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Definitions

• Adenosine is an ubiquitous modulator of numerous physiological activities, particularly within the cardiovascular and nervous systems. The effects of adenosine appear to be mediated by specific cell surface receptor proteins. Adenosine modulates diverse physiological functions including induction of sedation, vasodilation, suppression of cardiac rate and contractility, inhibition of platelet aggregability, stimulation of gluconeogenesis and inhibition of lipolysis. In addition to its effects on adenylate cyclase, adenosine has been shown to open potassium channels, reduce flux through calcium channels, and inhibit or stimulate phosphoinositide turnover through receptor- mediated mechanisms (See for example, C.E. Muller and B.
• Two receptor subtypes exhibit affinity for adenosine in the nanomolar range while two other known subtypes A 2b and A 3 are low-affinity receptors, with affinity for adenosine in the low-micromolar range.
• a x and A 3 adenosine receptor activation can lead to an inhibition of adenylate cyclase activity, while A 2a and A 25 activation causes a stimulation of adenylate cyclase.
• a few Ai antagonists have been developed for the treatment of cognitive disease, renal failure, and cardiac arrhythmias. It has been suggested that A 2a antagonists may be beneficial for patients suffering from Morbus Parkinson (Parkinson's disease) . Particularly in view of the potential for local delivery, adenosine receptor antagonists may be valuable for treatment of allergic inflammation and asthma. Available information (for example, Nyce & Metzger "DNA antisense Therapy for Asthma in an Animal Model" Na ture (1997) 385:
• Aj antagonists may block contraction of smooth muscle underlying respiratory epithelia, while A 2J -, or A 3 receptor antagonists may block mast cell degranulation, mitigating the release of histamine and other inflammatory mediators.
• a 2b receptors have been discovered throughout the gastrointestinal tract, especially in the colon and the intestinal epithelia. It has been suggested that A 2b receptors mediate cAMP response
• Adenosine receptors have also been shown to exist on the retinas of various mammalian species including bovine, porcine, monkey, rat, guinea pig, mouse, rabbit and human (See, Blazynski et al . , Discrete Distributions of Adenosine .Receptors in Mammalian .Retina, Journal of Neurochemis try, volume 54, pages 648-655 (1990); Woods et al . ,
• the present invention is based on compounds which selectivelv bind to adenosine A : receptor, thereby treating a disease associated with Ai adenosine receptor in a subject by administering to the subject a therapeutically effective amount of such compounds.
• the disease to be Treated are associated with cognitive disease, renal failure, cardiac arrhythmias, respiratory epithelia, transmitter release, sedation, vasoconstriction, bradycardia, negative cardiac inotropy and dromotropy, branchoconstriction, neutropil chemotaxis, reflux condition, or ulcerative condition.
• N-6 substituted 7-deazapurines include those which have an acetamide, carboxamide, substituted cyclohexyl, e . g. , cyclohexanol, or a urea moiety attached to the N-6 nitrogen through an alkylene chain.
• the present invention pertains to methods for modulating an adenosine receptor (s) in a mammal by administering to the mammal a therapeutically effective amount of a N-6 substituted 7-deazapurine, such that modulation of the adenosine receptor's activity occurs.
• Suitable adenosine receptors include the families of A 1# A 2 , or A 3>
• the N-6 substituted 7-deazapurine is a adenosine receptor antagonist .
• the invention further pertains to methods for treating N-6 substituted 7-deazapurine disorders, e . g.
• N-6 substituted 7 deazapurines include those illustrated by the general formula I :
• R x and R 2 are each independently a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety or together form a substituted or unsubstituted heterocyclic ring.
• R 3 is a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety.
• R 4 is a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety.
• R5 and R 6 are each independently a halogen atom, e . g.
• R 5 is carboxyl, esters of carboxyl, or carboxamides, or R 4 and R 5 or R 5 and R 6 together form a substituted or unsubstituted heterocyclic or carbocyclic ring.
• 1 ⁇ and R 2 can each independently be a substituted or unsubstituted cycloalkyl or heteroarylalkyl moieties.
• R 3 is a hydrogen atom or a substituted or unsubstituted heteroaryl moiety.
• R ⁇ s a hydrogen atom
• R 2 is a cyclohexanol, e . g .
• R 3 is phenyl
• R ⁇ is a ' hydrogen atom
• Rg is a methyl group.
• R is a hydrogen atom
• R 2 is
• R 3 is phenyl
• R 4 is a hydrogen atom
• R 5 and R are methyl groups .
• the invention further pertains to pharmaceutical compositions for treating a N-6 substituted 7-deazapurine responsive state in a mammal, e. g. , asthma, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease, renal disorders, gastrointestinal disorders, and eye disorders.
• the pharmaceutical composition includes a therapeutically effective amount of a N-6 substituted 7-deazapurine and a pharmaceutically acceptable carrier.
• the present invention also pertains to packaged pharmaceutical compositions for treating a N-6 substituted 7- deazapurine responsive state in a mammal.
• the packaged pharmaceutical composition includes a container holding a therapeutically effective amount of at least one N-6 substituted 7-deazapurine and instructions for using the N-6 substituted 7-deazapurine for treating a N-6 substituted 7- deazapurine responsive state in a mammal .
• the invention further pertains to compounds of formula 1 wherein
• R ] _ is hydrogen;
• R 2 is substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkyl, or R 1 and R 2 together form a substituted or unsubstituted heterocyclic ring;
• R 3 is unsubstituted or substituted aryl
• R 4 is hydrogen
• R 5 and R 6 are each independently hydrogen or alkyl, and pharmaceutically acceptable salts thereof.
• the deazapurines of this embodiment may advantageously be selective A- receptor antagonists. These compounds may be useful for numerous therapeutic uses such as, for example, the treatment of asthma, kidney failure associated with heart failure, and glaucoma.
• the deazapurine is a water soluble prodrug that is capable of being metabolized in vivo to an active drug by, for example, esterase catalyzed hydrolysis.
• the invention features a method for inhibiting the activity of an adenosine receptor (e.g., adenosine receptor),
• N-6 substituted 7- deazapurine e.g., preferably, an adenosine receptor antagonist
• the invention features a method for treating damage to the eye of an animal (e.g., a human) by administering to the animal an effective amount of an N-6 substituted 7-deazapurine of formula I.
• the N-6 substituted 7-deazapurine is an antagonist of A 3 adenosine receptors in cells of the animal.
• the damage is to the retina or the optic nerve head and may be acute or chronic.
• the damage may be the result of, for example, glaucoma, edema, ischemia, hypoxia or trauma.
• the invention also features a pharmaceutical composition comprising a N-6 substituted compound of formula I.
• the pharmaceutical preparation is an ophthalmic formulation (e . g. , an periocular, retrobulbar or intraocular injection formulation, a systemic formulation, or a surgical irrigating solution) .
• the invention features a compound having the formula II:
• R ⁇ and R 2 are each independently hydrogen, or substituted or unsubstituted alkoxy, aminoalkyl, alkyl, aryl, or alkylaryl, or together form a substituted or unsubstituted heterocyclic ring, provided that both R ⁇ and R 2 are both not hydrogen;
• R 3 is substituted or unsubstituted alkyl, arylalkyl, or aryl;
• R 4 is hydrogen or substituted or unsubstituted C x - C 6 alkyl;
• L is hydrogen, substituted or unsubstituted alkyl, or R 4 and L together form a substituted or unsubstituted heterocyclic or carbocyclic ring;
• R 6 is hydrogen, substituted or unsubstituted alkyl, or halogen;
• Q is CH 2 , O, S, or NR 7 , wherein R 7 is hydrogen or substituted or unsubstituted C 1 - C 6 alkyl
• X is CR 6 and Q is CK 1( C, S, or NK in formula II, wherein R f is as defined above.
• X is N.
• the invention further pertains to a method for inhibiting the activity of an adenosine receptor (e.g., an A 2b adenosine receptor) in a cell by contacting the cell with a compound of the invention.
• the compound is an antagonist of the receptor.
• the invention also pertains to a method for treating a gastrointestinal disorder (e.g., diarrhea) or a respiratory disorder (e.g., allergic rhinitis, chronic obstructive pulmonary disease) in an animal by administering to an animal an effective amount of a compound of formula II (e.g., an antagonist of A ;c ) .
• a gastrointestinal disorder e.g., diarrhea
• a respiratory disorder e.g., allergic rhinitis, chronic obstructive pulmonary disease
• a compound of formula II e.g., an antagonist of A ;c
• the animal is a human.
• This invention also features a compound having the structure:
• R is trans-4 -hydroxy cyclohexyl, 2-methylamino carbonylamino cyclohexyl, 2-methylamino carbonylamino cyclohexyl, acetamido ethyl, or methylamino carbonylamino ethyl; wherein Ar is a substituted or unsubstituted four to s x membered ring.
• Ar is phenyl, pyrrole, thiophene, furan, thiazole, imidazole, pyrazcle, 1,2,4- triazole, pyridine, 2 (IH) -pyridone, 4 (IK) -pyridone , pyrazme, pyrimidine, pyridazine, isothiazole, isoxazole, oxazole, tetrazole, naphthalene, tetralin, naphthyridine, benzofuran, benzothiophene, indole, 2, 3-dihydroindole, IK-indole, indoline, benzopyrazole, 1, 3-benzodioxole, benzoxazoie, purine, coumarin, chromone, quinoline, tetrahydroquinoline, isoguinoline, benzimidazole, quinazoline, pyridine
• Y is carbon or nitrogen
• R2 and R: ' are independently H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, halogen, methoxy, methyl amino, or methyl thio;
• R3 is H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, wherein said substituted alkyl is -C(R ⁇ ) (Rs)XRs.
• X is 0, S, or NRe
• R? and Re are each independently H or alkyl, wherein Rs and Rt are each independently alkyl or cycloalkyl, or Rs, Rt and the nitrogen together form a substituted or unsubstituted ring of between 4 and 7 members; 11
• R*. is H, alkyl, substituted alkyl, cycloalkyl. or a pharmaceutically acceptable salt, or a prcdruc derivative, or a biologically active metabolite; w t: the proviso that when R: is acetylamino ethyl, Ar is no: 4-pyridyl .
• This invention also pertains to a compound having the structure :
• R is aryl, substituted aryl, or heteroaryl
• R is H, alkyl, substituted alkyl, or cycloalkyl; wherein R.- is H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, wherein said substituted alkyl is -C(R ⁇ ) wherein Rt and Ri are each H or alkyl, wherein Ri and Rs are each alkyl or cycloalkyl, or R * R: and the nitrogen together form a ring system of between 4 and 7 members .
• This invention also features a method for inhibiting the activity of an h- ⁇ adenosine receptor in a cell, which comprises contacting said cell with the above-mentioned compounds .
• the present invention pertains to methods for treating a N-6 substituted 7-deazapurine responsive state in a mammal.
• the methods include administration of a therapeutically effective amount 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.
• N-6 substituted 7-deazapurine responsive state is intended to include a disease state or condition characterized by its responsiveness to treatment with a N-6 substituted 7-deazapurine of the invention as described infra , e . g . , the treatment includes a significant diminishment of at least one symptom or effect of the state achieved with a N-6 substituted 7-deazapurine of the invention.
• adenosine typically such states are associated with an increase of adenosine within a host such that the host often experiences physiological symptoms which include, but are not limited to, release of toxins, inflammation, coma, water retention, weight gain or weight loss, pancreatitis, emphysema, rheumatoid arthritis, osteoarthritis, multiple organ failure, infant and adult respiratory distress syndrome, allergic rhinitis, chronic obstructive pulmonary disease, eye disorders, gastrointestinal disorders, skin tumor ' promotion, immunodeficiency and asthma.
• physiological symptoms include, but are not limited to, release of toxins, inflammation, coma, water retention, weight gain or weight loss, pancreatitis, emphysema, rheumatoid arthritis, osteoarthritis, multiple organ failure, infant and adult respiratory distress syndrome, allergic rhinitis, chronic obstructive pulmonary disease, eye disorders, gastrointestinal disorders, skin tumor ' promotion, immunodeficiency and asthma.
• a N-6 substituted 7-deazapurine responsive state includes those disease states which are mediated by stimulation of adenosine receptors, e.g., A ! , A 2 a . A? b , A- , etc., such that calcium concentrations in cells and/or activation of PLC (phospholipase C) is modulated.
• a N-6 substituted 7-deazapurine responsive state is associated with adenosine receptor (s), e . g.
• the N-6 substituted 7-deazapurine acts as an antagonist.
• suitable responsive states which can be treated by the compounds of the invention e.g., adenosine receptor subtypes which mediate biological effects, include central nervous system (CNS) effects, cardiovascular effects, renal effects, respiratory effects, immunological effects, gastro-intestinal effects and metabolic effects.
• CNS central nervous system
• the relative amount of adenosine in a subject can be associated with the effects listed below; that is increased levels of adenosine can trigger an effect, e.g., an undesired physiological response, e.g., an asthmatic attack.
• CNS effects include decreased transmitter release (A x ) , sedation IA 1 ) , decreased locomotor activity (A 2a ) , anticonvulsant activity, chemoreceptor stimulation (A 2 ) and hyperalgesia.
• Therapeutic applications of the inventive compounds include treatment of dementia, Alzheimer's disease and memory enhancement .
• Cardiovascular effects include vasodilation (A 2a ) , (A 2b ) and
• a 3 vasoconstriction (A x ) , bradycardia (A x ) , platelet inhibition (A 2a ) , negative cardiac inotropy and dromotropy
• Therapeutic applications of the inventive compounds include, for example, prevention of ischaemia-induced impairment of the heart and cardiotonics, myocardial tissue protection and restoration of cardiac function.
• Renal effects include decreased GFR (A x ) , mesang al cell contraction (AX , antidiuresis .A 1 ) and inhibition of rerun release (A : ) .
• Suitable therapeutic applications of the inventive compounds include use of the inventive compounds as diuretic, natriuretic, potassium-sparing, kidney- protective/prevention of acute renal failure, antihypertensive, anti-oedematous and anti-nephritic agents.
• Respiratory effects include bronchodilation (A 2 ), bronchoconstriction (A : ) , chronic obstructive pulmonary disease, allergic rhinitis, mucus secretion and respiratory depression (A 2 ) .
• Suitable therapeutic applications for the compounds of the invention include anti -asthmatic applications, treatment of lung disease after transplantation and respiratory disorders.
• Immunological effects include immunosuppression (A 2 ) , neutrophil chemotaxis (A x ) , neutrophil superoxide generation
• Therapeutic applications of antagonists include allergic and non allergic inflammation, e.g., release of histamine and other inflammatory mediators.
• Gastrointestinal effects include inhibition of acid secretion (A : ) therapeutic application may include reflux and ulcerative conditions Gastrointestinal effects also include colonic, intestinal and diarrheal disease, e.g., diarrheal disease associated with intestinal inflammation (A 2b ) .
• Eye disorders include retinal and optic nerve head injury and trauma related disorders (A 3 ) .
• the eye disorder is glaucoma.
• Other therapeutic applications of the compounds cf the invention include treatment of obesity (lipclyt r properties), hypertension, treatment cf depression, sedative, anxiolytic, as antileptics and as laxatives, e.g., effecting motility without causing diarrhea.
• disease state is intended to include tnose conditions caused by or associated with unwanted levels of adenosine, adenylyl cyclase activity, increased physiological activity associated with aberrant stimulation of adenosine receptors and/or an increase in cAMP.
• the disease state is, for example, asthma, chronic obstructive pulmonary disease, allergic rhinitis, bronchitis, renal disorders, gastrointestinal disorders, or eye disorders. Additional examples include chronic bronchitis and cystic fibrosis.
• Suitable examples of inflammatory diseases include non-lymphocytic leukemia, myocardial ischaemia, angina, infarction, cerebrovascular ischaemia, intermittent claudication, critical limb ischemia, venous hypertension, varicose veins, venous ulceration and arteriosclerosis.
• Impaired reperfusion states include, for example, any post- surgical trauma, such as reconstructive surgery, thrombolysis or angioplasty.
• treatment of a N-6 substituted 7-deazapurine responsive state or “treating a N-6 substituted 7- deazapurine responsive state” is intended to include changes in a disease state or condition, as described above, such that physiological symptoms in a mammal can be significantly diminished or minimized.
• the language also includes control, prevention or inhibition of physiological symptoms or effects associated with an aberrant amount of adenosine.
• the control of the disease state or condition is such that the disease state or condition is eradicated.
• the control is selective such that aberrant levels of adenosine receptor activity are controlled while other physiologic systems and parameters are unaffected.
• N-6 substituted 7-deazapurine is art reco and is intended to include those compounds having zhe f I:
• N-substituted 7-deazapurine includes pharmaceutically acceptable salts thereof, and, in one embodiment, also includes certain N-6 substituted purines described herein.
• the N-6 substituted 7-deazapurine is not N-6 benzyl or N-6 phenylethyl substituted.
• R 4 is not benzyl or phenylethyl substituted.
• R- and R 2 are both not hydrogen atoms.
• R 3 is not a hydrogen atom.
• terapéuticaally effective amount of an N-6 substituted 7-deazapurine is that amount of a therapeutic compound necessary or sufficient to perform its intended function within a mammal, e.g., treat a N-6 substituted 7-deazapurine responsive state, or a disease state in a mammal.
• An effective amount of the therapeutic compound can vary according to factors such as the amount of the causative agent already present in the mammal, the age, sex, and weight of the mammal, and the ability of the therapeutic compounds of the present invention to affect a N- 6 substituted 7-deazapurine responsive state in the mammal.
• an in vi zro or in vi vc assay also can be used to determine an "effective amount" of the therapeutic compounds described infra .
• the ordinarily skilled artisan would select an appropriate amount of the therapeutic compound for use in the aforementioned assay or as a theraDeutic treatment .
• a therapeutically effective amount preferably diminishes at least one symptom or effect associated with the N-6 substituted 7-deazapurine responsive state or condition being treated by at least about 20%, (more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80%) relative to untreated subjects.
• Assays can be designed by one skilled in the art to measure the diminishment of such symptoms and/or effects. Any art recognized assay capable of measuring such parameters are intended to be included as part of this invention. For example, if asthma is the state being treated, then the volume of air expended from the lungs of a subject can be measured before and after treatment for measurement of increase in the volume using an art recognized technique. Likewise, if inflammation is the state being treated, then the area which is inflamed can be measured before and after treatment for measurement of diminishment in the area inflamed using an art recognized technique.
• cell includes both prokaryotic and eukaryotic cells.
• animal includes any organism with adenosine receptors or any organism susceptible to a N-6-substituted 7- deazapurine responsive state. Examples of animals include yeast, mammals, reptiles, and birds. It also includes transgenic animals .
• mammal is art recognized and is intended to include 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-6 substituted 7-deazapurine responsive state, inflamma ion, emphysema, asthma, central nervous system conditions, or acute respiratory distress syndrome, for example, are included as part of this invention.
• the present invention pertains to methods for modulating an adenosine receptor (s) in a mammal by administering to the mammal a therapeutically effective amount of a N-6 substituted 7-deazapurine, such that modulation of the adenosine receptor in the mammal occurs.
• adenosine receptors include the families of A 1# A 2 , or A 3#
• the N-6 substituted 7- deazapurine is an adenosine receptor antagonist.
• modulating an adenosine receptor is intended to include those instances where a compound interacts with an adenosine receptor (s), causing increased, decreased or abnormal physiological activity associated with an adenosine receptor or subsequent cascade effects resulting from the modulation of the adenosine receptor.
• Physiological activities associated with adenosine receptors include induction of sedation, vasodilation, suppression of cardiac rate and contractility, inhibition of platelet aggregbility, stimulation of gluconeogenesis, inhibition of lipolysis, opening of potassium channels, reducing flux of calcium channels, etc.
• modulate is intended to include preventing, eradicating, or inhibiting the resulting increase of undesired physiological activity associated with abnormal stimulation of an adenosine receptor, e.g., in the context of the therapeutic methods of the invention.
• the term modulate includes antagonistic effects, e . g. , diminishment cf the activity or production of mediators of allergy and allergic inflammation which results from the overstimulatio cf adenosine receptor(s).
• the therapeutic deazapurines of the invention can interact with ar. adenosine receptor to inhibit, for example, adenylate cyclase activity.
• condition characterized by aberrant adenosine receptor activity is intended to include those diseases, disorders or conditions which are associated with aberrant stimulation of an adenosine receptor, in that the stimulation of the receptor causes a biochemical and or physiological chain of events that is directly or indirectly associated with the disease, disorder or condition.
• This stimulation of an adenosine receptor does not have to be the sole causative agent of the disease, disorder or condition but merely be responsible for causing some of the symptoms typically associated with the disease, disorder, or condition being treated.
• the aberrant stimulation of the receptor can be the sole factor or at least one other agent can be involved in the state being treated.
• Examples of conditions include those disease states listed supra , including inflammation, gastrointestinal disorders and those symptoms manifested by the presence of increased adenosine receptor activity.
• Preferred examples include those symptoms associated with asthma, allergic rhinitis, chronic obstructive pulmonary disease, emphysema, bronchitis, gastrointestinal disorders and glaucoma.
• treating or treatment of a condition characterized by aberrant adenosine receptor activity is intended to include the alleviation of or diminishment of at least one symptom typically associated with the condition.
• the treatment also includes alleviation or diminishment of more than one symptom.
• the treatment cures, e.g., substantially eliminates, the symptoms associated with the condition.
• the present invention pertains to compounds, N-6 subs: .t ted 7-deazapurines, having the formula I:
• R j ⁇ and R 2 are each independently a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety or together form a substituted or unsubstituted heterocyclic ring;
• R 3 is a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety;
• R 4 is a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety.
• R 5 and R 6 are each independently a halogen atom, e.g., chlorine, fluorine, or bromine, a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety or R 4 and R 5 or R 5 and R 6 together form a substituted or unsubstituted heterocyclic or carbocyclic ring. Also included, are pharmaceutically acceptable salts of the N-6 substituted 7-deazapurines.
• R j and R 2 can each independently be a substituted or unsubstituted cycloalkyl or heteroarylalkyl moieties.
• R 3 is a hydrogen atom or a substituted or unsubstituted heteroaryl moiety.
• R 4 , R 5 and R 6 can each be independently a heteroaryl moiety.
• R is a hydrogen atom
• R 2 is a substituted or unsubstituted cyclohexane, cyclopentyl, cyclobutyl or cyclopropane moiety
• R 3 is a substituted or unsubstituted phenyl moiety
• R 2 is ' a cyclohexanol. a cyclohexanediol, a cyclohexylsulfonamide, a cycichexanam de , a cyclohexylester, a cyclohexene, a cyclopentanol or a cyclopentanediol and R 3 is a phenyl moiety.
• R j is a hydrogen atom
• R 2 is a cyclohexanol
• R 3 is a substituted or unsubstituted phenyl, pyridine, furan, cyclopentane, or thiophene moiety
• R 4 is a hydrogen atom, a substituted alkyl, aryl or arylalkyl moiety
• R5 and R 6 are each independently a hydrogen atom, or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety.
• R- is a hydrogen atom
• R is substituted or unsubstituted alkylamine, arylamine, or aikylarylamine, a substituted or unsubstituted alkylamide, arylamide or alkylarylamide, a substituted or unsubstituted alkylsulfonamide, arylsulfonamide or alkylarylsulfonamide, a substituted or unsubstituted alkylurea, arylurea or alkylarylurea, a substituted or unsubstituted alkylcarbamate, arylcarbamate or alkylarylcarbamate, a substituted or unsubstituted alkylcarboxylic acid, arylcarboxylic acid or alkylarylcarboxylic acid, is substituted or unsubstituted phenyl moiety
• R 4 is a hydrogen atom and R 5 and R 6 are methyl groups .
• R 2 is guanidine, a modified guanidine, cyanoguanidine, a thiourea, a thioamide or an amidine .
• R can be any organic radical
• R a -R c are each independently a hydrogen atom or a saturated or unsaturated alkyl, aryl or alkylaryl moiety and R 2d is a hydrogen atom or a saturated or unsaturated alkyl, aryl, or alkylaryl moiety, NR e R 2f , or OR ⁇ , wherein R? e " R 2c are each independently a hydrogen atom or a saturated or unsaturated alkyl, aryl or alkylaryl moieties.
• R 2a and R 2t) together can form a carbocyclic or heterocyclic ring having a ring size between about 3 and 6 members, e . g. , cyclopropyl, cyclopentyl, cyclohexyl groups.
• both R 5 and R 6 are not methyl groups, preferably, one of R 5 and R 6 is an alkyl group, e.g., a methyl group, and the other is a hydrogen atom.
• heterocyclic rings include between 4 and 12 carbon atoms, preferably between 5 and 7 carbon atoms, and car. be either aromatic or aliphatic.
• the heterocyclic ring car be further substituted, including substitution of one or more carbon atoms of the ring structure with one or more heteroatoms .
• Preferred amino acid side chains include those of glycine, alanine, valine, leucine, isoleucine, lysine, ⁇ - methylalanine, aminocyclopropane carboxylic acid, azetidine- 2-carboxylic acid, ⁇ -alanine, ⁇ -aminobutyric acid, alanine- alanine, or glycine-alanine .
• R 3 may be substituted or unsubstituted heteroaryl, preferably when R 5 and Rg are each alkyl.
• heteroaryl groups include pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, pyrrolyl, triazolyl, thioazolyl, oxazolyl, oxadiazolyl, furanyl, methylenedioxyphenyl and thiophenyl.
• R 3 is 2-pyridyl, 3-pyridyl, 4- pyridyl, 2-pyrimidyl or 3- pyrimidyl.
• R 5 and Rg are each hydroge . In another, R 5 and R 6 are each methyl.
• the deazapurine is 4- (trans-4- hydroxycyclohexyl) amino-2- (3-fluorophenyl) - 7H-pyrrolo [2 , 3d] pyrimidine .
• R 1 is hydrogen
• R : is substituted or unsubstituted C : -C ⁇ alkyl
• R 3 is substituted or unsubstituted phenyl
• R 4 is hydrogen
• L is hydrogen or substituted or unsubstituted C l -C 6 alkyl
• Q is 0, S or NR 7
• R is hydrogen or substituted or unsubstituted Ci-C 6 alkyl
• W is substituted or unsubstituted aryl.
• A may be CH 2 CH 2 .
• the deazapurine is 4- (2-acetylaminoethyl) amino-6- (4-chlorophenoxy)methyl-2- phenyl- 7H-pyrrolo [2 , 3d] pyrimidine .
• the deazapurine is 4- (2-acetylaminoethyl) amino-6- (N-phenylamino) methyl -2 -phenyl- 7H- yrrolo [2 , 3d] pyrimidine .
• the invention further pertains to a method for inhibiting the activity of an adenosine receptor (e.g., an ⁇ adenosine receptor) in a cell by contacting the cell with a compound cf the invention.
• an adenosine receptor e.g., an ⁇ adenosine receptor
• the compound is an antagonist of the receptor.
• alkyl refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
• alkyl further includes alkyl groups, which can further include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen, sulfur or phosphorous atoms.
• alkylaryl is an alkyl substituted with an aryl ( e . g. , phenylmethyl (benzyl)).
• alkyl also includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyis described above, but that contain at least one double or triple bond respectively.
• aryl refers to the radical of aryl groups, including 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, benzoxazole, benzothiazole, triazole, tetrazole, pyrazole, pyridine, pyrazme, pyridazine and pyrimidine, and tne l ⁇ e.
• Aryl groups also include polycyclic fused aromatic groups such as naphthyl, qumolyl, mdolyl, and the like.
• aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles” , “heteroaryls” or “heteroaromatics” .
• the aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl , alkoxycarbonyl, ammocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, ammo (including alkyl ammo, dialkylammo, arylamino, diarylam o, and alkylarylammo) , acylam o (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and
• Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, 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, sulf
• heteroato as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.
• the invention further pertains to pharmaceutical compositions for treating a N-6 substituted 7-deazapurine responsive state in a mammal, e.g., respiratory disorders (e.g., asthma, bronchitis, chronic obstructive pulmonary disorder, and allergic rhinitis) , renal disorders, gastrointestinal disorders, and eye disorders.
• anticancer agent is art recognized and is intended to include those agents which diminish, eradicate, or prevent growth of cancer cells without, preferably, adversely affecting other physiological functions.
• Representative examples include cisplatin and cyclophosphamide.
• the compounds of the present invention are administered as pharmaceuticals, to humans and mammals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
• pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound (s) of the present invention within or to the subject such that it can performs its intended function. Typically, such compounds are carried or transported from one organ, or portion cf the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of- being compatible with the other ingredients of the formulation and not injurious tc the patient.
• materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose,- starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes,- oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar,- buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;
• the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases.
• pharmaceutically acceptable salts refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in si tu during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine.
• esters refers to the relatively non-toxic, esterified products of the compounds of the present invention. These esters can be prepared in si tu during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent.
• Carboxylic acids can be converted into esters via treatment with an alcohol in the presence of a catalyst.
• Hydroxyl containing derivatives can be converted into esters via treatment with an esterifying agent such as alkanoyl halides.
• the term is further intended to include lower hydrocarbon groups capable of being solvated under physiological conditions, e.g., alkyl esters, methyl, ethyl and propyl esters. (See, for example, Berge et al . , supra . )
• the invention further contemplates the use of prodrugs which are converted in vivo to the therapeutic compounds of tne invention (see, e.g., R.B. Silverman, 1992, “The Organic Chemistry of Drug Design and Drug Action", Academic Press, Chapter 8) .
• prodrugs can be used to alter the biodistribution (e.g., to allow compounds which would not typically enter the reactive site of the protease) or the pharmacokinetics of the therapeutic compound.
• a carboxylic acid group can be esterified, e.g., with a methyl group or an ethyl group to yield an ester.
• the ester When the ester is administered to a subject, the ester is cleaved, enzymatically or non-enzymatically, reductively or hydrolytically, to reveal the anionic group.
• An anionic group can be esterified with moieties (e.g., acyloxymethyl esters) which are cleaved to reveal an intermediate compound which subsequently decomposes to yield the active compound.
• the prodrug is a reduced form of a sulfate or sulfonate, e.g., a thiol, which is oxidized in vivo to the therapeutic compound.
• an anionic moiety can be esterified to a group which is actively transported in vivo, or which is selectively taken up by target organs.
• the ester can be selected to allow specific targeting of the therapeutic moieties to particular reactive sites, as described below for carrier moieties.
• antioxidants examples include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabis lfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (3KA. , butylated hydroxytoluene (BHT) , lecithin, propyl gallate, alpha- tocopherol, and the like,- and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA) , sorbitol, tartaric acid, phosphoric acid, and the like.
• water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabis lfite, sodium sulfite and the like
• oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (3KA. , butylated hydroxytoluen
• Formulations of the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublmgual, rectal, vaginal and/or parenteral administration.
• the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
• the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
• Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients.
• the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
• Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth) , powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-cil liquid emulsion, or as an elixir or syrup, or as pastilles .
• an inert base such as gelatin and glycerin, cr sucrose and acacia
• a compound of the present invention may also be administered as a bolus, electuary or paste.
• the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: 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 glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, al ' ginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and gly
• compositions may also comprise buffering agents.
• Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
• a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
• Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, , m ⁇ rn 02/057267
• Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
• the tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical - formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
• Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
• suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
• Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
• the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required .
• Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
• Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
• Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body.
• dosage forms can be made by dissolving or dispersing the compound in the proper medium.
• Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active compound in a polymer matrix or gel.
• An appropriate buffer system e.g., sodium phosphate, sodium acetate or sodium borate
• sodium phosphate, sodium acetate or sodium borate may be added to prevent pH drift under storage conditions.
• Ophthalmic products are typically packaged in multidose form. Preservatives are thus required to prevent microbial contamination during use. Suitable preservatives include: benzalkonium chloride, thi erosal, chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium, sorbic acid, polyquaternium-1 , or other agents known to those skilled in the art. Such preservatives are typically employed at a level of from 0.001 to 1.0% weight/volume ("% w/v") .
• BSS ® Sterile Irrigating Solution and BSS Plus ® Sterile Intraocular Irrigating Solution are examples of physiologically balanced intraocular irrigating solutions.
• the latter type of solution is described in U.S. Pat. No. 4,550,022 (Garabedian, e ⁇ al . ) , the entire contents of which are hereby incorporated in the present specification by reference.
• Retrobulbar and periocular injections are known to those skilled in the art and are described in numerous publications including, for example, Ophthalmic Surgery: Principles of Practi ce, Ed . , G. L. Spaeth. W. B. Sanders Co., Philadelphia, Pa., U.S.A., pages 85-87 (1990) .
• Ophthalmic conditions which may be treated include, but are not limited to, retinopathies, macular degeneration, ocular ischemia, glaucoma, and damage associated with injuries to ophthalmic tissues, such as ischemia reperfusion injuries, photochemical injuries, and injuries associated with ocular surgery, particularly injuries to the retina or optic nerve head by exposure to light or surgical instruments.
• the compounds may also be used as an adjunct to ophthalmic surgery, such as by vitreal or subconjunctival injection following ophthalmic surgery.
• the compounds may be used for acute treatment of temporary conditions, or may be administered chronically, especially in the case of degenerative disease.
• the compounds may also be used prophylactically, especially prior to ocular surgery or noninvasive ophthalmic procedures, or other types of surgery.
• compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions' or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
• aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) , and suitable mixtures thereof, vegetable oils, such as ciive oil, and injectable organic esters, such as ethyl oleate .
• polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
• vegetable oils such as ciive oil
• injectable organic esters such as ethyl oleate .
• Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
• compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
• adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
• Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride
• the absorption of the drug in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
• Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides) . Depot injectable formulations are also prepared by entrapping the drug m liposomes or microemulsions which are compatible with body tissue.
• the preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administration is preferred.
• systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
• These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
• any suitable route of administration including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
• the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art .
• a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of • the pharmaceutical composition required
• the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
• a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
• intravenous and subcutaneous doses of the compounds of this invention for a patient when used for the indicated analgesic effects, will range from about 0.0001 to about 200 mg per kilogram of body weight per day, more preferably from about 0.01 to about 150 mg per kg per day, and still more preferably from about 0.2 to about 140 mg per kg per day.
• the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
• the present invention also pertains to packaged pharmaceutical compositions for treating a N-6 substituted 7 deazapurine responsive state, e.g., undesirable increased adenosine receptor activity in a mammal .
• the packaged pharmaceutical compositions include a container holding a therapeutically effective amount of at least one deazapurine as described supra and instructions for using the deazapurine for treating the deazapurine responsive state in the mammal.
• R is trans-4-hydroxy cyclohexyl, 2-methylamino carbonylamino cyclohexyl, acetylamino ethyl, or methylamino carbonylamino ethyl ;
• Ar is a substituted or unsubstituted four to six membered ring, phenyl, pyrrole, thiophene, furan, thiazole, imidazole, pyrazole, 1, 2, 4-triazole, pyridine, 2 (IH) -pyridone, 4 (IH) -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 (IH) -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 a structure :
• R: and R: ' are independently H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, halogen, methoxy, methyl amino, or methyl thio; wherein R 3 is H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, wherein said substituted alkyl is -C(R?) (R ⁇ )XR;, wherein X is 0, S, or NR * , wherein R- and Ri are each independently H or alkyl, wherein R ⁇ and Re are each independently alkyl or cycloalkyl, or NRsRe is a substituted or unsubstituted ring of between 4 and 7 members ,-
• NRsRe is a substituted or unsubstituted ring of between 4 and 7 members which is selected from the group consisting of:
• Ar has the structure :
• Y is carbon or nitrogen; wherein R: is H, or halogen, -O-alkyl group, amine group, or sulfide group;
• R 3 is H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, wherein said substituted alkyl is -C(R->) (Re)NRsRe, wherein Ri and R ⁇ are each independently H or alkyl, wherein Rs and R ⁇ are each independently alkyl or cycloalkyl, or Rs, Rb and the nitrogen together form a substituted or unsubstituted ring of between 4 and 7 members.
• Y is carbon
• R is hydrogen
• R* is hydrogen
• R3 and R4 are each methyl .
• R3 is -C(R-) (Re)NRsRe, wherein R? and R ⁇ are each independently H or alkyl, wherein Rs and Re are each independently alkyl or cycloalkyl, or R , R and the nitrogen together form a substituted or unsubstituted ring of between 4 and 7 members .
• Rz is halogen
• Y is nitrogen
• R is hydrogen
• Rs and R ⁇ are each hydroge .
• This invention also provides a compound having the structure
• R is aryl, substituted aryl, or heteroaryl
• Rz is H, alkyl, substituted alkyl, or cycloalkyl; wherein R 3 is H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, wherein said substituted alkyl is -C(Re) (RONR ⁇ Rs, wherein Re and R7 are each H or alkyl, wherein R « and Rs are each alkyl or cycloalkyl, or R ⁇ Rs and the nitrogen together form a ring system of between 4 and 7 members .
• the compound has the following structure:
• the compound has the structure :
• the compound has the structure:
• the compound has the structure :
• the compound has the structure:
• R 2 is a 5-6 membered aromatic ring; wherein R 3 and R ⁇ are independently H, or alkyl.
• the compound has tne structure :
• the compound has the structure :
• the compound has structure :
• the compound has the structure:
• the compound has the structure :
• This invention also provides a compound having the structure .-
• R 2 is a 5-6 membered aromatic ring; wherein R ; , and R ⁇ are independently H, or alkyl; alkyl; with the proviso that R 2 is not 4 -pyridyl.
• the compound has the structure:
• R 2 is a substituted 5-6 membered aromatic ring; wherein R : . and R, are independently H, or alkyl.
• the compound has the structure :
• R 2 is a 5-6 membered aromatic ring; wherein X is oxygen, or sulfur.
• the compound has the structure :
• R z is a 5-6 membered aromatic ring; wherein X is oxygen, or sulfur.
• the compound has the structure :
• This invention further provides a method for treating a disease associated with Az. adenosine receptor in a subject, comprising administering to the subject a therapeutically effective amount of a compound having. the formula IV, V, VI, VII, VIII, IX, or X.
• the subject is a mammal. In another embodiment of the method, the mammal is a human.
• the Ai adenosine receptor is associated with cognitive disease, renal failure, cardiac arrhythmias, respiratory epithelia, transmitter release, sedation, vasoconstriction, bradycardia, negative cardiac inotropy and dromotropy, branchoconstriction, neutropil chemotaxis, reflux condition, or ulcerative condition.
• This invention also provides a combination therapy for asthma, comprising compounds IV and V, and a steroid, ⁇ 2 agonist, glucocoticoid, lucotriene antagonist, or anticolinegic agonist.
• Diseases associated with adenosine Ai, ⁇ a, 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 entire content of which are fully incorporate herein by reference.
• This invention also provides a water-soluble prodrug of a compound having the structures IV, V, VI, VII, VIII, IX, or X, wherein said water-soluble prodrug that is metabolized in vivo to an active drug which selectively inhibit Ai adenosine receptor.
• said prodrug is metabolized in vivo by esterase catalyzed hydrolysis.
• This invention also provides a pharmaceutical composition comprising the prodrug and a pharmaceutically acceptable carrier.
• This invention further provides a method for inhibiting the activity of an Ai adenosine receptor in a cell, which comprises contacting said cell with a compound having the structures IV, V, VI, VII, VIII, IX, pr X.
• the compound is an antagonist of said Ai adenosine receptor.
• This invention also provides for a method for treating a gastrointestinal disorder in an subject, comprising administering to the an effective amount of a compound having the structures IV, V, VI, VII, VIII, IX, or X.
• said disorder is diarrhea.
• the subject is a human.
• the compound is an antagonist of Ai adenosine receptors.
• This invention also provides a method for treating respiratory disorder in a subject, comprising administering to the subject an effective amount of a compound having the structures IV, V, VI, VII, VIII, IX, or X.
• said disorder is asthma, chronic obstructive pulmonary disease, allergic rhinitis, or an upper respiratory disorder.
• the subject is a human.
• said compound is an antagonist of Ai adenosine receptors .
• This invention further provides a method for treating damage to the eye of a subject which comprises administering to said subject an effective amount of a compound having the structures IV, V, VI, VII, VIII, IX, or X.
• said damage comprises retinal or optic nerve head damage.
• said damage is acute or chronic.
• said damage is the result of glaucoma, edema, ischemia, hypoxia or trauma.
• the subject is a human.
• the compound is an antagonist of A ⁇ adenosine receptors.
• This invention also provides a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutically effective amount of a compound having the structures IV, V, VI, VII, VIII, IX, or X, and a pharmaceutically acceptable carrier.
• said therapeutically effective amount is effective to treat a respiratory disorder or a gastrointestinal disorder.
• said gastrointestinal disorder is diarrhea.
• said respiratory disorder is asthma, allergic rhinitis, or chronic obstructive pulmonary disease.
• said pharmaceutical composition is an ophthalmic formulation.
• said pharmaceutical composition is an periocular, retrobulbar or intraocular injection formulation.
• said pharmaceutical composition is a systemic formulation.
• said pharmaceutical composition is a surgical irrigating solution.
• This invention also provides a packaged pharmaceutical composition for treating a disease associated with Ai adenosine receptor in a subject, comprising: (a) a container holding a therapeutically effective amount of an adenosine Al specific compound; and (b) instructions for using said compound for treating said disease in a subject.
• a compound is k x selective. means that a compound has a binding constant to adenosine Al receptor of at least ten time higher then that to adenosine A 2a , A 2b or A 3 .
• This invention also provides a method of preparing the compound having structure IV, comprising the steps of
• step a) treating the product of step a) under cyclization conditions to provide
• step b) treating the product of step b) under suitable conditions to provide
• step d) treating the chlorinated product of step c) with NH2R] to provide
• Ri is trans-4 -hydroxy cyclohexyl, 2-methylamino carbonylamino cyclohexyl, acetylamino ethyl, or methylamino carbonylamino ethyl;
• Ar is a substituted or unsubstituted four to six membered ring
• R ⁇ is H, alkyl, substituted alkyl, cycloalkyl; or a pharmaceutically acceptable salt, or a prodrug derivative, or a biologically active metabolite; with the proviso that when R: is acetylamino ethyl , Ar is not 4 -pyridyl.
• This invention also provides a method of preparing the compound having structure V, comprising the steps of
• step a) treating the product of step a) under cyclization conditions to provide
• step b) treating the product of step b) under suitable conditions to provide
• R is aryl, substituted aryl, heteroaryl
• Rr is H, alkyl, substituted alkyl, or cycloalkyl; wherein R3 is H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, wherein said substituted alkyl is -C(Re) (R-)NR ⁇ Rs, wherein R ⁇ and R- are each H or alkyl, wherein R ⁇ and Rs are each alkyl or cycloalkyl, or NR ⁇ Rs is a ring system of between 4 and 7 members .
• the deazapurines of the invention can be prepared using standard methods for organic synthesis. Deazapurines can be purified by reverse phase HPLC, chromatography, recrystallization, etc. and their structures confirmed by mass spectral analysis, elemental analysis, IR and/or NMR spectroscopy.
• R 3 , R5 and Rg are as defined above.
• a protected 2-amino-3-cyano-pyrrole can be treated with an acyl halide to form a carboxyamido-3-cyano- pyrrole which can be treated with acidic methanol to effect ring closure to a pyrrolo [2, 3d] pyrimidine-4 (3H) -one (Muller,
• N- (l-dl-phenylethyl) -2- amino-3-cyano-pyrrole was treated with an acyl halide in pyridine and dichloromethane.
• the resultant N- (l-d2- phenylethyl) -2-phenylcarboxyamido-3-cyano-pyrrole was treated with a 10:1 mixture of methanol/sulfuric acid to effect ring closure, resulting in a dl- 7H-7- (1- phenylethyl) pyrrolo [2, 3d] pyrimidine-4 (3H) -one.
• R ⁇ through R5 are as defined above.
• R 1 through Rg are defined as above and R is a removable protecting group.
• Schemes IV and V depict methods for preparing the deazapurines 1 and 2 of the invention.
• R 5 and Rg are as described above, e . g. , CH-.
• Knoevengel condensation of malononitrile and an excess ketone, 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 in chloroform yielded a mixture of starting material, mono- (9), and di-brominated products (5/90/5) after distillation (70%) .
• the mixture was reacted with an ⁇ -methylalkylamine or ⁇ - methylarylamine, e . g. , ⁇ -methylbenzylamine, to deliver the aminopyrrole (10) .
• Scheme VIII depicts the synthesis of compound (18).
• Table 2 Selected list of 5- and 6 -substituted pyrrolopyrimidines .
• [2, 3d] pyrimidin-4 (3H) -one (1.0 g, 2.91 mmol) was suspended in polyphosphoric acid (30.0 mL) . The mixture was heated at 100"C for 4 hr. The hot suspension was poured onto ice water, stirred vigorously to disperse suspension, and basified to pH 6 with solid KOH. The resulting solid was filtered and collected to give 0.49 g (69%) of 5, 6 -dimethyl-2 -phenyl - 1H- pyrrolo [2, 3d] pyrimidin-4 ( 3H) -one.
• Preparation 12 To a solution of dl-l, 2-diaminopropane (1.48 g, 20.0 mmol) and sodium carbonate (2.73 g, 22.0 mmol) in dioxane (100.0 mL) and water (100.0 mL) was added di- ert-dicarbonate (4.80 g, 22.0 mmol) at room temperature. The resulted mixture was stirred for 14 hr. Dioxane was removed in vacuo . The precipitate was filtered off and the filtrate was concentrated in vacuo to dryness. The residue was triturated with EtOAc and then filtered.
• 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) and triethylamine (50.4 mg, 0.498 mmol) in dichloromethane (2.0 mL) was added propionyl chloride (25.6 mg, 0.024 ml, C.2 ⁇ 4 mmol) at 0°C. After 1 hr, the mixture was concentrated ir.
• the first fraction was dl -4- (l-methyl-2- (1, 1-dimethylethoxy) carbonylaminoe hyl) amino-5, 6 -dimethyl -2 -phenyl -7H- pyrrolo [2, 3d] pyrimidine: "H NMR (200 MHz, CDCl,) ⁇ 1.29 - 1.38
• 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 mg, 0.153 mmol) was treated with trifluoroacetic acid (0.5 mL) in dichloromethane (2.0 mL) for 14 hr. The organic solvent was removed in vacuo to dryness. The residue was dissolved in N,N-dimethylformamide (2.0 mL) and triethylamine (2.0 mL) . To the solution at 0°C was added acetic anhydride (17.2 mg, 0.016, 0.169 mmol).
• Example 16 To 10 mL of dimethylformamide (DMF) at room temperature were added 700 mg of 4 - ci s- 3 -hydroxycyclopentyl) amino-2 -phenyl- 5, 6-dimethyl- 7H-pyrrolo [2, 3d] pyrimidine followed by 455 mg of
• N-Boc glycine 20 mg of N,N-dimethylaminopyridine (DMAP) , 293 mg of hydroxybenzotriazole (HOBT) and 622 mg of 1- (3- dimethylaminopropyl) -3 -ethylcarboiimide hydrochloride (EDCl) .
• DMAP N,N-dimethylaminopyridine
• HBT hydroxybenzotriazole
• EDCl 1- (3- dimethylaminopropyl) -3 -ethylcarboiimide hydrochloride
• N-Bromosuccinimide (508 mg, 2.86 mmol) and AIBN (112 mg, C.55 mmol ) were added to a solution containing (22) ( 935 mg, 2.71 mmol ) and CC1 4 (50 mL) .
• the solution was heated to reflux. After 2 h the reaction was cooled to room temperature and concentrated in vacuo to yield a ' white solid.
• Flash chromatography (Si0 2 ; 1/1 CH : C1 : /Hexanes, R f 0.30) yielded 960 mg (84%)of a white solid (23).
• Example 18 Synthesis of adenosine Antagonists.
• Compound 1319 and Compound 1320 (Table 13 below) can be synthesized by the general procedures herein.
• Example 19 Synthesis of adenosine A : Antagonist.
• Compound 1 321 (Table 13 below) can be synthesized by 5 general procedures given below.
• Example 20 Synthesis of adenosine A x Antagonist.
• Compound 1504 (Table 15 below) can be synthesized by the general procedures given below.
• JU Compound 31 (200 mg, 0.47 mmol) was dissolved in DCM (4 mL) . Triethylamine (51 mg, 0.5mmol) and thiomorpholine (52 mg, O.Smmol) were added sequentially. The solution was mixed for several minutes and allowed to stand for 72 hours. The reaction was diluted with DCM and H : 0 and the layers were
• Yeast ⁇ -Galactosidase reporter gene assays for human adenosine A x and A 2a receptor Yeast strains (S. cerev ⁇ s ⁇ ae> were transformed with human adenosine A : (A : R; CADUS strain
• test compounds (5 ' -N-ethylcarboxamidoadenosine) , a potent adenosine receptor agonist with similar affinity for A, and A 2a receptors, was used as a ligand for all assays. Test compounds were examined at 8 concentrations (0.1 - 10,000 nM) for ability to inhibit NECA-induced ⁇ -Galactosidase activity by CY12660 or
• Yeast A, R and A 2a R Assay One vial each of CY8362 and CY12660 yeast/glycerol stock was thawed and used to inoculate Supplemented LT liquid media, pH 6.8 (92 ml LT liquid, to which 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 16-18 hr (overnight) at 30°C.
• Assays were conducted with a final volume of 100 ul in 96- well microtiter plates, such that a final concentration of 2% DMSO was achieved in all wells.
• 1-2 concen t rations of test compounds were utilized (10 uM, l ⁇ M ) .
• concentrations were tested (10000, 1000 , 500, 100, 50, 10, 1 and 0.1 nM) .
• 10 ul of 20% DMSO was added to "Control" and "Total” wells while 10 ul of Test Compound (in 20% DMSO) was added to "Unknown" wells.
• ⁇ -Galactosidase activity can be quantitated using either colorimetric (e.g., ONPG, CPRG) , luminescent (e.g., Galacton- Star) or fluorometric substrates (e.g., FDG, Resorufin) substrates.
• colorimetric e.g., ONPG, CPRG
• luminescent e.g., Galacton- Star
• fluorometric substrates e.g., FDG, Resorufin
• fluorescence detection is preferred on the basis of superior signal :noise ratio, relative freedom from interference and low cost .
• Fluorescein digalactopyranoside FDG, Molecular Probes or Marker Gene Technologies
• Yeast strains Saccharo yces cerevisiae strains CY12660 [far l*1 442 tbtl-1 fusl-HIS3 canl Stel4 : : trpl : :LYS2 ste3*1156 gpa l ( 4 1) -G ⁇ i3 lys2 ura3 leu2 trpl: his3; L ⁇ U2 PGKp-
• LT (Leu-Trp supplemented) media is composed of lOOg DIFCO yeast nitrogen base, supplemented with the following: l.Og valine, l.Og aspartic acid, 0.75g phenylalanine, 0.9g lysine, 0.45g tyrosine, 0.45g isoleucine, 0.3g methionine, 0.6g adenine, 0.4g uracil, 0.3g serine, 0.3g proline, 0.3g cysteine, 0.3g arginine, 0.9g histidine and l.Og threonine .
• yeast strains expressing a human A, adenosine receptor functionally integrated into the yeast pheromone system pathway is described.
• the A, adenosine receptor cDNA was obtained by reverse transcriptase PCR of human hippocampus mRNA using primers designed based on the published sequence of the human A : adenosine receptor and standard techniques. The PCR product was subcloned into the Ncol and Xbal sites of the yeast expression plasmid pMP15.
• the pMP15 plasmid was created from pLPXt as follows: The Xbal site of YEP51 (Broach, J.R. et al. (1983) "Vectors for high-level, inducible expression of cloned genes in yeast” p. 83-117 in M. Inouye (ed.), Experimental Manipulation of Gene Expression. Academic Press, New York) was eliminated by diges t ion, end-fill and religation to create YepSLNccDXba.
• pLPX (1990) Mol. Cell. Biol. l_Q:2582-2590) with PCR primers containing Bglll and Ncol sites.
• the resulting plasmid is called pLPX .
• pLPXt was modified by inserting the coding region of the a-factor pre- pro leader into the Ncol site. The prepro leader was inserted so that the Ncol cloning site was maintained at the 3' end of the leader, but not regenerated at the 5' end. In this way receptors can be cloned by digestion of the plasmid with Ncol and Xbal. The resulting plasmid is called pMP!5.
• the pMP15 plasmid into which was inserted the human A j adenosine receptor cDNA was designated p5095.
• the receptor cDNA is fused to the 3' end of the yeast a-factor prepro leader.
• the prepro peptide sequences are cleaved to generate mature full-length receptor. This occurs during processing of the receptor through the yeast secretory pathway.
• This plasmid is maintained by Leu selection (i.e., growth on medium lacking leucine) .
• Leu selection i.e., growth on medium lacking leucine
• yeast strain CY7967 was used as the starting parental strain.
• the genotype of CY7967 is as follows:
• MATa Mating type a The endogenous yeast G-protein GPAl has been deleted.
• gpal (41) -Gai3 was integrated into the . " . yeast genome.
• This chimeric Ga protein is composed of the first 41 amino acids of the endogenous yeast Ga subunit GPAl fused to the mammalian G-protein Gai3 in which the cognate N-terminal amino acids have been deleted.
• HIS3 coding region (thereby creating a pheromone inducible HIS3 gene) .
• yeast YS2.... methyltransferase (thereby lowering basal signaling through the pheromone pathway) .
• ste3D1156 endogenous yeast STR the a factor pheromone receptor (STE3) was disrupted.
• lys2 defect in 2-aminoapidate reductase yeast need lysine to grow.
• ura3 defect in orotidine- 5 ' -phosphate decarboxylase yeast need uracil to grow leu2 defect in b-isopropylmalate dehydrogenase
• yeast need leucine to grow
• yeast need tryptophan to grow.
• yeast need histidine to grow.
• Two plasmids were transformed into strain CY ⁇ 9 ⁇ by electroporation: plasmid p5095 (encoding human A : adenosine receptor; described above) and plasmid pl584, which is a
• Plasmid pl584 was derived from plasmid pRS426 (Christianson, T.W. et al .
• Plasmid pRS426 contains a polylinker site at nucleotides 2004-2016. A fusion between the FUS1 promoter and the ⁇ -galactosidase gene was inserted at the restriction sites Eagl and Xhol to create plasmid pl584. The pl584 plasmid is maintained by Trp selection
• the resultant strain carrying p5095 and pl584, referred to as CY12660 expresses the human A : adenosine receptor.
• CY12660 expresses the human A : adenosine receptor.
• To perform a growth assay on plates (assaying FUS1-HIS3) the plates were at pH 6.8 and contained 0.5-2.5 mM 3 -amino-1, 2 , 4 -triazole and lacked leucine, tryptophan and histidine.
• As a control for specificity a comparison with one or more other yeast-based seven transmembrane receptor screens was included in all experiments .
• yeast strains expressing a human A2a adenosine receptor functionally integrated into the yeast pheromone system pathway is described.
• the human A2a receptor cDNA was obtained from Dr. Phil Murphy (NIH) . Upon receipt of this clone, the A2a receptor insert was sequenced and found to be identical to the published sequence (GenBank accession # S46950) . The receptor cDNA was excised from the plasmid by PCR with VENT polymerase and cloned into the plasmid pLPBX, which drives receptor expression by a constitutive Phosphoglycerate Kinase
• yeast strain CY8342 was used as the starting parental strain.
• the genotype of CY8342 is as follows:
• MATa farlD1442 tbtl-1 lys2 ura3 leu2 trpl his3 fusl-HIS3 canl ste3D1156 gpaD1163 stel4 : :trpl : :LYS2 gpalp-rG ⁇ s E10K (or gpalp- rG ⁇ s D229S or gpalp-rG ⁇ s E10K+D229S)
• yeast strains were utilized in which the endogenous yeast G protein GPAl had been deleted and replaced by a mammalian G ⁇ s .
• Three rat G ⁇ s mutants were utilized. These variants contain one or two point mutations which convert them into proteins which couple efficiently to yeast ⁇ y. They are identified as G 3 «E10K (in which the glutamic acid at position ten is replaced with lysine) , G habitD229S (in which the aspartic ac d at position 229 is replaced with serine) and G complicat ⁇ 10K+D229S (which contains both point mutations) .
• Strain CY8342 (carrying one of the three mutant rat G as proteins) was transformed with either the parental vector pLPBX (Receptor-) or with pLPBX-A2a (Receptor * ) .
• a plasmid with the FUS1 promoter fused to ⁇ -galactosidase coding sequences was added to assess the magnitude of activation of the pheromone response pathway.
• Adenosine a natural agonist for this receptor, as well as two other synthetic agonists were utilized for development of this assay.
• Adenosine reported to have an EC 5C of approximately 75 nM, and ( - ) -N6- (2-phenylisopropyl) -adenosine
• PIA polypeptide A
• NECA -N-ethylcarboxamido- adenosine
• A, adenosine receptor to functionally couple in a heterologous yeast system was assessed by introducing the A : receptor expression vector (p5095, described above) into a series of yeast strains that expressed different G protein subunits. The majority of these transformants expressed G ⁇ subunits of the G ⁇ or G ⁇ o subtype. Additional G ⁇ proteins were also tested for the possible identification of promiscuous receptor-G ⁇ protein coupling. In various strains, a STE18 or a chimeric STE18- G ⁇ 2 construct was integrated into the genome of the yeast .
• Transformants were isolated and monolayers were prepared on media containing 3-amino-l, 2, 4-triazole, 4 U/ml adenosine deaminase and lacking histidine.
• Five microliters of various concentrations of ligand e.g., NECA at 0, 0.1, 1.0 and 10 mM was applied. Growth was monitored for 2 days. Ligand- dependent growth responses were tested in this manner in the various yeast strains. The results are summarized in Table 1 below. The symbol (-) indicates that ligand-dependent receptor activation was not detected while (+) denotes ligand-dependent response.
• the term "LIRMA" indicates ligand independent receptor mediated activation.
• fusl -LacZ Assay To characterize activation of the pheromone response pathway more fully, synthesis of ⁇ -galactosidase through fuslLacZ in response to agonist stimulation was measured. To perform the ⁇ -galactosidase assay, increasing concentrations of ligand were added to mid-log culture of human A ⁇ adenosine receptor expressed in a yeast strain co-expressing a Stel8-G ⁇ 2 chimera and GPA 4 ⁇ -G ⁇ i . Transformants were isolated and grown overnight in the presence of histidine and 4 U/ml adenosine deaminase.
• the A, adenosine receptor assay was further characterized by measurement of the receptor's radioligand binding parameters. Displacement binding of [ H] CPX by several adenosine receptor 146
• the natural ligand adenosine, as well as other thoroughly characterized and commercially available ligands were used for study of the human A2a receptor functionally expressed in yeast.
• Three ligands have been used in the establishment of this assay. They include:
• adenosine deaminase (4U/ml) was added to all assays .
• A2a receptor agonists were tested for the capacity to stimulate the pheromone response pathway in yeast transformed with the A2a receptor expression plasmid and expressing either G ⁇ s E10K, G ⁇ s D229S or G ⁇ s E10K+D229S.
• the ability of ligand to stimulate the pheromone response pathway in a receptor dependent manner was indicated by an alteration in the yeast phenotype .
• Receptor activation modified the phenotype from histidine auxotrophy to histidine prototrophy
• A2a receptor ligand spot assay An alternative approach to measuring growth and one that can be miniaturized for high throughput screening is an A2a receptor ligand spot assay.
• A2a receptor ligand spot assay A G QS E10K strain expressing the A2a receptor (A2aR+) or lacking the receptor (R-) was grown overnight in the presence of histidine and 4 U/ml adenos
• Yeast strains expressing G as E10K, G aS D229S or G O ..E10K+D229S were transformed with a plasmid encoding the human A2a receptor (R+) or with a plasmid lacking the receptor (R-). Transformants were isolated and grown overnight in the presence of histidine and 4 U/ml adenosine deaminase. 1 x 10 cells were diluted to 1 x 10 cells/ml and exposed to increasing concentrations of NECA for 4 hours, followed by determination of the ⁇ -galactosidase activity in the cells.
• the G relieD229S construct was the second-most potent G os construct for the A2a receptor, while the G relieE10K+D229S construct was the least potent of the three G as constructs tested, although even the G hinder ⁇ 10K»D225 ⁇ construct stimulated readily detectable amounts of ⁇ - galactosidase activity.
• Membranes from HEK-293 cells stably expressing the human Adenosine 2a [RB-HA2a] ,- Adenosine 2b [RB-HA2b] or Adenosine 3 [RB-HA3] receptor subtypes, respectively were purchased from Receptor Biology (Beltsville, MD) . Cell culture reagents were from Life Technologies (Grand Island, NY) except for serum that was from Hyclone (Logan, UT) .
• Yeast strains Saccharo yces cerevisiae strains CY12660
• Yeast culture Transformed yeast were grown in Leu-Trp [LT. 1 media (pH 5.4) supplemented with 2% glucose.
• LT. 1 media pH 5.4
• membranes 250 ml of LT medium were inoculated with start titer of 1-2 x 10 cells/ml from a 30 ml overnight culture and incubated at 30 C under permanent oxygenation by rotation. After 16 h growth the cells were harvested by centrifugation and membranes were prepared as described below.
• HEK-293 cells stably expressed human Adenosine 2a receptor subtype (Cadus clone # 5) were grown in Dulbeco"s minimal essential media (DMEM) supplemented with 10% fetal bovine serum and IX penicillin/streptomycin under selective pressure using 500 mg/ml G418 antibiotic, at 37°C in a humidified 5% C0 2 atmosphere .
• DMEM Dulbeco
• IX penicillin/streptomycin under selective pressure using 500 mg/ml G418 antibiotic, at 37°C in a humidified 5% C0 2 atmosphere .
• Yeast Cell Membrane Preparations 250 ml cultures were harvested after overnight incubation by centrifugation at 2,000 x g in a Sorvall RT6000 centrifuge. Cells were washed in ice-cold water, centrifuged at 4°C and the pellet was resuspended in 10 ml ice-cold lysis buffer [5 mM Tris-HCl, pH 7.5; 5 mM EDTA; and 5 mM EGTA] supplemented with Protease inhibitor cocktail tablets (1 tablet per 25 ml buffer) . Glass beads (17 g; Mesh 400-600; Sigma) were added to the suspension and the cells were broken by vigorous vortexing at 4°C for 5 min.
• HEK-293 cell membranes were prepared as described previously (Duzic E et ai .
• the cell lysate was then centrifuged at 36,000 x g (Sorvall RC5B, type SS34 rotor) for 45 min and the pellet resuspended in 5 ml membrane buffer [50 mM Tris-HCl, pH 7.5; 0.6 mM EDTA; 5 mM MgCl 2 ; 0.1 mM Phenylmethylsulfonyl fluoride, 10 mg/ml pepstatin A; and 10 mg/ml aprotinin) and stored at -80 °C for further experiments .
• 5 ml membrane buffer [50 mM Tris-HCl, pH 7.5; 0.6 mM EDTA; 5 mM MgCl 2 ; 0.1 mM Phenylmethylsulfonyl fluoride, 10 mg/ml pepstatin A; and 10 mg/ml aprotinin
• Bio-Rad protein assay kits based on the Bradford dye- binding procedure, (Bradford, M. : Anal. Biochem. 72:248 (1976) ) were used to determine total protein concentration in yeast and mammalian membranes.
• Adenosine 1 receptor subtype saturation and competi tion radioligand binding Saturation and competition binding on membranes from yeast cell transformed with human A x receptor subtype were carried out using antagonist [ H] DPCPX as a radioactive ligand. Membranes was diluted in binding buffer [50 mM Tris-HCl, pH 7.4; containing 10 mM MgCl 2 ; 1.0 mM EDTA; 0.25% BSA; 2 U/ml adenosine deaminase and 1 protease inhibitor cocktail tablet/50 ml] at concentrations of 1.0 mg/ml .
• Adenosine 2a receptor subtype competi tion radiol igand binding Competition binding on membranes from H ⁇ K293 cell stably expressing the human A2a receptor subtype were carried out using agonist [ H] CGS-21680 as a radioactive ligand.
• Membranes was diluted in binding buffer [50 mM Tris-HCl, pH 8.
• Membranes (10 ⁇ g/well) were incubate with [ I] AB-MECA (0.75 nM) in a final volume of 100 ⁇ l of binding buffer at 25°C for 1 hr in the absence and presence of 10 ⁇ M unlabeled IB-MECA or increasing concentrations of competing compounds in a 96-well microtiter plate.
• the A 1# A 2a and A 3 receptor subtypes radioligand binding assays was terminated by the addition of ice-cold 50 mM Tris-HCl (pH 7.4) buffer supplemented with 10 mM MgCl 2 , followed by rapid filtration over glass fiber filters (96-well GF/B UniFilters, Packard: previously presoaked in 0.5% polyethylenimine in a Filtermate 196 cell harvester (Packard) . The filter plates were dried coated with 50 ⁇ l /well scintillation fluid (MicrcScint-20, Packard) and counted in a TopCount (Packard) . Assays were performed in triplicate. Non-specific binding was 5.6 * 0.5%,
• Adenosine 2b receptor subtype competi tion radioligand binding Competition binding on membranes from HEK293 cell stably expressing the human A2b receptor subtype were carried out using Ai receptor antagonist [ H] DPCPX as a radioactive ligand. Membranes was diluted in binding buffer [10 mM Hepes- KOH, pH 7.4; containing 1.0 mM EDTA; 0.1 mM Benzamidine and 2 U/ml adenosine deaminase] at concentrations of 0.3 mg/ml. Membranes (15 ⁇ g/well) were incubate with [ H] DPCPX (15 nM) in .
• a primary function of certain cell surface receptors is to recognize appropriate ligands. Accordingly, we determined ligand binding affinities to establish the functional integrity of the Adenosine 1 receptor subtype expressed m yeast. Crude membranes prepared from Saccharomyces cerevisiae transformed with human Adenosine l receptor subtype construct exhibited specific saturable binding of [ H] DPCPX with a K D of 4.0 + 0.19 nM. The K D and B max value were calculated from the saturation isotherm and Scatchard transformation of the data indicated a single class of binding sites. The densities of adenosine binding sites in the yeast membrane preparations were estimated to 716.8 + 43.4 fmol/mg membrane protein.
• the pharmacological subtype characteristics of the recombinant yeast cells transformed with human A : receptor subtype were investigated with subtype selective adenosine ligands (XAC, DPCPX; CGS-15943; Compound 600; Compound 1002; NECA, (R)-PIA; IB-MECA and Alloxazine) that competed with [ 3 H] DPCPX in the expected rank order. Displacement curves recorded with these compounds show the typical steepness with all the ligands, and the data for each of the ligands could be modeled by a one-site fit. The apparent dissociation constants estimated for the individual compound from the curves (Table 5) are consistent with value published for the receptor obtained from other sources. Table 5 Ki values for membranes from yeast cells transformed with human A ⁇ receptor subtype
• Tables 6 through 12 demonstrate the efficacy and structure activity profiles of deazapurines of the invention.
• Tables 13 and 14 demonstrate selectivity can be achieved for human adenosine receptor sites by modulation of the functionality about the deazapurine structure.
• Table 14 also demonstrates the surprising discovery that the compounds set forth therein have subnanomolar activity and higher selectivity for the A 3b receptor as compared to the compounds in Table 13.
• R is 3-chlorophcnyl;' R, is 4-pyndyl; ' % activity :2> 10 ⁇ M
• Table 14 Profile of Selective A 2b Antagonists

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• 2003
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