosine receptor in a subject byadministering to the subject a therapeutically effectiveamount of such compounds. The disease to be treated areassociated with cognitive disease, rénal failure, cardiacarrhythmias, respiratory epithelia, transmitter release,sédation, vasoconstriction, bradycardia, négative cardiacinotropy and dromotropy, branchoconstriction, neutropilchemotaxis, reflux condition, or ulcerative condition.

The présent invention is based, at least in part, on thediscovery that certain N-6 substituted /-deazapurines,described infra, can be used to treat a N-6 substituted 7-deazapurine responsive State. Examples of such Statesinclude those in which the activity of the adenosinereceptors is increased, e.g., bronchitis, gastrointestinaldisorders, or asthma. These States can be characterized inthat adenosine receptor activation can lead to the inhibitionor stimulation of adenylate cyclase activity. Compositionsand methods of the invention include enantiomerically ordiastereomerically pure N-6 substituted 7-deazapurines.Preferred N-6 substituted 7-deazapurines include those whichhâve an acetamide, carboxamide, substituted cyclohexyl,e.g.,cyclohexanol, or a urea moiety attached to the N-6 nitrogenthrough an alkylene chain.

The présent invention pertains to methods for modulating anadenosine receptor(s) in a mammal by administering to themammal a therapeutically effective amount of a N-6substituted 7-deazapurine, such that modulation of theadenosine receptor's activity occurs. Suitable adenosinereceptors include the families of Ax, A2, or A 3 In apreferred embodiment, the N-6 substituted 7-deazapurine is aadenosine receptor antagonist.

The invention further pertains to methods for treating N-6 5 013295 substituted 7-deazapurine disorders, e.g., asthma,bronchitis, allergie rhinitis, chronic obstructive pulmonarydisease, rénal disorders, gastrointestinal disorders, and eyedisorders, in a mammal by administering to the mammal atherapeutically effective amount of a N-6 substituted 7-deazapurine, such that treatment of the disorder in themammal occurs. Suitable N-6 substituted 7 deazapurinesinclude those illustrated by the general formula I:

(I) and pharmacéutically acceptable salts thereof. R^ and areeach independently a hydrogen atom or a substituted orunsubstituted alkyl, aryl, or alkylaryl moiety or togetherform a substituted or unsubstituted heterocyclic ring. isa substituted or unsubstituted alkyl, aryl, or alkylarylmoiety. R is a hydrogen atom or a substituted orunsubstituted alkyl, aryl, or alkylaryl moiety. R g and Rgare each independently a halogen atom, e.g., chlorine,fluorine, or bromine, a hydrogen atom or a substituted orunsubstituted alkyl, aryl, or alkylaryl moiety or R^ and Rgor Rg and Rg together form a substituted or unsubstitutedheterocyclic or carbocyclic ring.

In certain embodiments, Rj_ and R2 can each independently bea substituted or unsubstituted cycloalkyl or heteroarylalkylmoieties. In other embodiments, Rg is a hydrogen atom or asubstituted or unsubstituted heteroaryl moiety. In stillother embodiments, R^, Rg and Rg can each be independently a 6 013295 heteroaryl moïeties. In a preferred embodiment, R is ahydrogen atom, R2 is a cyclohexanol, e.g., trans-cyclohexanol, R3 is phenyl, R 4 is a hydrogen atom, Rg is amethyl group and R g is a methyl group. In still anotherembodiment, R^ is a hydrogen atom, R2 is NHMe , R 3 is phenyl, R^ is a hydrogen atom and Rg and Rg are methylgroups.

The invention further pertains to pharmaceutical compositionsfor treating a N-6 substituted 7-deazapurine responsive Statein a mammal, e.g., asthrna, bronchitis, allergie rhinitis,chronic obstructive pulmonary disease, rénal disorders,gastrointestinal disorders, and eye disorders. Thepharmaceutical composition includes a therapeuticallyeffective amount of a N-6 substituted 7-deazapurine and apharmaceutically acceptable carrier.

The présent invention also pertains to packagedpharmaceutical compositions for treating a N-6 substituted 7-deazapurine responsive State in a mammal. The packagedpharmaceutical composition includes a container holding atherapeutically effective amount of at least one N-6substituted 7-deazapurine and instructions for using the N-6substituted 7-deazapurine for treating a N-6 substituted 7-deazapurine responsive State in a mammal. 013295

The invention further pertains to compounds of formula Iwherein R2 is hydrogen; R2 is substituted or unsubstituted cycloalkyl, substituted orunsubstituted alkyl, or and R2 together form a substitutedor unsubstituted heterocyclic ring; R 2 is unsubstituted or substituted aryl; R4 is hydrogen; and R5 and Rg are each independently hydrogen or alkyl, andpharmaceutically acceptable salts thereof. The deazapurinesof this embodiment may advantageously be sélective A3 receptorantagonists. These compounds may be useful for numeroustherapeutic uses such as, for example, the treatment ôfasthma, kidney failure associated with heart failure, andglaucoma. In a particularly preferred embodiment, thedeazapurine is a water soluble prodrug that is capable ofbeing metabolized in vivo to an active drug by, for example,esterase catalyzed hydrolysis.

In yet another embodiment, the invention features a methodfor inhibiting the activity of an adenosine receptor (e.g.,A3) in a cell, by contacting the cell with N-6 substituted 7-deazapurine (e.g., preferably, an adenosine receptorantagonist).

In another aspect, the invention features a method fortreating damage to the eye of an animal (e.g., a human) byadministering to the animal an effective amount of an N-6substituted 7-deazapurine of formula I. Preferably, the N-6substituted 7-deazapurine is an antagonist of A3 adenosinereceptors in cells of the animal. The damage is to theretina or the optic nerve head and may be acute or chronic.The damage may be the resuit of, for example, glaucoma,edema, ischemia, hypoxia or trauma. 8 013295

The invention also features a pharmaceutical compositioncomprising a N-6 substituted 7-deazapurine of formula I.Preferâbly, the pharmaceutical préparation is an ophthalmicformulation (e.g., an periocular, retrobulbar or intraocularinjection formulation, a systemic formulation, or a surgicalirrigating solution).

In yet another embodiment, the invention features adeazapurine having the.formula II:

N

R4 (II) wherein X is N or CR6; R-χ and R2 are each independentlyhydrogen, or substituted or unsubstituted alkoxy,aminoalkyl, alkyl, aryl, or alkylaryl, or together forma substituted or unsubstituted heterocyclic ring,provided that both R3 and R2 are both not hydrogen; R3is substituted or unsubstituted alkyl, arylalkyl, oraryl; R4 is hydrogen or substituted or unsubstituted Cx-C6 alkyl; L is hydrogen, substituted or unsubstitutedalkyl, or R4 and L together form a substituted orunsubstituted heterocyclic or carbocyclic ring; R6 ishydrogen, substituted or unsubstituted alkyl, orhalogen; Q is CH2, O, S, or NR7, wherein R7 is hydrogenor substituted or unsubstituted Cx- C6 alkyl; and W isunsubstituted or substituted alkyl, cycloalkyl, aryl,arylalkyl, biaryl, heteroaryl, substituted carbonyl,substituted thiocarbonyl, or substituted sulfonyl; 013295 - 9 - provided that if R3 is pyrrolidino, then R 4 is notmethyl. The invention also pertains to pharmaceuticallyacceptable salts and prodrugs of the compounds of theinvention.

In an advantageous embodiment, X is CR6 and Q is CH2, O, S,or NH in formula II, wherein R6 is as defined above.

In another embodiment of formula II, X is N.

The invention further pertains to a method for inhibiting theactivity of an adenosine receptor ( e.g., an A2b adenosinereceptor) in a cell by contacting the cell with a compound ofthe invention. Preferably, the compound is an antagonist ofthe receptor.

The invention also pertains to a method for treating agastrointestinal disorder (e.g., diarrhea) or a respiratorydisorder (e.g., allergie rhinitis, chronic obstructivepulmonary disease) in an animal by administering to an animalan effective amount of a compound of formula II ( e.g., an antagonist of A2b) . Preferably, the animal is a human.

This invention also features a compound having the structure:

wherein Ri is trans-4-hydroxy cyclohexyl, 2-methylaminocarbonylamino cyclohexyl, 2-methylamino carbonylaminocyclohexyl, acetamido ethyl, or methylaminocarbonylamino ethyl; 10 013295 wherein R3 is a substituted or unsubstituted four to sixmembered ring; wherein Rs is H, alkyl, substituted alkyl, aryl,5 arylalkyl, amino, substituted aryl, wherein said substituted alkyl is -C (R7) (Re) XRs>, wherein X is O, S, or NR10, wherein R7 and Rs are each independently H or alkyl,wherein R9 and Rio are each independently alkyl orcycloalkyl, or R9, Rio and the nitrogen together form a 10 substituted or unsubstituted ring of between 4 and 7 members; wherein Re is H, alkyl, substituted alkyl, cycloalkyl;or a pharmaceutically acceptable sait, or a prodrug 15 dérivative, or a biologically active métabolite, with the proviso that when Ri is acetylamino ethyl, R3 is not4-pyridyl.

In one embodiment of the compound, R3 is phenyl, pyrrole, 20 thiophene, furan, thiazole, imidazole, pyrazole, 1,2,4- triazole, pyridine, 2(1H)-pyridone, 4(1H)-pyridone, pyrazine,pyrimidine, pyridazine, isothiazole, isoxazole, oxazole,tetrazole, naphthalene, tetralin, naphthyridine, benzofuran,benzothiophene, indole, 2,3-dihydroindole, lH-indole, 25 indoline, benzopyrazole, 1,3-benzodioxole, benzoxazole,purine, coumarin, chromone, quinoline, tetrahydroquinoline,isoquinoline, benzimidazole, quinazoline, pyrido[2,3-bjpyrazine, pyrido[3,4-b]pyrazine, pyrido[3,2-c]pyridazine,pyrido[3,4-b]-pyridine, ΙΗ-pyrazole[3,4-d]pyrimidine, 30 pteridine, 2(1H)-quinolone, 1(2H)-isoquinolone, 1,4-benzisoxazine, benzothiazole, quinoxaline, quinoline-N-oxide,isoquinoline-N-oxide, quinoxaline-N-oxide, quinazoline-N-oxide, benzoxazine, phthalazine, cinnoline, or having astructure: 35 013295 11

wherein Y is carbon or nitrogen; wherein R2 and R2' are independently H, substituted orunsubstituted alkyl, substituted or unsubstituted aryl,halogen, methoxy, methyl amino, or methyl thio;

This invention also pertains to a compound having thestructure: wherein R3 is aryl, substituted aryl, or heteroaryl; wherein Rê is H, alkyl, substituted alkyl, orcycloalkyl; wherein Rs is H, alkyl, substituted alkyl,aryl, arylalkyl, amino, substituted aryl, wherein saidsubstituted alkyl is -C (Ri) (Rs)NR9Rio, wherein R7 and Reare each H or alkyl, wherein R9 and Rio are each alkyl orcycloalkyl, or R9, Rio and the nitrogen together form aring System of between 4 and 7 members.

This invention also features a method for inhibiting theactivity of an A1 adenosine receptor in a cell, whichcomprises contacting said cell with the above-mentionedcompounds. 013295

Detailed Description

The features and other details of the invention will now bemore particularly described and pointed out in the daims.It will be understood that the particular embodiments of theinvention are shown by way of illustration and not aslimitations of the invention. The principle features of thisinvention can be employed in various embodiments withoutdeparting from the scope of the invention.

The présent invention pertains to methods for treating a N-6substituted 7-deazapurine responsive State in a mammal. Themethods include administration of a therapeutically effectiveamount of a N-6 substituted 7-deazapurine, described infra,to the mammal, such that treatment of the N-6 substituted 7-deazapurine responsive State in the mammal occurs.

The language "N-6 substituted 7-deazapurine responsive State"is intended to include a disease State or conditioncharacterized by its responsiveness to treatment with a N-6substituted 7-deazapurine of the invention as describedinfra, e.g., the treatment includes a significantdiminishment of at least one symptom or effect of the Stateachieved with a N-6 substituted 7-deazapurine of theinvention. Typically such States are associated with anincrease of adenosine within a host such that the host oftenexpériences physiological symptoms which include, but are notlimited to, release of toxins, inflammation, coma, waterrétention, weight gain or weight loss, pancreatitis,emphysema, rheumatoid arthritis, osteoarthritis, multipleorgan failure, infant and adult respiratory distresssyndrome, allergie rhinitis, chronic obstructive pulmonarydisease, eye disorders, gastrointestinal disorders, skintumor promotion, immunodeficiency and asthma. (See forexample, C.E. Muller and B. Stein "Adenosine ReceptorAntagonists: Structures and Potential Thérapeutic

Applications," Current Pharmaceutical Design, 2.-501 (1996)and C.E. Muller "A^-Adenosine Receptor Antagonists," Exp.Opin. Ther. Patents 7(5):419 (1997) and I. Feoktistove, R. 13 013295

Polosa, S. T. Holgate and I. Biaggioni "Adenosine A 23receptors: a novel therapeutic target in asthma?" TiPS 19;148 (1998) ) . The effects often associated with such symptomsinclude, but are not limited to, fever, shortness of breath,nausea, diarrhea, weakness, headache, and even death. In oneembodiment, a N-6 substituted 7-deazapurine responsive Stateincludes those disease States which are mediated bystimulation of adenosine receptors, e.g., A^, A2a, A2b' A3'etc., such that calcium- concentrations in cells and/oractivation of PLC (phospholipase C) is modulated. In apreferred embodiment, a N-6 substituted 7-deazapurineresponsive State is associated with adenosine receptor(s),e.g., the N-6 substituted 7-deazapurine acts as anantagonist. Examples of suitabie responsive States which canbe treated by the compounds of the invention, e.g. , adenosinereceptor subtypes which médiate biological effects, includecentral nervous System (CNS) effects, cardiovascular effects,rénal effects, respiratory effects, immunological effects,gastro-intestinal effects and metabolic effects. Therelative amount of adenosine in a subject can be associatedwith the effects listed below; that is increased levels ofadenosine can trigger an effect, e.g., an undesiredphysiological response, e.g., an asthmatic attack. CNS effects include decreased transmitter release (Ax) ,sédation (Ai) , decreased locomctor activity (A2a) ,anticonvulsant activity, chemoreceptor stimulation (AJ andhyperalgesia. Therapeutic applications of the inventivecompounds include treatment of dementia, Alzheimer's diseaseand memory enhancement.

Cardiovascular effects include vasodilation (A2a) , (A2b) and (A3) , vasoconstriction (Αχ), bradyeardia (AJ , plateletinhibition (A2a), négative cardiac inotropy and dromotropy(AJ, arrhythmia, tachycardie and angiogenesis. Therapeuticapplications of the inventive compounds include, for example,prévention of ischaemia-induced impairment of the heart andcardiotonics, myocardial tissue protection and restoration 14 013295 of cardiac functïon. Rénal effects include decreased GFR (Ai), mesangial cellcontraction (Ai) , antidiuresis (AJ and inhibition of reninrelease . Suitable therapeutic applications of theinventive compounds include use of the inventive compounds asdiuretic, natriuretic, potassium-sparing, kidney-protective/prevention of acute rénal failure,antihypertensive, anti-oedematous and anti-nephritic agents.

Respiratory effects include bronchodilation (A2) ,bronchoconstriction (Ai), chronic obstructive pulmonarydisease, allergie rhinitis, mucus sécrétion and respiratorydépréssion (A2) . Suitable therapeutic applications for thecompounds of the invention include anti-asthmaticapplications, treatment of lung disease after transplantationand respiratory disorders.

Immunological effects include immunosuppression (A2),neutrophil chemotaxis (AJ , neutrophil superoxide génération(A2J and mast cell degranulation (A2b and AJ Therapeuticapplications of antagonists include allergie and non allergieinflammation, e.g., release of histamine and otherinflammatory mediators.

Gastrointestinal effects include inhibition of acid sécrétion(AJ therapeutic application may include reflux and ulcerativeconditions Gastrointestinal effects also include colonie,intestinal and diarrheal disease, e.g., diarrheal diseaseassociated with intestinal inflammation (A2b) .

Eye disorders include retinal and optic nerve head in jury andtrauma related disorders (A3) . In a preferred embodiment, theeye disorder is glaucoma.

Other therapeutic applications of the compounds of theinvention include treatment of obesity (lipolyticproperties) , hypertension, treatment of dépréssion, sédative, 15 013295 anxiolytic, as antileptics and as laxatives, e.g., effectingmotility without causing diarrhea.

The term "disease State" is intended to include those5 conditions caused by or associated with unwanted levels ofadenosine, adenylyl cyclase activity, increaséd physiologicalactivity associated with aberrant stimulation of adenosinereceptors and/or an increase in cAMP. In oné embodiment, thedisease State is, for example, asthma, chronic obstructive 10 pulmonary disease, allergie rhinitis, bronchitis, rénaldisorders, gastrointestinal disorders, or eye disorders.Additional examples include chronic bronchitis and cysticfibrosis. Suitable examples of inflammatory diseases includenon-lymphocytic leukemia, myocardial ischaemia, angina, 15 infarction, cerebrovascular ischaemia, intermittent claudication, critical limb ischemia, venous hypertension,varicose veins, venous ulcération and arteriosclerosis.Impaired reperfusion States include, for example, any post-surgical trauma, such as reconstructive surgery, thrombolysis 20 or angioplasty.

The language "treatment of a N-6 substituted 7-deazapurineresponsive State" or "treating a N-6 substituted 7-deazapurine responsive State" is intended to include changes 25 in a disease State or condition, as described above, suchthat physiological symptoms in a mammal can be significantlydiminished or minimized. The language also includes control,prévention or inhibition of physiological symptoms or effectsassociated with an aberrant amount of adenosine. In one 30 preferred embodiment, the control of the disease State orcondition is such that the disease State or condition iseradicated. In another preferred embodiment, the control issélective such that aberrant levels of adenosine receptoractivity are controlled while other physiologie système and 35 parameters are unaffected. 16 013295

The term "N-6 substituted 7-deazapurine" is art recognizedand is intended to include those compounds having the formula I:

"7 deaza site" (i) "N-substituted 7-deazapurine"acceptable salts thereof, and,includes certain N-6 substituted includes pharmaceuticallyin one embodiment, also purines described herein.

In certain embodiments, the N-6 substituted 7-deazapurine isnot N-6 benzyl or N-6 phenylethyl substituted. In otherembodiments, is not benzyl or phenylethyl substituted. Inpreferred embodiments, R^ and R2 are both not hydrogen atoms.In still other preferred embodiments, R 3 is not a hydrogenatom.

The language "therapeutically effective amount" of an N-6substituted 7-deazapurine, described infra, is that amount ofa therapeutic compound necessary or sufficient to perform itsintended function within a mammal, e.g., treat a N-6substituted 7-deazapurine responsive State, or a diseaseState in a mammal. An effective amount of the therapeuticcompound can vary according to factors such as the amount ofthe causative agent already présent in the mammal, the âge,sex, and weight of the mammal, and the ability of thetherapeutic compounds of the présent invention to affect a N-6 substituted 7-deazapurine responsive State in the mammal. 013295 - 17 -

One of ordinary skill in the art would be able to study theaforementioned factors andmake a détermination regarding theeffective amount of the therapeutic compound without undueexpérimentation. An in vitro or in vivo assay also Can beused to détermine an "effective amount" of the therapeuticcompounds described infra. The ordinarily skilled artisanwould select an appropriate amount of the therapeuticcompound for use in the af orementioned assay or as atherapeutic treatment. A therapeutically effective amount preferably diminishes atleast one symptom or ef fect associated with the N-6substituted 7-deazapurine responsive State or condition beingtreated by at least about 20%, (more preferably by at leastabout 40%, even more preferably by at least about 60%, andstill more preferably by at least about 80%) relative tountreated subjects. Assays can be designed by one skilled inthe art to measure the diminishment of such symptoms and/oreffects. Any art recognized assay capable of measuring suchparameters are intended to be included as part of thisinvention. For example, if asthma is the State beingtreated, then the volume of àir expended from the lungs of asubject can be measured before and after treatment formeasurement of increase in the volume using an art recognizedtechnique. Likewise, if inflammation is the State beingtreated, then the area which is inflamed can be measuredbefore and after treatment for measurément of diminishment inthe area inflamed using an art recognized technique.

The term "cell" includes both prokaryotic and eukaryoticcells.

The term "animal" includes any organism with adenosinereceptors or any organism susceptible to a N-6-substituted 7-deazapurine responsive State. Examples of animais includeyeast, mammals, reptiles, and birds. It also includestransgenic animais. 18 013295

The terni "mammal" is art recognized and is intended toinclude an animal, more preferably a warm-blooded animal,most preferably cattle, sheep, pigs, horses, dogs, cats,rats, mice, and humans. Mammals susceptible to a N-6substituted 7-deazapurine responsive State, inflammation,emphysema, asthma, central nervous System conditions, oracute respiratory distress syndrome, for example, areincluded as part of this invention.

In another aspect, the présent invention pertains to methodsfor modulating an adenosine receptor(s) in a mammal byadministering to the mammal a therapeutically effectiveamount of a N-6 substituted 7-deazapurine, such thatmodulation of the adenosine receptor in the mammal occurs.Suitable adenosine receptors include the families of Ax, A2,or A3 In a preferred embodiment, the N-6 substituted 7-deazapurine is an adenosine receptor antagonist.

The language "modulating an adenosine receptor" is intendedto include those instances where a compound interacts withan adenosine receptor(s), causing increased, decreased orabnormal physiological activity associated with an adenosinereceptor or subséquent cascade effects resulting from themodulation of the adenosine receptor. Physiologicalactivities associated with adenosine receptors includeinduction of sédation, vasodilation, suppression of cardiacrate and contractility, inhibition of platelet aggregbility,stimulation of gluconeogenesis, inhibition of lipolysis,opening of potassium channels, reducing flux of calciumchannels, etc.

The terms "modulate", "modulating" and "modulation" areintended to include preventing, eradicating, or inhibitingthe resulting increase of undesired physiological activityassociated with abnormal stimulation of an adenosinereceptor, e.g., in the context of the therapeutic methods ofthe invention. In another embodiment, the term modulate 013295 - 19 - includes antagonistic effects, e.g., diminishment of theactivity or production of mediators of allergy and allergieinflammation which results from the overstimulation ofadenosine receptor(s). For example, the therapeuticdeazapurines of the invention can interact with an adenosinereceptor to inhibit, for example, adenylate cyclase activity.

The language "condition characterized by aberrant adenosinereceptor activity" is intended to include those diseases,disorders or conditions which are associated with aberrant'stimulation of an adenosine receptor, in that the stimulationof the receptor causes a biochemical and or physiologicalchain of events that is directly or indirectly associatedwith the disease, disorder or condition. This stimulation cfan adenosine receptor does not hâve to be the sole causativeagent of the disease, disorder or condition but merely beresponsible for causing some of the symptoms typicallyassociated with the disease, disorder, or condition beingtreated. The aberrant stimulation of the receptor can be thesole factor or at least one other agent can be involved inthe State being treated. Examples of conditions includethose disease States listed supra, including inflammation,gastrointestinal disorders and those symptoms manifested bythe presence of increased adenosine receptor activity.Preferred examples include those symptoms associated withasthma, allergie rhinitis, chronic obstructive pulmonarydisease, emphysema, bronchitis, gastrointestinal disordersand glaucoma.

The language "treating or treatment of a conditioncharacterized by aberrant adenosine receptor activity" isintended to include the alleviation of or diminishment of atleast one symptom typically associated with the condition.The treatment also includes alleviation or diminishment ofmore than one symptom. Preferably, the treatment cures,e.g., substantially éliminâtes, the symptoms associated withthe condition. 013295 - 20 -

The présent invention pertains to compounds, N-6 substituted7-deazapurines, having the formula I:

(I) wherein and R2 are each independently a hydrogen atomor a substituted or unsubstituted alkyl, aryl, oralkylaryl moiety or together form a substituted orunsubstituted heterocyclic ring; R 3 is a hydrogen atomor a substituted or unsubstituted alkyl, aryl, oralkylaryl moiety; is a hydrogen atom or a substitutedor unsubstituted alkyl, aryl, or alkylaryl moiety. Rgand Rg are each independently a halogen atom, e.g.,chlorine, fluorine, or bromine, a hydrogen atom or asubstituted or unsubstituted alkyl, aryl, or alkylarylmoiety or R 4 and R5 or R5 and R6 together form asubstituted or unsubstituted heterocyclic or carbocyclicring. Also included, are pharmaceutically acceptablesalts of the N-6 substituted 7-deazapurines.

In certain embodiments, R1 and R2 can each independently be asubstituted or unsubstituted·cycloalkyl or heteroarylalkylmoieties. In other embodiments, R3 is a hydrogen atom or asubstituted or unsubstituted heteroaryl moiety. In stillother embodiments, R4, R5 and R6 can each be independently aheteroaryl moiety.

In one embodiment, R2 is a hydrogen atom, R2 is a substitutedor unsubstituted cyclohexane, cyclopentyl, cyclobutyl or 21 013295 cyclopropane moiety, R3 is a substituted or unsubstitutedphenyl moiety, R4 is a hydrogen atom and R5 and R6 are bothmethyl groups.

In another embodiment, R2 is a cyclohexanol, acyclohexanediol, a cyclohexylsulfonamide, a cyclohexanamide,a cyclohexylester, a cyclohexene, a cyclopentanol or acyclopentanediol and R3 is a phenyl moiety.

In still another embodiment, R3 is a hydrogen atom, R2 is acyclohexanol, R3 is a substituted or unsubstituted phenyl,pyridine, furan, cyclopentane, or thiophene moiety, R4 is ahydrogen atom, a substituted alkyl, aryl or arylalkyl moiety,and R5 and R6 are each indépendantly a liydrogen atom, or asubstituted or unsubstituted alkyl, aryl, or alkylaryl moiety.

In yet another embodiment, R3 is a hydrogen atom, R2 issubstituted or unsubstituted alkylamine, arylamine, oralkylarylamine, a substituted or unsubstituted alkylamide,arylamide or alkylarylamide, a substituted or unsubstitutedalkylsulfonamide, arylsuifonamide or alkylarylsulfonamide, asubstituted or unsubstituted alkylurea, arylurea oralkylarylurea, a substituted or unsubstituted alkylcarbamate,arylcarbamate or alkylarylcarbamate, a substituted orunsubstituted alkylcarboxylic acid, arylcarboxylic acid oralkylarylcarboxylic acid, R3 is a substituted or unsubstitutedphenyl moiety, R4 is a hydrogen atom and R5 and R6 are methylgroups.

In still another embodiment, R2 is guanidine, a modifiedguanidine, cyanoguanidine, a thiourea, a thioamide or anamidine.

In one embodiment, R2 can be

?2C

O 22 013295 wherein R2a-R2c are each independently a hydrogen atom or asaturated or unsaturated alkyl, aryl or alkylaryl moiety andR2d is a hydrogen atom or a saturated or unsaturated alkyl,aryl, or alkylaryl moiety, NR2eR2f, or 0R2g, wherein R2e-R2g areeach independently a hydrogen atom or a saturated orunsaturated alkyl, aryl or alkylaryl moieties.Alternatively, R2a and R2b together can form a carbôcyclic orheterocyclic ring having a ring size between about 3 and 8members, e.g., cyclopropyl, cyclopentyl, cyclohexyl groups.

In one aspect of the invention, both R5 and Rê are not methylgroups, preferably, onê of R5 and R6 is an alkyl group, e.g.,a methyl group, and the other is a hydrogen atom.

In another aspect of the invention, when R4 is 1-phenylethyland R3 is a hydrogen atom, then R3 is not phenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 3,4-dichlorophenyl, 3-methoxyphenyl or 4-methoxyphenyl or when R4and Ri are 1-phenylethyl, then R3 is not a hydrogen atom orwhen R4 is a hydrogen atom and R3 is a phenyl, then Rx is notphenylethyl.

In another aspect of the invention, when R5 and R6 togetherform a carbôcyclic ring, e.g.,

or pyrimido [4,5-6] indole, then R3 is not phenyl when R4 is 1-(4-methylphenyl)ethyl, phenylisopropyl, phenyl or 1-phenylethyl or when R3 is not a hydrogen atom when R4 is 1-phenylethyl. The carbôcyclic ring f ormed by R5 and Re can beeither aromatic or aliphatic and can hâve between 4 and 12carbon atoms, e.g., naphthyl, phenylcyclohexyl, etc.,preferably between 5 and 7 carbon atoms, e.g., cyclopentyl or 23 013295 cyclohexyl. Alternatively, R5 and R6 together can form. aheterocyclic ring, such as those disclosed below. Typicalheterocyclic rings include between 4 and 12 carbon atoms,preferably between 5 and 7 carbon atoms, and can be eitheraromatic or aliphatic. The heterocyclic ring can be furthersubstituted, including substitution of one or more carbonatoms of the ring structure with one or more heteroatoms.

In still another aspect of the invention, Rx and R2 form aheterocyclic ring. Représentative examples include, but arenot limiüed to, those heterocyclic rings listed below, suchas morpholino, piperazine and the like, e.g., 4- hydroxypiperidines, 4-aminopiperidines. Where Ri and Rztogether form a piperazino group, Ç»7

wherein R7 can be a hydrogen atom or a substituted orunsubstituted alkyl, aryl or alkylaryl moiety.

In yet another aspect of the invention R4 and Rs together canform a heterocyclic ring, e.g.,

wherein the heterocyclic ring can be either aromatic oraliphatic and can form a ring having between 4 and 12 carbonatoms, e.g., naphthyl, phenylcyclohexyl, etc. and can beeither aromatic or aliphatic, e.g., cyclohexyl, cyclopentyl.

The heterocyclic ring can be further substituted, including 24 - 013295 substitution of carbon atoms of the ring structure with oneor more heteroatoms. Alternatively, R4 and R5 together can'form a heterocyclic ring, such as those disclosed below.

In certain embodiments, the N-6 substituted 7-deazapurine isnot N-6 benzyl or N-6 phenylethyl substituted. In otherembodiments, R4 is not benzyl or phenylethyl substituted. Inpreferred embodiments, Ri and R2 are both not hydrogen atoms.In sti.ll other preferred embodiments, R3 is not H.

The compounds of the invention may comprise water-solubleprodrugs which are described in WO 99/33815, InternationalApplication No. PCT/US98/04595, filed March 9, 1998 and publislied July 8, 1999. The entire content of WO 99/33815 isexpressly incorporated herein by reference. The water-solubie prodrugs are metabolized in vivo to an active drug,e.g., by esterase catalyzed hydrolysis. Examples ofpotential prodrugs include deazapurines with, for example, R2as cycloalkyl substituted with -OC(O)(Z)NH2, wherein Z is aside chain of a naturally or unnaturally occurring aminoacid, or analog thereof, an α, β, γ, or ω amino acids, or adipeptide. Preferred amino acid side chains include those ofglycine, alanine, valine, leucine, isoleucine, lysine, ci-me thy la lanine, aminocyclopropane carboxylic acid, azetidine-2-carboxylic acid, β-alanine, γ-aminobutyric acid, alanine-alanine, or glycine-alanine.

In a further embodiment, the invention features deazapurinesof the formula (I) , wherein R! is hydrogen; R2 is substitutedor unsubstituted cycloalkyl, substituted or unsubstitutedalkyl, or R3 and R2 together form a substituted orunsubstituted heterocyclic ring; R3 is unsubstituted orsubstituted aryl; R4 is hydrogen; and R5 and Re are eachindependently hydrogen or alkyl, and pharmaceuticallyacceptable salts thereof. The deazapurines of thisembodiment may potentially be sélective A3 receptorantagonists. 25 013295

In One embodiment, R2 is substituted (e.g., hydroxysubstituted) or unsubstituted cycloalkyl. In an advantageoussubembodiment, Rx and R4are hydrogen, R3 is unsubstituted orsubstituted phenyl, and R5 and R6 are each alkyl. PreferablyR2 is mono-hydroxycyclopentyl or mono-hydroxycyclohexyl.also may be substituted with -NH-C(=O)E, wherein E issubstituted or unsubstituted Cx-C4 alkyl (e.g., alkylamine,e.g., ethylamine.).

Rx and R2 may also together form a substituted orunsubstituted heterocyclic ring, which may be substitutedwith an amine or acetamido group.

In cuiother aspect, R2 may be -A-NHC(=ü)o, wherein A isunsubstituted C1-C4 alkyl (e.g., ethyl, propyl, butyl), and Bis substituted or unsubstituted Cx-C4 alkyl (e.g., methyl,aminoalkyl, e.g., aminomethyl or aminoethyl, alkylamino,e.g., methylamino, ethylamino), preferably when Rx and R4 arehydrogen, R3 is unsubstituted or substituted phenyl, and R5and R6 are each alkyl. B may be substituted or unsubstitutedcycloalkyl^ e.g., cyclopropyl or 1-amino-cyclopropyl.

In another embodiment, R3may be substituted or unsubstitutedphenyl, preferably when R5 and R5 are each alkyl. Preferably, R3 may hâve one or more substituents (e.g., o-, m- or p- chlorophenyl, o-, m- or p- fluorophenyl).

Advantageously, R 3 may be substituted or unsubstitutedheteroaryl, preferably when R5 and R6 are e^ch alkyl.Examples of heteroaryl groups include pyridyl, pyrimidyl,pyridazinyl, pyrazinyl, pyrrolyl, triazolyl, thioazolyl,oxazolyl, oxadiazolyl, furanyl, methylenedioxyphenyl andthiophenyl. Preferably, £is 2-pyridyl, 3-pyridyl, 4-pyridyl,2-pyrimidyl or 3- pyrimidyl.

Preferably in one embodiment, R5 and R6 are each hydrogen. Inanother, R5 and R6 are each methyl. 26 013295

In a particularly preferred embodiment, the deazapurines ofthe invention are water-soluble prodrugs that can bemetabolized in vivo to an active drug, e.g. by esterasecatalyzed hydrolysis. Preferably the prodrug comprises an R2group which is cycloalkyl substituted with -OC (0) (Z)NH2,wherein Z is a side chain of a naturally or unnaturallyoccurring amino acid, an analog thereof, an a, β, γ, or ωamino acid, or a dipeptide. Examples of preferred sidechains include the side chains of glycine, alanine, valine,leucine, isoleucine, lysine, a-methylalanine, aminocyclopropane carboxylic acid, azetidine-2-carboxylicacid, β-alanine, γ-aminobutyric acid, alanine-alanine, orglycine-alanine.

In a particularly preferred embodiment, Z is a side chain ofglycine, R2 is cyclohexyl, R 3 is phenyl, and Rg and Rg aremethyl.

In another embodiment, the deazapurine is 4-( cis-3- hydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl- 7H-pyrrolo[2,3d]pyrimidine.

In another embodiment, the deazapurine is 4-(cis-3-(2-aminoacetoxy) cyclopentyl)amino-5,6-dimethyl-2-phenyl- 7H-pyrrolo[2,3d] pyrimidine trifluoroacetic acid sait.

In another embodiment, the deazapurine is 4—(3—acetamido) piperidinyl-5,6-dimethyl-2-phenyl7ii-pyrrolo [2,3d]pyrimidine.

In another embodiment, the deazapurine is 4-(2-N'-methylureapropyl)amino-5,6-dimethyl-2-phenyl7iî-pyrrolo[2,3d]pyrimidine.

In another embodiment, the deazapurine is 4-(2-acetamidobutyl) amino-5,6-dimethyl-2-phenyl-7tf-pyrrolo [2,3d]pyrimidine. 27 013295

In another embodiment, the deazapurine is 4-(2-N'-methylureabutyl ) amino-5,6-dimethyl-2-phenyl-7Ji-pyrrolo [2,3d]pyrimidine. 5 In another embodiment, the deazapurineaminocyclopropylacetamidoethyl)amino-2-phenyl-[2,3d]pyrimidine. is 4-(2-7H-pyrrolo

In another embodiment, the deazapurine is 4-(trans-4-10 hydroxycyclohexyl)amino-2- (3-chlorophenyl) - 7if-pyrrolo [2,3d] pyrimidine.

In another embodiment, the deazapurine is 4-(trans-4-hydroxycyclohexyl) amino-2 - (3-f luorophenyl) -7H-pyrrolo [2,3d] 15 pyrimidine.

In another embodiment, the deazapurine is 4-(trans-4-hydroxycyclohexyl) amino-2 - (4-pyridyl) - 7H-pyrrolo [2,3d]pyrimidine. 20

In yet another embodiment, the invention features a methodfor inhibiting the activity of an adenosine receptor (e.g.,Ax, A2a, A2b, or, preferably, A3) in a cell, by contacting thecell with N-6 substituted 7-deazapurine (e.g., preferably, an 25 adenosine receptor antagonist).

In another aspect, the invention features a method fortreating damage to the eye of an animal ( e.g., a human) byadministering to the animal an effective amount of an N-6 30 substituted 7-deazapurine. Preferably, the N-6 substituted7-deazapurine is an antagonist of A3 adenosine receptors incells of the animal. The damage is to the retina or theoptic nerve head and may be acute or chronic. The damage maybe the resuit of, for example, glaucoma, edema, ischemia, 35 hypoxia or trauma.

In a preferred embodiment, the invention features adeazapurine having the formula II, supra, wherein X is N or 28 013295 CR6; Rt and R2 are each independently hydrogen, or substitutedor unsubstituted alkoxy, aminoalkyl, alkyl, aryl, oralkylaryl, or together form a substituted or unsubstitutedheterocyclic ring, provided that both Rx and R2 are both nothydrogen; R3 is substituted or unsubstituted alkyl,arylalkyl, or aryl; R4 is hydrogen or substituted orunsubstituted C^-Cg alkyl; L is hydrogen, substituted orunsubstituted alkyl, or R4 and L together form a substitutedor unsubstituted heterocyclic or carbocyclic ring; R6 ishydrogen, substituted or unsubstituted alkyl, or halogen; Qis CH2, 0, S, or NR7, wherein R7 is hydrogen or substituted orunsubstituted C1-C6 alkyl; and W is unsubstituted orsubstituted alkyl, cycloalkyl, alkynyl, aryl, arylalkyl,biaryl, heteroaryl, substituted carbonyl, substitutedthiocarbonyl, or substituted sulfonyl, provided that if R3 ispyrrolidino, then R4 is not methyl.

In one embodiment, in compounds of formula II, X is CR6 andQ is CH2, 0, S, or NH. In another embodiment, X is N.

In a further embodiment of compounds of formula II, W issubstituted or unsubstituted aryl, 5- or 6- memberheteroaryl, or biaryl. W may be substituted with one or moresubstituents. Examples of substituents include: halogen,hydroxy, alkoxy, amino, aminoalkyl, aminocarboxyamide, CN,CF3, CO2R8, CONHR8, CONRgRg, SORg, SO2RB, and SO2NR8R9, whereinR8 and Rgare'each independently hydrogen, or substituted orunsubstituted alkyl, cycloalkyl, aryl, or arylalkyl.Preferably, W may be substituted or unsubstituted phenyl,e.g., methylenedioxyphenyl. W also may be a substituted orunsubstituted 5-membered heteroaryl ring, e.g., pyrrole,pyrazole, oxazole, imidazole, triazole, tetrazole, furan,thiophene, thiazole, and oxadiazole. Preferably, W may be a6-member heteroaryl ring, e.g., pyridyl, pyrimidyl, pyridazinyl, pyrazinal, and thiophenyl. In a preferrédembodiment, W is 2-pyridyl, 3- pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl,,or 5-pyrimidyl. 29 013295

In one advantageous embodiment of compounds of formula II, Qis NH and W is a 3-pyrazolo ring which is unsubstituted or N-substituted by substituted or unsubstituted alkyl,cycloalkyl, aryl, or arylalkyl.

In another embodiment of compounds of formula II, Q isoxygen, and W is a 2-thiazolo ring which is unsubstituted orsubstituted by substituted or unsubstituted alkyl,cycloalkyl, aryl, or arylalkyl.

In another embodiment of compounds of formula II, W issubstituted or unsubstituted alkyl, cycloalkyl e.g.,cyclopentyl, or arylalkyl. Examples of substituents includehaloyeu, hydxoxy , substituted or unsubstituted alkyl,cycloalkyl, aryl, arylalkyl, orNHR10, wherein R10 is hydrogen,or substituted or unsubstituted alkyl, cycloalkyl, aryl, orarylalkyl.

In yet another embodiment, the invention features adeazapurine of formula II wherein W is - (CH2) a-C (=0) Y or- (CH2) a-C (=S) Y, and a is an integer from 0 to 3, Y is aryl,alkyl, arylalkyl, cycloalkyl, heteroaryl, alkynyl, NHRUR12,or, provided that Q is NH, OR13, wherein Rn, R12 and R13 areeach independently hydrogen, or unsubstituted or substitutedalkyl, aryl, arylalkyl, or cycloalkyl. Preferably, Y is a 5-or 6- member heteroaryl ring.

Furthermore, W may be - (CH2) b-S (=0) jY, wherein j is 1 or 2, bis 0, 1, 2, or 3, Y is aryl, alkyl, arylalkyl, cycloalkyl,alkynyl, heteroaryl, NHR14R15, provided that when b is 1, Q isCH2, , and wherein R14, R15, and R16 are each independentlyhydrogen, or unsubstituted or substituted alkyl, aryl,arylalkyl, or cycloalkyl.

In another embodiment, R3 is selected from the groupconsisting of substituted and unsubstituted phenyl, pyridyl,pyrimidyl, pyridazinyl, pyrazinal, pyrrolyl, triazolyl, 30 013295 thioazolyl, oxazolyl, oxadiazolyl, pyrazolyl, furanyl,methylenedioxyphenyl, and thiophenyl. When R3 is phenyl, itmay be substituted with, for example, hydroxyl, alkoxy (e.g.,methoxy), alkyl (e.g., tolyl), and halogen,(e.g., o-, ia-, orp- fluorophenyl or o-, m-, or p- chlorophenyl).Advantageously, R3 may be 2-, 3-, or 4- pyridyl or 2- or 3-pyrimidyl.

The invention also pertains to a deazapurine wherein R6 ishydrogen or C1-C3 alkyl. Preferably, R6 is hydrogen.

The invention also includes deazapurines wherein R3 ishydrogen, and R2 is substituted or unsubstituted alkyl oralkoxy, substituted or unsubstituted alkylamine, ax y lamine,or alkylarylamine, substituted or unsubstituted aminoalkyl,amino aryl, or aminoalkylaryl, substituted or unsubstitutedalkylamide, arylamide or alkylarylami.de, substituted orunsubstituted alkylsulfonamide, arylsulfonamide or alkylarylsulfonamide, substituted or unsubstituted alkylurea,arylurea or alkylarylurea, substituted or unsubstitutedalkylcarbamate, arylcarbamate or alkylarylcarbamate, orsubstituted or unsubstituted alkylcarboxylic acid,arylcarboxylic acid or alkylarylcarboxylic acid.

Preferably, R2 is substituted or unsubstituted cycloalkyl,e.g., mono- or dihydroxy-substituted cyclohexyl orcyclopentyl (preferably, monohydroxy-substituted cyclohexylor monohydroxy-substituted cyclopentyl). 31 013295

Advantageously, R2 may be of the following formula:

or -A Â, wherein A is C^-Cg alkyl, C3-C7 cycloalkyl, a chain of one toseven atoms, or a ring of three to seven atoms, optionallysubstituted with Cj-Cg alkyl, halogens, hydroxyl, carboxyl,thiol, or amino groups; wherein B is methyl, N(Me)2, N(Et)2, NHMe, NHEt, (CH2)rNH3+, NH(CH2)rCH3, (CH^j-NH^ (CH2)rCHCH3NH2,(CHJrNHMe, (CH2)rOH, CH2CN, (CH2)inCO2H, CHR18Rig, or CHMeOH, 18λ19 ’ wherein r is an integer from 0 to 2, m is 1 or 2, R18 isalkyl, R19 is NH3 + or CO2H or R18 and Rig together are:

CH-NH \ /(CH2)p wherein p is 2 or 3; and R17 is C-^-Cg alkyl, C3-C7cycloalkyl, a chain of one to seven atoms, or a ring ofthree to seven atoms, optionally substituted with C1-C6alkyl, halogens, hydroxyl, carboxyl, thiol, or aminogroups.

Advantageously, A is unsubstituted or substituted Cj^-Cg alkyl.B may be unsubstituted or unsubstituted C-^-Cg alkyl.

In a preferred embodiment, R2 is of the formula -A-NHC(=O)B.In a particularly advantageous embodiment, A is -CH2CH2- andB is methyl.

The compounds of the invention may comprise water-solubleprodrugs which are mebabolized in vivo to an active drug,e.g., by esterase catalyzed hydrolysis. Examples ofpotential prodrugs include deazapurines with, for example, R2as cycloalkyl substituted with -OC(0) (Z)NH 2 , wherein Z is a 32 013295 side chain of a naturally or unnaturally occurring aminoacid, or analog thereof, an a, (3, γ, or ω amino acid, or adipeptide. Preferred amino acid side chains inclu.de those ofglycine, alanine, valine, leucine, isoleucine, lysine, a-methylalanine, aminocyclopropane carboxylic acid, azetidine-2-carboxylic acid, β-alanine, γ-aminobutyric acid, alanine-alanine, or glycine-alanine.

In another embodiment, Rj and R2 together are:

wherein n is 1 or 2, and wherein the ring may be optionallysubstituted with one or more hydroxyl, amino, thiol,carboxyl, halogen, CH2OH, CH2NHC(=0)alkyl, orCH2NHC(=0)NHalkyl groups. Preferably, n is 1 or 2 and saidring is substituted with -NHC(=0)alkyl.

In one advantageous embodiment, R! is hydrogen, R2 issubstituted or unsubstituted C^-Cg alkyl, R3 is substituted orunsubstituted phenyl, R4 is hydrogen, L is hydrogen orsubstituted or unsubstituted C^-Cg alkyl, Q is 0, S or NR7,wherein R7 is hydrogen or substituted or unsubstituted C^-Cgalkyl, and W is substituted or unsubstituted aryl.Preferably, R2 is -A-NHC(=O)B, wherein A and B are eachindependently unsubstituted or substituted C-j_-C4 alkyl. Forexample, A may be CH2CH2. B may be, for example, alkyl (e.g.,methyl), or aminoalkyl (e.g., aminomethyl). Preferably, R3is unsubstituted phenyl and L is hydrogen. R6 may be methylor preferably, hydrogen. Preferably, Q is 0, S, or NR7wherein R7 is hydrogen or substituted or unsubstituted Cj- C6alkyl, e.g., methyl. W is unsubstituted or substitutedphenyl (e.g., alkoxy, halogen substituted). Preferably, W is 33 013295 p-fluorophenyl, p-chlorophenyl, or p-methoxyphenyl. W mayalso be heteroaryl, e.g. , 2-pyridyl.

In a particularly prefêrred embodiment, the deazapurine is 4- (2-acetylaminoethyl) amino-6-phenoxymethyl-2-phenyl- 7H- pyrrolo[2,3d]pyrimidine.

In a particularly preferred embodiment, the deazapurine is 4-(2-acetylaminoethy1} amino-6-(4-fluorophenoxy)methyl-2- phenyl- 7Ji-pyrrolo [2,3d]pyrimidine.

In a particularly preferred embodiment, the deazapurine is4-(2-acetylaminoethyl) amino-6-(4-chlorophenoxy)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

In a particularly preferred embodiment, the deazapurine is 4-(2-acetylaminoethyl) amino-6-(4-methoxyphenoxy)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

In a particularly preferred embodiment, the deazapurine is 4-(2-acetylaminoethyl) amino-6-(2-pyridyloxy)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

In a particularly preferred embodiment, the deazapurine is 4-(2-acetylaminoethyl) amino-6-(N-phenylamino)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

In a particularly preferred embodiment, the deazapurine is 4-(2-acetylaminoethyl) amino-6- (N-methyl-N-phenylamino)methyl-2-phenyl-77ί-pyrrolo[2,3d]pyrimidine.

In a particularly preferred embodiment, the deazapurine is 4-(2-N'-methylureaethyl) amino-6-phenoxymethyl-2-phenyl- 7H-pyrrolo[2,3d]pyrimidine.

The invention further pertains to a method for inhibiting theactivity of an adenosine receptor (e.g., an A2b adenosinereceptor) in a cell by contacting the cell with a compound of 34 013295 the invention. Preferably, the compound is an antagonist ofthe receptor.

The invention also pertains to a method for treating agastrointestinal disorder (e.g., diarrhea) in an animal byadministering to an animal an effective amount of a compoundof the invention (e.g., an antagonist of A2b) . Preferably,the animal is a human.

In another embodiment, the invention relates to apharmaceutical composition containing an N-6 substituted 7-deazapurine of the invention and a pharmaceuticallyacceptable carrier.

The invention also pertains to a method for treating a N-6substituted 7-deazapurine responsive State in an animal, byadministering to a mammal a therapeutically effective amountof a deazapurine of the invention, such that treatment of aN-6 substituted 7-deazapurine responsive State in the animaloccurs. Advantageously, the disease State may be a disordermediated by adenosine. Examples of preferred disease Statesinclude: central nervous System disorders, cardiovasculardisorders, rénal disorders, inflammatory disorders, allergiedisorders, gastrointestinal disorders, eye disorders, andrespiratory disorders.

The term "alkyl" refers to the radical of saturated aliphaticgroups, including straight-chain alkyl groups, branched-chainalkyl groups, cycloalkyl (alicyclic) groups, alkylsubstituted cycloalkyl groups, and cycloalkyl substitutedalkyl groups. The term alkyl further includes alkyl groups,which can further include oxygen, nitrogen, sulfur orphosphorous atoms replacing one or more carbons of thehydrocarbon backbone, e.g., oxygen, nitrogen, sulfur orphosphorous atoms. In preferred embodiments, a straightchain or branched chain alkyl has 30 or fewer carbon atoms inits backbone ( e.g., C^-C^q for straight chain, C3-C3Q forbranched chain), and more preferably 20 or fewer. Likewise, 35 013295 preferred cycloalkyls hâve from 4-10 carbon atoms in theirring structure, and more preferably hâve 5, 6 or 7 carbons inthe ring structure.

Moreover, the term alkyl as used throughout the spécificationand daims is intended to include both "unsubstituted alkyls"and "substituted alkyls", the latter of which refers to alkylmoieties having substituents replacing a hydrogen on one ormore carbons of the hydrocarbon backbone. Such substituentscan include, for example, halogen, hydroxyl,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, antino (includingalkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino,sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl,cyano, azido, heterocyclyl, alkylaryl, or an aromatic orheteroaromatic moiety. It will be understood by thoseskilled in the art that the moieties substituted on thehydrocarbon chain can themselves be substituted, ifappropriate. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An "alkylaryl" moietyis an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). The term "alkyl" also includes unsaturatedaliphatic groups analogous in length and possiblesubstitution to the alkyls described above, but that containat least one double or triple bond respectively.

The term "aryl" as used herein, refers to the radical of arylgroups, including 5- and 6-merabered single-ring aromaticgroups that may include from zéro to four heteroatoms, forexample, benzene, pyrrole, furan, thiophene, imidazole,benzoxazole, benzothiazole, triazole, tetrazole, pyrazole,pyridine, pyrazine, pyridazine and pyrimidine, and the like. - 36 013295

Aryl groups also include polycyclic fused aromatic groupssuch as naphthyl, quinolyl, indolyl, and the like. Thosearyl groups having heteroatoms in the ring structure may alsobe referred to as "aryl heterocycles'1, "heteroaryls" or"heteroaromatics”. The aromatic ring can be substituted atone or more ring positions with such substituents asdescribed above, as for example, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino,sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl,cyano, azido, heterocyclyl, alkylaryl, or an aromatic orheteroaromatic moiety. Aryl groups can also be fused orbridged with alicyclic or heterocyclic rings which are notaromatic so as to form a polycycle (e.g., tetralin).

The terms "alkenyl" and "alkynyl" refer to unsaturatedaliphatic groups analogous in length and possiblesubstitution to the alkyls described above, but that containat least one double or triple bond respectively. Forexample, the invention contemplâtes cyano and propargylgroups.

Unless the number of carbons is otherwise specified, "loweralkyl" as used hêrein means an alkyl group, as defined above,but having from one to ten carbons, more preferably from oneto six carbon atoms in its backbone structure, even morepreferably one to three carbon atoms in its backbonestructure. Likewise, "lower alkenyl" and "lower alkynyl"hâve similar chain lengths.

The terms "alkoxyalkyl", "polyaminoalkyl" and"thioalkoxyalkyl" refer to alkyl groups, as described above, 37 013295 which further include oxygen, nitrogen or sulfur atomsreplacing one or more cardons of the hydrocarbon backbone,e.g., oxygen, nitrogen or sulfur atoms.

The terms "polycyclyl" or "polycyclic radical" refer to theradical of two or more cyclic rings ( e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) inwhich two or more carbons are common to two adjoining rings,e.g., the rings are "fused rings". Rings that are joinedthrough non-adjacent atoms are termed "bridged" rings. Eachof the rings of the polycycle can be substituted with suchsubstituents as described above, as for example, halogen,hydroxyl, alkylcarbonyloxy, ary1carbony1oxy, alkoxycarbonyloxy, aryloxÿcarbonyloxy, carboxylate,alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino,carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkyl, alkylaryl, or an aromatic orheteroaromatic moiety.

The term "heteroatom" as used herein means an atom of anyelement other than carbon or hydrogen. Preferred heteroatomsare nitrogen, oxygen, sulfur and phosphoruS.

The term "amino acids" includes naturally and unnaturallyoccurring amino acids found in proteins such as glycine,alanine, valine, cysteine, leucine, isoleucine, serine,threonine, méthionine, glutamic acid, aspartic acid,glutamine, asparagine, lysine, arginine, proline, histidine,phenylalanine, tyrosine, and tryptophan. Amino acid analogsinclude amino acids with lengthened or shortened side chainsor variant side chains with appropriate functional groups. 38 013295

Amino acids also include D and L stereoisomers of an aminoacid when the structure of the amino acid admits ofstereoisomeric forme. The term "dipeptide" includes two ormore amino acids linked together. Preferably, dipeptides aretwo amino acids linked via a peptide linkage. Particularlypreferred dipeptides include, for example, alanine-alanineand glycine-alanine.

It will be noted that the structure of some of the compoundsof this invention includes asymmetric carbon atoms and thusoccur as racemates and racemic mixtures, single enantiomers,diastereomeric mixtures and individual diastereomers. Ailsuch isomeric forms of these compounds are expressly includedin this invention. Each stereogenic carbon may be of the Ror S configuration. It is to be understood accordingly thatthe isomers arising from such asymmetry (e.g., ailenantiomers and diastereomers) are included within the scopeof this invention, unless indicated otherwise. Such isomerscan be obtained in substantially pure form by classicalséparation techniques and by stereochemically controlledsynthesis.

The invention further pertains to pharmaceutical compositionsfor treating a N-6 substituted 7-deazapurine responsive Statein a mammal, e.g., respiratory disorders ( e.g., asthma, bronchitis, chronic obstructive pulmonary disorder, andallergie rhinitis), rénal disorders, gastrointestinaldisorders, and eye disorders . The pharmaceutical compositionincludes a therapeutically effective amount of a N-6substituted 7-deazapurine, described supra, and apharmaceutically acceptable carrier. It is to be understood,that ail of the deazapurines described above are included fortherapeutic treatment. It is to be further understood thatthe deazapurines of the invention can be used alone or incombination with other deazapurines of the invention or incombination with additional therapeutic compounds, such asantibiotics, antiinflammatories, or anticancer agents, for 39 013295 example.

The term "antibiotic" is art recognized and is intended toinclude those substances produced by growing microorganisms 5 and synthetic dérivatives thereof, which eliminate or inhibitgrowth of pathogens and are selectively toxic to the pathogenwhile producing minimal or no deleterious effects upon theinfected host subject. Suitable examples of antibioticsinclude, but are not limited to, the principle classes of 10 aminoglycosides, cephalosporins, chloramphenicols, fuscidicacids, macrolides, penicillins, polymixins, tetracyc'lines andstreptomycins.

The term "antiinflammatory" is art recognized ana is intended 15 to include those agents which act on body mechanisms, withoutdirectly antagonizing the causative agent of the inflammationsuch as glucocorticoids, aspirin, ibuprofen, NSAIDS, etc.

The term "anticancer agent" is art recognized and is intended 20 to include those agents which diminish, eradicate, or preventgrowth of cancer cells without, preferably, adverselyaffecting other physiological functions. Représentativeexamples include cisplatin and cyclophosphamide. 25 When the compounds of the présent invention are administeredas pharmaceuticals, to humans and mammals, they can be givenper se or as a pharmaceutical composition containing, forexample, 0.1 to 99.5% (more preferably, 0.5 to 90%) of activeingrédient in combination with a pharmaceutically acceptable 30 carrier.

The phrase "pharmaceutically acceptable carrier" as usedherein means a pharmaceutically acceptable material,composition or vehicle, such as a liquid or solid filler, 35 diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of theprésent invention within or to the subject such that it canperforms its intended function. Typically, such compounds 40 013295 are carried or transported from one organ, or portion of thebody, to another orgari, or portion of the body. Each carriermust be "acceptable" in the sense of being compatible withthe other ingrédients of the formulation and not injurious tothe patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: sugars, such aslactose, glucose and sucrose; starches, such as corn starchand potato starch; cellulose, and its dérivatives, such assodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatin; talc;excipients, such as cocoa butter and suppository waxes; oils,such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil and soybean oil; glycols, such aspropylene glycol; polyols, such as giycerin, sorbitol,mannitol and polyethylene glycol; esters, such as ethyloleate and ethyl laurate; agar; buffering agents, such asmagnésium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonie saline; Ringer's solution; ethylalcohol; phosphate buffer solutions; and other non-toxiccompatible substances employed in pharmaceuticalformulations.

As set out above, certain embodiments of the présentcompounds can contain a basic functional group, such as aminoor alkylamino, and are, thus, capable of formingpharmaceutically acceptable salts with pharmaceuticallyacceptable acids. The term "pharmaceutically acceptablesalts" in this respect, refers to the relatively non-toxic,inorganic and organic acid addition salts of compounds of theprésent invention. These salts can be prepared in situduring the final isolation and purification of the compoundsof the invention, or by separately reacting a purifiedcompound of thé invention in its free base form with asuitable organic or inorganic acid, and isolating the saitthus formed. Représentative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stéarate, laurate,benzoate, lactate, phosphate, tosylate, citrate, maleate, 41 013295 fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts andthe like. (See, e.g., Berge et al. (1977) "PharmaceuticalSalts", J. Pharm. Scï. 66:1-19).

In other cases, the compounds of the présent invention maycontain one or more acidic functional groups and, thus, arecapable of forming pharmaceutically acceptable salts withpharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these instances refersto the relatively non-toxic, inorganic and organic baseaddition salts of compounds of the présent invention. Thesesalts can likewise be prepared in situ during the finalisolation and purification of the compounds, or by separatelyreacting the purified compound in its free acid form with asuitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable métal cation,with ammonia, or with a pharmaceutically acceptable organicprimary, secondary or tertiary amine. Représentative alkalior alkaline earth salts include the lithium, sodium,potassium, calcium, magnésium, and aluminum salts and thelike. Représentative organic amines useful for the formationof base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine andthe like.

The term "pharmaceutically acceptable esters" refers to therelatively non-toxic, esterified products of the compounds ofthe présent invention. These esters can be prepared in situduring the final isolation and purification of the compounds,or by separately reacting the purified compound in its freeacid form or hydroxyl with a suitable esterifying agent.Carboxylic acids can be converted into esters via treatmentwith an alcohol in the presence of a catalyst. Hydroxylcontaining dérivatives can be converted into esters via treatment with an esterifying agent such as alkanoyl halides.The term is further inÇended to include lower hydrocarbongroups capable of being solvated under physiological 42 - 013295 conditions, e.g., alkyl esters, methyl, ethyl and propylesters, (Seë,: for example, Berge et al., supra.)

The invention further contemplâtes the use of prodrugs whichare converted in vivo to the therapeutic compounds of theinvention (see, e.g., R.B. Silverman, 1992, "The OrganicChemistry of Drug Design and Drug Action", Academie Press,Chapter 8) . Such prodrugs can be used to alter thebiodistribution (e.g., to allow compounds which would nottypically enter the reactive site of the protease) or thepharmacokinetics of the therapeutic compound. For example,a carboxylic acid group, can be esterified, e.g., with amethyl group or an ethyl group to yield an ester. When theester is administered to a subject, the ester is cleaved,enzymatically or non-enzymatically, reductively orhydrolytically, to reveal the anionic group. An anionicgroup can be esterified with moieties (e.g., acyloxymethylesters) which are cleaved to reveal an intermediate compoundwhich subseguently décomposés to yield the active compound.

In another embodiment, the prodrug is a reduced form of asulfate or sulfonate, e.g., a thiol, which is oxidized invivo to the therapeutic compound. Furthermore, an anionicmoiety can be esterified to a group which is activelytransported in vivo, or which is selectively takën up bytarget organs. The ester can be selected to allow spécifietargeting of the therapeutic moieties to particular reactivesites, as described below for carrier moieties.

Wetting agents, emulsifiers and lubricants, such as sodiumlauryl sulfate and magnésium stéarate, as well as coloringagents, release agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can alsobe présent in the compositions.

Examples of pharmaceutically acceptable antioxidants include:water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodiumsulfite and the like; oil-soluble antioxidants, such as 43 013295 ascorbyl palmitate, butylated hydroxyanisole (BHA) , butylatedhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and métal chelating agents, such ascitric acid, ethylenediamine te’traacetic acid (EDTA) ,sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations of the présent invention include those suitablefor oral, nasal, topical, transdermal, buccal, sublingual,rectal, vaginal and/or parentéral administration. Theformulations may conveniently be presented in unit dosageform and may be prepared by any methods well known in the artof pharmacy. The amount of active ingrédient which can becombined with a carrier material to produce a single dosageform will generally be that amount of the compcund whichproduces a therapeutic effect. Generally, out of one hundredper cent, this amount will range from about 1 per cent toabout ninety-nine percent of active ingrédient, preferablyfrom about 5 per cent to about 70 per cent, most preferablyfrom about 10 per cent to about 30 per cent.

Methods of preparing these formulations or compositionsinclude the step of bringing into association a compound ofthe présent invention with the carrier and, optionally, oneor more accessory ingrédients. In general, the formulationsare prepared by uniformly and intimately bringing intoassociation a compound of the présent invention with liquidcarriers, or finely divided solid carriers, or both, andthen, if necessary, shaping the product.

Formulations of the invention suitable for oraladministration may be in the form of capsules, cachets,pills, tablets, lozenges (using a flavored basis, usuallysucrose and acacia or tragacanth), powders, granules, or asa solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquidémulsion, or as an élixir or syrup, or as pastilles (using aninert base, such as gelatin and glycerin, or sucrose andacacia) and/or as mouth washes and the like, each containing 44 013295 a predetermined amount of a compound of the présent inventionas an active ingrédient. A compound of the présent inventionmay also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration (capsulés, tablets, pills, dragees, powders,granules and the like), the active ingrédient is mixed withone or more pharmaceutically acceptable carriers, such assodium citrate or dicalcium phosphate, and/or any of thefollowing: fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; binders,such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia;humectants, such as glyceroi; disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate;solution retarding agents, such as paraffin; absorptionaccelerators, such as quaternary ammonium compounds; wettingagents, such as, for example, cetyl alcohol and glyceroimonostearate; absorbents, such as kaolin and bentonite clay;lubricants, such a talc, calcium stéarate, magnésiumstéarate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof; and coloring agents. In the case ofcapsules, tablets and pills, the pharmaceutical compositionsmay also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft andhard-filled gelatin capsules using such excipients as lactoseor milk sugars, as well as high molecular weight polyethyleneglycols and the like. A tablet may be made by compression or molding, optionallywith one or more accessory ingrédients. Compressed tabletsmay be prepared using binder (for example, gelatin orhydroxypropylmethyl cellulose), lubricant, inert diluent,preservative, disintegrant (for example, sodium starchglycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may bemade by molding in a suitable machine a mixture of the 01329 5 45 powdered compound moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of thepharmaceutical compositions of the présent invention, such asdragees, capsules, pills and granules, may optionallÿ bescored or prepared with coatings and shells, such as entericcoatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provideslow or controlled release of the active ingrédient thereinusing, for example, hydroxypropylmethyl cellulose in varyingproportions to provide the desired release profile, otherpolymer matrices, liposomes and/or microspheres. They may besteriîized by, for example, filtration through a bacteria-retaining fiïter, or by ineorporatiixg üterilizing agents inthe form of stérile solid compositions which can be dissolvedin stérile water, or some other stérile injectable mediumimmediately before use. These compositions may alsooptionallÿ contain opacifying agents and may be of acomposition that they release the active ingrédient(s) only,or preferentially, in a certain portion of thegastrointestinal tract, optionallÿ, in a delayed manner.Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingrédient canalso be in micro-encapsulated form, if appropriate, with oneor more of the above-described excipients.

Liquid dosage forms for oral administration of the compoundsof the invention include pharmaceutically acceptableémulsions, microemulsions, solutions, suspensions, syrups andélixirs. In addition to the active ingrédient, the liquiddosage forms may contain inert dilutents commonly used in theart, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylèneglycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol,tetrahydrofuryl alcohol, polyethylene glycols and fatty acid 46 013295 esters of sorbitan, and mixtures thereof.

Besides inert dilutents, the oral compositions can alsoinclude adjuvants such as wetting agents, emulsifying andsuspending agents, sweetening, flavoring, coloring, perfumingand preservative agents.

Suspensions, in addition to the active compounds, may containsuspending agents as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations ot the pharmaceutical compositions of theinvention for rectal or vaginal administration may bepresented as a suppository, which may be prepared by mixingone or more compounds of the invention with one or moresuitable nonirritating excipients or carriers comprising, forexample, cocoa butter, polyethylene glycol, a suppository waxor a salicylate, and which is solid at room température, butliquid at body température and, therefore, will melt in therectum or vaginal cavity and release the active compound.

Formulations of the présent invention which are suitable forvaginal administration also include pessaries, tampons,creams, gels, pastes, foams or spray formulations containingsuch carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration ofa. compound of this invention include powders, sprays,ointments, pastes, creams, lotions, gels, solutions, patchesand inhalants. The active compound may be mixed under stérileconditions with a pharmaçeutically acceptable carrier, andwith any preservatives, buffers, or propellants which may berequired.

The ointments, pastes, creams and gels may contain, inaddition to an active compound of this invention, excipients, 47 013295 such as animal and vegetable fats, oils, waxes, paraffins,starch, tragacanth, cellulose dérivatives, polyethyleneglycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof. wïw-r*·®*''

Powders and sprays can contain, in addition to a compound ofthis invention, excipients such as lactose, talc, silicicacid, aluminum hydroxide, calcium silicates and polyamidepowder, or mixtures of these substances. Sprays canadditionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane.

Transdermal patches hâve the added advantage of providingcontrolled delivery of a compound of the présent invention tothe body. Such dosage forms can be made by dissolving ordispersing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of thecompound across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane ordispersing the active compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutionsand the like, are also contemplated as being within the scopeof this invention. Preferably, the pharmaceutical préparation is an ophthalmic formulation (e.g., anperiocular, retrobulbar or intraocular injection formulation,a systemic formulation, or a surgical irrigating solution).

The ophthalmic formulations of the présent invention.may include one or more deazapurines and a pharmaceuticallyacceptable vehicle. Various types of vehicles may be used.

The vehicles will generally be aqueous in nature. Aqueoussolutions are generally preferred, based on case offormulation, as well as a patient's ability to easilyadminister such compositions by means of instilling one totwo drops of the solutions in the affected eyes. However,the deazapurines of the présent invention may also be readily 48 013295 incorporated into other types of compositions, such assuspensions, viscous or semi-viscous gels or other types ofsolid or semi-solid compositions. The ophthalmic compositions of the présent invention may also includevarious other ingrédients, such as buffers, preservatives,co-solvents and viscosity building agents.

An appropriate buffer System (e.g., sodium phosphate, sodiumacetate or sodium borate) may be added to prevent pH driftunder storage conditions.

Ophthalmic products are typically packaged in multidose form.Preservatives are thus required to prevent microbialcontamination during use. Suitable preservatives includeibenzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propyl paraben, phenylethyl alcohol, edetatedisodium, sorbic acid, polyquaternium-1, or other agentsknown to those skilled in the art. Such preservatives aretypically employed at a level of from 0.001 to 1.0%weight/volume ("% w/v").

When the deazapurines of the présent invention areadministered during intraocular surgical procedures, such asthrough retrobulbar or periocular injection and intraocularperfusion or injection, the use of balanced sait irrigatingsolutions as vehicles are most preferred. BSS® StérileIrrigating Solution and BSS Plus® Stérile IntraocularIrrigating Solution (Alcon Laboratories, Inc., Fort Worth,Texas, USA) are examples of physiologically balancedintraocular irrigating solutions. The latter type ofsolution is described in U. S. Pat. No. 4,550,022 (Garabedian,et al.), the entire contents of which are hereby incorporatedin the présent spécification by reference. Retrobulbar andperiocular injections are known to those skilled in the artand are described in numerous publications including, forexample, Ophthalmic Surgery: Principles of Practice,Ed. , G.L. Spaeth. W. B. Sanders Co., Philadelphia, Pa. , U.S.A.,pages 85-87 (1990). 49 013295

As indicated above, use of deazapurines to prevent or reducedamage to retinal and optic nerve head tissues at thecellular level is a particularly important aspect of oneembodiment of the invention. Ophthalmic conditions which maybe treated include, but are not limited to, rétinopathies,macular degeneration, ocular ischemia, glaucoma, and damageassociated with injuries to ophthalmic tissues, such asischemia reperfusion injuries, photochemical injuries, andinjuries associated with ocular surgery, particularlyinjuries to the retina or optic nerve head by exposure tolight or surgical instruments. The compounds may also beused as an adjunct to ophthalmic surgery, such as by vitrealor subconjunctival injection following ophthalmic surgery.The compounds may be used for acate · treaLnieiit of temporaryconditions, or may be administered chronically, especially inthe case of degenerative disease. The compounds may also beused prophylactically, especially prior to ocular surgery ornoninvasive ophthalmic procedures, or other types of surgery.

Pharmaceutical compositions of this invention suitable forparentéral administration comprise one or more compounds ofthe invention in combination with one or morepharmaceutically acceptable stérile isotonie aqueous ornonaqueous solutions, dispersions, suspensions or émulsions,or stérile powders which may be reconstituted into stérileinjectable solutions or dispersions just prior to use, whichmay contain antioxidants, buffers, bacteriostats, solutéswhich render the formulation isotonie with the blood of theintended récipient or suspending or thickening agents. Ëxamples of suitable aqueous and nonaqueous carriers whichmay be employed in the pharmaceutical compositions of theinvention include water, éthanol, polyols (such as glycerol,propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil,and injectable organic esters, such as ethyl oleate. Properfluidity can be maintained, for example, by the use ofcoating materials, such as lecithin, by the maintenance of - 50 013295 the required particle size in the case of dispersions, and bythe_ use of surfactants.

These compositions may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents anddispersing agents. Prévention of the action of microorganismsmay be ensured by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol,phénol sorbic acid, and the like. It may also be désirable toinclude isotonie agents, such as sugars, sodium chloride, andthe like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may bebrought about by the inclusion of agents which delayabsorption such as aluminum monostearate and gelâtin.

In some cases, in order to prolong the effect of a drug, itis désirable to slow the absorption of the drug fromsubeutaneous or intramuscular injection. This may beaccomplished by the use of a liquid suspension of crystallineor amorphous material having poor water solubility. The rateof absorption of the drug then dépends upon its rate ofdissolution which, in turn, may dépend upon crystal size andcrystalline form. Alternatively, delayed absorption of aparenterally-administered drug form is accomplished bydissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsulematrices of the subject compounds in biodégradable polymerssuch as polylactide-polyglycolide. Depending on the ratio ofdrug to polymer, and the nature of the particular polymeremployed, the rate of drug release can be controlled.Examples of other biodégradable polymers includepoly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug inliposomes or microemulsions which are compatible with bodytissue.

The préparations of the présent invention may be given - 51 013295 orally, parenterally, topically, or rectally. They are ofcourse given by forms suitable for each administration route.For example, they are administered in tablets or capsuleform, by injection, inhalation, eye lotion, ointment,suppository, etc. administration by injection, infusion orinhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administration is preferred.

The phrases "parentéral administration" and "administeredparenterally" as used herein means modes of administrationother than enterai and topical administration, usually byinjection, and includes, without limitation, intravenous,intramuscular, intraarterial, intrathécal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal,transtrachéal, subcutaneous, subcuticular, intraarticular,subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

The phrases "systemic administration," "administeredsystematically," "peripheral administration" and"administered peripherally" as used herein mean theadministration of a compound, drug or other material otherthan directly into the central nervous System, such that itenters the patient's System and, thus, is subject tometabolism and other like processes, for example,subcutaneous administration.

These compounds may be administered to humans and otheranimais for therapy by any suitable route of administration,including orally, nasally, as by, for example, a spray,rectally, intravaginally, parenterally, intracisternally andtopically, as by powders, ointments or drops,, includingbuccally and sublingually.

Regardless of the route of administration selected, thecompounds of the présent invention, which may be used in asuitable hydrated form, and/or the pharmaceuticalcompositions of the présent invention, are formulated into 52 013295 pharmaceutically acceptable dosage forms by conventionalmethods known to those of skill in the art.

Actual dosage levels of the active ingrédients in thepharmaceutical compositions of this invention may be variedso as to obtain an amount of the active ingrédient which iseffective to achieve the desired therapeutic response for aparticular patient, composition, and mode of administration,without being toxic to the .patient.

The selected dosage level will dépend upon a variety offactors including the activity of the particular compound ofthe présent invention employed, or the ester, sait or amidethereof, the route of administration, the time ofadministration, the rate of excrétion of the particularcompound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination withthe particular compound employed, the âge, sex, weight,condition, general health and prior medical history of thepatient being treated, and like factors well known in themedical arts. A physician or veterinarian having ordinary skill in the artcan readily détermine and prescribe the effective amount ofthe pharmaceutical composition reguired. For example, thephysician or veterinarian coula start doses of the compoundsof the invention employed in the pharmaceutical compositionat levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosageuntil the desired effect is achieved.

In general, a suitable daily dose of a compound of theinvention will be that amount of the compound which is thelowest dose effective to produce a therapeutic effect. Suchan effective dose will generally dépend upon the factorsdescribed above. Generally, intravenous and subcutaneousdoses of the compounds of this invention for a patient, whenused for the indicated analgésie effects, will range from 53 013295 about 0.0001 to about 200 mg per kilogram of body weight perday, more preferably from about 0.01 to about 150 mg per kgper day, and still more preferably from about 0.2 to about140 mg per kg per day.

If desired, the effective daily dose of the active compoundmay be administered as two, three, four, five, six or moresub-doses administered separately at appropriate intervalsthroughout the day, optionally, in unit dosage forms.

While it is possible for a compound of the présent inventionto be administered alone, it is préférable to administer thecompound as a pharmaceutical composition.

The présent invention also pertains to packagedpharmaceutical compositions for treating a N-6 substituted 7deazapurine responsive state, e.g., undesirable increasedadenosine receptor activity in a mammal. The packagedpharmaceutical compositions include a container holding atherapeutically effective amount of at least one deazapurineas described supra and instructions for using the deazapurinefor treating the deazapurine responsive State in the mammal.

The deazapurines of the invention can be prepared usingstandard methods for organic synthesis. Deazapurines can bepurified by reverse phase HPLC, chromatography,recrystallization, etc. and their structures confirmed bymass spectral analysis, elemental analysis, IR and/or NMRspectroscopy.

Typically, synthesis of the intermediates as well as thedeazapurines of the invention is performed in solution. Theaddition and removal of one or more protecting group is also 54 013295 typical practice and is known to those skilled in the art.Typical synthetic schemes for the préparation of deazapurineintermediates of the invention are outlined below in Scheme I.

This invention further provides a compound having thestructure (IV):

wherein Ri is trans-4-hydroxy cyclohexyl, 2-methylaminocarbonylamino cyclohexyl, acetylamino ethyl, ormethylamino carbonylamino ethyl; wherein R3 is a substituted or unsubstituted four to sixmembered ring, phenyl, pyrrole, thiophene, furan,thiazole, imidazole, pyrazole, 1,2,4-triazole, pyridine,2(1H)-pyridone, 4(1H)-pyridone, pyrazine, pyrimidine,pyridazine, isothiazole, isoxazole, oxazole, tetrazole,naphthalene, tetralin, naphthyridine, benzofuran,benzothiophene, indole, 2,3-dihydroindole, lH-indole,indoline, benzopyrazole, 1,3-benzodioxole, benzoxazole,purine, coumarin, chromone, quinoline,tetrahydroquinoline, isoquinoline, benzimidazole,quinazoline, pyrido[2,3-b]pyrazine, pyrido[3,4- b]pyrazine, pyrido[3,2-c]pyridazine, purido(3,4-b]- pyridine, lH-pyrazole[3,4-d]pyrimidine, pteridine,2(1H)-quinolone, 1(2H)-isoquinolone, 1,4-benzisoxazine,benzothiazole, quinoxaline, quinoline-N-oxide,isoquinoline-N-oxide, quinoxaline-N-oxide, quinazoline-N-oxide, benzoxazine, phthalazine, cinnoline, or havinga structure: 55 □13295

Y. K2’ wherein Y is carbon or nitrogen; wherein R2 and R2' are independently H, substituted orunsubstituted alkyl, substituted or unsubstituted aryl,halogen, methoxy, methyl amino, or methyl thio; whereinRs is H, alkyl, substituted alkyl, aryl, arylalkyl,amino, substituted aryl, wherein said substituted alkylis -C(R?) (R8)XR9, wherein X is 0, S, or NR10, wherein R7and Rb are each independently H or alkyl, wherein R9 andRio are each independently alkyl or cycloalkyl, or NR9R10is a substituted or unsubstituted ring of between 4 and7 members; wherein R6 is H, alkyl, substituted alkyl, cycloalkyl;or a pharmaceutically acceptable sait, a prodrugdérivative, or a biologically active métabolite, withproviso that when R 1 acetylamino ethyl, R3 is not 4-pyridyl.

In one embodiment of the compound having structure IV, NR9R10is a substituted or unsubstituted ring of between 4 and 7members which is selected from the group consisting of: 56 013295

wherein n is 0, 1, 2, or 3 ; wherein Re- is hydrogen, -OH,-CH2OH, -C ( =0) NR9R10, NHR11; wherein R11 is -C(=O)CH3, or-SO2Me, or

wherein R is H, alkyl, or aryl. 57 013295

In another embodiment of the compound having structure IV, R3has the structure:

wherein Y is carbon or nitrogen; wherein R 2 is H, orhalogen, -O-alkyl group, amine group, or sulfide group; wherein Rs is H, alkyl, substituted alkyl, aryl,arylalkyl, amino, substituted aryl, wherein saidsubstituted alkyl is -C (R?) (RslNRsRio, wherein R7 and Rsare each independently H or alkyl, wherein R9 and Rio areeach independently alkyl or cycloalkyl, or R 9, Rio andthe nitrogen together form a substituted orunsubstituted ring of between 4 and 7 members.

In another embodiment of the compound, Y is carbon. In another embodiment of the compound, R2 is hydrogen. In another embodiment of the compound, R6 is hydrogen. In another embodiment of the compound, Rs is hydrogen.

In another embodiment of the compound, Rs and R6 are eachmethyl.

In another embodiment of the compound, Rs is -C(R7) (Rs)NR9Rio,wherein R7 and Rs are each independently H or alkyl, wherein 013295 58 R.9 and Rio are each independently alkyl or cycloalkyl, or R9,Rio and the nitrogen together form a substituted orunsubstituted ring of between 4 and 7 members.

In another embodiment of the compound, R2 is halogen.

In another embodiment of the compound, Y is nitrogen.

In yet another embodiment of the compound, R2 is hydrogen.

In a further embodiment of the compound, R 5 and Re are eachhydrogen.

This invention also provides a compound having the structure(V) :

V wherein R3 is aryl, substituted aryl, or heteroaryl; wherein R6 is H, alkyl, substituted alkyl, or cycloalkyl;wherein Rs is H, alkyl, substituted alkyl, aryl,arylalkyl, amino, substituted aryl, wherein saidsubstituted alkyl is -C (R7) (Re)NR9Rio, wherein R7 and Reare each H or alkyl, wherein R9 and Rio are each alkyl orcycloalkyl, or R9, Rio and the nitrogen together form aring System of between 4 and 7 members. ü 1 329 5 59

In one embodiment of the compound having structure V, R7 andR.8 are each H; wherein Rs is H and Rio is -R12C ( =0) R13.

In another embodiment of the compound having structure V, R7and Rs are each H; wherein the ring System is morpholino,thiomorpholino, N-4-substituted piperazino, 2-substitutedpiperazine, or R8, substituted pyrrolidino, piperadine,wherein R8, is H, OH, CH2OH, -C (=0)NR9Rio, NR11, wherein R11 is-C(=O)CH3, -SO2Me.

In another embodiment of the compound, the compound has thefollowing structure:

(Compound 706)

In another embodiment of the compound, the compound has thestructure:

- 60 -

In another embodiment of the compound, the compound bas the structure: 013295

(Compound 1318-a)

In another embodiment of the compound, the compound has thestructure:

(Compound 1318-b) 61 013295

In another embodiment of the compound, the compound has the structure:

(Compound 1319)

In another embodiment of the compound, the compound has thestructure : 20

(Compound 1320) 35 013295 - 62 -

In another embodiment of the compound, the compound has thestructure:

(Compound 1321) A compound having the structure:

wherein R3 is a 5-6 membered aromatic ring; wherein R5and R6 are independently H, or alkyl. 63 013295

In one embodiment of the compound, the compound has the structure:

(Compound 1500)

In one embodiment of the compound, the compound has thestructure :

013295 64

In another embodiment of the compound, the compound has thestructure:

In another embodiment of compound 1500, the compound has the

In a further embodiment of the compound, the compound has thestructure:

013295 - 65 -

This invention also provides a compound having the structure:

wherein R3 is a 5-6 membered aromatic ring; whereiri R5and R6 are independently H or alkyl; with the provisothat R3 is not 4-pyridyl.

In one embodiment of the compound, the compound has thestructure :

(Compound 1501) 66 013295

This invention further provides a compound having thestructure:

VIII wherein R3 is a substituted 5-6 membered aromatic ring;wherein R5 and R6 are independently H, or alkyl.

In one embodiment of the compound, the compound has thestructure:

(Compound 1520)

This invention also provides a compound ’naving the structure: 013295 67

IX wherein R3 is a 5-6 membered aromatic ring; wherein X isoxygen, or sulfur.

In one embodiment of the compound, the compound has thestructure :

(Compound 1503) - 68 -

This invention also provides a compound having the structure: 013295

wherein R3 is a 5-6 membered aromatic ring; wherein X isoxygen, or sulfur.

In one embodiment of the compound, the compound has thestructure :

(Compound 1504) 013295 - 69 -

This invention further provides a method for treating . adisease associated with Ai adenosine receptor in a subject,comprising administering to the subject a therapeuticallyeffective amount of a compound having the formula IV, V, VI,VII, VIII, IX, or X.

In one embodiment of the method, the subject is a mammal. Inanother embodiment of the method, the mammal is a human.

In another embodiment of the method, the Ai adenosine receptoris associated with cognitive disease, rénal failure, cardiacarrhythmias, respiratory epithelia, transmitter release,sédation, vasoconstriction, bradycardia, négative cardiacinotropy and dromotropy, branchoconstriction, neutropilchemotaxis, reflux condition, or ulcerative condition.

This invention also provides a combination therapy forasthma, comprising compounds IV and V, and a steroid, β2agonist, glucocoticoid, lucotriene antagonist, oranticolinegic agonist. Diseases associated with adenosine.,AA2a, A2b and A3 receptors are disclosed in WO 99/06053 and WO-09822465, WO-09705138, WO-09511681, WO-09733879, JP-09291089,PCT/US98/16053 and U.S. Patent No. 5,516,894, the entirecontent of which are fully incorporate herein by reference.

This invention also provides a water-soluble prodrug of acompound having the structures IV, V, VI, VII, VIII, IX, orX, wherein said water-soluble prodrug that is metabolized invivo to an active drug which selectively inhibit Ai adenosinereceptor.

In one embodiment of the prodrug, said prodrug is metabolizedin vivo by esterase catalyzed hydrolysis.

This invention also provides a pharmaceutical compositioncomprising the prodrug and a pharmaceutically acceptablecarrier. 013295 - 70 -

This invention further provides a method for inhibiting theactivity of an Al adenosine receptor in a cell, whichcomprises contacting said cell with a compound having thestructures IV, V, VI, VII, VIII, IX, or X.

In one embodiment of the method, the compound is anantagonist of said Ai adenosine receptor.

This invention also provides for a method for treating agastrointestinal disorder in an subject, comprisingadministering to the an effective amount of a compound havingthe structures IV, V, VI, VII, VIII, IX, or X.

In one embodiment of the method, said disorder is diarrhea.

In another embodiment of the method, the subject is a human.

In another method of the method, the compound is anantagonist of Ai adenosine receptors.

This invention also provides a method for treatingrespiratory disorder in a subject, comprising administeringto the subject an effective amount of a compound having thestructures IV, V, VI, VII, VIII, IX, or X.

In one embodiment of the method, said disorder is asthma,chronic obstructive pulmonary disease, allergie rhinitis, oran upper respiratory disorder.

In another embodiment of the method, the subject is a human.

In another embodiment of the method, said compound is anantagonist of Al adenosine receptors.

This invention further provides a method for treating damageto the eye of a subject which comprises administering to saidsubject an effective amount of a compound having thestructures IV, V, VI, VII, VIII, IX, or X. 71 013295

In one embodiment of the method, said damage comprisesretinal or optic nerve head damage.

In another embodiment of the method, said damage is acute orchronic.

In another embodiment of the method, wherein said damage isthe result of glaucoma, edema, ischemia, hypoxia or trauma.

In another embodiment of the method, the subject is a human.

In another embodiment of the method, the compound is anantagonist of Ai adenosine receptors.

This invention also provides a pharmaceutical compositioncomprising a therapeutically effective amount of a compoundhaving the structures IV, V, VI, VII, VIII, IX, or X, and apharmaceutically acceptable carrier.

In another embodiment of the pharmaceutical composition, saidtherapeutically effective amount is effective to treat arespiratory disorder or a gastrointestinal disorder.

In another embodiment of the pharmaceutical composition, saidgastrointestinal disorder is diarrhea.

In another embodiment of the pharmaceutical composition, saidrespiratory disorder is asthma, allergie rhinitis, or chronicobstructive pulmonary disease.

In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is an ophthalmic formulation.

In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is an periocular, retrobulbar orintraocular injection formulation. _Ί2_ 01 329 5'

In yet another embodiment of the pharmaceutical composition,said pharmaceutical composition is a systemic formulation.

In a further embodiment of the pharmaceutical préparation, 5 said pharmaceutical composition is a surgical irrigatingsolution.

This invention also provides a packaged pharmaceuticalcomposition for treating a disease associated with A 1 10 adenosine receptor in a subject, comprising: (a) a containerholding a therapeutically effective amount of an adenosine Alspécifie compound; and (b) instructions for using saidcompound for treating said disease in a subject. 15 As used herein, "A compound is Α2 sélective." means that acompound has a binding constant to adenosine Al receptor ofat least ten time higher then that to adenosine A2a, A2b or A3.

This invention also provides a method of preparing the 20 compound having structure IV, comprising the steps of 73 013295

wherein P is a removable protecting group; b) treaîing the product of step a) under cycüzation conditions to provide

c) treating the product of step b) under suitable conditions to provide

d) treating the chlorinated product of step c) with NH2R1 to provide

74 013295 wherein Ri is trans-4-hydroxy cyclohexyl, 2-methylaminocarbonylamino cyclohexyl, acetylamino ethyl, ormethylamino carbonylamino ethyl; wherein R3 is a substituted or unsubstituted four to sixmembered ring; wherein Rs is H, alkyl, substituted alkyl, aryl,arylalkyl, amino, substituted aryl, wherein said substituted alkyl is -C(R7) (R8)XR9, wherein X is O, S, orNRio, wherein R7 and Rs are each independently H or alkyl,wherein R9 and Rio are each independently alkyl orcycloalkyl, or NR9R10 is a substituted or unsubstitutedring of between 4 and 7 members; wherein R6 is H, alkyl, substituted alkyl, cycloalkyl;or a pharmaceutically acceptable sait, or a prodrugdérivative, or a biologically active métabolite; withthe proviso that when Ri is acetylamino ethyl, R3 is not4-pyridyl.

This invention also provides a method of preparing thecompound having structure V, comprising the steps of 75 013295

wherein P is a removable protecting group; b) treating the product of step a) under cyclization conditions îo provide

c) treating the product of step b) under suitable conditions to provide

to provide 76 013295 wherein R3 is aryl, substituted aryl, heteroaryl; wherein R.6 is H, alkyl, substituted alkyl, or cycloalkyl;wherein Rs is H, alkyl, substituted alkyl, aryl,arylalkyl, amino, substituted aryl, wherein saidsubstituted alkyl is -C(R7) (Rs)NR9Rio, wherein R7 and Rsare each H or alkyl, wherein R9 and Rio are each alkyl orcycloalkyl, or NR9R10 is a ring System of between 4 and7 members.

Compounds represented by formula VI, VII, and VIII can besynthesized by any of the Schemes I-VIII. Compoundsrepresented by formula I7X, and X can be prepared by SchemeIX.

The invention is further illustrated by the followingexamples which in no way should be construed as being furtherlimiting. The contents of ail references, pending patentapplications and published patent applications, citedthroughout this application, including those referenced inthe background section, are hereby incorporated by reference.It should be understood that the models used throughout theexamples are accepted models and thât the démonstration ofefficacy in these models is prédictive of efficacy in humans.

This invention will be better understood from theExperimental Details which follow. However, one skilled inthe art will readily appreciate that the spécifie methods andresults discussed are merely illustrative of the invention asdescribed more fully in the daims which follow thereafter. 77 013295

EXPERIMENTAL DETAILS

The deazapurines of the invention can be prepared usingstandard methods for organic synthesis. Deazapurines can bepurified by reverse phase HPLC, chromatography,recrystallization, etc. and their structures confirmed bymass spectral analysis, elemental analysis, IR and/or NMRspectroscopy.

Typically, synthesis of the intermediates as well as thedeazapurines of the invention is performed in solution. Theaddition and removal of one or more protecting group is alsotypical practice and is known to those skilled in the art.Typical synthetic schemes for the préparation of deazapurineintermediates of the invention are outlined below in SchemeI.

Scheme I

POCk

105° C

.78_ 01329 5 wherein R 3, Rg and Rg are as defined above.

In general, a protected 2-amino-3-cyano-pyrrole can betreated with an acyl halide to form a carboxyamido-3-cyano-pyrrole which can be treated with'acidic methanol to effectring closure to a pyrrolo [2,3d]pyrimidine-4 (3H)-one (Muller,C.E. et al. J. Med. Chem. 40:4396 (1997)). Removal of thepyrrolo protecting group followed by treatment with achlorinating reagent,e.g., phosphorous oxychloride, producedsubstituted or unsubstituted 4-chloro-7H- pyrrolo[2,3d]pyrimidines. Treatment of the chloropyrimidinewith amines afforded 7-deazapurines.

For example, as shown in Scheme I, a N-(1-dl-phenylethyl)-2-amino-3-cyano-pyrrole was treated with an acyl halide inpyridine and dichloromethane. The résultant N-(l- dl-phenylethyl)-2-phenylcarboxyamido-3-cyano-pyrrole was treatedwith a 10:1 mixture of methanol/suifuric acid to effect ringclosure, resulting in a dl —Ί Η-Ί - (1- phenylethyl)pyrrolo[2,3d]pyrimidine-4(3 H) -one. Removal ofthe phenylethyl group by treatment of the pyrimidine withpolyphosphoric acid (PPA) followed by POC1 3 afforded a keyintermediate, the 4-chloro-7H-pyrrolo[2,3d]pyrimidine.

Further treatment of the 4-chloro-7H-pyrrolo(2,3d]pyrimidinewith various amines listed in Table 1 gives compounds offormula (I) and (II). 79 013295 TABLE 1 R M + H R M + H Zq 343.2 0 N νΛΛΛ, 351.27 1 343.18 rxrr 430.35 +o 337.21 il --V” 359.44 Q's, 364.19 404.3.2 1 M 330.18 >ΫΎ%, h 330.45 *vw* 1 347.22 01+ 339.47 0-\ \ —J '-NH 1 **wv 1 350.28 vvw » 353.41 013295 80 X\ /NH 5r X/* *Wv I V 344.19 ___L i 1 H 324.45 dX' 394.16 4V-.J0 359.38 O NH- 371.12 yNxx\ k/\ 379.40 -h\ XJ 'oh 359.39 σ> 387.41 H XX ^x» 403.33 H J vVj*'V 344.48 c ^NH—<— t 351.49 HO*'' 337.53 O 330.37 295.2 013295 81 X O O 407.23 r H 321.2 355.45 ï NH. /X ΌΗ 337.53 441.33 4 H 350.2 “"h 413.24 H P 343.2 Γ N H 372.48 Z H 373.2 -P 307.2 A general approach to préparé 6-substituted pyrroles isdepicted in the following scheme (Scheme II). 82 013295

Scheme II

wherein through are as defined above.

Transestérification and alkylation of ethyl cyanoacetate withan α-haloketone affords a ketomethylester. Protection of theketone followed by treatment with an amidine ( e.g. , alkyl,aryl or alkylaryl) hydrochloride produced the résultant ketalprotected pyrimidine. Removal of the protecting group,followed by cyclization and treatment with phosphorousoxychloride afforded the chloride intermediate which could befurther treated with an amine to afford an amine 6- 83 013295 substituted pyrrole. Additionally, alkylation of the pyrrolenitrogen can be achieved under art recognized conditions. A general approach to préparé 5-substituted pyrroles isdepicted in the following scheme (Scheme III). 10 15

Scheme III

20 25

H 30 wherein through Rg are defined as above and R is a removable protecting group.

Condensation of malononitrile and an excess of a ketonefollcwed by brcmination of the product. afforded a mixture of 35 starting material, monobrominated and dibrominated productswhich were treated with an alkylamine, arylamine oralkylarylamine. The résultant amine product was acylatedwith an acid chloride and the monacylated pyrrole was 84 01329 5 cyclized m the presence of acid to afford the correspondingpyrimidine. The pyrrole protecting group was removed withpolyphosphoric acid and treated with phosphorous oxychlorideto produce a chlorinated product. The chlorinated pyrrolecould subséquently be treated with an amine to produce anamino 5-substituted pyrrole. Alkylation of the pyrrolenitrogen can be achieved under art recognized conditions.

Schemes IV and V depictdeazapurines 1 and 2 of the methods for preparmg theinvention.

1

wherein Rg and Rg are as described above, e.g., CH^.

Spécifie Préparation of 6-methvl pvrrolopvrimidines :

The key reaction toward 6-methylpyrrolopyrimidines (1) [Rg =CHj] was cyclization of a cyanoacetate with benzamidine to apyrimidine. It was believed methyl cyanoacetate wouldcyclize more efficiently with benzamidine to a pyrimidinethan the corresponding ethyl ester. Therefore, transestérification and alkylation of ethyl cyanoacetate inthe presence of NaOMe and an excess of an a-haloacetylmoiety, e.g., chloroacetone, gave the desired methyl ester(3) in 79% yield (Scheme IV). The ketoester (3) wasprotected as the acetal (4) in 81% yield. A new cyclizationmethod to the pyrimidine (5) was achieved with an amidinehydrochloride, e.g., benzamidine hydrochloride, with 2 013295 - 85 - équivalents of DBU to afford the 5 in 54% isolated yield.This method improves the yield from 20% using the publishedconditions, which utilizes NaOMe during the cyclization withguanidine. Cyclization to the pyrrole-pyrimidine (6) wasachieved via deprotection of the acetal in aqueous HCl in 78%yield. Reaction of (6) with phosphorous oxychloride at refluxgave the corresponding 4-chloro dérivative (7). Couplingwith trans-4-aminocyclohexanol in dimethyl sulfoxide at 135°Cgave (1) in 57% from (7). One skilled in the art willappreciate that choice of reagents allows for greatflexibility in choosing the desired substituent R^.

Scheme IV

1 86 013295

Spécifie Préparation of 5-me thvlpyrrolopvrimidines

Knoevengel condensation of malononitrile and an excessketone, e.g., acetone in refluxing benzene gave 8 in 50%yield after distillation. Bromination of 8 with N- bromosuccinimde in the presence of benzoyl peroxide inchloroform yielded a mixture of starting material, mono- (9) ,and di-brominated products (5/90/5) after distillation (70%) .The mixture was reacted with an α-methylalkylamine or a-methylarylamine, e.g., α-methylbenzylamine, to deliver theaminopyrrole (10) . After passing through a short silica gelcolumn, the partially purified amine (31% yield) was acylatedwith an acid chloride, e.g., benzoyl chloride to delivermono- (11), and diacylated (12) pyrroles, which wereseparated by flash chromatography. Acid hydrolysis of thedisubstituted pyrrole (12) generated a combined yield of 29%for the acylpyrrole (11) . Cyclization in the presence ofconcentrated sulphuric acid and DMF yielded (13) (23%) , whichwas deprotected with polyphosphoric acid to (14) . Reactionof (14) with phosphorous oxychloride at reflux gave thecorresponding 4-chloro dérivative (15) . Coupling withtrans-4-aminocyclohexanol in dimethyl sulfoxide at 135°C gave (2)[Rg = CH^] in 30% from (14) (See Scheme V). One skilled inthe art will appreciate that choice of reagents allows forgreat flexibility in choosing the desired substituent R6. - 87 0J 329 5

Scheme V

15 013295 - 88 -

Alternative Synthetic Route to R^-Substituted Pvrroles. e.g.,5-methvl pyrrolopyrimidlnes:

This alternative route to Rg-substituted pyrroles, e.g., 5-methylpyrrolopyrimidines, involves transestérification andalkylation of ethyl cyanoacetate to (16) (Scheme VI). Thecondensation of (16) with benzamidine hydrochloride with 2équivalents of DBU affords the pyrimidine (17). Cyclizationto the pyrrole-pyrimidine (14) will be achieved viadeprotection of'the acetal in aqueous HCl. Reaction of (14)with phosphorous oxychloride at reflux gave the corresponding4-chloro dérivative (15). Coupling with trans-4- aminocyclohexanol in dimethyl suifoxide at 135°C gives 2.This procedure reduces the number of synthetic reactions tothe target compound (2) from 9 to 4 steps. Moreover, theyield is dramatically improved. Again, one skilled in theart will appreciate that choice of reagents allows for greatflexibility in choosing the desired substituent Rg. 89 . 013295

Scheme VI

5 A general approach to préparédes-methyl pyrrole is depictedin the following scheme(Scheme VII) 10

90 013295

Scheme VII

wherein R^ through are defined as above.

Alkylation of an alkyl cyanoacetate with a diethyl acetal inthe presence of a base afforded a cyano diethyl acetal whichwas treated with an amidine sait to produce a methylpyrrolopyrimidine precursor. The precursor was chlorinatedand treated with an amine to form the des-methylpyrrolopyrimidine target as shown above.

For example, Scheme VIII depicts the synthesis of compound(18). 91 013295

Scheme VIII

OH

Commercially available methyl cyanoacetate was alkylated withbromoacetaldehyde diethyl acetal in the presence of potassiumcarbonate and Nal to yield (19). Cyclization to thepyrimidine (20) was achieved in two steps. Initially, thepyrimidine-acetal was formed via reaction of (19) withbenzamidine hydrochloride with 2 équivalents of DBU. Therésultant pyrimidine-acetal was deprotected without 01329 5 92 purification with aqueous 1 N HCl and the résultant aldéhydecyclized to the pyrrolo-pyrimidine (20), which was isolatedby filtration. Reaction of (20) with phosphorous oxychlorideat reflux afforded the corresponding 4-chloro dérivative(21). Coupling of the chloro dérivative with trans-%-aminocyclohexanol in DMSO at 135°C gave compound (18) fromcompound (21).

Schemes II-VIII demonstrate that it is possible tofunctionalize the 5- and 6-position of the pyrrolopyrimidinering. Through the use of different starting reagents andslight modifications of the above reaction schemes, variousfunctional groups can be introduced at the 5- and 6-positionsin formula (I) and (II). Table 2 illustrâtes some examples.

Table 2. Selected list of 5- and 6-substitutedpyrrolopyrimidînes.

Starting Reagent R, Rf; z°Y°\ cr H 0 H Substituted Ar ο O XV H CH2C(O)OCH3 O O X Cl C(O)OCH3 ch3 O 0 X Cl C(O)NHCH3 ch3 93 013295 A skiiled artisan will know that metabolism of the compoundsdisclosed herein in a subject produces certain biologicallyactive métabolites which can serve as drugs. 5 The invention is further illustrated by the followingexamples which in no way should be construed as being furtherlimiting. The contents of ail references, pending patentapplications and published patent applications, citedthroughout this application, including those referenced in 10 the background section, are hereby incorporated by reference.It should be understood that the models used throughout theexamples are accepted models and that the démonstration ofefficacy in these models is prédictive of efficacy in humans. 15 20 94 013295

Exemplification,

Préparation 1: A modification of the alkylation method of Seela and Lüpkewas used. 1 To an ice-cooled (0°C) solution of ethylcyanoacetate (6.58 g, 58.1 mmol) in MeOH (20 mL) was slowlyadded a solution of NaOMe (25% w/v; 58.1 mmol) . After 10min, chloroacetone (5 mL; 62.8 mmol) was slowly added. After4 h, the solvent was removed. The brown oil was diluted theEtOAc (100 mL) and washed with H2O (100 mL) . The organicfraction was dried, filtered, and concentrated to a brown oil(7.79 g; 79%). The oil (3) (Scheme IV) was a mixture ofmethyl/ethyl ester products (9/1), and was used withoutfurther purification. 1H NMR (200 MHz, CDCl^) δ_4.24 (q, J = 7.2 Hz, OCH2), 3.91 (dd, 1H, J = 7.2, 7.0 Hz, CH), 3.62 (s,3H, OCH3), 3.42 (dd, 1H, J =15.0, 7.1Hz, lxCH2); 3.02 (dd,1H, J = 15.0, 7.0 Hz, 1 x CH2); 2.44 (s, 3H, CH3) , 1.26 (t, J = 7.1 Hz, ester-CH3) . 1Seela, F.,· Lüpke, U. Chem. Ber. 1977, 110, 1462-1469.

Préparation 2 :

The procedure of Seela and Lüpke was used.1 Thus, protectionof the ketone (3) (Scheme IV; 5.0 g, 32.2 mmol) with ethyleneglycol (4 mL, 64.4 mmol) in the presence of TsOH (100 mg)afforded (4) as an oil (Scheme IV; 5.2 g, 81.0) after flashchromatography (SiO2; 3/7 EtOAc/Hex, R^-0.35). Still contains~5% ethyl ester: XH NMR (200 MHz, CDClg) δ_4.24 (q, J = 7.2Hz, OCH2), 3.98 (s, 4H, 2 x acetal-CH2), 3.79 (s, 3H, OCH3), 3.62 (dd, 1H, J = 7. 2, 7.0 Hz, CH), 2.48 (dd, 1H, J = 15.0, 7.1 Hz, 1 x CH2), 2. 32 (dd, 1H, J = 15.0, 7.0 Hz, 1 x CH2 ) ; 1.35 (s, 3H, CH3), 1 .26 (t, J = 7.1 Hz, ester-CH3) ; MS (ES) : 200.1 (M++l).""Seela, F.; Lüpke, U . Chem. Ber . 1977, 110, 1462- 1469. 95 013295

Préparation 3: A solution of acetal (4) (Scheme IV, 1 g, 5.02 mmol) ,benzamidine (786 mg, 5.02 mmol) , and DBU (1.5 mL, 10.04 mmol)in dry DMF (15 mL) was heated to 85°C for 15 h. The mixturewas diluted with CHC13 (30 mL) and washed with 0.5 N NaOH (10mL) and H20 (20 mL) . The organic fraction was dried, filteredand concentrated to a brown oil. Flash chromatography (SiQ;1/9 EtOAc/CH2Cl2, Rf 0.35) was attempted, but materialcrystallized on the column. The silica gel was washed withMeOH. Fractions containing the product (5) (Scheme IV) wereconcentrated and used without further purification (783 mg,54.3%): 'H NMR (200 MHz, CDC13) δ 8.24 (m, 2H, Ar-H), 7.45 (m, 3H, Ar-H), 5.24 (br s, 2H, NH2) , 3.98 (s, 4H, 2 x acetal-CH2) , 3.60-3.15 (m, 2H, CH2) , 1.38 (s, 3H, CH3) ; MS (ES): 288.1(M++l) .

Préparation of compound (20) (Scheme VIII): A solution ofacetal (19) (4.43 g, 20.6 mmol)1, benzamine hydrochloride (3.22 g, 20.6 mmol), and DBU (6.15 mL, 41.2 mmol) in dry DMF(20 mL) was heated to 85°C for fifteen hours. The mixturewas diluted with lOOmL of CHC13, and washed with H2O (2 x 50mL). The organic fraction was dried, filtered, andconcentrated to a dark brown oil. The dark brown oil wasstirred in IN HCl (100 mL) for 2 hours at room température.The resulting slurry was filtered yielding the HCl sait of(20) as a tan solid (3.60g, 70.6%); XH NMR (200 MHz, DMSO-d6)11.92 (s 1H) , 8.05 (m, 2H, Ar-H), 7.45 (m, 3H, Ar-H) , 7.05(s, 1H, pyrrole-H); MS(ES): 212.1 (M++l).

Préparation 4: A solution of acetal (5) (700 mg, 2.44 mmol) in 1 N HCl (40mL) was stirred for 2 h at RT. The résultant slurry wasfiltered yielding the HCl sait of 2-phenyl-6-methyl-7Jî-pyrrolo [2,3d]pyrimidin-4 (3H)-one as a tan solid (498 mg,78.0%); lH NMR (200 MHz, DMSO-d6) δ 11.78 (s, 1H) , 8.05 (m,2H, Ar-H), 7.45 (m, 3Η, Ar-H), 6.17 (s, 1H, pyrrole-H), 2.25(s, 3H, CH3) ; MS (ES): 226.1 (M++l) . 96 013295

Préparation 5: A modification of the Chen et al. cyclization method wasused.1 To an ice-cooled (0°C) solution of bromide (9) , (SchemeV; 20.0 g, 108 mmol; 90% pure) in isopropyl alcohol (60 mL)was slowly added a solution of a-methylbenzylamine (12.5 mL, 97.3 mmol) . The black solution was allowed to warm to RT andstir for 15 h. The mixture was diluted with EtOAc (200 mL)and washed with 0.5 N NaOH (50 mL) . The organic fraction wasdried, filterëd, and concentrated to a black tar (19.2 g;94%). The residue was partially purified by flashchromatography (SiO2; 4/96 MeOH/CH2Cl2, Rf 0.35) to a blacksolid (6.38 g, 31%) as the compound dl-1-(1-phénylethyl)-2-amino-3-cyano-4-methylpyrrole: MS (ES): 226.1 (M++l) .xChen, Y. L. ; Mansbach, R. S.; Winter, S. M. ; Brooks, E.;Collins, J.; Corman, M. L.; Dunaiskis, A. R.; Faraci, W. S.;Gallaschun, R. J.; Schmidt, A.; Schulz, D. W. J. Med. Chem.1997, 40, 1749-1754.

Préparation 6:

To a solution of dl-1-(1-phenylethyl)-2-amino-3-cyano-4, 5-dimethylpyrrole1 (14.9 g, 62.5 mmol) and pyridine (10.0 mL)in dichloromethane (50.0 mL) was added benzoyl ch.loride (9.37g, 66.7 mmol) at 0°C. After stirring at (fc for 1 hr, hexane(10.0 mL) was added to help précipitation of product.Solvent was removed in vacuo and the solid was recrystallizedfrom EtOH/H2O to give 13.9 g (65%) of dl-l-(l-phenylethyl)-2-phenylcarbonylamino-3-cyano-4,5-dimethylpyrrole. mp 218-2E1XH NMR (200 MHz, CDCl3) δ_1.72 (s, 3H), 1.76 (d, J = 7.3 Hz,3H) , 1.98 (s, 3H) , 5.52 (q, J = 7.3 Hz, 1H) , 7.14-7.54 (m,9H), 7.68-7.72 (dd, J = 1.4 Hz, 6.9 Hz , 2H), 10.73 (s, 1H) ;MS (ES): 344.4 (M++l).

Liebigs Ann. Chem. 1986, 1485-1505. .97_ 01 329 5

The following compounds were obtained in a similar manner.

Préparation 6A: dl-1- (1-phenylethyl) -2- (3-pyridyl)carbonylamino-3-cyano-4,5-5 dimethylpyrrole.Λί NMR (200 MHz, CDC13) δ_1.83 (d, J = 6.8 Hz, 3H) , 2.02 (s, 3H) , 2.12 (s, 3H) , 5.50 (q, J = 6.8 Hz, 1H) ,7.14-7.42 (m, 5H), 8.08 (m, 2H), 8.75 (m, 3H); MS (ES): 345.2(M++l) . 10 dl-1-(1-phenylethyl)-2-(2-furyl)carbonylamino-3-cyano-4,5-dimethylpyrrole. XH NMR (200 MHz, CDC13) δ 1.84 (d, J = 7.4 Hz,3H) , 1.92 (s, 3H), 2.09 (s, 3H) , 5.49 (q, J = 7.4 Hz, 1H) ,6.54 (dd, J = 1.8 Hz, 3.6 Hz, 1H) , 7.12-7.47 (m, 7H) ; MS(ES): 334.2 (M++l), 230.1. 15 dl-1-(1-phenylethyl)-2-(3-furyl)carbonylamino-3-cyano-4,5-dimethylpyrrole. ΧΗ NMR (200 MHz, CDC13) δ 1.80 (d, J = 7 Hz3H) , 1.89 (s, 3H), 2.05 (s, 3H), 5.48 (q, J = 7 Hz, 1H) , 6.59(s, 1H) , 7.12-7.40 (m, 6H) , 7.93 (s, 1H) ; MS (ES): 334.1 20 (M++l), 230.0. dl-1- (1-phenylethyl) -2-cyclopentylcarbonylamino-3-cyano-4,5-dimethylpyrrole. XH NMR (200 MHz, CDC13) δ 1.82 (d, J = 7.4 Hz,3H), 1,88 (s, 3H), 2.05 (s, 3H), 1.63-1.85 (m, 8H), 2.63 (m, 25 1H), 5.43 (q, J = 7.4 Hz, 1H), 6.52 (s, 1H), 7.05-7.20 (m,5H); MS (ES): 336.3 (M++l). dl-1-(1-phenylethyl)-2-(2-thienyl)carbonylamino-3-cyano-4,5-dimethylpyrrole, 1H NMR (200 MHz, CDC13) δ 1.82 (d, J = 6.8 Hz, 30 3H) , 1.96 (s, 3H) , 2.09 (s, 3H) , 5.49 (q, J= 6.8 Hz, 1H) , 7.05-7.55 (m, 8H); MS (ES): 350.1 (M++l), 246.0. dl-1- (1-phenylethyl) -2- (3-thienyl) carbonylamino-3-cyano-4,5-dimethylpyrrole. 35 XH NMR (200 MHz, CDC13) δ 1.83 (d, J = 7.0 Hz, 3H) , 1.99 (s,3H) , 2.12 (s, 3H) , 5.49 (q, J = 7.0 Hz, 1H) , 6.90 (m, 1H) ,7.18-7.36 (m, 6H) , 7.79 (m, 1H) ; MS (ES): 350.2 (M++l), 246.1. 013295 - 98 - dl-1- (1-phenylethyl) -2- (4 —f luorophenyl ) carbonylamino-3-cyano- 4,5-dimethylpyrrole. XH NMR (200 MHz, CDCl3) δ 1.83 (d, J = 7.4 Hz, 3H) , 1.96 (s,3H), 2.08 (s, 3H), 5.51 (q, J = 7.4 Hz, 1H), 7.16-7.55 (m,9H); MS (ES): 362.2 (M++l), 258.1. dl-1- (1-phenylethyl) -2- (3-f luorophenyl) carbonylamino-3-cyano- 4,5-dimethylpyrrole. XH NMR (200 MHz, CDC13) δ 1.83 (d, J = 7.4 Hz 3H) , 1.97 (s,3H), 2.10(s, 3H), 5.50 (q, J = 7.4 Hz, 1H), 7.05-7.38 (m, 7H), 7.67-7.74 (m, 2H); MS (ES): 362.2 (M++l), 258.1. dl-1-(1-phenylethyl)-2-(2-fluorophenyl)carbonylamino-3-cyano- 4,5-dimethylpyrrole. XH NMR (200 MHz, CDC13) δ 1.85 (d, J = 7.2 Hz, 3H), 1.94 (s, 3H), 2.11 (s, 3H), 5.50 (q, J = 7.2 hz,1H), 7.12-7.35 (m, 6H) , 7.53 (m, 1H), 7.77 (m, 1H), 8.13 (m,1H); MS (ES): 362.2(M++1), 258.0. dl-1- (1-phenylethyl)-2-isopropylcarbonylamino-3-cyano-4,5-dimethylpyrrole.XH NMR (200 MHz, CDC13) δ 1.19 (d, J = 7.0Hz, 6H), 1.82(d, J = 7.2 Hz, 3H), 1.88 (s, 3H), 2.06 (s, 3H) ,2.46 (m, 1H) , 5.39 (m, J = 7.2 Hz, 1H), 6.64 (s, 1H), 7.11-7.36 (m, 5H); MS (ES): 310.2 (M++l) , 206.1 .

In the case of acylation of dl-1-(1-phenylethyl)-2-amino-3- cyano-4-methylpyrrole, monoacylated dl-1-(1-phenylethyl)-2- benzoylamino-3-cyano-4-dimethylpyrrole and diacylated pyrrole dl-1- (1-phenylethyl) -2-dibenzoylamino-3-cyano-4-irtethylpyrrole were obtained. Monoacylated pyrrole: H NMR (200 MHz, CDCI3) δ_7.69 (d, 2H, J = 7.8 Hz, Ar-H) , 7.58-7.12 (m, 8H, Ar-H) , 6.18 (s, 1H, pyrrole-H) , 5.52 (q, 1H, J= 7.2 Hz, CH-CH3), 2.05 (s, 3H, pyrrole-CHo), 1.85 (d, 3H, J = 7.2 Hz, CH-CH7);

+ J 1 J MS (ES): 330.2 (M+l); Diacylated pyrrole: H NMR (200 MHz,CDCI3) δ_7.85 (d, 2H, J = 7.7 Hz, Ar-H), 7.74 (d, 2H, J= 7.8Hz, Ar-H), 7.52-7.20 (m, 9H, Ar-H), 7.04 (m, 2H, Ar-H), 6.21(s, 1H, pyrrole-H), 5.52 (q, 1H, J= 7.2 Hz, CH-CH3), 1.77 (d, 013295 99 - 3H, J= 7.2 Hz, CH-CH3), 1.74 (s, 3H, pyrrole-CH3 ) ; MS (ES):434.1 (M++l).

Préparation 7 :

To a solution oÉîl-1-(1-phenylethyl)-2-phenylcarboxyamido-3-cyano-4,5-dimethylpyrrole (1.0 g, 2.92 mmol) in methanol(10.0 mL) was added concentrated sulfuric acid (1.0 mL) at0°C. The resulted mixture was refluxed for 15 hr and cooleddown to room température. The precipitate was filtered togive 0.48 g (48%) of dl-5,6-dimethyl-2-phenyl-7 H-7-( 1-phenylethyl)pyrrolo[2,3d] pyrimidin-4 (3i) -one. ^NMR (200 MHz,CDC13) δ_2.02 (d, J = 7.4 Hz, 3H) , 2.04 (s, 3H) , 2.41 (s, 3H) ,6.25 (q, J = 7.4 Hz, 1H), 7.22-7.50 (m, 9H) , 8.07-8.12 (dd,J = 3.4 Hz, 6.8 Hz, 2H) , 10.51 (s, 1H) ; MS (ES) : 344.2 (M++l) .

The following compounds were obtained in a similar manner asthat of Préparation 7 : dl -5,6-dimethyl-2-(3-pyridyl)-7 Η-Ί-(1-phenylethyl) pyrrolo[2,3d]pyrimidin-4(3H) -one. 1H NMR (200 MHz, CDC13)δ_2.03 (d, J = 7.2 Hz, 3H) , 2.08 (s, 3H), 2.42 (s, 3H), 6.24(q, J = 7.2 Hz, 1H) , 7.09-7.42 (m, 5H) , 8.48 (m, 2H) , 8.70(m, 3H); MS (ES): 345.1 (M++l). dl -5,6-dimethyl-2- (2-furyl) -7 H- 7 - ( 1 -pheny 1 e thy 1 ) pyrrolo [2,3d]pyrimidin-4 (3H)-one. NMR (200 MHz, CDC13) δ1.98 (d, J = 7.8 Hz, 3H), 1.99 (s, 3H) , 2.37 (s, 3H), 6.12(q, J = 7.8 Hz, 1H), 6.48 (dd, J=1.8 Hz, 3.6 Hz, 1H), 7.17-7.55 (m, 7H), 9.6 (s, 1H); MS (ES): 334.2 (M++l). dl -5,6-dimethyl-2- (3-furyl) -7 Η-Ί - (1-phenylethyl ) pyrrolo[2,3d]pyrimidin-4 (3H) -one. ’h NMR (200 MHz, CDC13) δ 1.99 (d,J = 7 Hz, 3H), 2.02 (s, 3H,, 2.42 (s, 3H>, 6.24 (q, J = 7 Hz,1H), 7.09 (s, 1H), 7.18-7.32 (m, 5H), 7.48 (s, 1H), 8.51 (s,1H); MS (ES): 334.2 (M++l). dl -5,6-dimethyl-2-cyclopentyl-7 H- 7 - ( 1-phenylethyl) 100 013295 pyrrolo[2,3d]pyrimidin-4(3H)-one. 2H NMR (200 MHz, CL>C13) δ 95 (d, J = 7.4 Hz, 3H), 2.00 (s, 3H), 2.33 (s. 3H) 88 (m, 8H), 2.97 (m, 1H), 6. 10 ( q, J = 7.4 Hz, 1H) 30 (m, 5H), 9.29 (s, 1H); MS (ES ): 336.3 (M ++1) dl-5, 6-dimethyl-2-(2-thienyl)-7Η-Ί- (1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3flj-one. 1H NMR (200 MHz, CDC13) δ2.02(d, J = 7.2 Hz, 3H), 2.06 (s, 3H), 2.41 (s, 3H), 6.13 (q,J = 7.2 Hz, 1H), 7.12 (dd, J = 4.8, 2.8 Hz, 1H), 7.26-7.32(m, 5H), 7.44 (d, J = 4.8 Hz, 1H), 8.01 (d, J = 2.8 Hz, 1H)11.25 (s, 1H); MS (ES): 350.2 (M++l). dl-5 , 6-dimethy1- 2 -(3 -thi eny1)-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3H)-one. nH NMR (200 MHz, CDC13) δ2.00 (d, J = 7.4 Hz, 3H), 2.05 (s, 3H), 2.43 (s, 3H), 6.24 (q,J = 7.4 Hz, 1H), 7.24-7.33 (m, 5H), 7.33-7.39 (m, 1H), 7.85(m, 1H), 8.47 (m, 1H), 12.01 (s, 1H); MS (ES): 350.2 (M++l). dl-5,6-dimethyl-2-(4-fluorophenyl)-7H-7-(1-phenylethyl )pyrrolo[2,3d]pyrimidin-4(3H)-one. 1H NMR (200 MHz, CDC13) δ2.01 (d, J = 6.8 Hz, 3H), 2.05 (s, 3H) , 2.42 (s, 3H), 6.26(q, J = 6.8 Hz, 1H) , 7.12-7.36 (m, 7H) , 8.23-8.30 (m, 2H) ,11.82 (s, 1H); MS (ES): 362.3 (M++l). dl-5,6-dimethyl-2-( 3-fluorophenyl)-ΊΗ-Ί-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3H)-one. 1H NMR (200 MHz, CDC13) δ2.02 (d, J = 7.4 Hz, 3H), 2.06 (s, 3H), 2.44 (s, 3H), 6.29(q, J = 7.4 Hz, 1H), 7.13-7.51(m, 7H), 8.00-8.04 (m, 2H) ,11.72 (s, 1H); MS (ES): 362.2 (M++l). dl-5,6-dimethyl-2-(2-fluorophenyl)-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3tf)-one. 1H NMR (200 MHz, CDC13) δ2.00(d, J = 7.2 Hz, 3H), 2.05 (s, 3H), 2.38 (s, 3H), 6.24 (q,J = 7.2 Hz, 1H), 7.18-7.45 (m, 8 H), 8.21 (m, 1H), 9.54 (s,1H); MS (ES): 362.2 (M++l). 013295 - 101 - dl-5, 6-dimethyl-2-isopropyl-7 Η-Ί - (1-phenylethyl)pyrrolo[2,3d]pyrimidin-4 (3 H) -one. τΗ NMR (200 MHz, CDC13) δ 1.30 (d, J = 6.8 Hz, 3H) , 1.32 (d,J = 7.0 Hz, 3H), 2.01 (s, 3H), 2.34 (s, 3H), 2.90 (m, 1H),6.13 (m, 1H) , 7.17-7.34 (m, 5H), 10.16 (s, 1H) ; MS (ES): 310.2 (M++l).

Préparation 8: A solution of dl-1-(1-phenylethyl)-2-benzoylamino-3-cyano-4- methylpyrrole (785 mg, 2.38 mmol) with concentrated H2SO4 (1 mL) in DMF (13 mL) was stirred at 130°C for 48 h. The black

solution was diluted with CHCI3 (100 mL) and washed with 1 N

NaOH (30 mL) , and brine (30 mL) . The organic fraction was dried, fiicered, concentrated, and purified by flash chromatography (SiC^; 8/2 EtOAc/Hex, Rf 0.35) to a brown solid (184 mg, 24%) as dl-5-methyl-2-phenyl-7H-7-( 1-1

phenylethyl) pyrrolo [2,3d] pyrimidm-4 (3tf) -one. H NMR (200 MHz, CDCI3) δ_8.18 (m, 2H, Ar-H), 7.62-7.44 (m, 3H, ΑΓ-Η), 7.40-7.18 (m, 5H, Ar-H), 6.48 (s, 1H, pyrrole-H) , 6.28 (q, 1H, J = 7.2 Hz, CH-CH3), 2.18 (s, 3H, pyrrole-CH3), 2.07(d, 3H, J = 7.2 Hz, CH-CH3); MS (ES): 330.2 (M+ + 1).

Préparation 9 : A mixture of dl-1-(1-phenylethyl)-2-amino-3-cyano-4,5-dimethylpyrrole (9.60 g, 40.0 mmol) and of formic acid (50.0mL, 98%) was refluxed for 5 hr. After cooling down to roomtempérature and scratching the sides of flask, copiousprecipitate was formed and filtered. The material was washedwith water until washings showed neutral pH to give dl-5,6-dimethyl-72ï-7- ( 1-phenylethyl ) pyrrolo [2,3d]pyrimidin-4 (3H) -one. :Η NMR (200 MHz, CDC13) δ 1.96 (d, J = 7.4 hz, 3H) , 2.00(s, 3H) , 2.38 (s, 3H) , 6.21 (q, J = 7.4 Hz, 1H) , 7.11-7.35(m, 5H), 7.81 (s, 1H), 11.71 (s, 1H); MS (ES): 268.2 (M++l). 102 013295

Préparation 10: dl-5,6-dimethyl-2-phenyl-7 Η-Ί- ( 1-phenylethyl ) pyrrolo[2,3d]pyrimidin-4 (3H)-one (1.0 g, 2.91 iranol) was suspended inpolyphosphoric acid (30.0 mL) . The mixture was heated at 100°Cfor 4 hr. The hot suspension was poured onto ice water,stirred vigorously to disperse suspension, and basified to pH6 with solid KOH. The resulting solid was filtered andcollected to give 0.49 g (69%) of 5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d]pyrimidin-4 (3H) -one. 1H NMR (200 MHz, DMSO-d6)δ_2.17 (s, 3H), 2.22 (s, 3H), 7.45 (br, 3H), 8.07 (br, 2H,),11.49 (s, 1H), 11.82 (s, 1H); MS (ES): 344.2 (M++l).

The following compounds were obtained in a similar manner asthat of Préparation 10: 5-methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidin-4(3H) -one. MS(ES): 226.0 (M++l). 5,6-dimethyl-2-(3-pyridyl)-7H-pyrrolo[2,3d]pyrimidin-4(3ff) -one. MS (ES): 241.1 (M++l). 5,6-dimethyl-2-(2-furyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one.:H NMR (200 MHz, DMSO-d6) δ 2.13 (s, 3H) , 2.18 (s, 3H) , 6.39(dd, J = 1.8, 3.6 Hz, 1H), 6.65 (dd, J = 1.8 Hz, 3.6 Hz, 1H),7.85 (dd, J = 1.8, 3.6 Hz, 1H, ) , 11.45 (s, 1H) , 11.60 (s, 1H); MS (ES): 230.1 (M++l). 5,6-dimethyl-2- (3-furyl) -7H-pyrrolo[2,3d]pyrimidin-4 (3H) -one.XH NMR (200 MHz, PMSO-dg) δ 2.14 (s, 3H) , 2.19 (s, 3H) , 6.66(s, 1H) , 7.78 (s, 1H) , 8.35 (s, 1H) , 11.3 (s, 1H) , 11.4 (s,1H); MS (ES): 230.1 (M++l). 5,6-dimethyl-2-cyclopentyl-7H~pyrrolo[2, 3d)pyrimidin-4(3H)-one. 1H NMR (200 MHz, DMSO-d6) δ 1.57-1.91 (m, 8 H), 2.12 (s,3H), 2.16 (s, 3H) , 2.99 (m, 1H) , 11.24 (s, 1H) , 11.38 (s, 1H); MS (ES): 232.2 (M++l). 103 013295 5 , 6-dimethyi-2- (2-thienyl) -7#-pyrrolo [2,3d]pyrimidin-4 (3#) -one. XH NMR (200 MHz, DMSO-d6) δ 2.14 (s, 3H) , 2.19 (s, 3H) ,7.14 (dd, J = 3.0, 5.2 Hz, 1H), 7.70 (d, J = 5.2 Hz 1H), 8.10(d, J=3.0 Hz, 1H), 11.50 (s, 1H); MS (ES): 246.1 (M++l) . 5.6- dimethyl-2- (3-thienyl) -7#-pyrrolo [2,3d] pyrimidin-4 (3#) -one. 2Η NMR (200 MHz, DMSO-d6) δ 2.17 (s, 3H) , 2.21(s, 3H) ,7.66(m, 1H), 7.75 (m, 1H), 8.43 (m, 1H) , 11.47 (s, 1H), 11.69(s, 1H); MS (ES): 246.1 (M++l). 5.6- dimethyl-2-( 4-fluorophenyl)-7#-pyrrolo[2,3d]pyrimidin- 4(3#)-one. 1H NMR (200 MHz, DMSO-d6) δ 2.17 (s, 3H) , 2.21 (s,3H) , 7.31 (m, 2H) , 8.12 (m, 2H) , 11.47 (s, 1H) ; MS (ES): 258.2 (M++l). 5.6- dimethyl-2-(3-fluorophenyl)-7#-pyrrolo[2,3d]pyrimidin-4(3#)-one. :Η NMR (200 MHz, DMSO-d6) δ 2.18 (s, 3H) , 2.21 (s,3H), 7.33 (m, 1H), 7.52 (m, 1H), 7.85-7.95 (m, 2H), 11.56 (s,1H), 11.80 (s, 1H); MS (ES): 258.1 (M++l). 5.6- dimethyl-2-(2-fluorophenyl)-7#-pyrrolo[2,3d]pyrimidin-4(3#)-one. ΤΗ NMR (200 MHz, DMSO-d6) δ 2.18 (s, 3H) , 2.22 (s,3H), 7.27-7.37 (m, 2H), 7.53 (m 1H), 7.68 (m, 1H), 11.54 (s,1H), 11.78 (s, 1H); MS (ES): 258.1 (M++l). 5.6- dimethyl-2-isopropyl-7#-pyrrolo[2,3d]pyrimidin-4 (3#) -one. XH NMR (200 MHz, DMSO-d6) δ 1.17 (d, J= 6.6 Hz, 6H) , 2.11 (s,3H) , 2.15 (s, 3H) , 2.81 (m, 1H) , 11.20 (s, 1H) , 11.39 (s, 1H); MS (ES): 206.1 (M++l). 5.6- dimethyl-7H-pyrrolo[2,3d]pyrimidin-4(3#)-one. 1H NMR(200 MHz, DMSO-d6) δ 2.13 (s, 3H) , 2.17 (s, 3H), 7.65 (s, 1H);MS (ES): 164.0 (M++l).

Préparation 11: A solution of 5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d} - 104 013295 pyrimidin-4 (3H) -one (1.0 g, 4.2 mmol) in phosphorusoxychloride (25.0 mL) was refluxed for 6 hr and thenconcentrated in vacuo to dryness. Water was added to theresidue to induce crystallization and the resulting solid wasfiltered and collected to give 0.90 g (83%) of 4-chloro-5,6-dimethyl-2-phenyl-TFi-pyrrolo [2,3d]pyrimidine. 1H NMR (200 MHz,DMSO-dg) δ_2.33 (s, 3H) , 2.33 (s, 3H) , 7.46-7.49 (m, 3H) ,8.30-8.35 (m, 2H), 12.20 (s, 1H); MS (ES): 258.1 (M++l).

The following compounds were obtained in a similar manner asthat of Préparation 11: 4-chloro-5-methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS(ES): 244.0 (M++l).

4-chloro-6-methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS (ES): 244.0 (M++l). 4-chloro-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. 1H NMR (200 MHz,DMSO-d6) 8.35 (2, 2H) , 7.63 (br s, 1H) , 7.45 (m, 3H) , 6.47 (br s, 1H); MS (ES): 230,0 (M++l). 4-chloro-5,6-dimethyl- 2 -(3-pyridyl)-7H-pyrrolo[2,3d]pyrimidine. MS (ES): 259.0 (M++l). 4-chloro-5,6-dimethyl-2- (2-furyl) -7H-pyrrolo[2,3d]pyrimidine.*H NMR (200 MHz, DMSO-d6) δ 2.35 (s, 3H), 2.35 (s, 3H) , 6.68(dd, J =1.8, 3.6 Hz, 1H), 7.34 (dd, J = 1.8 Hz, 3.6 Hz, 1H),7.89 (dd, J = 1.8, 3.6 Hz, 1H); MS (ES): 248.0 (M++l). 4-chloro-5,6-dimethyl-2- (3-furyl) -7H-pyrrolo [2,3d]pyrimidine.XH NMR (200 MHz, DMSO-dê) δ 2.31 (s, 3H) , 2.31 (s, 3H) , 6.62(s, 1H) , 7.78 (s, 1H) , 8.18 (s, 1H), 12.02 (s, 1H) ; MS (ES):248.1 (M++l). 4-chloro-5,6-dimethyl-2-cyclopentyl-7 H-pyrrolo [ 2 , 3d] pyrimidine. NMR (200 MHz, DMSO-d6) δ 1.61- 1.96 (m, 8H) , 013295 105 2.27 (s, 3H) , 2.27 (s, 3K) , 3.22 (m, 1H) , 11.37 (s, 1H) ; MS(ES): 250.1 (M++l). 4-chloro-5,6-dimethyl-2- (2-thienyl) -7 H-pyrrolo [ 2,3d] pyrimidine. 1H NMR (200 MHz, DMSO-dg) δ 2.29 (s, 3H) , 2.31 (s,3H), 7.14 (dd, J = 3.1 Hz, 4.0 Hz, 1H), 7.33 (d, J = 4.9 Hz,1H), 7.82 (d, J = 3.1 Hz, 1H), 12.19 (S, 1H); MS (ES): 264.1(M++l) . 4-chloro-5,6-dimethyl-2-(3-thienyl)-7 H-pyrrolo[2,3d]pyrimidine. NMR (200 MHz, DMSO-d6) δ 2.32 (s, 3H) , 2.32(s, 3H), 7.62 (dd, J = 3.0, 5.2 Hz, 1H), 7.75 (d, J = 5.2 Hz,1H), 8.20 (d, J = 3.0 Hz, 1H); MS (ES): 264.0 (M++l). 4-chloro-5,6-dimethyl-2-(4-fluorophenyl)-7 H-pyrrolo[2,3d]pyrimidine. hî NMR (200 MHz, DMSO-dg) δ 2.33(s, 3H), 2.33 (s,3H) , 7.30 (m, 2H) , 8.34 (m, 2H) , 12.11 (s, 1H) ; MS (ES) : 276.1. (M++l). 4-chloro-5,6-dimethyl-2-(3-fluorophenyl)-7 H-pyrrolo[2,3d]pyrimidine. 1H NMR (200 MHz, DMSO-d6) δ 2.31 (s, 3H) , 2.33 (s,3H), 7.29(m, 1H), 7.52 (m, 1H), 7.96 (m, 1H), 8.14(m, 1H),11.57 (s, 1H); MS (ES) : 276.1 (M++l) . 4-chloro-5,6-dimethyl-2- (2-f luorophenyl ) -7 Ji-pyrrolo [2,3d]pyrimidine. ΧΗ NMR (200 MHz, DMSO-d6) δ 2.34 (s, 3H) , 2.34(s, 3H), 7.33 (m, 2H), 7.44 (m, 1H), 7.99 (m, 1H), 12.23 (s,1H); MS (ES): 276.1 (M++l). 4-chloro-5,6-dimethyl-2-isopropyl-H-pyrrolo [2,3d]pyrimidine.1H NMR (200 MHz, DMSO-dg) δ 1.24 (d, J = 6.6 Hz, 6H) , 2.28 (s,3H), 2.28 (s, 3H), 3.08 (q, J = 6.6 Hz, 1H), 11.95 (s, 1H) ;MS (ES) : 224.0 (M++l) . 4-chloro-5,6-dimethyl-7H-pyrrolo[2,3d]pyrimidine. 1H NMR(200 MHz, DMSO-d6) δ 2.31 (s, 3H) , 2.32 (s, 3H) , 8.40 (s, 1H) ;MS (ES): 182.0 (M++l) . 106 013295 dI-4-chloro-5,6-dimethyl-2-phenyl-7ii-7- ( l-phenylethyl)pyrrolo '[2,3d]pyrimidine.

Préparation 12:

To a solution of dl-1,2-diaminopropane (1.48 g, 20.0 mmol)and sodium carbonate (2.73 g, 22.0 mmol) in dioxane (100.0mL) and water (100.0 mL) was added di-tert-dicarbonate (4.80g, 22.0 mmol) at room température. The resulted mixture wasstirred for 14 hr. Dioxane was removed in vacuo. Theprecipitate was filtered off and the filtrate wasconcentrated in vacuo to dryness. The residue was trituratedwith EtOAc and then filtered. The filtrate was concentratedin vacuo to dryness to give a mixture of dI-l-amino-2-(1,1-dimethylethoxy)carbonyiamino-propane and dl-2-amino-l-(1,1-dimethylethoxy) carbonylamino-propane which were not separableby normal chromatography method. The mixture was used for thereaction in Example 8.

Préparation 13:

To solution of Fmoc-P-Ala-OH (1.0 g, 3.212 mmol) and oxalylchloride (0.428 g, 0.29 mL, 3.373 mmol) in dichloromethane(20.0 mL) was added a few drops of N,N-dimethylformamide at0°C. The mixture was stirred at room température for 1 hrfollowed by addition of cyclopropylmethylamine (0.229 g, 0.28mL, 3.212 mmol) and triethylamine (0.65 g, 0.90 mL, 6.424mmol) . After 10 min, the mixture was treated with 1 Mhydrochloride (10.0 mL) and the agueous mixture was extractedwith dichloromethane (3 x 30.0 mL). The organic solution wasconcentrated in vacuo to dryness. The residue was treatedwith a solution of 20% piperidine in N,N-dimethylforamide(20.0 mL) for 0.5 hr. After removal of the solvent in vacuo,the residue was treated with 1 M hydrochloride (20.0 mL) andethyl acetate (20.0 mL) . The mixture was separated and theaqueous layer was basified with solid sodium hydroxide to pH= 8. The precipitate was removed by filtration and the aqueous solution was subjected to ion exchange column eluted 107 013295 with 20% pyridine to give 0.262 g (57%) of N-cyclopropylmethyl β-alanine amide. 1H NMR (200 MHz, CD 3OD)δ_0.22 (m, 2H), 0.49 (m, 2H), 0.96 (m, 2H) , 2.40 (t, 2H), 2.92(t, 2H), 3.05 (d, 2H); MS (ES): 143.1 (M++l).

Préparation 14: N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine.trans-1,4-cyclonexyldiamine (6.08 g, 53.2 mmol) was dissolvedin dichloromethane (lOOmL). A solution of di-t-

butyldicarbonate (2.32 g, 10.65 mmol in 40 mL dichloromethane) was added via cannula. After 20 hours, thereaction was partitioned between CHC13 and water. The layerswere separated and the aqueous layer was extracted with CHC13(3x). The combined organic layers were dried over MgSO 4,filtered and concentrated to yield 1.20 g of a white solid(53%). XH-NMR (200MHz, CDC13): δ 1.0-1.3 (m, 4H), 1.44 (s,9H), 1.8-2.1 (m, 4H), 2.62 (brm, 1H), 3.40 (brs, 1H), 4.37(brs, 1H0; MS (ES): 215.2 (M++l). 4-(N-acetyl)-N- tert-butoxycarbonyl- trans-1,4-cyclohexyldiamine. N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine (530 mg,2.47 mmol) was dissolved in dichloromethane (20 mL). Aceticanhydride (250 mg, 2.60 mmol) was added dropwise. After 16hours, the reaction was diluted with water and CHCl 3. Thelayers were separated and the aqueous layer was extractedwith CHC13 (3x) . The combined organic layers were dried overMgSO4, filtered and concentrated. Recrystallization (EtOH/H2O) yielded 190 mg of white crystals (30%) . JH NMR(200 MHz, CDC13) : δ 0.9 - 1.30 (m, 4H), 1.43 (s, 9H) , 1.96-2.10 (m, 7H), 3.40 (brs, 1H), 3.70 (brs, 1H), 4.40 (brs, 1H),4.40 (brs, 1H) ; MS (ES): 257.2 (M++l), 242.1 (M+ - 15), 201.1(M* - 56). 4-(4-trans-acetamidocyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-(1-phenylethyl) pyrrolo[2,3d]pyrimidine. 4-(N-acetyl)-N- tert -butoxycarbonyl- trans -1,4- 108 013295 cyclohexyldiamine (190 mg, 0.74 mmol), was dissolved indichloromethane (5 mL) and diluted with TFA (6 ml) . After 16hours, the reaction was concentrated. The crude solid, DMSO(2mL) , NaHCO3 (200 mg, 2.2 mmol) and4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (35 mg, 0.14 mmol) werecombined in a flask and heated to 130 °C. After 4.5 hours,the reaction was cooled to room température and diluted withEtOAc and water. The layers were separated and the aqueouslayer was extracted with EtOAc (3x). The combined organiclayers were dried over MgSO4, filtered and concentrated.Chromatography (silica preparatory plate; 20:1 CHCl3:EtOH)yielded 0.3 mg of a tan solid (1% yield) . MS (ES): 378.2(M++l) . 4-(N-methanesulfonyl)-N- tert-butoxycarbonyl- trans -1,4-cyclohexyldiamine. trans-1,4-cyclohexyldiamine (530 mg, 2.47 mmol) was dissolvedin dichloromethane (20 ml) and diluted with pyridine (233 mg,3.0 mmol). Methanesulfonyl chloride (300 mg, 2.60 mmol) wasadded dropwise. After 16 hours, the reaction was dilutedwith water and CHC13. The layers were separated and theaqueous layer was extracted with CHC13 (3x). The combinedorganic layers were dried over MgSO4, filtered andconcentrated. recrystallization (EtOH/H2O) yielded 206 mg ofwhite crystals (29%). XH-NMR (200MHz, CDC13) : δ 1.10-1.40 (m,4H) , 1.45 (s, 9H), 2.00-2.20 (m, 4H), 2.98 (s, 3H), 3.20-3.50(brs, 2H), 4.37 (brs, 1H); MS (ES) 293.1 (M++l), 278.1 (M+-15), 237.1 (M*-56). 4- (4- trans-methanesulfamidocyclohexyl) amino-5,6-dimethyl-2-phenyl-7H-(1-phenylethyl)pyrrolo[2,3d]pyrimidine. 4-(N-sulfonyl)-N- tert -butoxycarbonyl- trans -1,4-cyclohexyldiamine (206 mg, 0.71 mmol), was dissolved indichloromethane (5ml) and diluted with TFA (6 ml). After 16hours, the reaction was concentrated. The crude reactionmixture, DMSO (2 ml), NaHCO3 (100 mg, 1.1 mmol) and 1-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine were 109 013295 combined ffP*iâ*fi»sk -and heated to 130 °C. After 15 hours,the reaction was cooléd to room température, and diluted withEtOAc (3x). The combined organic layers were dried overMgSO4, filtered and concentrated. Chromatography (silicapreparatory plate, 20:1 CHCl3/EtOH) yielded 2.6 mg of a tansolid (5% yield). MS (ES): 414.2 (M++l) .

Example 1: A solution of 4-chloro-5,6-dimethyl-2-phenylH7pyrrolo [2,3d]pyrimidine (0.50 g, 1.94 mmol) and 4- trans-hydroxycyclohexylamine (2.23 g, 19.4 mmol) in methyl sulfoxide (10.0mL) was heated at 130°C for 5 hr. After cooling down to roomtempérature, water (10.0 mL) was added and the resultedaqueous solution was extracted with EtOAc (3 xlu.O mL). Thecombined EtOAc solution was dried (MgSO4) and filtered, thefiltrate was concentrated in vacuo to dryness, the residuewas chromatographed on silica gel to give 0.49 g (75%) of 4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7 H-pyrrolo [2,3d]pyrimidine . mp 197-199°C; XH NMR (200 MHz, CDC13)δ_1.25-1.59 (m, 8H) , 2.08 (s, 3H) , 2.29 (s, 3H) , 3.68-3.79 (m,1H), 4.32-4.38 (m, 1H) , 4.88 (d, J = 8 Hz, 1H), 7.26-7.49 (m,3H) , 8.40-8.44 (dd, J = 2.2, 8 Hz, 2H) , 10.60 (s, 1H) ; MS(ES): 337.2 (M++l).

The following compounds were obtained in a similar manner tothat of Example 1: 4 - ( 4 - trans-hydroxycyclohexyl ) amino-6-meth.yl-2-phenyl-7.H-pyrrolo[2,3d]pyrimidine. XH NMR (200 MHz, CDCI3) δ_11.37 (s,1H, pyrrole-NH), 8.45 (m, 2H, Ar-H), 7.55 (m, 3H, Ar-H), 6.17(s, 1H, pyrrole-H), 4.90 (br d, 1H, NH) , 4.18 (m, 1H, CH-O),3.69 (m, 1H, CH-N) , 2.40-2.20 (m, 2H) , 2.19-1.98 (m, 2H) ,2.25 (S, 3H, CH3) 1.68-1.20 (m, 4H); MS (ES): 323.2 (M++l). 4-(4- trans -hydroxycyclohexyl)amino-5-methyl-2-phenyl-7 H-pyrrolo[2,3d]pyrimidine. NMR (200 MHz, CDCI3) δ_11.37 (s,1H, pyrrole-NH), 8.40 (m, 2H, Ar-H), 7.45 (m, 3H, Ar-H), 5.96 110 013295 (s, 1H, pyrrole-H), 4.90 (brd, 1H, NH) , 4.18 (m, 1H, CH-O),3.69 (m, 1H, CH-N), 2.38-2.20 (m, 2H) , 2.18-1.98 (m, 2H) ,2.00 (s, 3H, CH3) 1.68-1.20 (m, 4H); MS (ES): 323.2 (M++l). 4- (4-trans-hydroxycyclohexyl) amino-2-phenyl-7i-pyrrolo [2,3d]pyrimidine. mp 245.5-246.5°C; NMR (200MHz, CD3OD) 5 8.33(m,2H, Ar-H) , 7.42 (m, 3H, Ar-H) , 7.02 (d, 1H, J=3.6 Hz, pyrolle-H), 6.53 (d, 1H, J=3.6 Hz, pyrrole-H), 4.26 (m, 1H,CH-O), 3.62 (m,lH, CH-N), 2.30-2.12 (m, 2H) , 2.12-1.96 (m,2H) , 1.64-1.34 (m, 4H> ; MS, M+l=309.3; Anal (C18H20N4O) C, H,N. 4-(4-trans-hydroxycyclohexy1)amino-5,6-dimethyl-2-(3-pyridyl)-7H-pyrrolo[2,3d]pyrimidine. XH NMR (200 MHz, CDC13)δ_1.21-1.54 (m, 8H) ; 2.28 (s, 3H) ; 2.33 (s, 3H) ; 3.70 (m,1H) , 4.31 (m, 1H) , 4.89 (d, 1H) , 7.40 (m, 1H) , 8.61 (m, 2H) ,9.64 (m, 1H); MS (ES): 338.2 (M++l). 4- (4- trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- (2-furyl) -7H-pyrrolo[2,3d]pyrimidine. 1H NMR (200 MHz, CDC13) δ 1.26-1.64(m, 8H), 2.22 (s, 3H) , 2.30 (s, 3H), 3.72(m, 1H), 4.23(m, 1H), 4.85 (d, 1H), 6.52(m, 1H), 7.12 (m, 1H), 7.53 (m,1H), 9.28 (s, 1H); MS (ES): 327.2 (M++l). 4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- (3-furyl) -7H-pyrrolo[2,3d]pyrimidine. 1H NMR (200 MHz, CDC13) δ 1.25-1.63 (m, 8 H), 2.11 (s, 3H), 2.27 (s, 3H), 3.71(m, 1H) , 4.20(m, 1H) , 4.84 (d, 1H) , 7.03 (m, 1H) , 7.45 (m, 1H) , 8.13(m,1H), 10.38 (m, 1H); MS (ES): 327.2 (M++l). 4 -(4 -trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-cyclopentyl-7H-pyrrolo[2,3d]pyrimidine. 1H NMR (200 MHz,CDC13) δ 1.26-2.04 (m, 16 H), 2.26 (s, 3H) , 2.27 (s, 3H) ,

3.15(m, 1H) , 3.70 (m, 1H) , 4.12 (m, 1H) , 4.75(d, 1H) ; MS (ES): 329.2 (M++l). 111 013295 4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- (2-thienyl)-77i-pyrrolo[2,3d]pyrimidin-4-amine. 1H NMR (200 MHz, CDC13) δ I. 28-1.59 (m, 8H), 2.19 (s, 3H), 2.29 (s, 3H), 3.74 (m, 1H) ,4.19 (m, 1H), 4.84 (d, 1H), 7.09 (m, 1H), 7.34 (m, 1H), 7.85(m, 1H), 9.02 (s, 1H); MS (ES): 343.2 (M++l). 4-(4-trans-hydroxycyclohexy1)amino-5,6-dimethyl-2-(3-thienyl)-7if-pyrrolo{2,3d]pyrimidine. 2H NMR (200 MHz, CDC13)δ 1.21-1.60 (m, 8H) , 1.98 (s, 3H), 2.23 (s, 3H) , 3.66 (m, 1H), 4.22 (m, 1H), 7.27 (m, 1H), 7.86 (m, 1H), 8.09 (m, 1H), II. 23 (s, 1H); MS (ES): 343.2 (M++l). 4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(4-fluorophenyl)-7H-pyrrolc[2,3 d]pyrimidine. 1K NMR (200 MHz,CDC13) δ 1.26-1.66 (m, 8H), 1.94 (s, 3H), 2.28 (s, 3H), 3.73(m, 1H) , 4.33 (m, 1H) , 4.92 (d, 1H) , 7.13 (m, 2H) , 8.41 (m,2H), 11.14 (s, 1H); MS (ES): 355.2 (M++l). 4-(4- trans-hydroxycyclohexyl)amino-5 , 6-dimethyl-2-(3-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidine. 1H NMR (200 MHz,CDC13) δ 1.26-1.71 (m, 8H), 2.06 (s, 3H), 2.30 (s, 3H), 3.72(m, 1H), 4.30 (m, 1H), 4.90 (d, 1H), 7.09 (m, 1H), 7.39 (m,1H) , 8.05 (m, 1H) , 8.20 (m, 1H) , 10.04 (s. 1H) ; MS (ES): 355.2 (M++l). 4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2 -(2 -fluorophenyl)-7H-pyrrolo[2,3d]pyrimidine. 1H NMR (200 MHz,CDC13) δ 1.30-1.64 (m, 8H), 2.17 (s, 3H), 2.31 (s, 3H), 3.73(m, 1H) , 4.24 (m, 1H) , 4.82 (d, 1H) , 7.28 (m, 2H) , 8.18 (m,1H) , 9.02 (m, 1H), 12.20 (s, 1H); MS (ES): 355.3 (M++l) . 4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-isopropyl-7H-pyrrolo [2,3d]pyrimidine 1H NMR (200 MHz, CDC13) δ 1.31 (d,J = 7.0 Hz, 6H) , 1.30-1.65 (m, 8H) , 2.27 (s, 3H) , 2.28 (s,3H) , 3.01 (m, J = 7.0 Hz, 1H) , 3.71 (m, 1H) , 4.14 (m, 1H) ,4.78 (d, 1H); MS (ES): 303.2. 013295 - 112 - dl -4- (2- crans -hydroxycyclohexyl ) amino-5 , 6-dimechyl-2-isopropyl-7H-pyrrolo [2,3d]pyrimidine H NMR (200 MHz, CDCI3) d 1.31-1.42 (br, 4H) , 1.75-1.82 (br, 4H) , 2.02 (s, 3H), 2.29 (s, 3H), 3. 53 (m, 1H) , 4.02 (m, 1H) , 5.08 (d, 1H) , 7.41-7.48 5 (m, 3H), 8. 30 (m, 2H) , 10.08 (s, 1H) ; MS (ES) : 337 .2 (M++l). 4- (3,4-trans-dihydroxycyclohexyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS (ES): 353.2 (M++l). 10 4-(3,4-cis-dihydroxylcyclohexyl)amino-5,6-dimethyl-2-phenyl- 7H-pyrrolo[2,3d]pyrimidine. MS (ES): 353.2 (M++l). 4-(2-acetylaminoethy1)amino-5 , 6-dimethyl-2-phenyl-7H-pvrrolo[2.3d]pyrimidine. 15 mp 196-199°C; XH NMR (200 MHz, CDC13) δ_1.72 (s, 3H) , 1.97 (s,3H), 2.31 (s, 3H), 3.59 (m, 2H), 3.96 (m, 2H), 5.63 (br, 1H) ,7.44-7.47 (m, 3H), 8.36-8.43 (dd, J = 1 Hz, 7 Hz, 2H), 10.76(s, 1H); MS (ES): 324.5 (M++l). 20 dI-4- (2-trans-hydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl- 7H-pyrrolo[2,3d]pyrimidine.1 :H NMR (200 MHz, CDC13) δ_1.62 (m, 2H) , 1.79 (br, 4H) , 1.92(s, 3H) , 2.29 (s, 3H), 4.11 (m, 1H) , 4.23 (m, 1H) , 5.28 (d, 25 1H), 7.41-7.49 (m, 3H), 8.22 (m, 2H), 10.51 (s, 1H); MS (ES): 323.2 (M++l). 1 For préparation of 2-trans-hydroxycyclopentylamine, see PCT9417090. 30 dl-4- (3-trans-hydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl- 7H-pyrrolo[2,3d]pyrimidine.1 XHNMR (200 MHz, CDC13) δ_1.58-1.90 (br, 6 H, ) , 2.05 (s, 3H) ,2.29 (s, 3H), 4.48-4.57 (m, 1H), 4.91-5.01 (m, 2H), 7.35-7.46(m, 3H), 8.42-8.47 (m, 2H) , 10.11 (s, 1H) ; MS (ES): 323.2 35 (M++l) . 113 01329 5 1 For préparation of 3-trans-hydroxycyclopentylamine, see 3ip-A-322242. dl-4- ( 3-cis-hydroxycyclopentyl) amino-5,6-dimethyl-2-pheny-l-7H-pyrrolo[2,3d]pyrimidine.1 ΧΗ NMR (200 MHz, CDC13) δ_1.82-2.28 (br, 6H) , 2.02 (s, 3H) ,2.30 (s, 3H) , 4.53-4.60 (m, 1H) , 4.95-5.08 (m, 1H) , 5.85-5.93(d, 1H), 7.35-7.47 (m, 3H), 8.42-8.46 (m, 2H), 10.05 (s, 1H) ;MS (ES): 323.2 (M++l). 1 For préparation of 3-cis-hydroxycyclopentylamine, see EP—A-322242. 4- ( 3,4-trans-dihydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl-7ff-pyrrolo [2,3d]pyrimidine.1 2Η NMR (2 00 MHz, CDC13, ô_1.92-1.99 (br, 2H), 2.14 (s, 3H), 2.20 (br, 2H), 2.30 (s, 3H) , 2.41-2.52 (br, 2H), 4.35 (m, 2H), 4.98 (m, 2H), 7.38-7.47 (m,3H), 8.38-8.42 (m, 2H), 9.53 (s, 1H); MS (ES): 339.2 (M++l).1 For préparation of 3,4-trans-dihydroxycyclopentylamine, seePCT 9417090. 4-(3-amino-3-oxopropy1)amino-5,6-dimethy1-2-phenyl-7 H-pyrrolo[2,3d]pyrimidine. XH NMR (200 MHz, CDC13) δ_2.02 (s, 3H) , 2.29 (s, 3H), 2.71 (t,2H) , 4.18 (m, 2H) , 5.75-5.95 (m, 3H) , 7.38-7.48 (m, 3H) , 8.37-8.41 (m, 2H) , 10.42 (s, 1H) ; MS (ES): 310.1 (M+-il). 4-(3-N-cyclopropylmethylamino-3-oxopropyl)amino-5,6-dimethyl- 2-phenyl-7H-pyrrolo [2,3d]pyrimidine. 1H NMR (200 MHz, CD3OD)δ_0.51 (q, 2H) , 0.40 (q, 2H) , 1.79-1.95 (br, 1H) , 2.36 (s,3H), 2.40 (s, 3H), 2.72 (t, 2H), 2.99 (d, 2H) , 4.04 (t, 2H) ,7.58-7.62 (m, 3H) , 8.22-8.29 (m, 2H); MS (ES): 364.2 (M++l). 4- (2-amino-2-oxoethyl) amino-5, 6-dimethyl-2-phenyl-7.fi-pyrrolo[2,3d]pyrimidine 1H NMR (200 MHz, CD3OD) δ 2.31 (s, 3H) , 2.38 (s, 3H), 4.26 (s, 2H), 7.36 (m, 3H), 8.33 (m, 2H);MS (ES): 396.1 (M++l). 114 ύ1 329 5 4-(2-N-methylamino-2-oxoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d]pyrimidine. XH NMR (200 MHz, CDC13) δ_1.99 (s,3H), 2.17 (s, 3H), 2.82 (d, 3H) , 4.39 (d, 2H), 5.76 (t, 1H) ,6.71 (br, 1H) , 7.41-7.48 (m, 3H) , 8.40 (m, 2H) , 10.66 (s,1H) ; MS (ES): 310.1 (M++l) . 4- (3-tert-butyloxyl-3-oxopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo(2,3d]pyrimidine. XH NMR (200 MHz, CDC13) δ_1.45 (s,9H), 1.96 (s, 3H), 2.29 (s, 3H), 2.71 (t, 2H), 4.01 (q, 2H) ,5.78 (t, 1H), 7.41-7.48 (m, 3H), 8.22-8.29 (m, 2H) ; MS (ES): 367.2 (M++l). 4-(2-hydroxyethyl)amino-5,6-dimethyl-2-phenyl-7 H- pyrrolo[2,3d]pyrimidine. ΧΗ NMR (200 MHz, CDC13) δ 1.92 (s,3H), 2.29 (s, 3H), 3.81-3.98 (br, 4H), 5.59 (t, 1H) , 7.39-7.48 (m, 3H), 8.37 (m, 2H), 10.72 (s, 1H); MS (ES): 283,1(M++l) . 4-( 3-hydroxypropyl)amino-5,6-dimethyl-2-phenyl-7 H- pyrrolo[2,3d]pyrimidine. 1H NMR (200 MHz, CDC13) δ 1.84 (m,2H) , 1.99 (s, 3H), 2.32 (s, 3H), 3.62 (t, 2H), 3.96 (m, 2H),3.35 (t, 1H), 7.39-7.48 (m, 3H), 8.36 (m, 2H), 10.27 (s, 1H) ;MS (ES) : 297.2 (M++l) . 4- ( 4-hydroxybutyl) amino-5,6-dimethyl-2-phenyl-7 H-pyrrolo[2,3d]pyrimidine. XH NMR (200 MHz, CDC13) δ 1.71-1.82 (m, 4H) ,1.99 (s, 3H), 2.31 (s, 3H), 3.68-3.80 (m, 4H), 5.20 (t, 1H),7.41-7.49 (m, 3H) , 8.41(m, 2H) , 10.37 (s, 1H) ; MS (ES): 311.2(M++l) . 4- (4-trans-acetylaminocyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. 4- ( 4-trans-methylsulfonylaminocyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. 013295 115 4-(2-acetylaminoethyl)amino-o,6-dimethyl-2-phenyl-7 Η-Ί-(1-phenylethyl)pyrrolo[2,3d]pyrimidine. 4- (4-trans-hydoxycyclohexyl)amino-5,6-dimethyl-2-phenyl-f#-l-phenylethyl)pyrrolo[2,3d]pyrimidine. 4-(3-pyridylmethyl)amino-5,6-dimethyl-2-phenyl-7 H-7 -(1-phenylethyl)pyrrolo[2,3d]pyrimidine. 4-(2-methylpropyl)amino-5,6-dimethyl-2-phenyl-7 Η-Ί-(1-phenylethyl)pyrrolo[2,3d]pyrimidine.

Example 2 :

To a stirred suspension of triphenylphosphine (0.047 g, 0.179mmol) and benzoic acid (0.022 g, 0.179 mmol) in THF (1.0 mL)cooled to 0°C was added 4-(4-fcrans-hydroxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7ü-pyrrolo [2,3d] pyrimidine (0.05 g,0.149 mmol) at 0°C. Diethyl azodicarboxylate (0.028 ml, 0.179mmol) was then added dropwise over 10 minutes. The reactionwas then allowed to warm to room température. After reactionwas complété by TLC the reaction mixture was quenched withaqueous sodium bicarbonate (3.0 mL). The aqueous phase wasseparated and extracted with ether (2 X 5.0 mL) . The organicextracts were combined, dried, and concentrated in vacuo todryness. To the residue was added ether (2.0 mL) and hexane(5.0 mL) whereupon the bulk of the triphenylphosphine oxidewas filtered off. Concentration of the filtrate gave aviscous oil which was purified by column chromatography(hexane :ethyl acetate=4:l) to give 5.0 mg (7.6%) of 4-(4-cis-benzoyloxycyclohexyl ) amino-5,6-dimethyl-2-phenyl-7.fi-pyrrolo[2,3d]pyrimidine. MS (ES): 441.3 (M++l). The reactionalso produced 50.0 mg (84%) of 4-(3-cyclohexenyl)amino-5,6-dimethyl-2-phenyl-7jci-pyrrolo [2,3d] pyrimidine. MS (ES): 319.2(M++l) . 116 013295

Exainple 3: Το a solution of 4-(4-cis-benzoyloxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine (5.0 mg, 0.0114mmol) in éthanol (1.0 mL) was added 10 drops of 2M sodiumhydroxide. After 1 hr, the reaction mixture was extractedwith ethyl acetate (3 x 5.0 mL) and the organic layer wasdried, filtered and concentrated in vacuo to dryness. Theresidue was subjected to column chromatography (hexane:ethylacetate=4:l) to give 3.6 mg (94%) of 4-(4-cis-hydroxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7 H- pyrrolo[2,3d]pyrimidine. MS (ES): 337.2 (M++l).

The following compounds were obtained in a similar manner asthat of Example 3 : 4- (3-N,N-dimethyl-3-oxopropyl) amino-5,6-dimethyl-2-phenylff?pyrrolo[2,3d]pyrimidine. 1H NMR (200 MHz, CDC13) δ 2.01 (s,3H), 2.31 (s, 3H), 2.73 (t, 2H), 2.97 (s, 6H), 4.08 (m, 2H) ,6.09 (t, 1H), 7.41-7.48 (m, 3H), 8.43 (m, 2H), 10.46 (s, 1H);MS (ES) : 338.2 (M++l) . 4-(2-formylaminoethyl)amino-5,6-dimethyl-2-phenyl-7 H- pyrrolo[2,3d]pyrimidine. NMR (200 MHz, CDC13) δ 2.26 (s,3H), 2.37 (s, 3H), 3.59-3.78 (m, 2H) , 3.88-4.01 (m, 2H) , 5.48-5.60 (m, 1H), 7.38-7.57 (m, 3H), 8.09 (s, 1H), 8.30-8.45(m, 2H), 8.82 (s, 1H); MS (ES): 310.1 (M++l). 4-(3-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7 H- pyrrolo[2,3d]pyrimidine. MS (ES): 338.2 (M++l). - 117 013295

Exemple 4 : 4- (3-tert-butyloxy-3-oxopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (70.0 mg, 0.191 mmol)) wasdissolved in trifluoroacetic acid:dichloromethane (1:1, 5.0mL) . The resulting solution was stirred at roomtempérature for 1 hr. and then refluxed for 2 hr. Aftercooling down to room température, the mixture wasconcentrated in vacuo to dryness. The residue was subjectedto préparative thin layer chromatography (EtOAc:hexane:AcOH=7 : 2.5 : 0.5) to give 40.0 mg (68%) of. 4- ( 3-hydroxy-3- oxopropyl) amino-5,6-dimethyl-2-phenyl-7 H-pyrrolo[2,3d]pyrimidine. ΧΗ NMR (200 MHz, CD3OD) δ 2.32 (s, 3H) , 2.38 (s,3H), 2.81 (t, 2H), 4.01 (t, 2H), 7.55 (m, 3H), 8.24 (m, 2H);MS (ES): 311.1 (M++l).

The following compound was obtained in a similar manner asthat of Example 4 : 4-(3-aminopropyl)amino-5,6-dimethyl-2-phenyl-7 H- pyrrolo[2,3d]pyrimidine. MS (ES): 296.1 (M ++1), 279.1 (M+- NH3) .

Example 5 : 4-(3-hydroxy-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7 H-pyrrolo[2,3d]pyrimidine (50.0 mg, 0.161 mmol) was dissolvedin a mixture of N,N-dimethylformamide (0.50 mL) , dioxane(0.50 mL) and water(0.25 mL) . To this solution was addedmethylamine (0.02 mL, 40% wt in water, 0.242 mmol),triethylamine (0.085 mL) and Ν,Ν,Ν'Ν'-tetramethyl uroniumtetrafluoroborate (61.2 mg, 0.203 mmol). After stirring atroom température for 10 min, the solution was concentratedand the residue was subjected to préparative thin layerchromatography (EtOAc) to give 35.0 mg (67%) of4-(3-N-methyl-

3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7 H-pyrrolo[2,3d]pyrimidine. JH NMR (200 MHz, CDC13) δ 1.92 (s,3H) , 2.30 (s, 3H), 2.65 (t, 2H), 4.08 (t, 2H), 5.90 (t, 1H) ,6.12 (m, 1H) , 7.45 (m, 3H)‘, 8.41 (m, 2H) , 10.68 (s, 1H) ; MS 118 013295 (ES): 311.1 (M++l).

The following compounds were obtained in a similar manner asthat of Example 5 : 4-(2-cyclopropanecarbonylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d]pyrimidine. MS (ES): 350.2 (M++l) . 4- (2-isobutyrylaminoethyl) amino-5,6-dimethyl-2-phenyl-7 H-pyrrolo [2,3d]pyrimidine. MS (ES): 352.2 (M++l) . 4- (3-propionylaminopropyl)amino-5,6-dimethyl-2-phenyl-7 H-pyrrolo[2,3d]pyrimidine. 1H NMR (200 MHz, CDC13) δ 1.00-1.08(t, 3H) , 1.71-2.03 (m, 4H) , 2.08 (s, 3H) , 2.37 (s, 3»), 3.26- 3.40 (m, 2H) , 3.79-3.96 (m, 2H), 5.53-5.62 (m, 1H),_6.17-b.33(m, 1H) , 7.33-7.57 (m, 3H) , 8.31-8.39 (m, 2H), 9.69 (s, 1H);MS (ES): 352.2 (M++l). 4- (2-methylsulfonyiaminoethyl)amino-5,6-dimethyl-2-phenylii?pyrrolo(2,3d]pyrimidine. XH NMR (200 MHz, CDC13) δ 2.18 (s,3H) , 2.27 (s, 3H), 2.92 (s, 3H) , 3.39-3.53 (m, 2H), 3.71-3.88(m, 2H), 5.31-5.39 (m, 1H), 6.17-6.33 (m, 1H), 7.36-7.43 (m,3H), 8.20-8.25 (m, 2H), 9.52 (s, 1H); MS (ES): 360.2 (M++l).

Example 6: A mixture of 4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (0.70 g, 2.72 mmol) and 1,2-diaminoethane (10.0mL, 150 mmol) was refluxed under inert atmosphère for 6 hr.The excess amine was removed in vacuo, the residue was washedsequentially with ether and hexanê to give 0.75 g (98%) of 4-(2-aminoethyl)amino-5 , 6-di met hy1- 2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS (ES); 282.2 (M++l), 265.1 (M+- NH3) .

Example 7 :

To a solution of 4-(2-aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (70.0 mg, 0.249 mmol) andtriethylamine (50.4 mg, 0.498 mmol) in dichloromethane (2.0 119 013295 mL) was added propionyl chloride (25.6 mg, 0.024 mL, 0.274mmol) at 0°C. After 1 hr, the mixture was concentrated invacuo and the residue was subjected to préparative thin layerchromatography (EtOAc) to give 22.0 mg (26%) of 4-(2-propi ony1aminoethy1)amino-5,6-dime thy1- 2-pheny1- 7 H-pyrrolo[2,3d]pyrimidine. MS (ES): 338.2 (M++l ) .

The following compounds were obtained in a similar manner asthat of Example 7 : 4- (2-N' -methylureaethyl) amino-5, 6-dimethyl-2-phényl-7Jï-pyrrolo [2,3d] pyrimidine. XH NMR (200 MHz, CDC13) δ 2.13 (s,3H), 2.32 (s, 3H), 3.53 (d, 3H), 3.55 (m, 2H) , 3.88 (m, 2H) , 4.29 (m, 1H) , 5.68 (t, 1H), 5.84 (m, 1H), 7.42 (m, 3H) , 8.36 (dd, 2H), 9.52 (s, 1H); MS (ES): 339.3 (M*+l). 4 -(2-N'-ethylureaethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS (ES): 353.2 (M++l).

Example 8:

To a solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide hydrochloride (41.1 mg, 0.215 mmol), dimethylamino-pyridine (2.4 mg, 0.020 mmol) and pyruvic acid (18.9 mg,0.015 mL, 0.215 mmol) in dichloromethane (2.0 mL) was added4- ( 2 - aminoe thy 1) amino-5,6-dimethyl-2-phenyl-7Ji-pyrrolo [2,3d]pyrimidine (55.0 mg, 0.196 mmol) . The mixture was stirred atroom température for 4 hr. Usual workup and columnchromatography (EtOAc) then gave 10.0 mg (15%) of 4-(2'-pyruvylamidoethy 1 ) amino-5 , 6 - dime thy 1 - 2-pheny 1 - 7//-pyrrolo[2,3d]pyrimidine. MS (ES): 352.2 (M++l).

Example 9 :

To a solution of 4-(2-aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (60.0 mg, 0.213 mmol) indichloromethane (2.0 mL) was added N-trimethylsilylisocyanate (43.3 mg, 0.051 mL, 0.320 mmol). The mixture was 013295 120 stirred at room température for 3 hr followed by addition ofaqueous sodium bicarbonate. After filtration through smallamount of silica gel, the filtrate was concentrated in vacuoto dryness to give 9.8 mg (14%) of 4-(2-ureaethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS (ES): 325.2(M++l) .

The following compounds were obtained in a similar manner asthat of Example 9 : d.Z-4- (2-acetylaminopropyl ) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d]pyrimidine. 1H NMR (200 MHz, CDC13) δ 1.28-1.32(d, J=8 Hz, 3 H), 1.66 (s, 3H) , 1.96 (s, 3H), 2.30 (s, 3H)3.76-3.83 (m, 2IÎ) , 4.104.30 (m, 1K) , 5.60-5.66 (t, J=6 Hz,1H), 7.40-7.51(m, 3H), 8.36-8.43 (m, 2H), 10.83 (s, 1H); MS(ES) : 338.2 (M++l) . (R)-4-(2-acetylaminopropyl)amino-5, 6-dimethyl-2-phenyl-7H-pyrrolo[2,3d] pyrimidine. XH NMR (200 MHz, CDC13) δ 1.31 (d,3H) , 1.66 (s, 3H) 1.99 (s, 3H), 2.31 (s, 3H), 3.78-3.83 (m,2H), 4.17-4.22 (m, 1H), 5.67 (t, 1H), 7.38-7.5 (m, 3H), 8.39(m, 2H), 10.81 (s, 1H); MS (ES): 338.2 (M++l). (R) -4-(l-methyl-2-acetylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d]pyrimidine. 1H NMR (200 MHz, CDC13) δ 1.41 (d, 3H), 1.68 (s, 3H), 2.21 (s, 3H), 2.34 (s, 3H), 3.46-3.52 (br, m, 2H) , 4.73 (m, 1H) , 5.22 (d, 1H) , 7.41-7.46 (m,3H), 8.36-8.40 (m, 2H), 8.93 (s, 1H); MS (ES): 338.2 (M++l). (S) -4-(2-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo (2,3d]pyrimidine. 1H NMR (200 MHz, CDC13) δ 1.31 (d,3H) , 1.66 (s, 3H) 2.26 (s, 3H), 2.35 (s, 3H) , 3.78-3.83 (m,2H), 4.17-4.22 (m, 1H), 5.67 (t, 1H) , 7.38-7.5 (m, 3H), 8.39(m, 2H), 8.67(s, 1H); MS (ES): 338.2 (M++l). (S)-4-(l-methyl-2-acetylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d]pyrimidine. 1H NMR (200 MHz, CDC13) δ - 121 - 013295 1.41 (d, 3H), 1.68 (s, 3H), 2.05 (s, 3H), 2.32 (s, 3H) , 3.46-3.52 (m, 2H) , 4.73 (m, 1Ή) , 5.22 (d, 1H), 7.41-7.46 (m, 3H), 8.36- 8.40 (m, 2H) , 10.13 (s, 1H); MS (ES): 338.2 ( M++l) .

Example 10:

Reaction of 4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine with the mixture of dl-l-amino-2-(1,1-dimethylethoxy)carbonylamino-propane and dl-2-amino-1-(1,1-dimethylethoxy)carbonylamino-propane was run in a similar manner asthat of Example 1. The reaction gave a mixture of dl-4-(1-methyl-2-(1,1-dimethylethoxy)carbonylamino)ethylamino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine and dl-4-(2-methyl-2-(1,1-dimethylethoxy)carbonylamino)ethylamino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine which wreseparated by column chromatography (EtOAc:hexanes=l:3). Thefirst fraction was dl -4-(l-methyl-2-(1,1-dimethylethoxy)c arbony1aminoethy1)amino - 5,6 -dimethy1-2-pheny1-7H-pyrrolo[2,3d]pyrimidine : 1H NMR (200 MHz, CDC13) δ 1.29 - 1.38(m, 12 H), 1.95 (s, 3H) , 2.31 (s, 3H) 3.34-3.43 (m, 2H) ,4.62-4.70 (m, 1H), 5.36-5.40 (d, J=8 Hz, 1H), 5.53 (br, 1H), 7.37- 7.49(m, 3H), 8.37-8.44(m, 2H) , 10.75 (s, 1H). MS 396.3(M++l); The second fraction was dl-4-(2-(1,1-dimethylethoxy)carbonylaminopropyl)amino-5,6-dimethy1-2 -phenyl-7H-pyrrolo[2,3d]pyrimidine: XH NMR (200 MHz, CDC13) δ1.26-1.40 (m, 12 H), 2.00 (s, 3H), 2.31 (s, 3H) 3.60-3.90 (m,2H), 3.95-4.10 (m, 1H), 5.41-5.44 (d, J=6.0 Hz, 1H), 5.65(br,1H) , 7.40-7.46(m, 3H) , 8.37-8.44(m, 2H) , 10.89 (s, 1H) ; MS(ES): 396.2 (M++l).

The following compounds were obtained in a similar manner asthat of Example 10: (S, S) -4- (2 -acetylaminocyclohexyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d]pyrimidine . XH NMR (200 MHz, CDC13) δ_1.43 (m, 4 H), 1.60 (s, 3 H) , 1.83 (m, 2 H), 2.18 (s, 3 H), 2.30 (m, 2 H), 2.32 (s, 3 H) , 3.73 (br, 1H), 4.25 (br, 1H), 5.29 (d, 1H), 7.43-7 .48 (m, 3H) , 8 .35-8.40 (m, 2H), 9. .05 (s, 1 H) . 122 013295 4- (2-methyl-2-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d]pyrimidine . XH NMR (200 MHz, CDC13) δ_1.51 (s,6H) , 1,56 (s, 3H), 2.07 (s, 3H), 2.36 (s, 3H) , 3.76 (d, 2H) ,5.78 (t, 1H), 7.41-7.48 (m, 3H), 7.93 (ε, 1H), 8.39 (m, 2H) , 5 10.07 (s, 1H); MS (ES): 352.3 (M++l).

Example 11: dl-4-(l-methyl-2-(1,1-dimethylethoxy) carbonyl aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (60.6 10 mg, 0.153 mmol) was treated with trifluoroacetic acid (0.5mL) in dichloromethane (2.0 mL) for 14 hr. The organicsolvent was removed in vacuo to dryness. The residue wasdissolved in N,N-dimethylformamide (2.0 mL) and triethylamine(2.0 mL ) . To the solution at 0°C was added acetic anhydride 15 (17.2 mg, 0.016, 0.169 mmol). The resulted mixture was stirred at room température for 48 hr and then concentratedin vacuo to dryness. The residue was subjected to préparativethin layer chromatography (EtOAc) to give 27.0 mg (52%) ofdl-4-(1-methyl-2-acetylaminoethyl)amino-5,6-dimethyl-2- 20 phenyl-7H-pyrrolo[2,3d]pyrimidine. 1H NMR (200 MHz, CDC13) δ 1.38-1.42 (d, J=8 Hz, 3 H), 1.69 (s, 3H) , 2.01 (s, 3H) , 2.32(s, 3H) 3.38-3.60 (m, 2H), 4.65-4.80 (m, 1H), 5.23-5.26 (d,J=6 Hz, 1H) , 7.40-7.51(m, 3H) , 8.37-8.43(m, 2H) , 10.44 (s, 1H); MS (ES): 338.2 (M++l). 25

Example 12: (R, R) -4- (2-aminocyclohexyl ) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d]pyrimidine, prepared in a similar manner asthat of Example 1 from 4-chloro-5,6-dimethyl-2-phenyl-7H- 30 pyrrolo[2,3d]pyrimidine (0.15 g, 0.583 mmol) and (IR, 2R)-(- )-1,2-diaminocyclohexane (0.63 g, 5.517 mmol), was treatedwith triethylamine (0.726 g, 7.175 mmol) and acetic anhydride(0.325 g, 3.18 mmol) in N,N-dimethylformamide (10.0 mL) atroom température for 2 hr. After removal of solvent in vacuo, 35 ethyl acetate (10.0 mL) and water (10.0 mL) were added to theresidue. The mixture was separated and the agueous layer was 013295 - 123 - extracted with ethyl acetate (2 x 10.0 mL) . The combinedethyl acetate solution was dried (MgSO4) and filtered. Thefiltrate was concentrated in vacuo to dryness and the residuewas subjected to column chromatography (EtOAc:Hexane=l: 1) togive 57.0 mg (26%) of (R,R)-4-(2-acetylaminocyclohexyl)amino-

5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. 1H NMR (200 MHz, CDC1, s) δ_1.43 (m, 4 H) , 1.60 (s, 3 H), 1.84 (m, 2 H) , 2 .22 (s, 3 H), 2.30 (m, 2 H) , 2.33 (s, 3 H), 3.72 (br, 1H) , 4.24 (br, 1H), 5.29 (d, 1H) , 7.43-7.48 (m, 3H), 8.35- 8.39 (m, 2H) , 8.83 (s, 1 H); MS (ES): 378.3 (M++l) .

Ex amp le 13 :

To a solution of 4-(2-hydroxyethyl)amino-5,6-dimethyl-2-pheny1-7#-pyrrolo[2,3d]pyrimidine (40.0 mg, 0.141 mmol) inpyridine (1.0 mL) was added acetic anhydride (0.108 g, 1.06mmol) at 0°C. The mixture was stirred at room température for4 hr and the solvent was removed in vacuo. The residue wassubjected to préparative thin layer chromatography(EtOAc:hexane=l: 1) to give 32.3 mg (71%) of 4-(2-acetyloxyethyl ) amino-5,6-dimethyl-2-phenyl-7#-pyrrolo [2,3d]pyrimidine. ’ή NMR (200 MHz, CDC13) δ_1.90 (s, 3H) , 2.08 (s,3H), 2.31 (s, 3H), 4.05 (m, 2H) , 4.45 (t, 2H) , 5.42 (m, 1H),7.41-7.49 (m, 3H), 8.42(m, 2H), 11.23 (s, 1H) .

Example 14 : A solution of Fmoc-p-Ala-OH (97.4 mg, 0.313 mmol) and oxalylchloride (39.7 mg, 27.3 pL, 0.313 mmol) in dichloromethane(4.0 mL) with 1 drop of N,N-dimethylformamide was stirred at0°C for 1 hr followed by addition of 4-(2-aminoethyl)amino- 5,6-dimethyl-2-phenyl-7#-pyrrolo[2,3d]pyrimidine (80.0 mg,0.285 mmol) and triethylamine (57.6 mg, 79.4 pL, 0.570 mmol)at 0°C. After 3 hr, the mixture was concentrated in vacuo andthe residue was treated with the solution of 20% piperidinein N,N-dimethylforamide (2.0 mL) for 0.5 hr. After removal ofthe solvent in vacuo, the residue was washed with diethylether:hexane (1:5) to give 3.0 mg (3%) of 4-(6-amino-3-aza-4- 013295 124 oxohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [ 2 , 3 d ]pyrimidine. MS (ES): 353.2 (M++l).

Example 15: A solution of 4-(2-aminoethyl) amino-5,6-dimethyl-2-phenyl-77f-pyrrolo[2,3d]pyrimidine (70.0 mg, 0.249 mmol) and succinicanhydride (27.0 mg, 0.274 mmol) in dichloromethane (4.0 mL)with 1 drop of N,N-dimethylformamide was stirred at roomtempérature for 4 hr. The reaction mixture was extracted with20% sodium hydroxide (3 x 5.0 mL). The aqueous solution wasacidified with 3 M hydrochloride to pH = 7.0. The wholemixture was extracted with ethyl acetate (3 x 10 mL) . Thecombined organic solution was dried (MgSO4) and filtered. Thefiltrate was concentrated in vacuo to dryness to give 15.0 mg(16%) of 4-(7-hydroxy-3-aza-4,7-dioxoheptyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS (ES): 382.2(M++l) .

Example 16 :

To 10 mL of dimethylformamide (DMF) at room température wereadded 700 mg of 4-(cis-3-hydroxycyclopentyl)amino-2-phenyl- 5,6-dimethyl-7H-pyrrolo[2,3d]pyrimidine followed by 455 mg ofN-Boc glycine, 20 mg of Ν,Ν-dimethylaminopyridine (DMAP) , 293mg of hydroxybenzotriazole (HOBT) and 622 mg of l-(3-dimethylaminopropyl)-3-ethylcarboiimide hydrochloride (EDC1) .The reaction mixture was left stirring overnight. DMF wasthen removed under reduced pressure and the reaction mixturewas partitioned between 20mL of ethyl acetate and 50mL ofwater. The aqueous portion was extracted further with 2x2OmLof ethyl acetate and the combined organic portions werewashed with brine, dried over anhydrous sodium sulfate,filtered and concentrated. Purification on silica gel,eluting with ethyl acetate/hexane gave 410 mg of the desiredproduct: 4-( cis-3-(N-t-butoxycarbonyl-2-aminoacetoxy)cyclopentyl) amino-2-phenyl-5,6, -dimethyl- 7H-pyrrolo [2,3d]pyrimidine, MS (ES) (M++l ) =480.2 . The ester was then treated 013295 - 125 - with 5 mL of 20% trifluoroacetic acid in dichloromethane atroom température, left over night and then concentrated.Trituration with ethyl acetate gave 300 mg of an off whitesolid; 4- ( cis-3- (2-aminoacetoxy) cyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine trifluoroacetic acidsait, MS (ES) (M++l)=380.1.

One skilled in the art will appreciate that the followingcompounds can be synthesized by the methods disclosed above: 4- ( cis-3-hydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d] pyrimidine MS (ES) (M++l)= 323.1. 4- ( cis-3 (2 -aminoacetoxy) cyclopentyl ) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d] pyrimidinetrifluoroacetic acid saitMS (ES) (M++l) = 380.1. 4 -(3-acetamido)piperidinyl-5,6-dimethyl-2-pheny1-7H-pyrrolo[2,3d]pyrimidineMS (ES) (M++l) = 364.2. 4-(2-N'-methylureapropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d] pyrimidine, MS (ES) (M++l)=353.4. 4 -(2-ace tami dobuty1)amino-5 , 6-dimethy1-2-pheny1-7H-pyrrolo[2,3d]pyrimidine, MS (ES) (M++l)= 352.4. 4-(2-N'-methylureabutyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidineMS (ES) (M++l)= 367.5 4-(2-aminocyclopropylacetamidoethyl)amino-2-pheny1-7H-pyrrolo[2,3d] pyrimidine MS (ES) (M++l)= 309.1. 4-(trans-4-hydroxycyclohexyl)amino-2-(3-chlorophenyl)-7H- - 12 6 013295 pyrrolo[2,3d] pyrimidine MS (ES) (M++l)=342.8. 4-(trans-4-hydroxycyclohexyl)amino-2-(3-fluorophenyl)- 7H- pyrrolo [2,3d] pyrimidine MS (ES) (M++l)=327.2. 4-( trans- 4-hydroxycyclohexyl)amino-2-(4-pyridyl)- 7H- pyrrolo[2,3d]pyrimidine MS (ES) (M++l)=310.2.

Example 17

Scheme IX

The pyrrole nitrogen of (7) (Scheme IX) was protected withdi-t-butyldicarbonate under basic conditions to yield thecorresponding carbamate (22) . Radical bromination of (22)proceeded regioselectively to yield bromide (23). Ingeneral, compound (23) served as a key electrophilicintermediate for various nucleophilic coupling partners.Displacement of the alkyl bromide with sodium phenolatetrihydrate yielded compound (24) . Subséquent displacement of - 127 013295 the aryl chloride and removal ofprotecting group occurred in onecompound (25). the t-butyl carbamatestep yielding desired

Detailed Synthesis of Coxnpounds (22)-(25) in Accordance withScheme IX

Di-t-butyl dicarbonate (5.37 g, 24.6 mmol) and dimethylaminopyridine (1.13 g, 9.2 mmol) were added to a solutioncontaining (7) (1.50 g, 6.15 mmol) and pyridine (30 mL ) .

Af ter 20 h the reaction was concentrated and the residue waspartitioned between CH2C12 and water. The CH2C12 layer wasseparated, dried over MgSO4, filtered and concentrated toyield a i»lack solid. Flash chromatography ( SiO2; 1/9EtOAc/Hexanes, Rf 0.40) yielded 1.70 g (80%) of a white solid(22). XH NMR (200 MHz, CDC13) δ_8.50 (m, 2H, Ar-H) , 7.45 (m,3H, Ar-H), 6.39 (s, 1H, pyrrole-H) , 2.66 (s, 3H, pyrrole-CHj) ,1.76 (s, 9H, carbamate-) ; MS, Μ + 1 = 344.1; Mpt = 175-177°C.

23 013295 - 128 - N-Bromosuccinimide (508 mg, 2.86 mmol) and AIBN (112 mg, 0.68mmol) were added to a solution containing (22) (935 mg, 2.71mmol) and CC14 (50 mL) . The solution was heated to reflux.After 2 h the reaction was cooled to room température andconcentrated in vacuo to yield a white solid. Flashchromatography (SiC^; 1/1 CH2C12/Hexanes, R^O.30) yielded 960mg (84%)of a white solid (23) . XH NMR (200 MHz, CDC13) δ_8.52(m, 2H, Ar-H), 7.48 (m, 3H, Ar-H), 6.76 (s, 1H, pyrrole-H),4.93 (s, 2H,pyrrole-CH2Br) , 1.79 (s, 9H, carbamate-CHJ ; MS,Μ + 1 = 423.9; Mpt = 155-157°C.

Sodium phenoxide trihydrate (173 mg, 1.02 mmol) was added inone portion to a solution of bromide (23) (410 mg, 0.97 mmol)dissolved in CH2C12 (5 mL) and DMF (10 mL) . After 2 h thereaction solution was partitioned between CH2C12 and water.The water layer was extracted with CH2C12. The combined CH2C12layers were washed with water, dried over MgSO4, filtered andconcentrated to yield a yellow solid. Flash chromatography(Si02; 1/6 EtOAc/Hexanes, Rf 0.30) yielded 210 mg (50%) of awhite solid (24). 1H NMR (200 MHz, CDC13) δ_8.53 (m, 2H, Ar-H) , 7.48 (m, 3H, Ar-H), 7.34 (m, 2H, Ar-H), 7.03 (m, 3H, Ar-H), 6.83 (s, 1H, pyrrole-H), 5.45 (s, 2H, ArCH2O) , 1.76 (s,9H, carbamate-CH3) ; MS, M+ = 436.2. - 129 013295

A solution containing (24) (85 mg, 0.20 mmol), N- acetylethylenediamine (201 mg, 1.95 mmol) and DMSO (3 mL) was heated to 100°C. After 1 h the température was raised to 130°C. After 3 h the reaction was cooled to room température and partitioned between EtOAc and water. The water layer was extracted with EtOAc (2x). The combined EtOAc layers are washed with water, dried over MgSO4, filtered and concentrated. Flash chromatography (S1O2; 1/10 EtOH/ CHC13,1

Rf 0.25) yielded 73 mg (93%)of a white foamy solid (25). HNMR (200 MHz, DMSO-d6) δ 11.81 (br s, 1H, N-H), 8.39 (m, 2H,Ar-H) , 8.03 (br t, 1H, N-H), 7.57 (br t, 1H, N-H), 7.20 -7.50 (m, 5H, Ar-H), 6.89-7.09 (m, 3H, Ar-H), 6.59 (s, 1H,pyrrole-H) , 5.12 (s, 2H, ArCH20) , 3.61 (m, 2H, NCH2) , 3.36 (m,2H, NCHJ , 1.79 (s, 3H,COCH3); MS, M+ 1 = 402.6

The following compounds were obtained in a manner similar tothat of Example 17 : 4-(2-acetylaminoethyl)amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. mp 196-197°C; MS (ES): 401.6 (M++l). 4-(2-acetylaminoethyl)amino-6-(4-fluorophenoxy)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS(ES): 420.1 (M++l). 130 013295 4-(2-acetylaminoethyl)amino-6-(4-chlorophenoxy)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS(ES): 436.1 (M++l). 4-(2-acetylaminoethyl)amino-6-(4-methoxyphenoxy)methyl-2-phenyl-72i-pyrrolo [2,3d]pyrimidine. MS(ES): 432.1 (M++l) . 4- (2-acetylaminoethyl) amino-6 - (N-pyridin-2-one)methyl-2-phenyl-7H-pyrrolo [2,3d]pyrimidine. MS(ES): 403.1 (M++l) . 4- (2-acetylaminoethyl)amino-6- (N-phenylamino)methyl-2-phenyl-7tf-pyrrolo[2,3]pyrimidine. MS(ES): 400.9 (M++l) . 4-(2-acetylaminoethyl)amino-6-(N-methyl-N-phenylamino)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS(ES): 414.8 (M++l). 4-(2-N'-methylureaethyl)amino-6-phenoxymethyl-2-phenyl- 7H-pyrrolo[2,3d]pyrimidine. MS (ES): 416.9 (M++l).

Example 18: Synthesis of adenosine AT Antagonists.

Compound 1319 and Compound 1320 (Table 13 below) can besynthesized by the general procedures herein. ci

Compound 26 X = FCompound 27 X = Cl

Compound 1319Compound 1320

Compound 1319 (81%) ^-NMR (d6-DMSO) d 1.37 (m, 4H) , 1.93 (m,2H) , 2.01 (m, 2H) , 4.11 (brs, 1H), 4.61 (d, 1H, J = 4.4 Hz),6.59 (m, 1H), 7.09 (m, 1H), 7.21 (m, 2H), 7.49 (dd, 1H, J =8Hz, 14Hz), 8.03 (m, 1H), 8.18 (d, 1H, J = 8 Hz), 11.55 (brs,1H). MS (ES): 327.0 (M++l). 131 013295

Compound 1320 (31%) MS (ES): 343.1 (M++l) .

Example 19: Synthesis of adenosine Antagonist.

Compound 1321 (Table 13 below) can be synthesized by thegeneral procedures given below.

Compound 28 (10.93g, 50.76 mmol) was dissolved in DMF (67mL) . 4-Amidinopyridine hydrochloride (8.0g, 50.76 mmol) andDBU (15.4 g, 101.5 mmol) were added seguentially and thereaction was heated to 85°C. After 22 hours, the reaction wascooled to room température and the DMF was removed in vacuo.The dark oil was diluted with 2M HCl (80 mL). The reactionwas allowed to stand. After 2 hours, the solution was cooledto 10°C and filtered. The solid was washed with cold waterand dried to yield 7.40g of a yellow solid, Compound 29(69%). 1H-NMR (200MHz, d6-DMSO) d6.58 (s, 1H) , 7.27 (s, 1H) ,8.53 (d, 2H, J = 5.6), 9.00 (d, 2H, J = 5.2Hz), 12.35 (brs,1H). MS (ES): 212.8 (M++l).

Compound 29 (7.4 mmol, 29.8 mmol) was diluted with POC13andheated to 105°C. After 18 hours, the reaction is cooled toroom température and the POC13 is removed in vacuo. The thickdark oil is diluted with MeOH (75mL) followed by ether(120mL) . The amorphous red solid is filtered and washed withether to yield 3.82 g of a red solid. The crude solid isapproximately 80% pure and used without further purificationin the next reaction. MS (ES): 230.7 (M++l). 013295 - 132 -

Compound 1321 ^-NMR (15%) (200MH, d6-DMSO) d 1.38 (m, 4H) , 1.92 (brs, 2H), 2.02 (brs, 2H) , 3.44 (brs, 1H) , 4.14 (brs,1H) , 4.56 (d, 1H, J = 4 Hz), 6.63 (m, 1H) , 7.15 (m, 1H) , 7.32(d, 1H, J = 6.2 Hz), 8.20 (d, 2H, J = 4.4 Hz), 8.65 (d, 2H,J = 4.4Hz), 11.67 (brs, 1H). MS (ES): 310.2 (M++l).

Compound 1501 (Table 15 below) ^-NMR (70%) (200MHz, CD3OD) d1.84 (s, 3H), 3.52 (t, 2H, J = 6.0 Hz), 3.83, t, 2H, J = 6.0Hz), 6.51 (d, 1H, J = 3.4Hz), 7.06 (d, 1H, J = 3.8 Hz), 7.42(m, 3H), 8.36 (m, 2H). MS (ES): 296.0 (M++l).

Compound 1502 (Table 15 below) MS (ES): 345.0 (M++l).

Compound 1500 (Table 15 below) 1H-NMR (200MHz, CDC13) d 1.40- 1.80 (m, 6H), 1.85 - 2.10 (m, 2H), 2.18 (s, 3H), 2.33 (s,3H), 2.50 (d, 3H), 3.90 - 4.10 (m, 2H) , 4.76 (m, 1H), 5.50(d, 1H), 6.03 (m, 1H), 7.40 (m, 3H), 8.37 (m, 2H), 9.15 (brs,1H). MS (ES): 393.3 (M++l).

Example 20: Synthesis of adenosine Ax Antagonist.

Compound 1504 (Table 15 below) can be synthesized by thegeneral procedures given below.

Compound 1504

Compound 31 (200 mg, 0.47 mmol) was dissolved in DCM (4 mL) .Triethylamine (51 mg, 0.5mmol) and thiomorpholine (52 mg,0.5mmol) were added sequentially. The solution was mixed forseveral minutes and allowed to stand for 72 hours. Thereaction was diluted with DCM and H 2O and the layers wereseparated. The aqueous layer was extracted with DCM. Thecombined DCM layers were dried over MgSO 4, filtered and 133 013295 concentrated. Ethyl ether was added' to the crude sample andthe resulting solid was filtered to yield lOOmg of a white solid, 32(62%). 1HNMR (200MHz, CDC13) d 1.76 (s, 9H) , 2.66 (brs, 2H), 2.79 (m, 2H). (brs, 2H), 3.86 (s, 2H), 7.46 (m, 3H) , 8.50 Compound 3 2 was combined with DMSO (3mL) and trans-4-aminocyclohexanol (144mg, 1.25 mmol) and heated to 130°C for 4 hours. The reaction was cooled to roomtempérature, and diluted with EtOAc and H2O. The layers wereseparated and the aqueous layer was extracted with EtOAc(2x) . The combined organic layers were washed with H2O andbrine, dried over MgSO4, filtered and concentrated.Chromatography (silica, 8:1 CHC13/EtOH) yields 32 mg of a tanoil. Ethyl ether was added and the resulting solid wasfiltered to yield 5 mg of a white solid (9%).OSIC-148265:hi-NMR (2 00MHz, CD3OD) : δ 1.44 (brm, 4H) , 2.03 (brm, 2H) , 2.21(brm, 2H), 2.70 (brm, 8H), 3.63 (m, 4H), 3.92 (m, 1H), 4.26(brs, 1H) , 6.42 (s, 1H) , 7.42 (m, 3H) , 8.33 (m, 2H) .

Examp le 21: Synthesis of adenosine A1 Antagonist.

Compound 1503 (Table 15 below) can be synthesized by thegeneral procedures given below.

Compound 1503

The bromide, compound 31 (220 mg, 0.47 mmol) was dissolved in1:1 DMF:Dichloromethane (5 mL) . To this was added K2CO3 (71mg, 0.52 mmol) and morpholine (0.047 mL, 0.47 mmol). Themixture was allowed to stir at room température overnight.Solvents were removed in vacuo and the residue waspartitioned betweenH2O and dichloromethane. The organic layer - 134 013295 was dried with MgSO4, filtered, and concentrated to give anoff white solid which upon trituration with ether/hexanesgave 175mg of a white solid, 33 (84%) . 1H-NMR (200MHz,CDC13) : ( 1.9 (9H, s), 2.54 (4H, s), 3.65 (4H, s), 3.85 (1H,s), 6.59 (1H, s), 7.45 (3H, m),8.5 (2H, m) .

Compound 33 (50 mg, 0.11 mmol) and trans-4-aminocyclohexanol (105 mg, 0.91 mmol) were taken up in DMSO (2mL) . Therésultant solution was sparged with N2 and then heated to100°C in an oil bath and stirred overnight. The crude reactionmixture was poured into water and extracted twice with ethylacetate (50mL). The combined organic layers were washed withH20. After drying with MgSQand filtering, the organic layerwas concentrated in vacuo to give an orange solid.Chromatography (silica, 10% CH3OH in CH2C12) yielded 15mg(33%). XH-NMR ( 200 MHz, CDC13) : ( 1.24 - 1.62 (4H, m) , 1.85(2H, m), 2.10 (2H, m), 2.26 (4H, m), 3.53 (4H, m), 4.22 (1H,m), 4.73 (1H, m), 5.85 (1H, d), 6.15 (1H, s), 7.25 (3H, m) ,8.42 (2H, M), 10.0 (1H, s). MS (ES): 408 (M+ + 1).

Compounds 1500, 1501, and 1502 can be synthesized usingsimilar préparation steps of Example 20 by treating compound32 with an appropriately substituted amine. 135 013295

Yeast β-Galactosidase reporter gene assays for humanadenosine Ax and A2a receptors Yeast strains (S. cerevisiae)were transformée! with human adenosine A! {A3R; CADUS strainCY12660) or human A2a (A2a; CADUS strain CY8362) and theaddition of a lacZ(β-Galactosidase) reporter gene to utilizeas a functional readout. A complété description of thetransformations is listed below (see Yeast Strains). NECA(5'-N-ethylcarboxamidoadenosine), a potent adenosine receptoragonist with similar affinity for Ax and A2a receptors, wasused as a ligand for ail assays. Test compounds wereexamined at 8 concentrations (0.1 - 10,000 nM) for ability toinhibit NECA-induced β-Galactosidase activity by CY12660 orCY8362.

Préparation of Yeast Stock Cultures: Each of the respectiveyeast strains, CY12660 and CY8362, were streaked onto an LTagar plate and incubated at 30°C until colonies wereobserved. Yeast from these colonies were added to LT liquid(pH 6.8) and grown overnight at 30°C. Each yeast strain wasthen diluted to an OD600 = 1.0 - 2.0 (approximately 1-2 X 107cells/ml), as determined spectrophotometrically (MolecularDevices VMAX) . For each 6 ml of yeast liquid culture, 4 ml of40% glycerol (1:1.5 vol:vol) was added ("yeast/glycerolstock"). From this yeast/glycerol stock, ten 1 ml aliquotswere prepared and stored at -80°C until required for assay.

Yeast AXR and A2aR Assay: One vial each of CY8362 and CY12660yeast/glycerol stock was thawed and used to inoculateSupplemented LT liquid media, pH 6.8 (92 ml LT liquid, towhich is added: 5 ml of 40% glucose, 0.45 ml of IM KOH and 2.5 ml of Pipes, pH 6.8). Liquid cultures were grown 16-18hr (overnight) at 30°C. Aliquots from overnight cultureswere then diluted in LT media, containing 4U/ml adenosinedeaminase (Type VI or VII from calf intestinal mucosa,Sigma), to obtain OD600 = 0.15 (1.5 X 106 cells/ml) for CY8362(A2aR) and OD600 = 0.50 ( 5X106 cells/ml) for CY12660 (AXR) . 136 - 013295

Assays were conducted with a final volume of 100 μΐ in 96-well microtiter plates, such that a final concentration of 2%DMSO was achieved in ail wells. For primary screening, 1-2concentrations of test compounds were utilized (10 uM, ΙμΜ ) .For compound profiling, 8 concentrations were tested (10000,1000, 500, 100, 50, 10, 1 and 0.1 nM) . To each microtiterplate, 10 μΐ of 20% DMSO was added to "Control" and "Total"wells while 10 μΐ of Test Compound (in 20% DMSO) was added to"Unknown" wells. Subséquently, 10 μΐ of NECA (5 μΜ for Aj^R,1 uM for A2aR) were added to "Total" and "Unknown" wells; 10μΐ of PBS was added to the "Control" wells. In the finaladdition, 80 ul of yeast strain, CY8362 or CY12660, wereadded to ail wells. Ail plates were then agitated briefly(LabLine orbital shaker 2-3 min) and allowed to incubate for4 hrs. at 30°C in a dry oven. β-Galactosidase activity can be quantitated using eithercolorimétrie (e.g., ONPG, CPRG), luminescent (e.g., Galacton-Star) or fluorometric substrates (e.g., FDG, Resorufin)substrates. Currently, fluorescence détection is preferredon the basis of superior signal:noise ratio, relative freedomfrom interférence and low cost. Fluoresceindigalactopyranoside (FDG, Molecular Probes or Marker GeneTechnologies), a fluorescent β-Galactosidase substrate, wasadded to ail wells at 20 ul/well (final concentration = 80uM) . Plates were shaken for 5-6 sec (LabLine orbital shaker)and then incubated at 37°C for 90 min (95% O2/5% CO2incubator). At the end of the 90 min incubation period, β-Galactosidase activity was stopped using 20 μΐ/well of IMNa2CO3 and ail plates shaken for 5-6 sec. Plates were thenagitated for 6 sec and relative fluorescence intensitydetermined using a fluorometer (Tecan Spectrafluor;excitation = 485 nm, émission = 535 nm).

Calculations: Relative fluorescence values for "Control" . 137 _ 01 329 5 wells were interpreted as background and subtxacted from"Total" and "Unknown" values. Compound profiles were analyzedvia logarithmic transformation (x-axis: compound concentration) followed by one site compétition curve fitting 5 to calculate IC50 values (GraphPad Prism).

Yeast strains: Saccharomyces cerevisiae strains CY12660[farl*1442 tbtl-1 fusl-HIS3 canl stel4: :trpl: :LYS2 ste3*1156gpal (41 ) -Gai3 lys2 ura3 leu2 trpl: his3; LEU2 PGKp- 10 MfalLeader-hAlR-PHO5term 2mu-orig REP3 Ampr] and CY8362[gpalp-rGasElOK farl*1442 tbtl-1 fusl-HIS3 canl stel4::trpl:LYS2 ste3*1156 lys2 ura3 leu2 trpl his3; LEU2 PGKp-hA2aR 2mu-ori REP3 Ampr] were developed. 15 LT Media: LT (Leu-Trp supplemented) media is composed of 100gDIFCO yeast nitrogen base, supplemented with the following:1.0g valine, 1.0g aspartic acid, 0.75g phenylalanine, 0.9glysine, 0.45g tyrosine, 0.45g isoleucine, 0.3g méthionine,0.6g adenine, 0.4g uracil, 0.3g serine, 0.3g proline, 0.3g 20 cysteine, 0.3g arginine, 0.9g histidine and 1.0g threonine.

Construction of Yeast Strains Expressing Human Ax AdenosineReceptor

In this example, the construction of yeast strains expressing 25 a human Ai adenosine receptor functionally integrated into theyeast pheromone System pathway is described. I. Expression Vector Construction 30 To construct a yeast expression vector for the human A!adenosine receptor, the Ax adenosine receptor cDNA wasobtained by reverse transcriptase PCR of human hippocampusmRNA using primers designed based on the published sequenceof the human ΑΣ adenosine receptor and standard techniques. 35 The PCR product was subcloned into the Ncol and Xbal sites ofthe yeast expression plasmid pMP15. 138

The pMP15 plasmid was created from pLPXt as follows: TheXbal site of YEP51 (Broach, J.R. et al. (1983) "Vectors forhigh-level, inducible expression of cloned genes in yeast" p.83-117 in M. Inouye (ed.), Experimental Manipulation of GeneExpression. Academie Press, New York) was eliminated bydigestion, end-fill and religation to create Yep51NcoDXba.Another Xbal site was created at the BamHI site by digestionwith BamHI, end-fill, linker (New England Biolabs, # 1081)ligation, Xbal digestion and re-ligation to generateYEP51NcoXt. This plasmid was digested with Esp31 and Ncoland ligated to Leu2 and PGKp fragments generated by PCR. The2 kb Leu2 PCR product was generated by amplification fromYEP51Nco using primers containing Esp31 and BglII sites. The660 base pair PGKp PCR product was generated by amplificationfrom pPGKas (Rang, Y.-S. et al. (1990) Mol. Cell. Biol..10:2582-2590) with PCR primers containing BglII and Ncolsites. The resulting plasmid is called pLPXt. pLPXt wasmodified by inserting the coding région of the a-factor pre-pro leader into the Ncol site. The prepro leader wasinserted so that the Ncol cloning site was maintained at the3' end of the leader, but not regenerated at the 5' end. Inthis way receptors can be cloned by digestion of the plasmidwith Ncol and Xbal. The resulting plasmid is called pMPl5.

The pMP15 plasmid into which was inserted the human A!adenosine receptor cDNA was designated p5095. In thisvector, the receptor cDNA is fused to the 3' end of the yeasta-factor prepro leader. During protein maturation the prepropeptide sequences are cleaved to generate mature full-lengthreceptor. This occurs during Processing of the receptorthrough the yeast secretory pathway. This plasmid ismaintained by Leu sélection (i.e., growth on medium lackingleucine). The sequence of the cloned coding région wasdetermined and found to be équivalent to that in thepublished literature (GenBank accession numbers S45235 andS56143). 139 013295 II. Yeast Strain Construction

To create a yeast strain expressing the human adenosinereceptor, yeast strain CY7967 was used as the startingparental strain. The génotype of CY7967 is as follows: MATa gpaD1163 gpal(41)Gai3 farlD1442 tbt-1 FUS1-HIS3canl stel4: :trpl: :LYS2 ste3DH56 lys2 ura3 leu2 trplhis3

The genetic markers are reviewed below:MATa.......... Mating type a. gpalD1163........... gpal(41)Gai3 farlD1442............

The endogenous yeast G-protein GPA1 hasbeen deleted. gpal(41)-Gai3 was integrated into theyeast genome. This chimeric Ga protein iscomposed of the first 41 amino acids ofthe endogenous yeast Ga subunit GPA1 fusedto the mammalian G-protein Gai3 in whichthe cognate N-terminal amino acids hâvebeen deleted. FAR1 gene (responsible for cell cyclearrest) has been deleted (therebypreventing cell cycle arrest uponactivation of the pheromone responsepathway). tbt-1.................... strain with high transformation efficiency by electroporation. FUS1-HIS3......... a fusion between the FUS1 promoter and the HIS3 coding région (thereby creating a pheromone inducible HIS3 gene). can 1.................. arginine/canavinine permease. stel4: :trpl: :L gene disruption of STE14, a C-farnesylYS2.... methyltransferase (thereby lowering basal signaling through the pheromone pathway).ste3D1156............. endogenous yeast STR, the a factor pheromone receptor (STE3) was disrupted.Iys2.................... defect in 2-aminoapidate reductase, yeast need lysine to grow. ura3.................... defect in orotidine-5'-phosphate decarboxylase, yeast need uracil to grow 140 - 01 329 5 leu2.................... defect in b-isôpropylmalate dehydrogenase, yeast need leucine to grow.trpl.................... defect in phosphoribosylanthranilate, yeast need tryptophan to grow.his3.................... defect in imidazoleglycerolphosphate dehydrogenase, yeast need histidine togrow.

Two plasmids were transformed into strain CY7967 byelectroporation: plasmid p5095 (encoding human A! adenosinereceptor; described above) and plasmid pl584, which is aFUSl-3-galactosidase reporter gene plasmid. Plasmid pl584was derived from plasmid pRS426 (Christianson, T.W. et al.(1992) Gene 110:119-1122) . Plasmid pRS426 contains apolylinker site at nucléotides 2004-2016. A fusion betweenthe FUS1 promoter and the β-galactosidase gene was insertedat the restriction sites EagI and Xhol to create plasmidpl584. The pl584 plasmid is maintained by Trp sélection(i.e., growth on medium lacking leucine).

The résultant strain carrying p5095 and pl584, referred to asCY12660, expresses the human A! adenosine receptor. To growthis strain in liquid or on agar plates, minimal medialacking leucine and tryptophan was used. To perform a growthassay on plates (assaying FUS1-HIS3), the plates were at pH6.8 and contained 0.5-2.5 mM 3-amino-l,2,4-triazole andlacked leucine, tryptophan and histidine. As a control forspecificity, a comparison with one or more other yeast-basedseven transmembrane receptor screens was included in ailexperiments.

Construction of Yeast Strains Expressing Human A2a AdenosineReceptor

In this example, the construction of yeast strains expressinga human A2a adenosine receptor functionally integrated intothe yeast pheromone System pathway is described. 141 013295 I. Expression Vector Construction

To construct a yeast expression vector for the human A2aadenosine receptor, the human A2a receptor cDNA was obtainedfrom Dr. Phi1 Murphy (NIH). Upon receipt of this clone, theA2a receptor insert was sequenced and found to be identicalto the published sequence (GenBank accession # S46950). Thereceptor cDNA was excised from the plasmid by PCR with VENTpolymerase and cloned into the plasmid pLPBX, which drivesreceptor expression by a constitutive Phosphoglycerate Kinase(PGK) promoter in yeast. The sequence of the entire insertwas once again sequenced and found to be identical with thepublished sequence. However, by virtue of the cloningstrategy employed there were three amino acids appended tothe carboxy-terminus of the receptor, GlySerVal. II. Yeast Strain Construction

To create a yeast strain expressing the human A2a adenosinereceptor, yeast strain CY8342 was used as the startingparental strain. The génotype of CY8342 is as follows: MATa farlDl442 tbtl-1 lys2 ura3 leu2 trpl his3 fusl-HIS3 canlste3Dll56 gpaD1163 stel4: :trpl: :LYS2 gpalp-rGasE10K (or gpalp-rGosD229S or gpalp-rGasE10K+D229S)

The genetic markers are as described above, except for the G-protein variation. For human A2a receptor-expression, yeaststrains were utilized in which the endogenous yeast G proteinGPAl had been deleted and replaced by a mammalian Gos. Threerat G„s mutants were utilized. These variants contain one ortwo point mutations which convert them into proteins whichcouple efficiently to yeast βγ. They are identified asGasE10K (in which the glutamic acid at position ten isreplaced with lysine) , GosD229S (in which the aspartic acid atposition 229 is replaced with serine) and GosE10K+D229S (whichcontains both point mutations).

Strain CY8342 (carrying one of the three mutant rat Gas 01329 5 - 142 - proteins) was transformed with eïther the parental vectorpLPBX (Receptor') or with pLPBX-A2a (Receptor+) . A plasmidwith the FUS1 promoter fused to β-galactosidase codingsequences (described in above) was added to assess themagnitude of activation of the pheromone response pathway.

Functional Assay using Yeast Strains Expressing Human AxAdenosine Receptor

In this example, the development of a functional screeningassay in yeast for modulators of the human Ax adenosinereceptor is described. I. Ligands Used in Assay

Adenosine, a naturel agonist for this receptor, as well astwo other synthetic agonists were utilized for development ofthis assay. Adenosine, reported to hâve an EC50 ofapproximately 75 nM, and (-)-N6-(2-phenylisopropyl)-adenosine(PIA) with a reported affinity of approximately 50 nM wereused in a subset of experiments. 5 ' -N-ethylcarboxamido-adenosine (NECA) was used in ail growth assays. To preventsignaling due to the presence of adenosine in the growthmedia, adenosine deaminase (4U/ml) was added to ail assays. II. Biological Response in Yeast

The ability of the Ax adenosine receptor to functionallycouple in a heterologous yeast System was assessed byintroducing the Ax receptor expression vector (p5095,described above) into a sériés of yeast strains thatexpressed different G protein subunits. The majority ofthese transformants expressed Ga subunits of the Goi or Ga0subtype. Additional Ga proteins were also tested for thepossible identification of promiseuous receptor-Ga proteincoupling. In various strains, a STE18 or a chimeric STE18-Gy2 construct was integrated into the genome of the yeast.The yeast strains harbored a defective HIS3 gene and anintegrated copy of FUS1-HIS3, thereby allowing for sélection 013295 - 143 - in sélective media containing 3-amino-l,2,4-triazole (testedat 0.2 , 0.5 and 1.0 mM) and lacking histidine. Transformantswere isolated and monolayers were prepared on mediacontaining 3-amino-l,2,4-triazole, 4 U/ml adenosine deaminase 5 and lacking histidine. Five microliters of variousconcentrations of ligand (e.g., NECA at 0, 0.1, 1.0 and 10mM) was applied. Growth was monitored for 2 days. Ligand-dependent growth responses were tested in this manner in thevarious yeast strains. The results are summarized in Table 10 1 below. The symbol (-) indicates that ligand-dépendent receptor activation was not detected while (+) dénotésligand-dependent response. The term "LIRMA" indicates ligandindependent receptor mediated activation. 144 013295

Table 3

Yeast strain Goî subunit Gy subunit Strain Variants Resuit CY1316 GPA-i STE18 - GPA41-Gail + GPA41-Gai2 + GPA41-Gai3 + GPA41-Gai2-GaOB LIRMA GPA41-GaSE10K - GPA41-GaSD229s - CY7967 GPA41-G„i3- integrated STE18 ' CY2120 GPA-, STE18 sst2û + GPA41-Gail + GPA41-G„i2 + GPA41-Gai3 + GPA41-Gai2-GaOB LIRMA GPA41-GaSE10K - GPA41-GaSD229s - CY9438 GPA2 STE18-Gy2 - GPA41-Gail + GPA41-Gai2 GPA41-Gai3 + GP A41 - Gai 2 - Ga0B LIRMA GPA41-GaSE10K - GPA41-GaSD229s - CY10560 GPA-] -integrated STE18-Gy2 sst2A ++

As indicated in Table 3, the most robust signaling was foundto occur in a yeast strain expressing the GPAX (41 )-Gai3chimère. III. fusl-LacZ Assay

To characterize activation of the pheromone response pathwaymore fully, synthesis of β-galactosidase through fuslLacZ inresponse to agonist stimulation was measured. To perform theβ-galactosidase assay, increasing concentrations of ligandwere added to mid-log culture of human A3 adenosine receptorexpressed in a yeast strain co-expressing a Stel8-Gy2 chimera

U » O LO U - 145 - and GPA41-Gai3. Transformants were isolated and grownovernight in the presence of histidine and 4 U/ml adenosinedeaminase. After five hours of incubation with 4 U/mladenosine deaminase and ligand, induction of β-galactosidasewas measured using CPRG as the substrate for β-galactoside.5 x 105 celle were used per assay.

The results obtained with NECA stimulation indicated that ata NECA concentration of 10'8 M approximately 2-foldstimulation of β-galactosidase activity was achieved.Moreover, a stimulation index of approximately 10-fold wasobserved at a NECA concentration of 10"5 M.

The utility of this assay was extonded by validation of cheactivity of antagonists on this strain. Two known adenosineantagonist, XAC and DPCPX, were tested for their ability tocompete against NECA (at 5 mM) for activity in the β-galactosidase assay. In these assays, β-galactosidase-induction was measured using FDG as the substrate and 1.6 x105 cells per assay. The results indicated that both XAC andDPCPX served as potent antagonists of yeast-expressed A!adenosine receptor, with IC50 values of 44 nM and 49 nM,respectively.

In order to détermine if this inhibitory effect was spécifieto the Ax subtype, a sériés of complementary experiments wereperformed with the yeast-based A2a receptor assay. Resultsobtained with the A2a yeast-based assay indicated that XAC wasa relatively effective A2a receptor antagonist, consistentwith published reports. In contrast, DPCPX was relativelyinert at this receptor, as expected from published reports. IV. Radioligand Binding

The Ai adenosine receptor assay was further characterized bymeasurement of the receptor's radioligand binding parameters. 013295 - 146 -

Displacement binding of [3H]CPX by several adenosine receptorreference compounds, XAC, DPCPX, and CGS, was analyzed usingmembranes prepared from yeast expressing the human A!adenosine receptor. The results with yeast membranesexpressing the human A2 adenosine receptor were compared tothose from yeast membranes expressing the human A2a adenosinereceptor or the human A3 receptor to examine the specificityof binding. To perform the assay, fifty mg of membranes wereincubated with 0.4 nM [3H]CPX and increasing concentrations ofadenosine receptor ligands. Incubation was in 50 mM Tris-HC1, pH 7.4, 1 mM EDTA, 10 mM MgCl2, 0.2 5 % BSA and 2 U/mladenosine deaminase in the presence of protease inhibitorsfor 60 minutes at room température. Binding was terminatedby addition of ice-cold 50 mM Tris-HCl, pH 7.4 plus 10 mMMgCl2, followed by rapid filtration over GF/B filterspreviously soaked with 0.5 % polyethyenimine, using a Packard96-well harvester. Data were analyzed by nonlinear leastsquare curve fitting procedure using Prism 2.01 software.The IC50 values obtained in this experiment are summarized inTable 4, below:

Table 4 IC™ InMl Comoound hAIR hA2aR hA3R XAC 6.6 11.7 53.1 DPCPX 8.5 326.4 1307.0 CGS-15943 13.1 15.8 55.5 NECA 215.5 294.9 34.9 R-PIA 67.6 678.1 23.6 IB-MECA 727.7 859.4 3.1 Alloxozine 1072.0 1934.0 8216.0

These data indicate that the reference compounds hâveaffinities consistent with those reported in the literature.The data further indicate that the yeast-based assays are ofsufficient sensitivity to discriminate receptor subtypespecificity. 147 013295

Functional Assay using Yeast Strains Expressing Human A2aAdenosine Receptor

In this example, the development of a functional screeningassay in yeast for modulators of the human Ax adenosinereceptor is described. I. Ligands Used in Assay

The natural ligand adenosine, as well as other thoroughlycharacterized and commercially available ligands were usedfor study of the human A2a receptor functionally expressed inyeast. Three ligands hâve been used in the establishment ofthis assay. They include:

Ligand

Adenosine 5'-N-ethylcarboxamidoadenosine(NECA) (-)-N6-(2-phenylisopropyl)-adenosine (PIA)

Reoorted Κ£500 nM10-15 nM

100-125 nM

Function agonistagonist agonist

To prevent signaling due to the presence of adenosine in thegrowth media, adenosine deaminase (4U/ml) was added to ailassays. II. Biological Response in Yeast A2a receptor agonists were tested for the capacity tostimulate the pheromone response pathway in yeast transformedwith the A2a receptor expression plasmid and expressingeither GasE10K, GasD229S or GasE10K+D229S. The ability ofligand to stimulate the pheromone response pathway in areceptor dépendent manner was indicated by an alteration inthe yeast phenotype. Receptor activation modified thephenotype from histidine auxotrophy to histidine prototrophy(activation of fusl-HIS3). Three independent transformantswere isolated and grown overnight in the presence ofhistidine. Cells were washed to remove histidine and diluted 148 013295 to 2 x 106 cells/ml. 5 μΐ of each transformant was spottedonto nonselective media (including histidine) or sélectivemedia (1 mM AT) in the absence or presence of 4 U/mladenosine deaminase. Plates were grown at 30 °C for 24hours. In the presence of histidine both Receptor+ (R+) andReceptor" (R') strains were capable of growth. However, inthe absence of histidine only R+ cells grew. Since no ligandhad been added to these plates two explanations were possiblefor this resuit. One possible interprétation was that thereceptor bearing yeast were at a growth advantage due toLigand Independent Receptor Mediated Activation (LIRMA).Alternatively the yeast could hâve been synthesizing theligand adenosine. To distinguish between these twopossibiiities, an enzyme which dégradés the ligand, adenosinedeaminase (ADA), was added to the growing yeast and plates.In the presence of adenosine deaminase R+ cells no longer grewin the absence of histidine, indicating that the yeast wereindeed synthesizing ligand.

This interprétation was confirmed by an A2a growth assay inliquid. In this experiment R+ yeast (a GasE10K strainexpressing the A2a receptor) were inoculated at threedensities (1 x 106 cell/ml; 3 x 105 cells/ml; or 1 x 105cells/ml) in the presence or absence of adenosine deaminase(4 U/ml) . The stringency of the assay was enhanced withincreasing concentrations (0, 0.1, 0.2 or 0.4 mM)of 3-amino-1,2,4-triazole (AT), a compétitive antagonist ofimidazoleglycerol-P dehydratase, the protein product of theHIS3 gene. In the presence of adenosine deaminase and 3-amino-1,2,4-triazole yeast grew less vigorously. However inthe absence of 3-amino-1,2,4-triazole, adenosine deaminasehad little effect. Thus adenosine deaminase itself had nodirect effect upon the pheromone response pathway.

An alternative approach to measuring growth and one that canbe miniaturized for high throughput screening is an A2a 013295 - 149 - receptor ligand spot assay. A GOSE10K strain expressing theA2a receptor (A2aR+) or lacking the receptor (R-) was grownovernight in the presence of histidine and 4 U/ml adenosinedeaminase. Cells were washed to remove histidine and dilutedto 5 x 106 cells/ml. 1 x 106 cells were spread onto sélectiveplates containing 4 U/ml adenosine deaminase and 0.5 or 1.0mM 3-amino-l, 2,4-triazole (AT) and allowed to dry for 1 hour.5 μΐ of the following reagents were applied to the monolayer:10 mM adenosine, 38.7 mM histidine, dimethylsulfoxide (DMSO),10 mM PIA or 10 mM NECA. Cells were grown 24 hours at 30°C.The results showed that cells without receptor could onlygrow when histidine was added to the media. In contrast, R+cells only grew in areas where the A2a receptor ligands PIAand NECA had been spotted. Since the plates containedadenosine deaminase, the lack of growth where adenosine hadbeen spotted confirmed that adenosine deaminase was active. III. fusl LacZ Assay

To quantitate activation of the yeast mating pathway,synthesis of β-galactosidase through fuslLacZ was measured.Yeast strains expressing GasEl0K, GasD229S or GosE10K+D229S weretransformed with a plasmid encoding the human A2a receptor(R+) or with a plasmid lacking the receptor (R-).Transformants were isolated and grown overnight in thepresence of histidine and 4 U/ml adenosine deaminase. 1 x 107cells were diluted to 1 x 106 cells/ml and exposed toincreasing concentrations of NECA for 4 hours, followed bydétermination of the β-galactosidase activity in the cells.The results demonstrated that essentially no β-galactosidaseactivity was detected in R- strains, whereas increasingamounts of β-galactosidase activity were detected in R+strains expressing either G^EIOK, GasD229S or GasE10K+D229S asthe concentration of NECA increased, indicating a dosedépendent increase in units of β-galactosidase detected inresponse to exposure to increased ligand concentration. Thisdose dependency was only observed in cells expressing the A2a 150 013295 receptor. Furthermore the most pocent Gos construct for theA2a receptor was GasEl0K. The GosD229S construct was thesecond-most potent Gas construct for the A2a receptor, whilethe GOSE10K+D229S construct was the least potent of the threeGas constructs tested, although even the GasE10K+D229Sconstruct stimulated readily détectable amounts of β-galactosidase activity.

For a further description of the assays identified, see U.S.Application Publication No. ÜS-2002-0015967-A1, publishedFebruary 7, 2002, entitled "Functional Expression ofAdenosine Receptors in Yeast", now abandoned, the entirecontents of which are hereby incorporated herein byreference.

Pharmacological Characterization of the Human AdenosineReceptor Subtypes

Materïal and Methods

Materials. [3H]-DPCPX [Cyclopentyl-1,3-dipropylxantine, 8-(120.0 Ci/mmol); [3H]-CGS 21680,(30 Ci/mmol) and [125I] -AB-MECA

[dipropyl-2,3-3H (N) ][carboxyethyl-3H (N) ] ( [125I] -4-Aminobenzyl-5 ' -N-Methylcarboxamideoadenosine) (2,200Ci/mmol) were purchased from New England Nuclear (Boston,MA). XAC (Xantine amine congener); NECA (5'-N-Ethylcarboxamidoadenosine); and IB-MECA from ResearchBiochemicals International (RBI, Natick, MA) . The AdenosineDeaminase and Complété protease inhibitor cocktail tabletswere purchased from Boehringer Mannheim Corp. (Indianapolis,IN) . Membranes from HEK-293 cells stably expressing the humanAdenosine 2a [RB-HA2a]; Adenosine 2b [RB-HA2b] or Adenosine3 [RB-HA3] receptor subtypes, respectively were purchasedfrom Receptor Biology (Beltsville, MD) . Cell culture reagentswere from Life Technologies (Grand Island, NY) except forsérum that was from Hyclone (Logan, UT). 151 013295

Yeast strains: Saccharomyces cerevisiae strains CY12660[farl*1442 tbtl-1 fusl-HIS3 canl stel4: :trpl: :LYS2 ste3*1156gpal(41)-Gai3 lys2 ura3 leu2 trpl: his3; LEU2 PGKp-MfoilLeader-hAlR-PHO5term 2mu-orig REP3 Ampr] and CY8362[gpalp-rGasElOK farl*1442 tbtl-1 fusl-HIS3 canl stel4: :trpl:LYS2 ste3*1156 lys2 ura3 leu2 trpl his3; LEU2 PGKp-hA2aR 2mu-ori REP3 Ampr] were developed as described above.

Yeast culture: Transformed yeast were grown in Leu-Trp [LT]media (pH 5.4) supplemented with 2% glucose. For thepréparation of membranes 250 ml of LT medium were inoculatedwith start titer of 1-2 x 106 cells/ml from a 30 ml overnightculture and incubated at 30°C under permanent oxygénation byrotation. After 16 h growth the cells were harvested bycentrifugation and membranes were prepared as describedbelow.

Mammalian Tissue Culture: The HEK-293 cells stably expréssedhuman Adenosine 2a receptor subtype (Cadus clone # 5) weregrown in Dulbeco's minimal essential media (DMEM)supplemented with 10% fêtai bovine sérum and IXpenicillin/streptomycin under sélective pressure using 500mg/ml G418 antibiotic, at 37°C in a humidified 5% CO2atmosphère.

Yeast Cell Membrane Préparations: 250 ml cultures wereharvested after overnight incubation by centrifugation at2,000 x g in a Sorvall RT6000 centrifuge. Cells were washedin ice-cold water, centrifuged at 4°C and the pellet wasresuspended in 10 ml ice-cold lysis buffer [5 mM Tris-HCl, pH7.5; 5 mM EDTA; and 5 mM EGTA] supplemented with Proteaseinhibitor cocktail tablets (1 tablet per 25 ml buffer).Glass beads (17 g; Mesh 400-600; Sigma) were added to thesuspension and the cells were broken by vigorous vortexing at4°C for 5 min. The homogenate was diluted with additional 30ml lysis buffer plus protease inhibitors and centrifuged at 152 013295 3,000 x g for 5 min. Subsequently the membranes were peletedat 36,000 x g (Sorvall RC5B, type SS34 rotor) for 45 min. Theresuiting membrane pellet was resuspended in 5 ml membranebuffer [50 mM Tris-HCl, pH 7.5; 0.6 mM EDTA; and 5 mM MgCl2]supplemented with Protease inhibitor cocktail tablets (1tablet per 50 ml buffer) and stored at -80 °C for furtherexperiments.

Mammalian Cell Membrane Préparations: HEK-293 cell membraneswere prepared as described previously (Duzic E et al.: J.Biol. Chem., 267, 9844-9851, 1992} Briefly, cells werewashed with PBS and harvested with a rubber policeman. Cellswere pelted at 4°C 200 x g in a Sorvall RT6000 centrifuge. Thepellet was resuspended in 5 ml/dish of lysis buffer at 4°C (5mM Tris-HCl, pH 7.5; 5 mM EDTA; 5 mM EGTA; 0.1 mMPhenylmethylsulfonyl fluoride, 10 mg/ml pepstatin A; and 10mg/ml aprotinin) and homogenized in a Dounce homogenizer.The cell lysate was then centrifuged at 36,000 x g (SorvallRC5B, type SS34 rotor) for 45 min and the pellet resuspendedin 5 ml membrane buffer [50 mM Tris-HCl, pH 7.5; 0.6 mM EDTA;5 mM MgCl2; 0.1 mM Phenylmethylsulfonyl fluoride, 10 mg/mlpepstatin A; and 10 mg/ml aprotinin) and stored at -80 °C forfurther experiments.

The Bio-Rad protein assay kits, based on the Bradford dye-binding procedure, (Bradford, M. : Anal. Biochem. 72:248(1976)) were used to détermine total protein concentration inyeast and mammalian membranes.

Adenosine 1 receptor subtype saturation and compétitionradioligand binding: Saturation and compétition binding onmembranes from yeast cell transformed with human Ai receptorsubtype were carried out using antagonist [3H] DPCPX as aradioactive ligand. Membranes was diluted in binding buffer[50 mM Tris-HCl, pH 7.4; containing 10 mM MgCl2; 1.0 mM EDTA;0.25% BSA; 2 U/ml adenosine deaminase and 1 protease 153 013295 inhibitor cocktail tablet/50 ml] at concentrations oi 1.0mg/ml.

In saturation binding membranes (50 pg/well) were incubate5 with increasing concentrations of [3H] DPCPX (0.05 - 25 nM) ina final volume of 100 μΐ of binding buffer at 25°C for 1 hr inthe absence and presence of 10 μΜ unlabeled XAC in a 96-well microtiter plate. 10 In compétition binding membranes (50 pg/well) were incubatewith [3H] DPCPX (1.0 nM) in a final volume of 100 ml ofbinding buffer at 25°C for 1 hr in the absence and presence of10 μΜ unlabeled XAC or increasing concentrations of competingcompounds in a 96-well microtiter plate. 15

Adenosine 2a receptor subtype compétition radioligandbinding: Compétition binding on membranes from HEK293 cellstably expressing the human A2a receptor subtype were carriedout using agonist [3H] CGS-21680 as a radioactive ligand. 20 Membranes was diluted in binding buffer [50 mM Tris-HCl, pH7.4; containing 10 mM MgCl2; 1.0 mM EDTA; 0.25% BSA; 2 U/mladenosine deaminase and 1 protease inhibitor cocktailtablet/50 ml] at concentrations of 0.2 mg/ml. Membranes (10pg/well) were incubate with [3H] CGS-21680 (100 nM) in a final 25 volume of 100 ml of binding buffer at 25°C for 1 hr in theabsence and presence of 50 μΜ unlabeled NECA or increasingconcentrations of competing compounds in a 96-well microtiterplate. 30 Adenosine 3 receptor compétition radioligand binding:Compétition binding on membranes from HEK293 cell stablyexpressing the human A3 receptor subtype were carried outusing agonist [125I] AB-MECA as a radioactive ligand. Membraneswas diluted in binding buffer [50 mM Tris-HCl, pH 7.4; 35 containing 10 mM MgCl2; 1.0 mM EDTA; 0.25% BSA; 2 U/mladenosine deaminase and 1 protease inhibitor cocktail - 154 013295 tablet/50 ml] at concentrations of 0.2 mg/ml. Membranes (10gg/well) were incubate with [125I] AB-MECA (0.75 nM) in a finalvolume of 100 μΐ of binding buffer at 25°C for 1 hr in theabsence and presence of 10 μΜ unlabeled IB-MECA or increasingconcentrations of competing compounds in a 96-well microtiterplate.

At the end of the incubation, the A1( A2a and A3 receptorsubtypes radioligand binding assays was terminated by theaddition of ice-cold 50 mM Tris-HCl (pH 7.4) buffersupplemented with 10 mM MgCl2, followed by rapid filtrationover glass fiber filters (96-well GF/B UniFilters, Packard)previously presoaked in 0.5% polyethylenimine in a Filtermate196 cell harvester (Packard). The filter plates were driedcoated with 50 μΐ /well scintillation fluid (MicroScint-20,Packard) and counted in a TopCount (Packard). Assays wereperformed in triplicate. Non-specific binding was 5.6 ±0.5%, 10.8 ± 1.4% and 15.1 ± 2.6% of the total binding in aAIR, A2aR and A3R binding assay, respectively.

Adenosine 2b receptor subtype compétition radioligandbinding: Compétition binding on membranes from HEK293 cellstably expressing the human A2b receptor subtype were carriedout using Ax receptor antagonist [3H] DPCPX as a radioactiveligand. Membranes was diluted in binding buffer [10 mM Hepes-KOH, pH 7.4; containing 1.0 mM EDTA; 0.1 mM Benzamidine and2 U/ml adenosine deaminase] at concentrations of 0.3 mg/ml.Membranes (15 μg/well) were incubate with [3H] DPCPX (15 nM)in a final volume of 100 μΐ of binding buffer at 2 5°C for 1 hrin the absence and presence of 10 μΜ unlabeled XAC orincreasing concentrations of competing compounds in a 96-wellmicrotiter plate. At the end of the incubation, the assaywas terminated by the addition of ice-cold 10 mM Hepes-KOH(pH 7.4) buffer followed by rapid filtration over glass fiberfilters (96-well GF/C UniFilters, Packard) previouslypresoaked· in 0.5% polyethylenimine in a Filtermate 196 cell 155 013295 harvester (Packard). The filter plates were aried coatedwith 50 μΐ/well scintillation fluid (MicroScint-20, Packard)and counted in a TopCount (Packard) . Assays were performedin triplicate. Non-specific binding was 14.3 ± 2.3% of thetotal binding.

Spécifie binding of [3H] DPCPX; [3H] CGS-21680 and [125I] AB-MECA was defined as the différence between the total bindingand non-specific binding. Percent inhibition of the compoundswas calculated against total binding. Compétition data wereanalyzed by itérative curve fitting to a one site model, andKj values were calculated from IC50values (Cheng and Prusof,Biochem. Pharmacol. 22, 3099-3109, 1973) using the GraphPadPrizm 2.01 software. Résulte A primary function of certain cell surface receptors is torecognize appropriate ligands. Accordingly, we determinedligand binding affinities to establish the functionalintegrity of the Adenosine 1 receptor subtype expressed inyeast. Crude membranes prepared from Saccharomycescerevisiae transformed with human Adenosine 1 receptorsubtype construct exhibited spécifie saturable binding of [3H]DPCPX with a KD of 4.0 ± 0.19 nM. The KD and Bmax value werecalculated from the saturation isotherm and Scatchardtransformation of the data indicated a single class ofbinding sites. The densities of adenosine binding sites inthe yeast membrane préparations were estimated to 716.8 ± 43.4 fmol/mg membrane protein.

The pharmacological subtype characteristics of therecombinant yeast cells transformed with human Ax receptorsubtype were investigated with subtype sélective adenosineligands (XAC, DPCPX; CGS-15943; Compound 600; Compound 1002;NECA, (R)-PIA; IB-MECA and Alloxazine) that competed with [3H]DPCPX in the expected rank order. Displacement curves 156 013295 recorded with these compounds show the typical steepness withail the ligands, and the data for each of the ligands couldbe modeled by a one-site fit. The apparent dissociationconstants estimated for the individual compound from thecurves (Table 5) are consistent with value published for thereceptor obtained from other sources.

Table 5

Ki values for membranes from yeast cells transformed withhuman receptor subtype

Ligands

Kx (nM) XAC 5.5 DPCPX 7.1 CGS-1594 10.8 NECA 179.6 (R)-PIA 56.3 IB-MECA 606.5

Alloxazine 894.1

Compound 600 13.9

Compound 1002 9.8

Tables 6 through 12 démonstrate the efficacy and structureactivity profiles of deazapurines of the invention. Tables13 and 14 demonstrate selectivity can be achieved for humanadenosine receptor sites by modulation of the functionalityabout the deazapurine structure. Table 14 also demonstratesthe surprising discovery that the compounds set forth thereinhâve subnanomolar activity and higher selectivity for the A2breceptor as compared to the compounds in Table 13. -157- 013295 TABLE 6

Effect of Ng-Substituent

Al Compound R Binding Ki (nM) Yeast IC50 (nM) 600 ........... 13.9 97.2 601 •Kl·-" 1423 >10,000 602 /H .......... 483.5 >10,000 603 -HÔ~ 196.6 4442.0 604 . K O I ΛΛ 1 II 1V-/ ü >10,000 >10000 605 1 /~\.........ï 5 >10000 >10000 606 297.9 >10000 013295 -158- 607 KD 309.7 >10000 608 ,ZH hO w 29.1 609 *OH kx, 193.9 610 ,,ç OH (i) 411.5 611 / v 0 Ph KDK 785.6 >10000 612 D NHAc Trans (S,S) 64.8 613 Dp NHAc Trans (R,R) 6726.0 - 614 η°Ύλ (dl) 32.1 -159- 013295

615 vO (dl) 816.9 2577.0 616 Z0H 34.3 •hC X TABLE 7

Effect of C2-Substituent

Al Compound R Binding Ki(nM) Yeast IC50 (nM) 700 σΝ 604.5 >10000 701 157.7 763.1 -160- 013295 702 Vi 198.5 2782.5 703 ο 443.6 >10000 704 61.1 297.0 705 (U 30.1 194.7 706 /Y Y F A 19.9 707 FÀ^ 62.8 708 2145 709 À Ύ A 48.7 -161- 013295 TABLE 8

Effect of Pyrrole Ring Substituent HI\T Λ/ R R”’ ry 1 R’ —R” Al Compound R R’ R" R’" Binding Ki(nM) Yeast IC50 (nM) 800 X Me Me Me 3311 >10000 801 θ' H Me H 22.3 148.3 802 O" H H Me 8.9 803 J?4 0 X> Me Me 2210 >10000 804 θ' X) Me Me 863.1 805 0" X2> Me Me 4512 ο 13295 -162- 806 q* Me Me 8451 Me' \=/ 807 &amp; Me -o Me Me 35.3 TABLE9

Al Compound R Binding Ki (nM) Yeast IC50 (nM) 900 863.1 901 ,jy 4512 902 8451 903 35.3 -163- 013295 TABLE 10

Effect of N6-Substituent

Al Compound R Binding Ki(nM) Yeast IC50 (nM) 1000 1789 >10000 1001 H 1 0 54.4 1865 1002 H 1 0 9.8 82.8 1003 O 26.7 195.7 1004 0 32.8 545.8 -164- 013295 1005 0 147.5 3972 1006 V—·”γΧ 0 151.7 2918 1007 0 1 L .ΝΗ-S-Me 1 0 692.5 >10000 1008 Η 0 93.1 3217 1009 ο 475.3 >10000 1010 674.9 9376.0 1011 121.9 2067.5 1012 233.9 3462 1013 Η 270.1 3009.5 1014 V^/0H 384.9 2005 1015 179.3 3712 1016 176.1 5054 -165- 013295 TABLE 11

Effect of N6-Substituent

Al Compound R Binding Ki(nM) Yeast IC50 (nM) 1100 0 9.8 115.4 1101 0 53.9 551.0 1102 0 10.3 101.3 1103 0 71.1 3217 1104 H χ-γ!γ- Me 0 (±) 6.5 58.7 1105 H νγ!γψ Me 0 (*) 105.4 472.1 -166- 013295 1106 Me H 0 (S) 27.8 162.4 1107 Me H 0 (4) 126.5 1297.0 1108 ^χ^ΗΑο 2.3 1109 ' s 9.0 1110 1 E 17.3 1111 ^χ/ΝΠΑο 2.5 1112 ^Λ^νπα. 213 -167- 013295 TABLE 12 "Retro-Amide" Analogues

Al Compound R Binding Ki (nM) Yeast IC50 (nM) 1200 o 16.5 189.4 1201 7.4 45.7 1202 O ''Λ'ν 95.8 3345.0 1203 V^^NMe2 529.1 4040.0 1204 0 1060.0 >10000 -168- 013295 1205 0 1272 >10000 1206 VyNH! 0 50.8 4028 1207 L ^NHMe K ï 0 48.5 701.5 TABLE 13

Profile of Sélective Adenosîne Antagoniste nhr Me 1 /—Μβ Binding Ki (nM) 0 " \ H Compound R Al A2a A2b A3 1300 9.8- 25.1 18.0- 48.6 80.3 513.0 1301 Me ,0^NHAc 27.8 50.7 84.6 429.8 1302 H 1 NHMe V Y 0 20.2 75.6 20.1 4.3 -169- 013295 1303 0 ^NHMe 17.4 111.3 120.6 44.6 1304 ,>°Η 13.9- 30.9 933.7 138.0 21.5 13051 ,>°Η 46.6 730.9 30% 9.9 13062 }.>0Η 16.4 766.3 168.3 71.7 1307 (dl) 29.1 190.6 1143.0 3.1 1308 /^Ζ\^ΟΗ (±) 180 230 670 1.0 -170- 013295 1309 H \ /Me { Y T sz ° 1 A/VW 1 40 109 109 0.3 1310 x i (CH2)3 JL ^NHMe 1 255 76% 275 £2.6 1311 x i \N/ 'Me! 531 981 736 5.3 1312 x I (CH2)4 JL \N/ ^NHMe 1 H 443 2965 375 <6.2 13133 JLx? . \N/ —nh3‘ 1 30% 65% 515 24 1314 x i ^NHEt 1 H 87 204 30 0.02 -171- 013295 1315 ï H 75,000 720,000 3,400 507 1316 1 H 333 710,000 710,000 97 1317 H 710,000 710,000 720,000 369 131S4 \ s >°H 3.7±0.5 630± 56.4 2307± 926 630±76 13194’5 X >0H 1.8 206 802 270 13204’6 X: „>°H 8.0 531 530 419 13214’7 X^ >0H 8.0 131 1031 54%s 12 2-thienyl-2-yl; C5-H;3 water soluble;4 Rs and Re are hydrogen;5 R3 is 3-fluorophenyl;6 R3is 3-chlorophenyl;7 R3 is 4-pyridyl;8 % activity @ 10 μΜ -172- 013295

Table 14:

Profile of Sélective Ajb Antagonists

Compound XRi *2 Binding Data K± (nM) A2a A2b a3 1400 -O-Ph Me 41.7 21 10.3 14.6 1401 -O-Ph(p)F Me 33 58 8.8 18 1402 -O-Ph(p)Cl Me 825 591 22 60 1403 -N-pyridin-2- Me 60 41 18 48 1404 one -NH-Ph Me 49 31 4.6 57 -173- 013295 TABLE 15. Adenosine Αχ Receptor Sélective Compounds * at least 10 times more sélective than other three subtypes.

Compound Structure Ki-Aj^ Relative Ki-A2a Relative Ki-A2b Relative Ki-A3 706 CHa * 1 rV —CHg A H 1318 X ^NH I H * d -H Pt H U ★ 1319 ΗΝΙ»Ή Q OH N iT Λ % N V H F ★ 1320 .......... o IOH NA- -> ΓΎ V H U 1 Cl -174- 013295

-175- 013295 1503 XX ★ 1504 Έ v VZ ★ 013295 -176-

Paoes 176-201 relate to compounds spécifie to the A2b receptor

Summarv of the Invention

The présent invention is also based on compounds whichselectively bind to adenosine A2a receptor, thereby treating adisease associated with A2a adenosine receptor in a subject byadministering to the subject a therapeutically effective amountof such compounds. The disease to be treated are associatedwith, for example, a central nervous System disorder, acardiovascular disorder, a rénal disorder, an inflammatorydisorder, a gastrointestinal disorder, an eye disorder, anallergie disorder or a respiratory disorder.

This invention also features a compound having the structure:

(VI) wherein NR2R2 is a substituted or unsubstituted 4-8 memberedring ; wherein R3 is a substituted or unsubstituted four to sixmembered ring; wherein Rs is H, alkyl, substituted alkyl, aryl, arylalkyl,amino, substituted aryl, wherein said substituted alkyl is -C(R?) (Rs)XRe, wherein X is O, S, or NRio, wherein R7 and Rs areeach independently H or alkyl, wherein R9 and Rio are each 013295 -177- independently alkyl or cycloalkyl, or R<>, Rio and the nitrogentogether form a substitutéd or unsubstituted ring of between 4and 7 members; wherein R6 is H, alkyl, substitutéd alkyl, or cycloalkyl; with the proviso that NR1R2 is not 3-acetamido piperidino, 3-hydroxy pyrrolidino, 3-methyloxy carbonylmethyl pyrrolidino, 3-aminocarbonylmethyl, or pyrrolidino; with the proviso that NR1R2is 3-hydroxymethyl piperidino only when R3 is 4-pyridyl.

This invention also features a method for inhibiting theactivity of an A2a adenosine receptor in a cell, whichcomprises contacting said cell with the above-mentionedcompounds.

This invention also provides a compound having the structure:

(VI) wherein NRXR2 is a substitutéd or unsubstituted 4-8membered ring; wherein R3 is a substitutéd or unsubstituted four to six -178- 013295 membered ring; wherein Rs is H, alkyl, substituted alkyl, aryl,arylalkyl, amino, substituted aryl, wherein saidsubstituted alkyl is -C (Ri) (Rs)XR9, wherein X is O, S, orNRio, wherein R7 and Rs are each independently H or alkyl,wherein R9 and Rio are each independently alkyl orcycloalkyl, or R9, Rio and the nitrogen together form asubstituted or unsubstituted ring of between 4 and 7members; wherein Re is H, alkyl- substituted alkyl, or cycloalkyl; with the proviso that NR1R2 is not 3-acetamido piperidino,3-hydroxy pyrrolidino, 3-methyloxy carbonylmethylpyrrolidino, 3-aminocarbonylmethyl, or pyrrolidino; withthe proviso that NR1R2 is 3-hydroxymethyl piperidino onlywhen R3 is 4-pyridyl.

In one embodiment of the compound, R3 is a substituted orunsubstituted four to six membered ring, phenyl, pyrrole,thiophene, furan, thiazole, imidazole, pyrazole, 1,2,4-triazole, pyridine, 2(1H)-pyridone, 4(1H)-pyridone, pyrazine,pyrimidine, pyridazine, isothiazole, isoxazole, oxazole,tetrazole, naphthalene, tetralin, naphthyridine, benzofuran,benzothiophene, indole, 2,3-dihydroindole, ΙΗ-indole, indoline,benzopyrazole, 1,3-benzodioxole, benzoxazole, purine, coumarin,chromone, quinoline, tetrahydroquinoline, isoquinoline,benzimidazole, quinazoline, pyrido[2,3-b]pyrazine, pyrido[3,4-bjpyrazine, pyrido[3,2-c]pyridazine, pyrido[3,4-b]-pyridine,lH-pyrazole[3,4-d]pyrimidine, pteridine, 2(1H)-quinolone,1 (2H)-isoquinolone, 1,4-benzisoxazine, benzothiazole. 013295 -179- quinoxaline, quinoline-N-oxide, isoquinoline-N-oxide,quinoxaline-N-oxide, quinazoline-N-oxide, benzoxazine,phthalazine, cinnoline, or having a structure:

wherein Y is carbon or nitrogen; wherein R3' is H, substituted or unsubstituted alkyl,substituted or unsubstituted aryl, halogen, methoxy, methylamino, methyl thio;

In another embodiment of the compound, the compound has thestructure:

wherein m is 1 or 2; wherein Ra and Rb are each independentlybe H, -ÔH, -CH2OH, -CH2CH2OH, -C(=O)NH2, a heteroatom, or—C (=O)NR11R11 ' ; wherein R1; is aryl, substituted aryl, orheteroaryl; wherein Ru' is alkyl or XRn", wherein X is O, orN and Rxl" is substituted alkyl or aryl.

In another embodiment of the compound, R1R2N is (D)-2-aminocarbonyl pyrrolidino, (D)-2-hydroxymethyl pyrrolidino, -180- 013295 (L>?-2-hydroxymethyl-trans-4-hydroxy pyrrolidino, piperazino, or3-hydroxymethyl piperidino.

In another embodiment of the compound, the compound has thestructure :

RB wherein m is 0, 1, 2, or 3; wherein Y is O, S, or NR, whereinR is Ra or Rb; wherein Ra and Rb are each independently be H, -OH, -CH2OH, -CH2CH2OH, -C(=O)NH2, a heteroatom, or —C (=O)NR11R11 ' ; wherein Rn is aryl, substituted aryl, orheteroaryl; wherein R1X' is alkyl or XRxl", wherein X is O, orN and Ru" is substituted alkyl or aryl.

In another embodiment of the compound, the compound has thestructure:

F

(Compound 1600) -181- 013295

In another structure :

In another structure : embodiment of the compound, the

embodiment of the compound, the

compound ha s the compound has the (Compound 1602) -182- 013295

In another embodiment of the compound, the compound has thestructure:

In another embodiment of the compound, the compound has the structure :

-183- d I 3295

In another structure : (Compound 1604) embodiment of the compound, the compound has the

In another structure : (Compound 1605) embodiment of the compound, the compound has the

(Compound 1606) -184- 013295

In another embodiment of the compound, the compound bas ttiestructure:

(Compound 1607)

In yet another embodiment of the compound, the compound has thestructure:

In a further embodiment of the compound, the compound has thestructure :

-185- 013295

This invention further provides a compound having the structure(V) :

wherein R5 is H, or methyl.

In one embodiment of the compound V, the compound has thestructure:

(Compound 1608) -186- 013295

In another embodiment of the compound V, the compound has thestructure:

This invention also provides a method for treating a diseaseassociated with A2a adenosine receptor in a subject, comprisingadministering to the subject a therapeutically effective amountof compounds IV, or V.

In one embodiment of the method, the compound treats saiddiseases by stimulating adenylate cyclase.

In another embodiment of the method, the subject is a mammal.

In another embodiment of the method, the mammal is a human.

In another embodiment of the method, said A2a adenosine receptoris associated with Parkinson's disease and diseases associatedwith locomotor activity, vasodilation, platelet inhibition,neutrophil superoxide génération, cognitive disorder, or seniledementia.

Diseases associated with adenosine Al, A2a, A2b and A3 receptors -187- 013295 are disclosed in WO 99/06053 and WO-09822465, WO-09705138, WO-095116&amp;L, WO-09733879, JP-09291089, PCT/US98/16053 and U.S.Patent No. 5,516,894, the entire content of which are fullyincorporate herein by reference.

This invention also provides a water-soluble prodrug ofcompounds IV, or V; wherein said water-soluble prodrug that ismetabolized in vivo to produce an active drug which selectivelyinhibit A2a adenosine receptor.

In one embodiment of the prodrug, said prodrug is metabolizedin vivo by esterase catalyzed hydrolysis.

This invention also provides a pharmaceutical compositioncomprising the prodrug and a pharmaceutically acceptablecarrier.

This invention also provides a method for inhibiting theactivity of an A2a adenosine receptor in a cell, which comprisescontacting said cell with compounds IV, or V.

In one embodiment of the method, the compound is an antagonistof said A2a adenosine receptor.

In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is an ophthalmic formulation.

In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is an periocular, retrobulbar orintraocular injection formulation.

In another embodiment of the pharmaceutical composition, said -188- 013295 pharmaceutical composition is a systemic formulation.

This invention also provides a method for treating agastrointestinal disorder in an subject, comprisingadministering to the an effective amount of compounds IV, or V.

In one embodiment of the method, said disorder is diarrhea.

In another embodiment of the method, the subject is a human.

In another embodiment of the method, the compound is anantagonist of A2a adenosine receptors.

This invention further provides a method for treatingrespiratory disorder in a subject, comprising administering tothe subject an effective amount of compounds IV, or V.

In one embodiment of the method, said disorder is asthma,chronic obstructive pulmonary disease, allergie rhinitis, or anupper respiratory disorder.

In another embodiment of the method, the subject is a human.

In another embodiment of the method, said compound is anantagonist of A2a adenosine receptors.

This invention also provides a method for treating damage tothe eye of a subject which comprises administering to saidsubject an effective amount of compounds IV, or V.

In one embodiment of the method, said damage comprises retinalor optic nerve head damage. -189- 013295

In another embodiment of the method, said damage is acute orchronic.

In another embodiment of the method, said damage is the resuitof glaucoma, edema, ischemia, hypoxia or trauma.

In another embodiment of the method, the subject is a human.

In another embodiment of the method, the compound is anantagonist of A2a adenosine receptors.

This invention also provide a pharmaceutical compositioncomprising a therapeutically effective amount of compounds IV,or V and a pharmaceutically acceptable carrier.

In one embodiment of the pharmaceutical composition, saidtherapeutically effective amount is effective to treatParkinson's disease and diseases associated with locomotoractivity, vasodilation, platelet inhibition, neutrophilsuperoxide génération, cognitive disorder, or senile dementia.

In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is an ophthalmic formulation.

In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is an periocular, retrobulbar orintraocular injection formulation.

In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is a systemic formulation.

In another embodiment of the pharmaceutical composition, said -190- 013295 pharmaceutical composition is a surgical irrigating solution.

This invention also provides a combination therapy forParkinson's disease comprising compounds IV and V, and any ofthe dopamine enhancers.

This invention further provides a combinational therapy forcancer comprising compounds IV and V, and any of the cytotoxicagents.

This invention further provides a combinational therapy forglaucoma, comprising compounds IV or V, and a prostaglandinagonist, a muscrinic agonist, or a β-2 antagonist.

This invention also provides a packaged pharmaceuticalcomposition for treating a disease associated with A2a adenosinereceptor in a subject, comprising: (a) a container holding atherapeutically effective amount of compounds IV, or V; and (b)instructions for using said compound for treating said diseasein a subject.

This invention also provide a method of preparing compound IV,comprising the steps of -191- 013295

wherein P is a removable protecting grnup; b) treating the product of step a) under cyclization conditions to provide

d) treating the chlorinated product of step c) with NHR1R2 to provide

013295 -192- wherein NR^Rj is a substituted or unsubstituted 4-8membered ring; wherein R3 is a substituted or unsubstituted four to sixmembered ring; wherein Rs is H, alkyl, substituted alkyl, aryl, arylalkyl,amino, substituted aryl, wherein said substituted alkyl is-C(R7) (Re)XR9, wherein X is O, S, or NRio, wherein R7 and Rsare each independently H or alkyl, wherein R9 and Rio areeach independently alkyl or cycloalkyl, or R9, Rio and thenitrogen together form a substituted or unsubstituted ringof between 4 and 7 members; wherein R6 is H, alkyl, substituted alkyl, or cycloalkyl; with the proviso that NR1R2 is not 3-acetamido piperidino,3-hydroxy pyrrolidino, 3-methyloxy carbonylmethylpyrrolidino, 3-aminocarbonylmethyl, or pyrrolidino; withthe proviso that NR1R2 is 3-hydroxymethyl piperidino onlywhen R3 is 4-pyridyl.

This invention further provides a method of preparing compoundV, comprising the steps of -193- 013295

wherein P is a removable protecting group; b) treating the product of step a) under cyclization conditions to provide

c) treating the product of step b) under suitable conditions to provide

d) treating the chlorinated product of step c) fïrst with dimethylamine and formaldéhyde,then with N-methyl benzylamine and fïnally with NH2R1 to provide

-194- 013295 wherein Ri is acetamido ethyl; wherein R3 is 4-pyridyl;wherein R5 is H or methyl; wherein R6 is N-methyl-N-benzylami nome thyl.

As used herein, "A compound is A2a sélective." means that acompound has a binding constant to adenosine A2a receptor of atleast five time higher then that to adenosine Ar, A2b, or A3.

The invention is further illustrated by the following exampleswhich in no way should be construed as being further limiting.The contents of ail references, pending patent applications andpublished patent applications, cited throughout thisapplication, including those referenced in the backgroundsection, are hereby incorporated by reference. It should beunderstood that the models used throughout the examples areaccepted models and that the démonstration of efficacy in thesemodels is prédictive of efficacy in humans.

This invention will be better understood from the ExperimentalDetails which follow. However, one skilled in the art willreadily appreciate that the spécifie methods and resultsdiscussed are merely illustrative of the invention as describedmore fully in the daims which follow thereafter.

Example 22: Synthesis of Adenosine A2b Antagoniste, compounds1601, 1602, and 1603.

Carpound26

Carpxmd27

Coupon! 28

Conpomd 1601 -195- 013295

Compound 26 (10.93g, 50.76 mmol) was dissolved in DMF (67 mL) .5 4-Amidinopyridine hydrochloride (8.0g, 50.76 mmol) and. DBU (15.4 g, 101.5 mmol) were added sequentially and the reactionwas heated to 85°C. After 22 hours, the reaction was cooled toroom température and the DMF was removed in vacuo. The darkoil was diluted with 2M HCl (80 mL). The reaction was allowed

10 to stand. After 2 hours, the solution was cooled to 10°C andfiltered. The solid was washed with cold water and dried toyield 7.40g of a yellow solid, compound 27 (69%). 1H-NMR (200MHz, d5-DMSO) d 6.58 (s, 1H) , 7.27 (c, 1H) , 8.53 (d, 2H. J.= 5.6), 9.00 (d, 2H, J = 5.2Hz), 12.35 (brs, 1H) . MS (ES): 15 212.8 (M++l) .

Compound 27 (7.4 mmol, 29.8 mmol) was diluted with POC13 and heated to 105°C. After 18 hours, the reaction is cooled to roomtempérature and the POC13 is removed in vacuo. The thick dark 20 oil is diluted with MeOH (75mL) followed by ether (120mL) . Theamorphous red solid is filtered and washed with ether to yield3.82 g of a red solid. The crude solid, compound 28, isapproximately 80% pure and used without further purification inthe next reaction. ^i-NMR (200MHz, d6-DMSO) d 6.58 (s, 1H) , 7.27 25 (s, 1H), 8.53 (d, 2H, J = 5.6), 9.00 (d, 2H, J = 5.2Hz), 12.35 (brs, 1H). MS (ES): 212.8 (M++l).

Compound 1601: DMSO (5 mL) and D-prolinol (500mg, 4.94 mmol)were added to compound 28 (500mg, 2.17 mmol) was added. The 30 reaction was heated to 120°C. After 18 hours, The reaction wascooled to room température and diluted with EtOAc and H2O. Thelayers were separated and the aqueous layer was extracted withEtOAc (2x) . The combined organic layers were washed with H2O(2x) , brine, dried over MgSO4, filtered and concentrated to 35 yield 200mg of a tan solid. The solid was recrystallized from -196- 013295

EtOAc to yield 82 mg of a tan solid (13%) . ^H-NMR (200 MHz,d6-DMSO) d 2.05 (m, 4H), 3.43 (m, 1H), 3.70-4.00 (m, 3H), 4.50(brs, 1H), 4.92 (brs, 1H), 6.62 (m, 1H) , 7.22 (m, 1H), 8.22 (d,2H, J = 6.0 Hz), 6.64 (d, 2H, J = 6.2 Hz), MS (ES): 296.0(M++l), mp = 210 - 220°C (decomp.).

Compound 1602: Chromatography (silica, 9:1 CHCl3/MeOH) yielded10 mg of a tan solid (2%) ^H-NMR (d6-DMSO) d 2.00 - 2.50 (m,4H), 4.05 (m, 1H), 4.21 (m, 1H), 6.71 (d, 1H, J = 3.2 Hz), 7.18(d, 1H, J = 3.2 Hz), 8.37 (d, 2H, J = 4.8 Hz), 8.56 (d, 2H, J= 5 .0 Hz). MS (ES): 309.1 (M++l).

Compound 1603. Chromatography (silica, 20:1 Hexanes /EtOAc)yielded 135 mg of a tan solid (53%). XH-NMR (d6-DMSO) d 2.00(m, 4H), 3.43 (brs, 1H), 3.74 (brs, 2H), 3.87 (brs, 1H), 4.49(brs, 1H) , 4.93 (m, 1H) , 6.56 (m, 1H) , 7.12 (m, 1H) , 7.40 (m,3H) , 8.34 (m, 2H), 11.62 (brs, 1H). MS (ES): 295.1 (M++l) .

Compound 1605. Into a 50mL RBF 60mg of 2-(4'-pyridyl)-4-Chloropyrimidinopyrrole HCl sait was dissolved in 2mL anhydrousDMSO. 3-(R)-Hydroy-(D)-prolinol TFA sait (380mg) and 500mgsodium bicarbonate were added thereto. The mixture was thenflashed with nitrogen gas for 5min and heated to 130° C, After2 hours, the reaction was cooled to room température and theDMSO was removed in vacuo. The residue was partitioned betweenEtOAc (15mL) and saturated sodium bicarbonate aqueous solution(15mL). The organic layer was séparated and washed with brine (15mL) and dried over Na2SO4. After removal of solvent, the * crude product was purified by préparative TLC (CH2Cl2/MeOH =95/5) to yield 35 mg (50%). 1H-NMR (200MHz, CDC13) ( 2.3-2.5(1H), 3.4-3.8 (3H), 4.4-4.6 (2H), 6.4 (1H); 7.1 (1H); 8.2 (d,2H); 8.7 (d, 2H); 11.0 (1H). MS (ES): 312 (M++l). -197- 013295

Exemple 23: Synthesis of Adenosine A2a Antagonist, compound1606.

Compound 28 (200mg) was treated with DMF (30mL) , (2,2-dimethylglycine methyl ester (73mg HCl sait in 2mL water) and500mg sodium bicarbonate. After 18 hours, the DMF was removedin vacuo. The residue was partitioned between EtOAc (30mL) andsaturated sodium bicarbonate aqueous solution (15mL). Theorganic layer was washed with brine (15mL), dried over sodiumsulfate, filtered and concentrated. Chromatography (silica,10:4 hexanes/EtOAc) yielded 150mg of pure product, compound 29(69%). Ή-NMR (200MHz, CDC13) , ( 1.4 (s, 6H) , 3.8 (s, 3H) ; 3.9(s, 2H) ; 6.4 (s, 1H) ; 7.4-7.5 (m, 3H) ; 8.4 (m, 2H) ; 9.8 (s,1H) .

Compound 1606:

Procedure is the same as Compound 1605 (72%) . Ή-NMR (200MHz,CDC13), ( 1.3 (s, 6H) , 1.7-1.9 (m, 2H) ; 2.05-2.30 (m, 2H) ;3.6-4.1 (m, 11H); 4.80-4.95 (m, 1H); 6.4 (s, 1H); 7.4-7.6 (m,3H); 8.3-8.4 (d, J = 8.5 Hz, 2H), 10 (s, 1H) . MS (ES): 424.0(M++l) .

The following compounds can be synthesized in the same manner.Compound 1600: (51%). MS (ES): 326.0 (M++l). -198- ( 1.40 - 1.80 (m, 5H) , 3H) , 6.66 (d, 1H, J =

6.3Hz), 8.65 (d, 2H, J 1 (M++l) . 013295

Compound 1607: 1H-NMR (200MHz, CDC13) ,2.80 - 3.50 (m, 3H) , 4.60 - 4.80 (m,6.2Hz)( 7.26 (m, 1H), 8.21 (d, 2H, J == 5.8Hz), 11.90 (s, 1H). MS (ES): 310

Compound 1608: (64%). ^-NMR (200MHz, d6-DMSO) , (1.75 (s, 3H) ,2.11 (s, 3H), 2.29 (s, 3H), 3.56 (m, 6H), 7.23 - 7.41 (m, 5H) ,8.00 (brs, 1H), 8.23 (d, 2H, J = 6.0Hz), 8.63 (d, 2H, J = 5.4Hz), 8.82 (brs, 1H), 11.56 (brs, 1H) . MS (ES): 444.Q (M++l) .

Compound 1604: XH-NMR (200MHz, CD3OD) ( 3.40 (m, 4H) , 4.29 (m,4H) , 6.99 (s, 1H), 7.5 - 7.2 (m, 3H), 7.90 (d, 2H), 8.39 (d,2H), 8.61 (d, 2H). MS (ES): 357.0 (M+ +1). TABLE 16. Adenosine A2a Receptor Sélective Compounds * at least 5 times more sélective than other three subtypes.

lompound Structure Relative Ki-Al Ki-A2a Relative Ki-A2b Relative Ri-A3 1600 Q nk2 Ύ * O H -199- 013295

-200- 013295 1604 H N w N it 1605 H\ N N\C ★ 1606 CM" N λΟλ rU v)~° * -201- 013295 1607 '\Χ * 1608 ΗΝ. Vr'^-O pjôcl·' * 20 -202- 013295

Pages 202-256 relate to compounds spécifie to the A? receptor

Summarv of the Invention

The présent invention is also based on compounds whichselectively bind to adenosine A3 receptor, thereby treating adisease associated with A3 adenosine receptor in a subject byadministering to the subject a therapeutically effective amountof such compounds. The disease to be treated are associatedwith, for example, asthma, hypersensitivity, rhinitis, hayfever, sérum sickness, allergie vasculitis, atopie dermatitis,dermatitis, psoriasis, eczema, idiopathic pulmonary fibrosis,éosinophilie chlorecystitis, chronic airway inflammation,hyperéosinophilie syndromes, éosinophilie gastroenteritis,edema, urticaria, éosinophilie myocardial disease, episodicangioedema with eosinophilia, inflammatory bowel disease,ulcerative colitis, allergie granulomatosis, carcinomatosis,éosinophilie granuloma, familial histiocytosis, hypertension,mast cell degranulation, tumor, cardiac hypoxia, cérébralischemia, diuresis, rénal failure, neurological disorder,mental disorder, cognitive disorder, myocardial ischemia,bronchoconstriction, arthritis, autoimmune disease, Crohn'sdisease, Grave's disease, diabètes, multiple sclerosis,anaemia, psoriasis, fertility disorders, lupus erthyematosus,reperfusion injury, brain arteriole diameter, the release ofallergie mediators, scleroderma, stroke, global ischemia,central nervous System disorder, cardiovascular disorder,rénal disorder, inflammatory disorder, gastrointestinaldisorder, eye disorder, allergie disorder, respiratorydisorder, or immunological disorder.

This invention also features a compound having the structure: 013295 -203- 10

15 20 25 wherein Ri is H and R2 is cyclopropyl methylaminocarbonylethyl, cis-3-hydroxy cyclopentyl, acetamido butyl,methylamino carbonylamino butyl, ethylamino carbonylaminopropyl, methylamino carbonylamino propyl, 2-acetyl amino-3-methyl butyl, N,N-diethylamino carbonylamino ethyl,thioacetamido ethyl, 3-amino acetyloxy cyclopentyl, 3-hydroxy cyclopentyl, 2-pyrrolyl carbonyl aminoethyl, 2-imidazolidinone ethyl, l-aminocarbonyl-2-methyl propyl, 1-aminocarbonyl-2-phenyl ethyl, 3-hydroxy azetidino, 2-imidazolyl ethyl, acetamido ethyl, 1-(R)-phenyl-2-hydroxyethyl, N-methylaminocarbonyl pyridyl-2- methyl,or Ri, R2 and the nitrogen together are 3-acetamidopiperadino, 3-hydroxy pyrrolidino, 3-methyloxycarbonylmethyl pyrrolidino, 3-aminocarbonylmethylpyrrolidino, or 3-hydroxymethyl piperidino. wherein R3 is a substituted or unsubstituted four to sixmenbered ring, pyrrole, thiophene, furan, thiazole,imidazole, pyrazole, 1,2,4-triazole, pyridine, 2(1H)-pyridone, 4(1H)-pyridone, pyrazine, pyrimidine,pyridazine, isothiazole, isoxazole, oxazole, tetrazole,naphthalene, tetralin, naphthyridine, benzofuran, 30

-204- benzothiophene, indole, 2,3-dihydroindole, lH-indole,indoline, benzopyrazole, 1,3-benzodioxole, benzoxazole,purine, coumarin, chromone, quinoline,tetrahydroquinoline, isoquinoline, benzimidazole,quinazoline, pyrido[2,3-b]pyrazine, pyrido[3,4-b]pyrazine,pyrido[3,2-c]pyridazine, pyrido[3,4-b]-pyridine, 1H-pyrazole[3,4-d]pyrimidine, pteridine, 2(1H)-quinolone,1(2H)-isoquinolone, 1,4-benzisoxazine, benzothiazole,quinoxaline, quinoline-N-oxide, isoquinoline-N-oxide,quinoxaline-N-oxide, quinazoline-N-oxide, benzoxazine,phthalazine, or cinnoline. wherein Rs is H, alkyl, substituted alkyl, aryl, orsubstituted aryl; wherein R6 is H, alkyl, substituted alkyl, or cycloalkyl.

This invention also features a method foractivity of an A3 adenosine receptor in a cell,contacting said cell with the above-mentioned inhibiting thewhich comprisescompounds.

Typical synthetic schemes for the préparation of deazapurineintermediates of the invention are outlined below in Scheme I.

This invention also provides a method of preparing compound IV,comprising the steps of 013295 a) reacting

P to provide

O

P wherein P is a removable protecting group; b) treating the product of step a) under cyclisation conditions to prov,id

c) treating the product of step b) under suitable conditions to provide

d) treating the chlorinated product of step c) with NHR]R2 to provide

-206- 013295 wherein Ri is H and R2 is cyclopropyl methylaminocarbonylethyl, cis-3-hydroxy cyclopentyl, acetamido butyl,methylamino carbonylamino butyl, ethylamino carbonylaminopropyl, methylamino carbonylamino propyl, 2-acetyl amino-3-methyl butyl, N,N-diethylamino carbonylamino ethyl,thioacetamido ethyl, 3-amino acetyloxy cyclopentyl, 3-hydroxy cyclopentyl, 2-pyrrolyl carbonyl aminoethyl, 2-imidazolidinone ethyl, l-aminocarbonyl-2-methyl propyl, 1-aminocarbonyl-2-phenyl ethyl, 3-hydroxy azetidino, 2-imidazolyl ethyl, acetamido ethyl, l-(R)-phenyl-2-hydroxyethyl, N-methylaminocarbonyl pyridyl-2- methyl,or Ri, R2 and the nitrogen together are 3-acetamidopiperadino, 3-hydroxy pyrrolidino, 3-methyloxycarbonylmethyl pyrrolidino, 3-aminocarbonylmethylpyrrolidino, or 3-hydroxymethyl piperidino. wherein R3 is a substituted or unsubstituted four to sixmembered ring; wherein Rs is H, alkyl, substituted alkyl, aryl, orsubstituted aryl; wherein R6 is H, alkyl, substituted alkyl, or cycloalkyl.

This invention also provides a method of preparing compound ofV, comprising the steps of 013295 -207- reacting

wherein P is a removable protecting group; treating thc piuduct of siep a) undci c^ciizaûon conditions to provide

c) treating the product of step b) under suitable conditions to provide

d) treating the chlorinated product of step c) with NH2CH2(CH2)mCH2NHC(=O)Rlprovide NH2CH2(CH2)mCH2NHC(=O)Rl 013295 -208- wherein m is 0, 1, or 2; wherein Ri is cyclopropyl methyl, methyl, methylamino, or5 aminomethyl ; wherein R3 is aryl, substituted aryl, heteroaryl; wherein Rs is H, alkyl, substituted alkyl, aryl,10 arylalkyl, amino, substituted aryl, wherein said substituted alkyl is -C(R7) (Re)NR9Rio, wherein R7 and Rs areeach H or alkyl, wherein R9 and Rio are each alkyl orcycloalkyl, or R9, Rio and the nitrogen together form a ring System of between 4 and 7 members; 15 wherein R6 is H, alkyl, substituted alkyl, or cycloalkyl.

This invention further provided a method of preparing compoundVI, comprising 20 013295 -209-

wherein P is a removable protecting group; b) treating the product of step a) under cyclization conditions to provide

c) treating the product of step b) under suitable conditions to provide

d) treating the chlorinated product of step c) with HN to provide

NHAc -210- 013295 wherein R3 is unsubstituted aryl. wherein Rs is H, alkyl, substituted alkyl, aryl,arylalkyl, amino, substituted aryl, wherein saidsubstituted alkyl is -C(R7) (Rs)NR9Rio, wherein R7 and Re areeach H or alkyl, wherein R9 and Rio are each alkyl orcycloalkyl, or R9, Rio and the nitrogen together form aring System of between 4 and 7 members; wherein Re is H, alkyl, substituted alkyl, or cycloalkyl.

This invention also provides a compound having the structure:

IV wherein Ri is H and R2 is cyclopropyl methylaminocarbonylethyl, cis-3-hydroxy cyclopentyl, acetamido butyl,methylamino carbonylamino butyl, ethylamino carbonylaminopropyl, methylamino carbonylamino propyl, 2-acetyl amino-3-methyl butyl, Ν,Ν-diethylamino carbonylamino ethyl,thioacetamido ethyl, 3-amino acetyloxy cyclopentyl, 3-hydroxy cyclopentyl, 2-pyrrolyl carbonyl aminoethyl, 2-imidazolidinone ethyl, 1-aminocarbonyl-2-methyl propyl, 1-aminocarbonyl-2-phenyl ethyl, 3-hydroxy azetidino, 2-imidazolyl ethyl, acetamido ethyl, 1-(R)-phenyl-2-hydroxyethyl, N-methylaminocarbonyl pyridyl-2- methyl, -211- 013295 or Ri, R2 and the nitrogen together are 3-acetamidopiperadino, 3-hydroxy pyrrolidino, 3-methyloxycarbonylmethyl pyrrolidino, 3-aminocarbonylmethylpyrrolidino, or 3-hydroxymethyl piperidino. wherein R3 is a substituted or unsubstituted benzene,pyrrole, thiophene, furan, thiazole, imidazole, pyrazole,1,2,4-triazole, pyridine, 2 ( 1H)-pyridone, 4(1H)-pyridone,pyrazine, pyrimidine, pyridazine, isothiazole, isoxazole,oxazole, tetrazole, naphthalene, tetralin, naphthyridine,benzofuran, benzothiophene, indole, 2,3-dihydroindole, 1H-indole, indoline, benzopyrazole, 1,3-benzodioxole,benzoxazole, purine, coumarin, chromone, quinoline,tetrahydroquinoline, isoquinoline, benzimidazole,quinazoline, pyrido[2,3-b]pyrazine, pyrido[3,4-b]pyrazine,pyrido[3,2-c]pyridazine, pyrido[3,4-b]-pyridine, 1H-pyrazole[3,4-d]pyrimidine, pteridine, 2(1H)-quinolone,1 (2H)-isoquinolone, 1,4-benzisoxazine, benzothiazole,quinoxaline, quinoline-N-oxide, isoquinoline-N-oxide,quinoxaline-N-oxide, quinazoline-N-oxide, benzoxazine,phthalazine, or cinnoline; wherein Rs is H, alkyl, substituted alkyl, aryl, orsubstituted aryl; wherein Re is H, alkyl, substituted alkyl, or cycloalkyl. -212- ü1 329 5

In one embodiment of the compound, the compound has thestructure:

In another embodiment of the compound, R3 is phenyl.

In another embodiment of the compound, Rê is hydrogen ormethyl.

In another embodiment of the compound, Rs is hydrogen, methyl,phenyl, 3-chlorophenyloxy methyl, or trans-2- phenylaminomethyl pyrrolidino methyl.

This inventionstructure : further provides a compound having the

V -213- 013295 wherein m is 0, 1, or 2; wherein Ri is cyclopropyl methyl, methyl, methylamino, oraminomethyl; wherein R3 is aryl, substituted aryl, or heteroaryl; wherein Rs is H, alkyl, substituted alkyl, aryl,arylalkyl, amino, substituted aryl, wherein saidsubstituted alkyl is -C(R?) (Rs)NR9Rio, wherein R7 and Rs areeach H or alkyl, wherein Rs and Rio are each alkyl orcycloalkyl, or R9, Rio and the nitrogen together form aring System of between 4 and 7 members; wherein R6 is H, alkyl, substituted alkyl, or cycloalkyl.

In one embodiment of compound V, m is 0 and

In another embodiment of compound V, m is 1 In another embodiment of compound V, m is 2 In another embodiment of compound V, Rs and In another embodiment of compound V, Rs and In another embodiment of compound V, Rs and In another structure : embodiment ; of compound V, the R3 is phenyl. and R3 is phenyl. and R3 is phenyl. R6 are methyl. R6 are methyl. R6 are methyl. compound has the 013295 -214-

(Compound 1316)

In another embodiment of compound V, the compound ha s the structure :

(Compound 1311) -215- 013295

In another embodiment of compound V, the compound has thestructure :

(Compound 1202)

In another embodiment of compound V, the compound has thestructure :

(Compound 1310) 013295 -216-

In another embodiment of compound V, the compound has the structure:

(Compound 1312)

This invention further provides a compound having thestructure:

(Compound 609) -217-

This invention also provides a compound having the structure: 013295

wherein Rj is unsubstituted aryl. wherein Rs is H, alkyl, substituted alkyl, aryl,15 arylalkyl, amino, substituted aryl, wherein said substituted alkyl is -C(R7) (Rs)NR9Rio, wherein R7 and Re areeach H or alkyl, wherein R9 and Rio are each alkyl orcycloalkyl, or R9, Rio and the nitrogen together form a ring System of between 4 and 7 members; 20 wherein R6 is H, alkyl, substituted alkyl, or cycloalkyl.

In one embodiment of compound VI, the compound has thestructure:

(Compound 1309) -218-

In one embodiment of compound 1309, the compound has the structure: 013295

In another embodiment of compound 1309, the compound has thestructure:

‘\X -219- 013295

This invention also provides a compound having the structure:

wherein Rx is 3-hydroxy cyclopentyl ethylaminocarbonylamino propyl, N,N-diethylamino carbonylaminoethyl, thioacetamido ethyl, 3-amino acetyloxy cyclopentyl,3-hydroxy cyclopentyl, 2-pyrrolyl carbonyl aminoethyl, 2-imidazolidinone ethyl, l-aminocarbonyl-2-methyl propyl, 1-aminocarbonyl-2-phenyl ethyl, 3-hydroxy azetidino, 2-imidazolyl ethyl, acetamido ethyl, 1-(R)-phenyl-2-hydroxyethyl, or N-methylaminocarbonyl pyridyl-2- methyl; wherein R5 and R6 are independently H, substituted orunsubstituted alkyl, or aryl.

In one embodiment of the compound, the compound has thestructure :

(Compound 1700) -220-

In another embodiment of the compound, the compound has the structure: 013295

(Compound 1701)

In another embodiment of the compound, the compound has thestructure:

(Compound 1702) -221- û13295 structure: the compound bas the

In another embodiment of the compound,

In another embodiment of the compound, the compound has thestructure:

(Compound 1705) -222-

In ariother embodiment of the compound, the compound has the structure: 013295

(Compound 1706)

In another embodiment of the compound, the compound has thestructure:

-223- 013295

In another embodiment of the compound, the compound has thestructure :

In another embodiment of the compound, the compound has the structure :

-224-

In anotherstructure : 013295 embodiment of the compound, the compound has the

In another structure : embodiment of the compound, the compound has the

(Compound 1707) -225-

In another embodiment of the compound, the compound has the structure: 013295

(Compound 1708)

In another embodiment of the compound, the compound has thestructure :

(Compound 1709) -226-

In another embodiment of the compound, the compound has the structure: 013295

(Compound 1710)

In another embodiment of the compound, the compound has thestructure :

(Compound 1712) -227-

In another embodiment of the compound, the compound has the structure: 013295

(Compound.1713)

In another embodiment of the compound, the compound has thestructure:

-228- 013295

In another embodiment of the compound, the compound has thestructure:

In another embodiment of the compound, the compound has thestructure :

-229-

In another embodiment of the compound, the compound has the structure: 013295

(Compound 1715)

In another embodiment of the compound,structure: the compound has the

In another embodiment of the compound,structure: the compound has the

-230-

This invention also provides a compound having the structure: 013295

wherein Ri, R2 and the nitrogen together are 3-hydroxypyrrolidino, 3-methyloxy carbonylmethyl pyrrolidino, 3-aminocarbonylmethyl pyrrolidino, or 3-hydroxymethylpiperidino; wherein Rs and R6 are independently H, substituted orunsubstituted alkyl, or aryl.

In one embodiment of the compound, the compound has thestructure :

(Compound 1711) -231- 013295

In another embodiment of the compound, the compound has thestructure :

(Compound 1703)

In another embodiment of the compound, the compound has thestructure :

-232- 013295

In anotherstructure : embodiment of the compound, the compound has the

In anotherstructure :

(Compound 1716) -233- 013295

In another embodiment of the compound, the compound has thestructure:

In another embodiment of the compound, the compound has thestructure :

-234- 013295

In another ernbodiment of the compound, the compound has thestructure:

013295 -235-

In another embodiment of the compound, the compound hasstructure: the

In another embodiment of the compound, the compound hasstructure: the

(Compound 1718) -236- 013295

In another embodiment of the compound, the compound has thestructure:

In another embodiment of the compound, the compound has thestructure :

This invention also provides a method for treating a diseaseassociated with A3 adenosine receptor in a subject, comprisingadministering to the subject a therapeuticaiiy effective amountof any of the compounds IV, V, VI, VII, or VIII. -237- 013295

In one embodiment of the method, the subject is a mammal.

In another embodiment of the method, the mammal is a human.

In another embodiment of the method, said A3 adenosine receptoris associated with a central nervous System disorder, acardiovascular disorder, asthma, hypersensitivity, rhinitis,hay fever, sérum sickness, allergie vasculitis, atopiedermatitis, dermatitis, psoriasis, eczema, idiopathic pulmonaryfibrosis, éosinophilie chlorecystitis, chronic airwayinflammation, hyperéosinophilie syndromes, éosinophiliegastroenteritis, edema, urticaria, éosinophilie myocardialdisease, episodic angioedema with eosinophilia, inflammatorybowel disease, ulcerative colitis, allergie granulomatosis,carcinomatosis, éosinophilie granuloma, familial histiocytosis,hypertension, mast cell degranulation, tumor, cardiac hypoxia,cérébral ischemia, diuresis, rénal failure, neurologicaldisorder, mental disorder, cognitive disorder, myocardialischemia, bronchoconstriction, arthritis, autoimmune disease,Crohn's disease, Grave' s disease, diabètes, multiple sclerosis,anaemia, psoriasis, fertility disorders, lupus erthyematosus,reperfusion injury, brain arteriole diameter, the release ofallergie mediators, scleroderma, stroke, global ischemia,central nervous System disorder, cardiovascular disorder,rénal disorder, inflammatory disorder, gastrointestinaldisorder, eye disorder, allergie disorder, respiratorydisorder, or immunological disorder.

Diseases associated with adenosine Al, A2a, A2b and A3receptors are disclosed in WO 99/06053 and WO-09822465, WO-09705138, WO-09511681, WO-09733879, JP-09291089, PCT/US98/16053and U.S. Patent No. 5,516,894, the entire content of which are -238- 013295 fully incorporate herein by reference.

This invention also provides a water-soluble prodrug of any ofthe compounds IV, V, VI, VII, or VIII; wherein said water-soluble prodrug that is metabolized in vivo to an active drugwhich selectively inhibit A3 adenosine receptor.

In one embodiment of the prodrug, said prodrug is metabolizedin vivo by esterase catalyzed hydrolysis.

This invention also provides a pharmaceutical compositioncomprising the prodrug and a pharmaceuti cal l.y acceptablecarrier.

This invention also provides a method for inhibiting theactivity of an A3 adenosine receptor in a cell, which comprisescontacting said cell with any of the compounds IV, V, VI, VII,or VIII.

In one embodiment of the method, the compound is an antagonistof said A3 adenosine receptor.

In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is an ophthalmic formulation.

In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is an periocular, retrobulbar orintraocular injection formulation.

In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is a systemic formulation. -239- 013295

This invention also provides a method forgastrointestinal disorder in an subject,administering to the an effective amount ofcompounds IV, V, VI, VII, or VIII. treating acomprisingany of the

In one embodiment of the method, said disorder is diarrhea.

In another embodiment of the method, the subject is a human.

In another embodiment of the method, the compound is anantagonist of A3 adenosine receptors.

This invention further provides a method for treatingrespiratory disorder in a subject, comprising administering tothe subject an effective amount of any of the compounds IV, V,VI, VII, or VIII.

In one embodiment of the method, said disorder is asthma,chronic obstructive pulmonary disease, allergie rhinitis, or anupper respiratory disorder.

In another embodiment of the method, the subject is a human.

In another embodiment of the method, said compound is anantagonist of A3 adenosine receptors.

This invention also provides a method for treating damage tothe eye of a subject which comprises administering to saidsubject an effective amount of any of the compounds IV, V, VI,VII, or VIII.

In one embodiment of the method, said damage comprises retinal -240- 013295 or optic nerve head damage.

In another embodiment of the method, said damage is acute orchronic.

In another embodiment of the method, said damage is the resuitof glaucoma, edema, ischémie, hypoxia or trauma.

In another embodiment of the method, the subject is a human.

In another embodiment of the method, the compound is anantagonist of A3 adenosine receptors.

This invention also provide a pharmaceutical compositioncomprising a therapeutically effective amount of any of thecompounds IV, V, VI, VII, or VIII and a pharmaceuticallyacceptable carrier.

In one embodiment of the pharmaceutical composition, saidtherapeutically effective amount is effective to treat arespiratory disorder or a gastrointestinal disorder.

In another embodiment of the pharmaceutical composition, saidgastrointestinal disorder is diarrhea.

In another embodiment of the pharmaceutical composition, saidrespiratory disorder is asthma, allergie rhinitis, or chronicobstructive pulmonary disease.

In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is an ophthalmic formulation. -241- 013295

In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is an periocular, retrobulbar orintraocular injection formulation.

In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is a systemic formulation.

In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is a surgical irrigating solution.

This inventio also provides a packaged pharmaceuticalcomposition for treating a disease associated with A3 adenosinereceptor in a subject, comprising: (a) a container holding atherapeutically effective amount of any of the compounds IV, V,VI, VII, or VIII; and (b) instructions for using said compoundfor treating said disease in a subject.

Compounds represented by the formula IV, V, VI, VII, and VIIIcan be synthesized by the Schemes I-IX.

As used herein, "A compound is A3 sélective." means that acompound has a binding constant to adenosine A3 receptor of atleast ten time higher then that to adenosine Alz A2a, or A2b.

The invention is further illustrated by the following exampleswhich in no way should be construed as being further limiting.The contents of ail references, pending patent applications andpublished patent applications, cited throughout thisapplication, including those referenced in the backgroundsection, are hereby incorporated by reference. It should beunderstood that the models used throughout the examples areaccepted models and that the démonstration of efficacy in these -242- 013295 models is prédictive of efficacy in humans. A skilled artisan will know that metabolism of the compoundsdisclosed herein in a subject produces certain biologicallyactive métabolites which can serve as drugs.

This invention will be better understood from the ExperimentalDetails which follow. However, one skilled in the art willreadily appreciate that the spécifie methods and resultsdiscussed are merely illustrative of the invention as describedmore fully in the daims which follow thereafter.

Example 24: Adenosine A3 Antagonist Expérimentais

Compound 1700 (Table 17 below): MS (ES) : 366.1 (M++l) . Compound 1710 (Table 17 below): MS (ES) : 381.1 (M++l) . Compound 1316 (Table 17 below): MS (ES) : 353.2 (M++l) . Compound 1703 (Table 17 below): MS (ES) : 357.1 (M++l) . Compound 1719 (Table 17 below): 1H-NMR (200MHz, d6-DMSO) ( 1.75 (m, 2H) , 3.11 (m, 2H), 3.35 (s, 3H) , 3.59 (m, 2H), 5.72 (m, 1H), 5.96 2H), 8.32 (m, (m, 1H) , 6.55 (s, 1H) , 7. 2H) . 15 (s, 1H) , 7.49 (m, Compound 1704 (Table 17 below): MS (ES) : 367.0 (M++l) . Compound 1706 (Table 17 below) : XH· -MMR 200MHz, CDC13) d 1.22 (m, 2H), 1.60-2 .40 (m, 4H), 4 .53 (m, 1H), 4. 94 (m, 1H), 5.70 (d, 1H, J = 8.2 Hz) , 6.35 (d, 1H, J = 2.8 Hz) , 6.97 (d, 1H, J = 2 .0 Hz), 7.50 (m, 3H), 8.40 (m, 2H) , 10.83 (brs, 1H) .

Compound 1707 (Table 17 below): MS(ES): 347.0 (M++l). 013295 -243-

Compound 1708 (Table 17 below): MS (ES) 399.0 (M++l) . Compound 1709 (Table 17 below): MS (ES) 385.9 (M++l) . Compound 1710 (Table 17 below) : MS (ES) 434.0 (M++l) . Compound 1711 (Table 17 below) : XH· -NMR (200MHz , CD3OD) d 3.95 (d, 2H, J = 5 . 8Hz), 4. 23 - 4. 31 (m, : 2H), 4. 53 (t, 2H, J = 8.8Hz), 6.30 (d, 1H, J = 3.0Hz), 6.98 (d, 1H, J = 3.0Hz), 7.45- 7.48 (m, 3H), 7.83 - 8.42 (m, 2H), 9.70 (brs, 1H). MS (ES):281.1 (M++l).

Compound 1712 (Table 17 below): ^-NMR (200MHz, CD3OD) d 3.02(m, 2H), 3.92 (m, 2H), 5.09 (2, 2H), 6.53 (s, 1H), 6.30-7.04(br s, 1H) , 6.92 (m, 2H) , 7.02 (m, 1H) , 7.21 (dd, 1H, J = 8.2Hz), 7.40 (m, 3H) , 7.50-7.80 (br s, 1H), 8.33 (m, 2H) . MS(ES): 445.1 (M++l).

Compound 1713 (Table 17 below): 1H-NMR (200MHz, CDC13) d 1.65-1.80( m, 7H) , 1.88-2.00 (m, 1H) , 2.10 - 2.40 (m, 1H) , 2.70-3.05 (m, 3H) , 3.09-3.14 (m, 2H) , 3.16-3. 38 (m, 1H) , 3.45 (d, 1H, J = 1 4Hz) , 3.53-3.60 (m, 2H) , 3.84-3. 92 (m, 2H) , 3.97 (d, 1H, J = 14Hz) , 5.55 (t, 1H, J = 5.8Hz) , 6.17 (s, 1H) , 6.55-6.59 (m, , 2H) , 6.64-6.71 (m, 1H), 7.1 1-7.19 (m, 2H) , 7.43-7.46 (m, 3H) , 8.38-8.42 (m, 2H) , MS (ES) : 484.0 (M++l) .

Compound 1714 (Table 17 below): MS (ES): 471.0 (M++l).

Compound 1715 (Table 17 below): MS (ES): 505.0 (M++l).

Compound 1716 (Table 17 below): 1H-NMR (200MHz, CD3OD) d 1.65 (m, 1H), 2.18 (m, 1H) , 2.49 (br d. 2H, J = 6.2Hz), 2.64 (m, 1H) , 3.38 (m. 1H) , 3.69 (s, 3H) , 3. 72 (m, 1H) , 3.93 (m, 1H) , 4.10 (m, 1H), 5.06 (2, 2H), 6.58 (s , 1H), 6.92 (m, 2H) , 7.02 (m, 1H), 7.23 (dd, 1H, J = 8. 1Hz) , 7 .39 (m, 3H), 8.32 (m, 2H) . -244- 013295 MS (ES): 477.1 (M++l) . Compound 1717 (Table 17 below): 1H-NMR (200MHz, CD3OD) d 1.69 (m, 1H) , 2.26 (m, 1H), 2.42 (d, 2H, J = 7.4Hz), 2.72 (m, 1H) , 3.53 (m, 1H) , 3.83 (m, 1H) , 4.02 (m, 1H), 4.14 (dd, 1H, J = 10.6, 7.0Hz), 5.14 (2, 2H), 6.69 (s, 1H), 6.96 (m, 2H) , 7.06 (m, 1H) , 7.25 (dd, 1H, J = 8.0Hz), 7.39 (m, 3H), 8.35 (m. 2H) . MS (ES) : 462.2 (M++l) . Compound 1718 (Table 17 below)s 1H-NMR (200MHz, CD3OD) d 1.40 -2.00 (m , 5H) , 3.52 (d, 2H, 7.6Hz), 3 .80 - 4.00 (m, 1H), 4.00 - 4.20 (m, 3H) i , 4.50 (m, 2H) , 6.36 - 6.50 ( in, 2H), 6.54 (s. 1H), 6.84 - 6. 92 (m, 1H), 7.05 (t, 1H, J = 8.2Hz), 7.30 - 7.45 (m, 3H), 8.24 (d, 2H, J = 9.8Hz). MS (ES): 449.0 (M++l) .15 20 25 -245- 013295 TABLE 17. Adenosine A3 Receptor Sélective Compounds * at least 10 times mcre sélective than other three subtypes.

Oompound Structure Ki-Al Ki-A2a Ki-A2b KÏ-A3 1202 X NH I ★ X ^NH 1 CH 3 "V»· θχί H 1700 T 1' ★ ^*NH 1 CH g C^' 5^“· H -246- 10 15 013295 1309 H /X/N\ X-CH3 [ J Y 0 1 /CHs * 1701 h3c^ HN. ^nh ai ρΛ· * 20 -247- 013295

-248- 013295

-249- 10 15 20 013295

1702 H3c. Z HN O NH ★ Zi CH 3 ^Xcn· H Ht ★ 1703 b flA_ ΓΎ Ό U -250- 013295

-251- 013295

013295 -252- 1708 <Ύ ^ΝΗ θΑ„Χ„/ν/ ★ 1709 CH, -- * -253- 013295

-254- 013295

-255- 013295

013295 -256-

25 -257-

This invention provides a compound having the structure: 013295

This invention also provides a compound having thc structure:

This invention further provides a compound having thestructure:

1507 -258-

This invention also provides a compound having the structure: 013295

1508

This invention further provides a compound having the structure :

This invention also provides a compound having t hic stirncùtiirc:

1510 -259-

This invention also provides a compound having the structure: 013295

1511

This invention further providesstructure: a compound having the

This invention also provides a compound having the structure: Ç^Çnh2

nAj—O

H 1513

This invention further provides a compound having thestructure: -260- 013295

This invention further provides a compound having the

1515

This invention also provides a compound having the structure

1516 -261- 013295

1517

This invention further provides a compound having the

•ï CiV, U. Ci J

This invention also provides a compound having the structure:

provides

O

OK

This invention further a compound having the structure: -262- 013295

1520

In a further embodiment the invention provides a method fortreating a disease associated with Ai adenosine receptor in asubject, comprising administering to the subject atherapeutically effective amount of compounds 1505, 1506, 1507,1508, 1509, 1510, 1511, 1512, 1513, 1514, 1516, 1517, 1518, 1519, or 1520.

In a further embodiment the invention provides the above method, wherein the subject is a mammal.

In a further embodiment the invention provides the above method, wherein the mammal is a human.

In a further embodiment the invention provides the abovemethod, wherein said Ai adenosine receptor is associated withcognitive disease, rénal failure, cardiac arrhythmias,respiratory epithelia, transmitter release, sédation,vasoconstriction, bradycardia, négative cardiac inotropy anddromotropy, branchoconstriction, neutropil chemotaxis, refluxcondition, or ulcerative condition. -263- 013295

In a further embodiment the invention provides a water-solubleprodrug of compound 1505, 1506, 1507, 1508, 1509, 1510, 1511,1512, 1513, 1514, 1516, 1517, 1518, 1519, or 1520, wherein thewater-soluble prodrug is metabolized in vivo to produce anactive drug which selectively inhibits Ai adenosine receptor.

In a further embodiment the invention provides, wherein saidprodrug is metabolized in vivo by esterase catalyzedhydrolysis.

In a further embodiment the invention provides a pharmaceuticalcomposition comprising the above prodrug and a pharmaceuticallyacceptable carrier.

In a further embodiment the invention provides a method forinhibiting the activity of an Al adenosine receptor in a cell,which comprises contacting the cell with compounds 1505, 1506,1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1516, 1517,1518, 1519, or 1520.

In a further embodiment the invention provides the above methodfor inhibiting the activity of an Al adenosine receptor in acell, wherein the compound is an antagonist of the Ai adenosinereceptor.

In a further embodiment the invention provides the above methodfor inhibiting the activity of an Al adenosine receptor in acell, wherein the cell is human cell.

In a further embodiment the invention provides the above methodfor inhibiting the activity of an Al adenosine receptor in ahuman cell, wherein the compound is an antagonist of Ai 013295 -264- adenosine receptors.

In a further embodiment the invention provides a method fortreating a disease associated with Ai adenosine receptor in asubject, wherein said disease is asthma, chronic obstructivepulmonary disease, allergie rhinitis, or an upper respiratorydisorder.

In a further embodiment the invention provides a method fortreating a disease associated with Ai adenosine receptor in asubject, wherein said disease is asthma, chronic obstructivepulmonary disease, allergie rhinitis, or an upper respiratorydisorder and wherein the subject is a human.

In a further embodiment the invention provides a method fortreating the above disease, wherein said compound is anantagonist of Al adenosine receptors.

In a further embodiment the invention provides a combinationtherapy for asthma, comprising the compound 1505, 1506, 1507,1508, 1509, 1510, 1511, 1512, 1513, 1514, 1516, 1517, 1518,1519, or 1520, and a steroid, β2 agonist, glucocorticoid,leukotriene antagonist, or anticolinergic agonist.

In a further embodiment the invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount ofthe compound 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512,1513, 1514, 1516, 1517, 1518, 1519, or 1520, and a pharmaceutically acceptable carrier.

In a further embodiment the invention provides a method fortreating a respiratory disorder with the compound 1505, 1506,1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1516, 1517, -265- 013295 1518, 1519, or 1520, wherein said respiratory disorder isasthma, allergie rhinitis, or chronic obstructive pulmonarydisease.

In a further embodiment the invention provides the abov.epharmaceutical composition(s) , wherein said pharmaceuticalcomposition is an periocular, retrobulbar or intraocularinjection formulation.

In a further embodiment the invention provides the abovepharmaceutical composition(s) , wherein said pharmaceuticalcomposition is a systemic formulation.

In a further embodiment the invention provides the abovepharmaceutical composition(s) , wherein said pharmaceuticalcomposition is a surgical irrigating solution.

In a further embodiment the invention provides a packagedpharmaceutical composition for treating a disease associatedwith Al adenosine receptor in a subject, comprising: (a) a container holding a therapeutically effective amount of the compounds 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1518, 1519, or 1520; and 1513, 1514, 1516, 1517, (b) instructions for using said compound said disease in a subject. for treating a further embodiment the invention provides a pharmaceutically acceptable sait of the compound 1505, 1506,1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1516, 1517,1518, 1519, or 1520. -266- 013295

In a further embodiment the invention provides the abovepharmaceutically acceptable sait, wherein the pharmaceuticallyacceptable sait, of the compound 1509, 1511, 1515, 1518, or 1519contains a cation selected from the group consisting of sodium,calcium and ammonium.

In yet a further embodiment the invention provides a method fortreating a disease associated with Ai adenosine receptor in asubject, wherein the Ai adenosine receptor is associated withcongestive heart failure.

Exemplification

Exemple 21: Synthesis of 1-[6-(4-Hydroxy-4-phenyl-piperidin-l-y1-methy1)- 2-pheny1-7H-pyrrolo[2,3-d]pyrimidin-4-y1]-pyrrolidine-2-carboxylic acid amide (1505).

Compound 1505 was synthesized in a manner similar to that ofExample 17 using synthesis scheme IX with L-prolineamide and 4-phenyl-piperidin-4-ol to obtain:

Ή-NMR (d6-DMSO) d 1.53 (s, 1H) , 1.60 (s, 1H) , 1.84-2.30 (m,6H) , 2.66 (m, 2H) , 3.60 (s, 2H) , 3.88 (m, 1H) , 4.02 (m, 1H) ,4.66 (d, 1H, J = 6.8Hz), 4.73 (s, 1H) , 6.44 (s, 1H) , 6.94 (s, -267- 013295 1H) , 7.12 - 7.50 (m, 10H) , 8.35 (m, 2H) , 11.6 (brs, 1H) ; MS(ES): 305.1 (M++l); mp = 234-235°C.

Example 22s Synthesis of [N-(2-Phenyl-7H-pyrrolo [2,3-d]pyrimidin-4-yl)(L)-prolinamide (1506)

Compound 1506 was synthesized using synthesis scheme VII withL-prolineamide to obtain: 1506 ^î-NMR (DMS0-d6) d 2.05 (m, 4H) , 3.85 (m, 1H) , 4.05 (m, 1H) ,4.70 (d, 1H, J=8.0Hz), 6.58 (brs, 1H), 6.95 (brs, 1H), 7.15 (d,1H, J=3.4Hz), 7.40 (m, 3H), 7.50 (brs, 1H), 8.40 (m, 2H), 11.6(brs, 1H); MS (ES): 308.3 (M++l). mp= 236-238°C.

Exemple 23: Synthesis of [27- (2-phenyl-6-methoxymethyl-7ii-pyrrolo[2,3-d]pyrimidin-4-yl)-(L)-prolinamide (1507)

Compound 1 was synthesized using precursor compound 23 ofsynthesis scheme IX to obtain: -268- 013295

Bromide 23 (4.23g, lOmmol) is dissolved in anhydrous methanol(60mL) and DCM (120mL) and treated with AgO2CCF3 under N2 at rtfor lh. The solid is removed by filtration and washed with DCM(2x20mL). The filtrate is concentrated in vacuo. The residue isredissolved in DCM (80mX.) , Tbe resulted solution is then washedwith saturated NaHCO3 solution and brine, dried over MgSO4,filtered and concentrated to give 3.71g (4, 99%) off whitesolid. ^-NMR (CDC13) d 1.75 (s, 9H) , 3.51 (s, 3H) , 4.83 (s,2H) , 6.70 (s, 1H), 7.47 (m, 3H), 8.52 (m, 2H) .

Aryl chloride 4 (2.448g, 6.55mmol), DMSO (15mL), L-prolineamide(4.0g, 35.0mmol) and NaHCO3 (2.9g) are combined and heated to120°C under nitrogen. After 4h, the reaction is cooled to roomtempérature and diluted with water (60ml) . The resulted slurryis extracted with DCM (lOx). The combined organic layers arewashed with saturated NaHCO3 solution and brine, dried over 013295 -269-

MgSO4, filtered and concentrated to give 2.48g brown solid. Pureproduct (1.86g, 81%) is obtained after flash column as whitesolid. White crystals are gotten from THF/hexane. M.p. = 213-215°C. ^-NMR (CDC13) d 2.15 (m, 3H) , 2.52 (m, 1H) , 3.26 (s,3H) , 3.92 (m, 1H) , 4.10 (m, 1H) , 4.42 (s, 2H) , 5.08 (d, 1H,J=8.2Hz), 5.49 (brs, 1H) , 6.48 (s, 1H) , 7.08 (brs, 1H) , 7.42(m, 3H), 8.38 (m, 2H), 9.78 (brs, 1H); MS (ES): 352.2 (M++l).

Example 24: Synthesis of 4-Hydroxy-l-(2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-pyrrolidine-2-carboxylic acid amide (15Q8)

Compound 1508 was obtained with synthesis scheme VII using cir-hydroxy prolineamide to obtain:

1H-NMR (dÊ-DMSO) d 1.90 (m, 1H), 3.85 (d, 1H, J = 9.2Hz), 4.08(m, 1H), 4.37 (s, 1H), 4.67 (dd, 1H, J = 8.8, 4.0Hz), 5.30 (s,1H) , 6.55 (s, 1H), 7.15 (s, 2H) , 7.37 (m, 3H) , 7.64 (s, 1H) ,8.37 (m, 2H), 11.65 (brs, 1H); MS (ES): 324.2 (M++l); mp = 268-271°C.

Example 25: Synthesis of 3-[4-((S)-2-Carbamoyl-pyrrolidin-l-yl)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl]-propionic acid(1509)

Compound 1509 was obtained using precursor compound 23 of -270- 013295 synthesis scheme IX to obtain:

The tert-butoxycarbonyl protected aryl bromide 23 (4.0g,9.5mmol), dry DMSO (25ml), NaH2PO4 (454mg, 3.79mmol) and Na2HPO4(1.62g, 11.4mmol) were combined and heated to 50°C under argonfor approximately 3.5h. The mixture was then poured into water(200ml) and extracted with three 100ml portions of EtOAc. Thecombined organic layers were thoroughly washed with water,brine, dried over MgSO4, filtered and concentrated to give ayellow solid which was purified by triturating with éthanol, togive 1.55g of a pale yellow solid (7). The mother liquor waspurified by flash chromatography (10% EtOAc in hexane) to givean additional 454mg (60%). 1H-NMR (CDC13) d 1.77 (s, 9H) , 7.25(s, 1H), 7.48 (m, 3H), 8.52 (m, 2H) 10.39 (s, 1H); m.p.= 156°C(dec).

Aldéhyde 7 (600mg, 1.7mmol) was dissolved in dry THF (20ml) andcooled to 0°C under argon. To this was added a 0°C solution of(tert-butoxycarbonylmethylene)-triphenylphosphorane (694mg, -271- 013295 1.8mmol) in 10ml of dry THF dropwise through a cannula. After3h the mixture was concentrated and purified by trituratingwith éthanol to give 565mg (73%) of a white solid (8) . 1HNMR(CDC13) d 1.58 (s, 9H) , 1.79 (s, 9H) , 6.46 (d, 1H) , 6.95 (s,1H), 7.48 (m, 3H) , 8.09 (d, 1H), 8.56 (m, 2H).

H2,Pd-CTHF, EtOAC

A solution of compound 8 (565mg 1.2mmol) in 5ml THF was dilutedto 100ml with EtOAc. After adding 600mg of catalyst (5% wt Pd,50% H20) and purging with argon, the mixture was hydrogenatedunder atmospheric pressure. After 8h the mixture was filtered,concentrated and purified with flash chromatography (10% EtOAc in hexane) to isolate 200mg (35%) of 9 as a clear oil crystallized upon standing. XHNMR (CDC13) d 1.42 (s, 9H) ,(s, 9H) , 2.65 (t, 2H), 3.32 (t, 2H), 6.41 (s, 1H) 7.45 (m,8.51 (m, 2H). that 1.753H) ,

prolinamide (440mg, 4.4mmol) were combined and heated to 85°Cunder argon. After 14 hours the mixture is cooled to roomtempérature and partitioned between water and ethyl acetate. -272- 013295

The layers were separated and the aqueous layer washed withEtOAc (3x) . The combined organic layers were thoroughly washedwith water (3x) , brine, dried over MgSO4, filtered andconcentrated to give 10 as a yellow film which was purified by 5 flash chromatography (2.5% MeOH in CH2C12) . 185mg (97%). MS(ES): 435.8 (M++l).

Ester 10 (30mg, mmol) in 5ml dioxane was hydrolyzed by adding15 0.5ml concentrated HCl. After 3 hours the mixture was concentrated in vacuo and recrystalized in EtOH/ EtOAc toobtain 1509 as a white solid (20mg, 61%) . MS (ES) : 380 (M++l) . 20 Exemple 26s Synthesis of [M-(2-phenyl-6-aminocarbonylmethoxymethyl-7.if-pyrrolo [2,3-d]pyrimidin-4-yl) - (L) -prolinamide (1510)

Compound 1510 was obtained using precursor compound 23 of25 synthesis scheme IX to obtain:

HOCH2CO2CH3, DCM, AgOTf, rt, 2h

30 -273- 013295

Bromide 23 (1.27g, 3mmol) and molecular sieve (5g) are stirredin anhydrous methyl glycolate (5.8g, 60mmol) and DCM (40mL).The solution is treated with AgOTf under N2 and allowed to stirfor 3h. The solid is removed by filtration and washed with DCM(2x20mL). The filtrate is concentrated in vacuo. The residue isredissolved in DCM (80mL). The resulted solution is then washedwith water, saturated NaHCO3 solution and brine, dried overMgSO4, filtered and concentrated to give 1.35g (99%) off whitesolid (12). ^i-NMR (CDC13) d 1.75 (s, 9H) , 3.80 (s, 3H) , 5.0 (s,

Aryl chloride 12 (177mg, 0.41mmol), DMSO (lOmL), L-prolinamide(466mg, 4mmol) and NaHCO3 (500mg) are combined and heated to120°C under nitrogen. After 4h, the reaction is cooled to roomtempérature and diluted with water (60ml). The resulted slurryis. extracted with DCM (5x30mL). The combined organic layersare washed with saturated NaHCO3 solution and brine, dried overMgSO4, filtered and concentrated to give brown solid. Pureproduct (154mg, 92%) is obtained after flash column as whitesolid (13). 1H-NMR (CDC13) d 2.15 (m, 3H) , 2.52 (m, 1H) , 3.55(s, 3H) , 4.58 (s, 2H), 5.08 (s, 1H, ), 5.85 (brs, 1H), 6.48 (s,1H) , 7.08 (brs, 1H) , 7.42 (m, 3H) , 8.40 (m, 2H) , 10.-58 (brs,1H); MS (ES): 410.1 (M++l). -274- 013295

Methyl ester 13 (124mg, 0.3iranol) is dissolved in HOCH3 (15mL).Ammonia is bubbled through the solution for 0.5h. The reactionmixture is then stirred for another 3h at rt. After removal ofsolvent lllmg of a white solid (1510, 93%) is obtained. 1H-NMR 5 (CDC13) d 1.82 (m, 3H), 2.20 (m, 1H), 2.80 (m, 1H), 3.10 (m, 1H) , 3.63 (dd, 2H, J1=13.8Hz, J2=19.4Hz), 3.87 (m, 1H) , 4.07 (m,1H), 4.97 (m, 1H), 5.96 (m, 2H), 6.35 (s, 1H), 6.86 (brs, 1H),7.11 (brs, 1H), 7.37 (m, 3H), 8.28 (m, 2H), 11.46 (brs, 1H); MS(ES): 394.8 (M++l). 0

Exemple 27: Synthesis of [4-(2-Carbamoylpyrrolidin-l-yl)-2-phenyl-7Ji-pyrrolo [2,3-d]pyrimidine-6-carboxylic acid] (1511) 15

Compound 1511 was synthesized using precursor compound 15 ofsynthesis scheme VII to obtain:

NaH, then PhSO2CI

DMF 0—>20°C, 4h

20 15 16 -275- 013295 Το a suspension of sodium hydride (780mg of a 60% oilsuspension, 19.5mmol) in dry DMF (20mL) , cooled by an ice/waterbath, under nitrogen, is added a solution of thepyrrolopyrimidine 15 (2.00g, 7.52mmol) in DMF (lOmL) over 5min. After 15 min, benzenesulfonyl chloride (1.2mL, 9.40mmol)is added, then the cooling bath is removed. After 4h, thereaction mixture is poured into a mixture of ice and sat. NaHCO3sol., the precipitated solid is filtered off and trituratedwith acetone (3 ) and methanol (2 ), yielding 2.37g of a beigesolid. This solid (16) contains approx. 10mol-% DMF (based onthat 83% yield) and can be used in the next step; a pure samplecar. ba obtained by chromatography on silica gel using acetoneas eluent. ^-NMR (CDC13) : d 6.70 (d, J = 4.2Hz, 1H) , 7.47- 7.68 (m, 6H), 7.76 (d, J=4.2Hz, 1H), 8.24-8.32 (m, 2H), 8.48- 8.56 (m, 2H) ; IR (solid): n = 3146 cm'1, 1585, 1539, 1506,1450, 1417, 1386, 1370, 1186, 1176, 1154, 1111, 1015, 919, 726,683, 616, 607; MS (ES): 372/370 (MH+) ; mp = 226-227 SC.

To a solution of the N-sulfonyl compound 16 (337mg, 0.911mmol)in dry THF (34mL) , cooled by dry ice/acetone, is added LDA-THF(l.OmL, 1.5M solution in cyclohexane, 1.5mmol). After 45min,carbon dioxide is bubbled into the solution for 5min, then thecooling bath is removed. When the solution has reached ambienttemp., the solvents are evaporated, yielding 398mg of the sait17, containing 0.5 equiv. of (iPr) 2NCO2Li, as yellow solid. The -276- 013295 sait is used without purification in the next step. ’-H-NMR (D6-DMSO) : d = 6.44 (s, 1H), 7.50-7.75 (m, 6H), 8.33-8.40 (m, 2H) ,8.53 (dd, J= 8.0, 1.6Hz, 2H).

A solution of the lithium sait 17 (50mg) and L-prolinamide(122mg, 1.07mmol) in DMSO (1.5mL) is heated under nitrogen to80 SC for 15.5h. 4% aq. acetic acid (lOmL) is added to thecooled solution, and the mixture is extracted with EtOAc(5'10mL). The combined organic layers are washed with 4% aq.acetic acid (lOmL), water (lOmL) and brine (lOmL) and are driedover MgSO4. Filtration and concentration gives 40mg of 18 asa yellowish solid, which is used without purification in thenext step. 1H-NMR (CD3OD) : d= 1.95-2.36 (m, 4H) , 3.85-3.95 (m,1H), 3.95-4.17 (m, 1H), 4.72 (brs, 1H), 7.14 (s, 1H), 7.35-7.45(m, 3H), 7.45-7.70 (m, 3H), 8.33-8.50 (m, 4H).

NaOH

MeOH20 SC, 2h

18 -277- 013295 A solution of sodium hydroxide in methanol (1.5mL, 5M, 7.5mmol)is added to a solution of the pyrrolopyrimidine 18 (40mg,0.081mmol) in methanol (2mL) . After 2h, the pH is adjusted to5, most of the methanol is evaporated, the mixture is extractedwith EtOAc (5 lOmL), the combined organic layers are washedwith brine and dried over MgSO4. Filtration and concentrationyields 24mg of a pale yellow solid, which is triturated withtoluene/EtOAc/MeOH to yield 15.6mg (55%) of the acid 1511 asslightly yellowish solid. ^-NMR (CD30D) : d = 2.05-2.20 (m,4H), 3.95-4.10 (m, 1H), 4.15-4.25 (m, 1H), 4.85 (brs, 1H), 7.14(s, 1H) , 7.35-7.42 (m, 3H), 8.38-8.45 (m, 2H); IR (solid): n =3192 cm'1, 2961, 2923. 2877, 1682, 1614. 1567, 1531, 1454, 1374,1352, 1295, 1262, 1190, 974, 754, 700; MS (ES): 352 (M++l); m.p.= 220 SC (decomp.).

Example 28s Synthesis of 1-(6-methyl-2-phenyl-7/i-pyrrolo [2,3-d]pyrimidine-4-yl)-(S)-pyrrolidine-2 -carboxylic acid amide(1512)

Compound 1512 was synthesized by the following steps:

Aryl chloride 20 (3g, 10.7 mmol) , DMSO (50ml) and (S)- prolinamide were combined and heated to 85°C under argon. Afterstirring overnight (14hrs), the mixture was cooled to room -278- 013295 temperature and poured into 800ml of water. This was extractedwith three 200ml portions of EtOAc. The combined organic layerswere thoroughly washed with water (3 x 300 ml), brine, driedover MgSO4, filtered and concentrated to give a dark brownsolid. The solid was recrystallized twice from EtOAc to yield1.95g (57%) of a tan solid (1512). 1HNMR(DMSO-d6) d 1.8-2.2 (m,4H), 2.3 (s, 3H), 3.8 (m, 1H), 4.0 (m, 1H), 4.6 (d, 1H) 6.2 (s,1H), 6.9 (s, 1H), 7.2 (m, 3H), 7.3 (s, 1H), 8.4 (m, 2H), 11.5(s, 1H); MS (ES): 322 (M++l)

Example 29: Synthesis of 1-[6-(2-Hydroxv-ethoxymethyl)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-pyrrolidine-2-carboxylic acid amide(1513)

Compound 1513 was synthesized in a manner similar to that ofExample 17 using synthesis scheme IX with L-prolineamide andethane-1,2-diol to obtain: MS (ES): 382 (M++l). 1513 -279- 013295

Exemple 30: Synthesis of 4-(6-Imidazol-l-ylmethyl- 2-phenyl-7H-pyrrolo[ 2,3-d]pyrimidin-4-ylamino)-cyclohexanol(1514).

Compound 1514 was synthesized in a manner similar to that ofExample 17 using synthesis scheme IX with N-6 aminocyclohexanol and imidazole to obtain:

OH 1514 MS (ES): 389 (M++l)

Example 31: Synthesis of 4-(4-Hydroxy-cyclohexylamino)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid(1515)

Compound 1515 was synthesized in a manner similar to that ofExample 27 using synthesis scheme IX with N-6 aminocyclohexanol to obtain: -280- □13295

1515 MS (ES): 353 (M++l)

Example 32: Synthesis of 4-[6-(2-Hydroxy-ethoxymethyl)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino]-cyclohexanol(1516)

Compound 1516 was synthesized in a manner similar to that ofCompound 1513 using synthesis scheme IX with N-6 aminocyclohexanol to obtain:

1516 -281- 013295 MS (ES): 383 (M++l)

Example 33s Synthesis of 4-(4-Hydroxy-cyclohexylamino)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid 5 methyl ester (1517)

A solution of the lithium sait 17 (0.13mmol) in dry DMF (4mL)10 is stirred with methyl iodide (O.lmL, 1.6mmol) at 20 SC underargon for 3h. DMF is evaporated, and aqueous ammonium chloridesolution is added (15mL). The mixture is extracted with EtOAc(3'15mL), the combined organic layers are washed with water(2'10mL) and brine (lOmL) and are dried over MgSO4. Filtration 15 and concentration gives 21mg (38%) of the methyl ester 22.

1. h2n-Q„oh DMSO, 80 SC, 5h,then 20 SC, 13.5h 2. NaOH, MeOH, 20 min 22

1517 -282- 013295 A solution of the methyl ester 22 (24.5mg, 0.057mmol) and 4-trans-aminocyclohexanol (66mg, 0.57mmol) in DMSO (1.5mL) isheated under nitrogen to 80 2C for 5h, then the heating isstopped, and stirring at 20 SC is continued for 13.5h. 4% aq.acetic acid (lOmL) is added to the cooled solution, and themixture is extracted with EtOAc (3'10mL). The combined organiclayers are washed with 4% aq. acetic acid (lOmL) , water (lOmL)2n NaOH (lOmL) , water (lOmL) , and brine (lOmL) and are driedover MgSO4. To a solution of the crude material obtained afterfiltration and concentration (1H NMR indicates about 50%removal of the benzenesulfonyl group) in THF (2mL) is added asolution of NaOH in MeOH (0.5mL of 5m solution, 2.5mmol) atambient température. After 20min, water and sat. NaHCO3solution (5mL each) are added, and the mixture is extractedwith EtOAc (4'15mL). The combined organic layers are washedwith 2n NaOH (lOmL), water (lOmL), and brine (lOmL) and aredried over MgSO4. Chromatography of the crude material obtainedafter filtration and concentration on silica gel, eluting withhexanes/EtOAc 1:1 ® 1:2 yields 8.6mg (41%) of 1517 as a whitesolid, mp. 225-227 aC. ^-NMR (CD3OD) : d = 1.38-1.62 (m, 4H) ,1.95-2.10 (m, 2H), 2.10-2.25 (m, 2H) , 3.55-3.70 (m, 1H), 3.91(s, 3H), 4.20-4.35 (m, 1H), 7.32 (s, 1H), 7.35-7.47 (m, 3H) ,8.35-8.42 (m, 2H) ; IR (solid): n = 3352 cm"1, 3064, 2935, 2860,1701, 1605, 1588, 1574, 1534, 1447, 1386, 1333, 1263, 1206,1164, 1074, 938, 756, 705; MS (ES): 367 (MH+) . -283- 01329 5

Exemple 34: Synthesis of [4-(2-Carbamoyl-pyrrolidin-l-yl)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-6-ylmethoxy]-acetic acidmethyl ester (1518) 5 Compound 1518 was synthesized in a manner similar to example 2 6using precursor compound 12 to obtain: 10 15

MS (ES): 410 (M++l)

Exemple 35: Synthesis of [4-(2-Carbamoyl-pyrrolidin-l-yl) 20 -2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-6-ylmethoxy]-acetic acid (1519) -284-

Compound 1519 was synthesized in a manner similar to compound1518 wherein the methyl ester group was hydrolized with a baseto obtain:

1519 MS (ES): 396 (M++l)

Example 36: Synthesis of 4-(4-Hydroxy-cyclohexylamino)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid amide(1520)

-285- 013295

Gaseous ammonia is condensed into a solution of thepyrrolopyrimidine 23 (7.8mg, 0.021mmol) in methanol (6mL), cooled by dry ice/acetone, until a total volume of 12mL isreached. After stirring for lOd at 20 aC, the solvents areevaporated, and the residue is purified by préparative TLC onsilica gel, eluting with 5% MeOH in CH2C12. The materiel thus -obtained is triturated with ether to yield 6.5mg (88%) of theamide 1520 as white solid, mp. 210-220 2C (decomp.). 1H-NMR(CD3OD) : d= 1.40-1.60 (m, 4H) , 2.00-2.15 (m, 2H) , 2.15-2.25 (m,2H) , 3.55-3.70 (m, 1H) , 4.20-4.35 (m, 1H) , 7.16 (s, 1H) , 7.35-7.47 (m, 3H), 8.34-8.40 (m, 2H); IR (solid): n = 3358 cm'1,3064, 3025, 2964, 2924, 2853, 1652, 1593, 1539, 1493, 1452,1374, 1326, 1251, 1197, 1113, 1074, 1028, 751, 699; MS (ES):352 (MH+) .

Activitv of Compounds

Adenosine 1 (Ax) receptor subtype saturation and compétitionradio ligand binding were carried out for compounds 1505, 1506,1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1516, 1517,1518, 1519, and 1520 as described herein and inter alia, onpages 152-153 of this spécification. Ail of the above-referenced compounds equaled or surpassed the Ax receptorbinding affinity of reference compounds 1318 or 1319 asdescribed herein and, inter alia, in Table 13, on page 171 ofthe spécification.

The water solubilities of the above compounds listed in Table18 are expected to be better than reference compounds 1318 or1319 due to their cLogP values, which were calculated using thecomputer program CS ChemDraw, ChemDraw Ultra ver. 6.0 ©1999 asprovided by CambridgeSoft Corporation, 100 Cambridge ParkDrive, Cambridge, MA 02140. 013295 -286-

The compounds spécifie to the Ax receptor listed in Table 18 hadlower cLogP values, between about 1.5 to about 3.4, as comparedto reference compounds 1318 or 1319 with a cLogP value about3.8. It was not predicted that the more polar A1 receptor 5 compounds listed in Table 18 having lower cLogP values than thereference compounds 1318 or 1319 would still retain the potencyand Ax receptor binding selectivity as compared to thosereference compounds. 10 15 -287- 10 15 013295

Table 18

Compound cLogP 1505 4.1 1506 3.0 1507 2.88 1508 2.1 1509 2.9 1510 1.5 1511 2.7 1 ci o U. J «J 3.37 1513 2.4 1514 2.8 1515 3.1 1516 2.8 1517 3.4 1518 2.4 1519 2.2 1520 2.4 20 013295 -288-

Paoes 288-293 relate to additional compounds spécifie to A2a receptor

This invention provides a compound having the structure:

This invention also provides a compound having thestructure :

In a further embodiment the invention provides a method fortreating a disease associated with A2a adenosine receptorin a subject, comprising administering to the subject atherapeutically effective amount of compounds 1609 or 1610. -289- 013295

The invention also provides the above method, wherein thesubject is a mammal.

The invention further provides the above method, whereinthe mammal is a human.

The invention also provides the method for treating adisease associated with A2a adenosine receptor in a subject,wherein the A2a adenosine receptor is associated withlocomotor activity, vasodilation, platelet inhibition,neutrophil superoxide génération, cognitive disorder,senile dementia, or Parkinson's disease.

The invention provides the above method, wherein thecompound treats the diseases by stimulating adenylatecyclase.

The invention also provides a water-soluble prodrug of thecompound 1609 or 1610, wherein the water-soluble prodrug ismetabolized in vivo to an active drug to selectivelyinhibit an A2a adenosine receptor.

The invention also provides a water-soluble prodrug of thecompound 1609 or 1610, wherein the prodrug is metabolizedin vivo by esterase catalyzed hydrolysis.

The invention also provides a pharmaceutical compositioncomprising the water-soluble prodrug of the compound 1609or 1610, and a pharmaceutically acceptable carrier. -290- 013295

The invention also provides a method for inhibiting theactivity of an A2a adenosine receptor in a cell, whichcomprises contacting the cell with compound 1609 or 1610.

The invention also provides a method for inhibiting theactivity of an A2a adenosine receptor in a cell, whichcomprises contacting the cell with compound 1609 or 1610,wherein the compound is an antagonist of said A2a adenosinereceptor.

The invention also provides the above method, wherein thecell is a human cell.

The invention also provides the above method, wherein thecell is a human cell and the compound is an antagonist ofA2a adenosine receptors.

The invention also provides a pharmaceutical compositioncomprising a therapeutically effective amount of thecompound 1609 or 1610 and a pharmaceutically acceptablecarrier.

The invention also provides the above pharmaceuticalcomposition, wherein the therapeutically effective amountis effective to treat Parkinson's disease and diseasesassociated with locomotor activity, vasodilation, plateletinhibition, neutrophil superoxide génération, cognitivedisorder, or senile dementia.

The invention also provides the above pharmaceuticalcomposition, wherein the pharmaceutical composition is anophthalmic formulation.

The invention also provides the above pharmaceutical -291- 013295 composition, wherein the pharmaceutical composition is anperiocular, retrobulbar or intraocular injectionformulation.

The invention also provides the above pharmaceuticalcomposition, wherein the pharmaceutical composition is asystemic formulation.

The invention also provides the above pharmaceuticalcomposition, wherein the pharmaceutical composition is asurgical irrigating solution.

The invention also provides a combination therapy forParkinson's disease, comprising the compounds 1609 or 1610,and any of the dopamine enhancers.

The invention also provides a combination therapy forcancer, comprising the compound 1609 or 1610, and any ofthe cytotoxic agents.

The invention also provides a combination therapy forglaucoma, comprising the compound 1609 or 1610, and aprostaglandin agonist, a muscrinic agonist, or a b-2antagonist.

The invention also provides a packaged pharmaceuticalcomposition for treating a disease associated with A2aadenosine receptor in a subject, comprising: (a) a container holding a therapeutically effectiveamount of the compound 1609 or 1610; and -292- 013295 (b) instructions for using the compound for treatingsaid disease in a subject.

Exemplification

Exemple 41: Synthesis of 1-(6-Phenyl-2-pyridin-4-yl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-pyrrolidine-2-carboxylic acidamide (1609) .

Compound 1609 was synthesized by reacting L-prolinamidewith the appropriate chloride intermediate described insynthesis scheme II on page 82 to obtain:

1H-NMR (d6-DMS0) d 1.95-2.15 (m, 4H) , 4.00 (brs, 1H) , 4.15(brs, 1H), 4.72 (brs, 1H), 6.90 (brs, 1H), 7.19 (brs, 1H),7.30 (t, 1H, J = 7.0Hz), 7.44 (t, 2H, J = 7.0Hz), 7.59 (s,1H), 7.92 (brs, 2H), 8.26 (d,2H, J = 6.2Hz), 8.65 (d, 2H,J = 6.2Hz); MS (ES): 384.9 (M++l) ; Mpt = 280-316°C (decomp.). -293- 013295

Exemple 42: Synthesis of 1-[6-(3-Methoxy-phenyl)-2-pyridin-4-yl- 7-H-pyrrolo [2 , 3-d]pyrimidin-4-yl] -pyrrolidine-2-carboxylic acid amide (1610) .

Compound 1610 was synthesized by reacting L-prolinamidewith the appropriate chloride intermediate described insynthesis scheme II on page 82 to obtain:

1610 ^-NMRidj-DMSO) d 2.07(m,4H), 3.85(s,3H), 4.02(m,lH), 4.17(m,lH), 4.75(m,lH), 6.89{m,lH), 7.00(s,lH), 7.23(S,1H),7.35(t,1H,J=8.2Hz), 7.53(s,2H), 7.60(s,lH),

8.28(d,2H,J=5.8Hz), 8.67(d,2H,J=5.8Hz), 12.37(s,lH); MS (ES) : 415.0 (M++l) .

Activitv of Compounds

Adenosine 2a (A2a) receptor subtype compétition radio ligandbinding were carried out for compounds 1609 and 1610 asdescribed herein and inter alia, on page 153 of thisspécification. Compounds 1609 and 1610 were found hâve A2areceptor binding affinity and selectivity. -294- 013295

Pages 294-300 relate to additional compounds spécifie to A? receptor

This invention also provides a compound having the structure:

In a further embodiment the invention provides a method forinhibiting the activity of an A3 adenosine receptor in a cell,which comprises contacting the cell with the compound 1720.

In a further embodiment the invention provides a method forinhibiting the activity of an A3 adenosine receptor in a cell,wherein the compound is an antagonist of the A3 adenosinereceptor.

In a further embodiment the invention provides the above methodfor inhibiting the activity of an A3 adenosine receptor in acell, wherein the cell is human cell.

In a further embodiment the invention provides the above methodfor inhibiting the activity of an A3 adenosine receptor in acell, wherein the cell is a human cell and wherein the compound -2S5- 013295 is an antagonist of A3 adenosine receptors.

In a further embodiment the invention provides a method oftreating damage to the eye of a subject which comprisesadministering to the subject a composition comprising atherapeutically effective amount of the compound 1720.

In a further embodiment the invention provides the abovemethod, wherein the damage comprises retinal or optic nervehead damage.

In a further embodiment the invention provides a therapy forglaucoma, comprising administering to a subject atherapeutically effective amount of the compound 1720.

In a further embodiment the invention provides a therapy forglaucoma comprising one or more adenosine receptor antagonists,preferably comprising an adenosine receptor A3 antagonist(preferably an N-6 substituted 7-deazapurine, most preferably[2-(3H-Imidazol-4-yl)-ethy1]-(2-phenyl-7H-pyrrolo[2,3—d]pyrimidin-4-yl)-amine).

In an alternative embodiment the invention provides acombination therapy for glaucoma, comprising an adenosinereceptor A3 antagonist (preferably an N-6 substituted 7-deazapurine, most preferably [2-(3H-Imidazol-4-yl)-ethyl]-(2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-amine)) and one or moreother compounds selected from the group consisting of betaadrenoceptor antagonists (i.e. beta adrenergic antagonists orb-blockers) (e.g. timolol maleate, betaxolol, carteolol,levobunolol, metipranolol, L-653328 (the acetate ester of L- -296- 013295 652698) , beta 1 adrenoceptor antagonists) , alpha-2 adrenoceptoragonists (e.g. aplaclonidine, brimonidine, AGN-195795, AGN-190837 (an analog of Bay-a-6781)), carbonic anhydraseinhibitors (brïnzolamide, dorzolamide, MK-927 (an inhibitor ofthe human carbonic anhydrase II isoenzyme), inhibitors ofcarbonic anhydrase IV isoenzyme), cholinergic agonists (e.g.muscarinic cholinergic agonists, carbachol, pilocarpine HCl,pilocarpine nitrate, pilocarpine, pilocarpine prodrugs (e.g.DD-22A)), prostaglandins and prostaglandin receptor agonists(e.g. latanoprost, unoprostone isopropyl, PGF2 alpha agonists,prostanoid-sélective FP receptor agonists, PG agonists such asthe hypotensive prostamides) , angiotensin converting enzyme(ACE) inhibitors (e.g. Spirapril, spiraprilat), AMPA receptorantagonists, 5-HT agonists (e.g. a sélective 5-HT IA receptoragonist such as MKC-242 (5-3-[((2S)-1,4-benzodioxan-2-ylmethyl)amino]propoxy-1,3-benxodioxole HCl), angiogenesisinhibitors (e.g. the steroid anecortave), NMDA antagonists(e.g. HU-211, memantine, the cannabinoid NMDA-receptor agonistdexanabinol, prodrugs and analogs of dexanabinol, NR2B-selective antagonists (e.g. eliprodil (SL-82.0715)), renininhibitors (e.g. CGP-38560, SR-43845), cannabinoid receptoragonists (e.g. tetrahydrocannabinol (THC) and THC analogs,sélective CB2 cannabinoid receptor agonists (e.g. L-768242, L-759787), compounds such as anandamide that bind to both brain-specific CB1 receptors and peripheral CB2 receptors),angiotensin receptor antagonists (e.g., angiotensin II receptorantagonists (e.g. CS-088), sélective angiotensin II AT-Ireceptor antagonists, such as iosartan potassium) ,hydrochlorothiazide (HCTZ), somatostatin agonists (e.g. thenon-peptide somatostatin agonist NNC-26-9100), glucocorticoidantagonists, mast cell degranulaticn inhibitors (e.g. -297- 013295 nedocromil), alpha-adrenergic receptor blockers (e.g.dapiprazole, alpha-2 adrenoceptor antagonists, alpha 1adrenoceptor antagonists (e.g. bunazosin)), alpha-2adrenoceptor antagonists, thromboxane A2 mimetics, proteinkinase inhibitors (e.g. H7), prostaglandin F dérivatives (e.g.S-1033), prostaglandin-2 alpha antagonists (e.g. PhXA-34),dopamine DI and 5-HT2 agonists (fenoldopam), nitric-oxide-releasing agents (e.g. NCX-904 or NCX-905, nitric-oxide-releasing dérivatives of timolol) , 5-HT 2 antagonists (e.g.sarpogrelate), NMDA antagonists (e.g. prodrugs and analogs ofdexanabinol), alpha 1 adrenoceptor antagonists (e.g.bunazosin), cyclooxygenase inhibitors (e.g. diclofenac, or thenon-steroidal compound nepafenac), inosine, dopamine D2receptor and alpha 2 adrenoceptor agonists (e.g. talipexole),dopamine DI receptor antagonist and D2 receptor agonists (e.g.SDZ-GLC-756), vasopressin receptor antagonists (e.g.vasopressin V2 receptor antagonists (e.g. SR-121463)),endothelin antagonists (e.g. TBC-2576), 1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) and related analogsand prodrugs, thyroid hormone receptor ligands (e.g. KB-130015), muscarinic Ml agonists, NMDA-receptor antagonists(e.g. the cannabinoid NMDA-receptor antagonist dexanabinol) , PGagonists such as the hypotensive lipids, prostamides, sodiumchannel blockers, NMDA antagonists, mixed-action ion channelblockers, beta adrenoceptor antagonist and PGF2 alpha agonistcombinations (e.g. latanoprost and timolol), guanylate cyclaseactivators (e.g. atrial natriuretic peptide (ANP) or non-peptide mimetics, inhibitors of ANP neutral endopeptidase,nitrovasodilators (e.g. nitroglycerin, hydralazine, sodiumnitroprusside), endothelin receptor modulators (e.g. ET-1 ornon-peptide mimetics, sarafotoxin· S5c), ethacrynic acid, cther -298- 013295 phenoxyacetic acid analogs (e.g. indacrinone, ticrynafen),actin disrupters (e.g. latrunculin), calcium channel blockers(e.g. verapamil, nifedipine, brovincamine, nivaldipine) andneuroprotective agents. A combination therapy for glaucoma, comprising the compound of1702, and one or more compounds selected from the groupconsisting of beta adrenoceptor antagonists, alpha-2adrenoceptor agonists, carbonic anhydrase inhibitors,cholinergic agonists and prostaglandin receptor agonists.

In a further embodiment the invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount ofthe compound 1720 and a pharmaceutically acceptable carrier.

In a further embodiment the invention provides a packagedpharmaceutical composition for treating a disease associatedwith A3 adenosine receptor in a subject, comprising: (a) a container holding a therapeutically effectiveamount of the compound 1720; and (b) instructions for using said compound for treatingsaid disease in a subject.

In a further embodiment the invention provides a method ofmaking a composition which comprises the compound 1720, themethod comprising admixing the compound 1702 with a suitablecarrier.

In a further embodiment the invention provides a 013295 -299- pharmaceutically acceptable sait of compound 1720, wherein thepharmaceutically acceptable sait contains an anion selectedfrom the group consisting of maleic, fumaric, tartaric,acetate, phosphate and mesylate.

Exemplification

Example 43: Synthesis of [2-(3H-Imidazol-4-yl) -ethyl] - (2-phenyl-7fi-pyrrolo [2,3-d]pyrimidin-4-yl) -amine (1720)

Compound 1720 was synthesized using precursor compound 1 ofsynthesis scheme VII to obtain:

Aryl chloride 1 (400mg, 1.50mmol) , DMSO (lOmL) and histamine(1.67g, lS.Ommol) are combined and heated to 12 0°C undernitrogen. After 6.5h, the reaction is cooled to roomtempérature and partitioned between EtOAc and water. Thelayers are separated and the aqueous layer is extracted withEtOAc (3x) . The combined organic layers are washed with brine(2x) , dried over MgSO4, filtered and concentrated to yield494mg of a brown solid. The solid is washed with cold MeOH andrecrystallized from MeOH to yield 197mg (43%) of an off whitesolid (1720). ^-NMR (CD3OD) d 3.05 (t, 2H, J = 7.0Hz), 3.94 013295 10 -300- (t, 2H, J = 7.0Hz), 6.50 (d, 1H, J = 3.5Hz), 6.88 (brs, 1H) ,7.04 (d, 1H, J = 3.5Hz), 7.42 (m, 3H) , 7.57 (s, 1H), 8.34 (m,2H) ; MS (ES): 305.1 (M++l) ; Mpt = 234-235°C. 5 Activitv of Compounds

Adenosine 3 (A3) receptor compétition radio ligand binding wascarried out for compound 1720 as described herein and interalia, on pages 153-154 of this spécification. Compound 1720was found to hâve an A3 receptor binding affinity greater than10 times that of reference compound 1308 as described hereinand, inter alia, ->n Table 13, on page 169 of the spécification. 013295 -301- 10

Incorporation by Reference

Ail patents, published patent applications and other referencesdisclosed herein are hereby expressly incorporated herein byreference.

Equivalents

Those skilled in the art will recognize, or be able toascertain, using no more than routine expérimentation, manyéquivalents to spécifie embodiments of the invention describedspecifically herein. Such équivalents are intended to beencompassed in the scope of the following daims. -302- 013295

This invention further provides compounds having the formula:

wherein R1NR2 together form a ring having the structure:

or Ri is H and R2 is:

OH

R5 is H, or substituted or unsubstituted alkyl oralkylaryl.

Claims (65)

1. -303- 013295 What is claimed is:

   1. A compound having the structure:

   wherein RJtfR.2 together form a ring having the structure

   nh2 or

   or Rj is H and R2 is: OH

   -3Ü4- wherein 013295 when R1NR2 together are

   R5 is H, -CH2 (NC5H8) (OH) (C6H5) , -CH2OCH3, - (CH2)2C(O)OH, -CH2OCH2C(O)NH2, -C(O)OH, -ch3,-CH2O(CH2)2OH,-CH2OCH2C(O)OCH3, or -CH2OCH2C (O) OH;or when Rl is H and R2 is OH

   R5 is -CH2 (N2C3H3) , -C(O)OH, -CH2O (CH2) 2OH, -C(O)OCH3, or -C(O)NH2, or a pharmaceutically acceptable sait thereof. -305- 013295
2. 2. The compound of claim 1, having the structure
3. 3. The compound of claim 1, having the structure
4. 4. The compound of claim 1, having the structure
5. 5. The compound of claim 1, having the structure

   -306- 013295
6. 7. The compound of claim 1, having the structure;

   Ν^"ΝH N-'-l ° NH,

   CONH; compound of claim 1, having the structure :

   -307- 013295
7. 9. The compound of claim 1, having the structure:
8. 10. The compound of claim 1, having the structure :
9. 11.. The compound of claim 1, having the structure :

   -308- 013295

   -309- 013295

   16

   structure :
10. 17. The compound of claim 1, having the structure:
11. 18. Use of the compound of claim 1 for manufacturing amédicament useful for treating a disease associatedwith an Ai adenosine receptor in a subject. -310- 013295
12. 19. The use of daim 18, wherein said Ai adenosine receptoris associated with cognitive disease, rénal failure,cardiac arrhythmias, respiratory epithelia, transmitter release, sédation,négative cardiacbronchoconstriction, vasoconstriction, bradycardia,inotropy and dromotropy,neutrophil chemotaxis, reflux condition, or ulcerative condition.
13. 20. A water-soluble prodrug of the compounds of claim 1,wherein the water-soluble prodrug is metabolized invivo to produce an active drug which selectivelyinhibits Ai adenosine receptor.
14. 21. Use of the compound of claim 1 for manufacturing amédicament useful for inhibiting the activity of an Aladenosine receptor in a cell.
15. 22. The use of claim 18, wherein said disease is asthma, chronic obstructive pulmonary disease, allergie rhinitis, or an upper respiratory disorder.
16. 23. A pharmaceutical composition comprising the compound ofclaim 1, a pharmaceutically acceptable carrier and atleast one of a steroid, β2 agonist, glucocorticoid,leukotriene antagonist, or an anticolinergic agonist.
17. 24. A pharmaceutical composition comprising atherapeutically effective amount of the compound ofclaim 1, and a pharmaceutically acceptable carrier.
18. 25. The use of claim 18, wherein the disease is asthma,allergie rhinitis, or chronic obstructive pulmonarydisease.
19. 26. A packaged pharmaceutical composition for treating a Ο Ί 329 5 -311- disease associated with an Al adenosine receptor in asubject, comprising: (a) a container holding a therapeutically effectiveamount of the compound of claim 1; and (b) instructions for using said compound for treatingsaid disease in a subject.
20. 27. A pharmaceutically acceptable sait of the compound ofclaim 6, 8, 12, 15, or 16 wherein the sait contains acation selected from the group consisting of sodium,calcium and ammonium.
21. 28. The use of claim 18, wherein the Ai adenosine receptoris associated with congestive heart failure. 013295 -312-
22. 29. A compound having the structure:
23. 30. A compound having the structura:
24. 31. Use of the compound of claim 29 or 30 for manufacturinga médicament useful for treating a disease associatedwith an A2a adenosine receptor in a subject.
25. 32. The use of claim 18 or 31, wherein the subject ishuman.
26. 33. The use of claim 32, wherein said A2a adenosine receptoris associated with locomotor activity, vasodilation,platelet inhibition, neutrophil superoxide génération,cognitive disorder, senile dementia, or Parkinson'sdisease.
27. 34. The use of claim 31, wherein the compound treats saiddiseases by stimulating adenylate cyclase. -313- 013295
28. 35. A water-soluble prodrug of the compound of claim 29 or30, wherein said water-soluble prodrug that ismetabolized in vivo to an active drug which selectivelyinhibit A2a adenosine receptor.
29. 36. The prodrug of claim 35, wherein said prodrug ismetabolized in vivo by esterase catalyzed hydrolysis.
30. 37. A pharmaceutical composition comprising the prodrug ofclaim 35 and a pharmaceutically acceptable carrier.
31. 38. Use of the compound of claim 29 or 30 for manufacturinga médicament useful for inhibiting the activity of anA2a adenosine receptor in a cell.
32. 39. A pharmaceutical composition comprising atherapeutically effective amount of the compound ofclaim 29 or 30 and a pharmaceutically acceptablecarrier.
33. 40. The pharmaceutical composition of claim 39, whereinsaid therapeutically effective amount is effective totreat Parkinson's disease and diseases associated withlocomotor activity, vasodilation, platelet inhibition,neutrophil superoxide génération, cognitive disorder,or senile dementia.
34. 41. The pharmaceutical composition of claim 39, wherein said pharmaceutical formulation. composition is an ophthalmic 42. The pharmaceutical composition of claim 24 or 39, wherein said pharmaceutical composition is anperiocular, retrobulbar or intraocular injectionformulation. -314- 013295
35. 43. The pharmaceutical composition of claim 24 or 39,wherein said pharmaceutical composition is a systemicformulation.
36. 44. The pharmaceutical composition of claim 24 or 39,wherein said pharmaceutical composition is a surgicalirrigating solution.
37. 45. A pharmaceutical composition comprising the compoundsof claim 29 or 30, a pharmaceutically acceptablecarrier and any of the dopamine enhancers.
38. 46. A pharmaceutical composition comprising the compound ofclaim 29 or 30, a pharmaceutically acceptable carrierand any of the cytotoxic agents.
39. 47. A pharmaceutical composition comprising the compound ofclaim 29 or 30, a pharmaceutically acceptable carrierand at least one of a prostaglandin agonist, amuscrinic agonist, or a (3-2 antagonist.
40. 48. A packaged pharmaceutical composition for treating adisease associated with an A2a adenosine receptor in asubject, comprising: (a) a container holding a therapeutically effectiveamount of the compound of claim 29 or 30; and (b) instructions for using said compound for treatingsaid disease in a subject.
41. 49. A pharmaceutically acceptable sait of the compound ofclaim 29 or 30. -315- 013295
42. 50. A compound having the structure:

    or a pharmaceutically acceptable sait thereof.
43. 51. Use of the compound of claim 50 for manufacturing amédicament useful for inhibiting the activity of an A3adenosine receptor in a cell.
44. 52. The use of claim 21, 38 or 51, wherein the cell is ahuman cell.
45. 53. Use of the compound of claim 50 for manufacturing amédicament useful for treating damage to the eye of asubject.
46. 54. The use of claim 53, wherein the damage comprisesretinal or optic nerve head damage.
47. 55. Use of the compound of claim 50 for manufacturing amédicament useful for treating glaucoma in a subject.
48. 56. The use of claim 55, wherein the médicament furthercomprises one or more compounds selected from the groupconsisting of beta adrenoceptor antagonists, alpha-2adrenoceptor agonists, carbonic anhydrase inhibitors,cholinergic agonists, prostaglandins and prostaglandinreceptor agonists, angiotensin converting enzyme (ACE) -316- 013295 inhibitors, AMPA receptor antagoniste, 5-HT agonists,angiogenesis inhibitors, NMDA antagoniste, renininhibitors, cannabinoid receptor agonists, angiotensinreceptor antagonists, hydrochlorothiazide (HCTZ),somatostatin agonists , glucocorticoid antagonists,mast cell degranulation inhibitors, alpha-adrenergicreceptor blockers, alpha-2 adrenoceptor antagonists,thromboxane A2 mimetics, protein kinase inhibitors,prostaglandin F dérivatives, prostaglandin-2 alphaantagonists, dopamine DI and 5-HT2 agonists, nitric-oxide-releasing agents, 5-HT 2 antagonists,cyclooxygenase inhibitors, inosine, dopamine D2receptor and alpha 2 adrenoceptor agonists, dopamine DIreceptor antagonist and D2 receptor agonists,vasopressin receptor antagonists, endothelinantagonists, l-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) and relatedanalogs and prodrugs, thyroid hormone receptor ligands,muscarinic Ml agonists, sodium channel blockers, mixed-action ion channel blockers, beta adrenoceptorantagonist and PGF2 alpha agonist combinations,guanylate cyclase activators, nitrovasodilators,endothelin receptor modulators, ethacrynic acid, otherphenoxyacetic acid analogs, actin disrupters, calciumchannel blockers and neuroprotective agents.
49. 57. The use of claim 55, wherein the médicament furthercomprises one or more compounds selected from the groupconsisting of beta adrenoceptor antagonists, alpha-2adrenoceptor agonists, carbonic anhydrase inhibitors,cholinergic agonists and prostaglandin receptoragonists. A pharmaceutical composition comprising atherapeutically effective amount of the compound ofclaim 50 and a pharmaceutically acceptable carrier. 58 -317- 013295
50. 59. A packaged pharmaceutical composition for treating adisease associated with an A3 adenosine receptor in asubject, comprising: (a) a container holding a therapeutically effectiveamount of the compound of claim 50; and (b) instructions for using said compound for treatingsaid disease in a subject.
51. 60. A method of making a composition which comprises thecompound of claim 50, the method comprising admixingthe compound with a suitable carrier.
52. 61. A pharmaceutically acceptable sait of the compound of claim 29, 30 or 50, wherein the pharmaceutically acceptable sait contains an anion selected from thegroup consisting of maleic, fumaric, tartaric, acetate,phosphate and mesylate.
53. 62. The pharmaceutical composition of claim 58, furthercomprising one or more compounds selected from thegroup consisting of beta adrenoceptor antagonists,alpha-2 adrenoceptor agonists, carbonic anhydraseinhibitors, cholinergic agonists, prostaglandins andprostaglandin receptor agonists, angiotensin convertingenzyme (ACE) inhibitors, AMPA receptor antagonists, 5-HT agonists, angiogenesis inhibitors, NMDA antagonists,renin inhibitors, cannabinoid receptor agonists,angiotensin receptor antagonists, hydrochlorothiazide(HCTZ), somatostatin agonists, glucocorticoidantagonists, mast cell degranulation inhibitors, alpha-adrenergic receptor blockers, alpha-2 adrenoceptorantagonists, thromboxane A2 mimetics, protein kinaseinhibitors, prostaglandin F dérivatives, prostaglandin-2 alpha antagonists, dopamine DI and 5-HT2 agonists, -318- 013295 nitric-oxide-releasing agents, 5-HT 2 antagonists,cyclooxygenase inhibitors, inosine, dopamine D2receptor and alpha 2 adrenoceptor agonists, dopamine DIreceptor antagonist and D2 receptor agonists,vasopressin receptor antagonists, endothelinantagonists, l-(3-hydroxy-2- phosphonylmethoxypropyl)cytosine (HPMPC) and relatedanalogs and prodrugs, thyroid hormone receptor ligands,muscarinic Ml agonists, sodium channel blockers, mixed-action ion channel blockers, beta adrenoceptorantagonist and PGF2 alpha agonist combinations,guanylate cyclase activators, nitrovasodilators,endothelin receptor modulators, ethacrynic acid, otherphenoxyacetic acid analogs, actin disrupters, calciumchannel blockers and neuroprotective agents.
54. 63. The pharmaceutical composition of claim 58, furthercomprising one or more compounds selected from thegroup consisting of beta adrenoceptor antagonists,alpha-2 adrenoceptor agonists, carbonic anhydraseinhibitors, cholinergic agonists and prostaglandinreceptor agonists.
55. 64. Use of the compound of claim 50 for manufacturing amédicament useful for treating a disease associatedwith an A3 adenosine receptor in a subject in need ofsuch treatment, wherein the disease associated with theA3 adenosine receptor is asthma, bronchitis, chronicobstructive pulmonary disorder, or bronchoconstriction.
56. 65. A process of manufacturing the compound of claim 50,comprising the steps of: -319- 013295 (a) reacting the compound

    with histamine in the presence of dimethyl sulfoxide; (b) heating the reaction mixture under an inertatmosphère, (c) cooling the reaction mixture and separating theorganic layer from the aqueous layer; (d) washing the organic layer with brine, drying, andfiltering, to yield the compound.
57. 66. The process of claim 65, wherein in step (b) thereaction mixture is heated under a nitrogen atmosphère.
58. 67. The process of claim 65, wherein in step (d) the organiclayer is dried with MgSO4.
59. 68. Use of the compound of claim 1 for manufacturing amédicament useful for treating a disease associated withan Ai adenosine receptor in a subject in need of suchtreatment, wherein the disease is antidiuresis,bradycardia, bronchitis, bronchoconstriction,Alzheimer's disease, cardiac arrythmias, cardiachypoxia, hypertension, inflammation, négative cardiacinotropy and dromotropy, rénal failure, sédation or isassociated with transmitter release, respiratoryepithelia, contraction of smooth muscle underlying -320- 013295 respiratory epithelia, vasoconstriction or mast celldegranulation.
60. 69. Use of the compound of daim 1 for enhancing the memoryof a subject.
61. 70. Use of the compound of claim 29 or 30 for manufacturinga médicament useful for treating a disease associatedwith an A2a adenosine receptor in a subject in need ofsuch treatment, wherein the disease associated with theA2a adenosine receptor is Parkinson's disease orglaucoma.
62. 71. Use of the compound of claim 50 for manuf acturing amédicament useful for treating a disease associated withan A3 adenosine receptor in a subject in need of suchtreatment, wherein the disease associated with the A3adenosine receptor is myocardial ischemia, bronchitis,or bronchoconstriction.
63. 72. Use of the compound of claim 50 for manuf acturing amédicament useful for treating inflammation of the eyeassociated with an A3 adenosine receptor in a subject.
64. 73. Use of the compound of claim 50 for manuf acturing amédicament useful for treating a disease associated withan A3 adenosine receptor in a subject in need of suchtreatment, wherein the disease associated with the A3adenosine receptor is associated with mast celldegranulation.
65. 74. Use of the compound of claim 50 for manufacturing amédicament useful for treating a disease associated withan A3 adenosine receptor in a subject in need of suchtreatment, wherein the disease associated with the A3adenosine receptor is asthma, glaucoma, retinopathy,ocular ischemia, or macular degeneration.