NO327207B1 - Compounds specific for adenosine A1, A2A and A3 receptor as well as their use in the preparation of drugs and methods for the preparation of the compounds - Google Patents

Description

The present invention relates to compounds as stated in claims 1, 28, 29 and 44, use thereof as stated in claims 18, 20, 30, 34, 44 and 58, pharmaceutical compositions containing such compounds as stated in claims 22, 25, 35, 41 and 50 as well as methods for producing such compounds as stated in claim 55.

Background for the invention

Adenosine is a general modulator of many physiological activities, especially within the cardiovascular and nervous systems. The effects of adenosine appear to be mediated by specific cell surface receptor proteins. Adenosine modulates many physiological functions including induction of anesthesia, vasodilation, suppression of heart rate and contractility, inhibition of platelet aggregation, stimulation of gluconeogenesis and inhibition of lipolysis. In addition to its effects on adenylate cyclase, adenosine has been shown to open calcium channels, reduce influx through calcium channels, and inhibit or stimulate phosphoinositide consumption via receptor-mediated mechanisms (see, for example, CE. Muller and B. Stein "Adenosine Receptor Antagonists: Structures and Potential Therapeutic Applications," Current Pharmaceutical Design, 2, 501 (1996) and CE. Muller "Ai-Adenosine Receptor Antagonists," Exp. Opin. Ther. Patents 7(5):419 (1997)).

Adenosine receptors belong to the superfamily of purine receptors which are currently divided into Pi (adenosine) and P2 (ATP, ADP and other nucleotides) receptors. Four receptor subtypes for the nucleoside adenosine have so far been cloned from different species including humans. Two receptor subtypes (Ai and A2a) show affinity for adenosine in the nano-molar range, while two other known subtypes A2b and A3 are low-affinity receptors with an affinity for adenosine in the low-micromolar range. Ai and A3 adenosine receptor activation

can lead to an inhibition of adenylate cyclase activity,

while A2a and A2b activation causes a stimulation of adenylate cyclase.

Some Ai antagonists have been developed for the treatment of cognitive disease, renal failure and cardiac arrhythmia. It has been suggested that A2a 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 the treatment of allergic inflammation and asthma. Available information (for example Nyce and Metzger "DNA antisense Therapy for Asthma in an Animal Model" Nature (1997) 385:721-5) indicates that in this pathophysiological context Ai antagonists can block contraction of smooth muscle underlying the respiratory epithelium , while A2b or A3 receptor antagonists can block mast cell degranulation, which moderates the release of histamine and other inflammatory mediators. A2b receptors have been discovered throughout the gastrointestinal tract, particularly in the intestine and intestinal epithelium. It has been suggested that A2b receptors mediate cAMP responsiveness (Strohmeier et al., J. Bio. Chem. (1995) 270:2387-94).

Adenosine receptors have also been shown to exist on the retina 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 Neurochemistry, volume 54, pages 648-655 (1990); Woods et al., Characterization of Adenosine A!- Receptor Binding Sites in Bovine Retinal Membranes, Experimental Eye Research, volume 53, pages 325-331

(1991) and Braas et al., Endogenous adenosine and adenosine receptors localized to ganglion cells of the retina, Proceedings of the National Academy of Science, volume 84, pages 3906-3910 (1987). Recently, Williams reported the observation of adenosine transport sites in a cultured human retinal cell line (Williams et al., Nucleoside Transport Sites in a

Cultured Human Retinal Cell Line Established By SV-40 T Antigen Gene, Current Eye Research, volume 13, pages 109-118

(1994)).

Compounds that regulate adenosine uptake have previously been proposed as potential therapeutic agents for the treatment of retinal and optic nerve head damage. In US Patent No. 5,780,450 to Shade, Shade discusses the use of adenosine uptake inhibitors in the treatment of eye disorders. Shade does not describe the use of specific A3 receptor inhibitors.

Additional adenosine receptor antagonists are needed as pharmacological tools and are of great interest as drugs in the above-mentioned diseases and/or conditions.

Summary of the invention

The present invention is based on compounds as stated in claim 1 and which selectively bind to adenosine Ai receptor, thereby treating disease associated with Ax adenosine receptor in an individual, by administering to the individual a therapeutically effective amount of such compounds. The disease to be treated is associated with cognitive impairment, renal failure, cardiac arrhythmia, respiratory epithelium, transmitter release, anesthesia, vasoconstriction, bradycardia, negative cardiac inotropy and dromotropic, bronchoconstriction, neutrophil chemotaxis, regurgitation or peptic ulcer conditions.

The present invention is based, at least in part, on the discovery that certain N-6 substituted 7-deazapurines, described below, can be used to treat an N-6 substituted 7-deazapurine responsive condition. Examples of such conditions include those in which the activity of the adenosine receptors is increased, for example bronchitis, gastrointestinal disorders or asthma. These conditions can be characterized by the fact that adenosine receptor activation can lead to the inhibition or stimulation of adenylate cyclase activity. Compositions and methods according to the invention include enantiomerically or diastereomerically pure N-6-substituted 7-deazapurines. Preferred N-6 substituted 7-deazapurines include those having an acetamide, carboxamide, substituted cyclohexyl, for example cyclohexanol or a urea unit attached to the N-6 nitrogen via an alkylene chain.

The present invention relates to compounds having the structure:

wherein R1NR2 together form a ring having the structure: or R1 is H and R2 is: and, when RXNR2 together are

R5 is H, -CH2 (NC5H8) (OH) (C6H5) , -CH2OCH3, - (CH2) 2C (0) OH, - CH2OCH2C (0)NH2, -C(0)OH, -CH3, -CH20 (CH2 ) 20H, - CH2OCH2 (0) OCH3 or -CH2OCH2C (0) OH, or

when R 1 is H and R 2 is

is R5 -CH2 (N2C3H3), -C(0)OH, CH2O (CH2)2OH, -C(0)0CH3 or -C(0)NH2

or a pharmaceutically acceptable salt thereof.

The invention further relates to pharmaceutical compositions for treating an N-6-substituted 7-deazapurine responsive condition in a mammal, for example asthma, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease, kidney diseases, gastrointestinal diseases and eye disorders. The pharmaceutical composition comprises a therapeutically effective amount of an N-6-substituted 7-deazapurine and a pharmaceutically acceptable carrier.

The present invention also relates to packaged pharmaceutical compositions for treating an N-6-substituted 7-deazapurine responsive condition in a mammal. The packaged pharmaceutical composition includes a container containing a therapeutically effective amount of at least one N-6-substituted 7-deazapurine and instructions for using the N-6-substituted 7-deazapurine to treat an N-6-substituted 7- deazapurine-responsive state in a mammal.

The deazapurines according to this embodiment can advantageously be selective A1 receptor antagonists. These compounds may be useful for several therapeutic applications, such as, for example, the treatment of asthma, renal failure associated with heart failure, and glaucoma.

In yet another embodiment, compounds according to the invention can inhibit the activity of an adenosine receptor (for example A3) in a cell where the cell is brought into contact with N-6-substituted 7-deazapurine (for example preferably an adenosine receptor antagonist).

The compounds can also be used for the production of drugs to treat damage to the eye of an animal (for example a human). Preferably, the N-6-substituted 7-deazapurine is an antagonist of A3 adenosine receptors in mammalian cells. The damage is to the retina or the optic nerve head and can be acute or chronic. The damage may for example be the result of glaucoma, oedema, ischaemia, hypoxia or trauma.

The invention also relates to a pharmaceutical composition comprising a compound according to claim 1. Preferably, the pharmaceutical preparation is an ophthalmic formulation (for example a periocular, retrobulbar or intraocular injection formulation, a systemic formulation or a surgical injection solution).

Detailed description

The features and other details of the invention will now be more specifically described and pointed out in the claims.

The term "N-6-substituted 7-deazapurine responsive condition" is intended to include a disease state or condition, which is characterized by its sensitivity to treatment with a compound of the invention as described below. Typically, such conditions are associated with an increase in adenosine in a host organism such that the host organism often experiences physiological symptoms that include, but are not limited to, release of toxins, inflammation, coma, water retention, weight gain or loss, pancreatitis, emphysema, rheumatoid arthritis, osteoarthritis, multiple organ failure, respiratory distress syndrome in children and adults, allergic rhinitis, chronic obstructive pulmonary disease, eye diseases, gastrointestinal diseases, promotion of skin tumors, immunodeficiency and asthma. (See, for example, CE. Muller and B. Stein (Adenosine Receptor Antagonists: Structures and Potential Therapeutic Applications," Current Pharmaceutical Design, 2:501 (1996) and CE. Muller "Ai-Adenosine Receptor Antagonists," Exp. Opin. Ther . Patents 7( 5) : 419 (1997) and I. Fekotistove, R. Polosa, S. T. Holgate and I. Biaggioni "Adenosine A2B receptors: a novel therapeutic target in asthma?2 TIPS 19: 148 (1998)). The effects that often associated with such symptoms include, but are not limited to, fever, shortness of breath, nausea, diarrhea, weakness, headache, and even death.In one embodiment, an N-6-substituted 7-deazapurine-responsive state includes those disease states that are promoted by stimulation of adenosine receptors, for example Ai, A2a, A2b, A3 etc. so that calcium concentrations in cells and/or activation of PLC (phospholipase C) are modulated.

In a preferred embodiment, a condition is associated with adenosine receptor(s). For example, the compound acts as an antagonist. Examples of suitable responsive conditions that can be treated with the compounds of the invention, for example adenosine receptor subtypes that mediate biological effects, include central nervous system (CNS) effects, cardiovascular effects, renal effects, respiratory effects, immunological effects, gastrointestinal effects and metabolic effects . The relative amount of adenosine in an individual can be associated with the effects listed below, i.e. increased levels of adenosine can initiate an effect, for example an unwanted physiological response, for example an asthma attack.

CNS effects include decreased transmitter release (Ai), anesthesia (Ai), decreased locomotor activity (A2a), anticonvulsant activity, chemoreceptor stimulation (A2), and hyperalgesia. Therapeutic uses of the compounds of the invention include treatment of dementia, Alzheimer's disease and memory enhancement.

Cardiovascular effects include vasodilatation (A2a), (A2b) and (A3), vasoconstriction (Ai), bradycardia (Ai), platelet inhibition (A2a), negative cardiac inotropy and dromotropic (Ai), arrhythmia, tachycardia and angiogenesis. Therapeutic uses of the compounds according to the invention include, for example, prevention of ischemia-induced weakening of the heart and cardiotonia, protection of myocardial tissue and restoration of cardiac function.

Renal effects include decreased GFR (Ai), mesangial cell contraction (Ai), antidiuresis (Ai) and inhibition of renal excretion (Ax). Suitable therapeutic uses of the compounds according to the invention include the use of the inventive compounds as diuretic, natriuretic, potassium sparing, kidney protective/preventive for acute renal failure, antihypertensive, anti-edematous and anti-nephritic agents.

Respiratory effects include bronchodilation (A2), bronchoconstriction (Ai), chronic obstructive pulmonary disease, allergic rhinitis, mucus secretion and respiratory impairment (A2). Suitable therapeutic uses for the compounds of the invention include anti-asthmatic uses, treatment of post-transplant lung disease and respiratory disorders.

Immunological effects include immunosuppression (A2), neutrophil chemotaxis (Ai), neutrophil superoxide production (A2a) and mast cell degranulation (A2b and A3). Therapeutic applications of antagonists include allergic and non-allergic inflammation, for example the release of histamine and other inflammatory mediators.

Gastrointestinal effects include inhibition of acid secretion (Ai) where therapeutic use may include inhibitions, regurgitation and ulcerative conditions. Gastrointestinal effects also include colonic, small intestinal and diarrheal disease, for example diarrheal disease associated with inflammation of the small intestine (A2b).

Eye disorders include head damage to the retina and optic nerve, and trauma-related disorders (A3) . In a preferred embodiment, the eye disorder is cataract.

Other therapeutic uses of the compounds of the invention include treatment of obesity (lipolytic properties), hypertension, treatment of depression, relaxing anxiolytics as antileptics and as laxatives, for example by providing motility without causing diarrhoea.

The term "disease state" is intended to include such conditions which are caused by or associated with undesirable levels of adenosine, adenylyl cyclase activity, increased physiological activity associated with abnormal stimulation of adenosine receptors and/or an increase in cAMP. In one embodiment, the disease state is, for example, asthma, chronic obstructive pulmonary disease, allergic rhinitis, bronchitis, kidney diseases, gastrointestinal diseases or eye diseases. Additional diseases include chronic bronchitis and cystic fibrosis. Suitable examples of inflammatory diseases include non-lymphocytic leukemia, myocardial ischemia, angina, infarction, cerebrovascular ischemia, intermittent claudication, critical limb ischemia, venous hypertension, varicose veins, venous ulceration and arteriosclerosis. Conditions with impaired reperfusion include, for example, any post-surgical trauma, such as reconstructive surgery, thrombolysis or angioplasty.

The term "treatment of an N-6 substituted 7-deazapurine responsive condition" or "treatment of an N-6 substituted 7-deazapurine responsive condition" is intended to include changes in a disease state or condition as described above such that physiological symptoms in a mammals may be significantly reduced or minimized. The term also includes control, prevention or inhibition of physiological symptoms or effects associated with an abnormal amount of adenosine. In a preferred embodiment, the control of the disease state or condition is such that the disease state or condition is removed. In another preferred embodiment, the control is selective so that abnormal levels of adenosine receptor activity are controlled, while other physiological systems and parameters are unaffected.

The term "N-6 substituted 7-deazapurine" is known in the art and is intended to include such compounds having formula I:

"N-substituted 7-deazapure" includes pharmaceutically acceptable salts thereof and, in one embodiment, also certain N-6-substituted purines described herein.

The term "therapeutically effective amount" of a compound of the invention as described below indicates the amount of a therapeutic compound necessary or sufficient to exert its intended function in a mammal. An effective amount of the therapeutic compound may 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 particular disease state in the mammal.

One of ordinary skill in the art will be able to study the aforementioned factors and make a determination as to the effective amount of the therapeutic compound without undue experimentation. An in vitro or in vivo assay may also be used to determine an "effective amount" of the therapeutic compound described below. The person of ordinary skill in the art would select an appropriate amount of the therapeutic compound for use in the aforementioned assay or as a therapeutic treatment.

A therapeutically effective amount preferably reduces a symptom or effect associated with the particular condition 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 even more preferably by at least 80%) relative to untreated individuals. Assays may be designed by the skilled person to measure the reduction of such symptoms and/or effects. Any assay known in the art and capable of measuring such parameters is intended to be used. For example, if asthma is the condition being treated, then the volume of air pushed out from the lungs of an individual can be measured before and after treatment to measure the increase in volume using a technique known in the art. Likewise, if inflammation is the condition being treated, the area that is inflamed can be measured before and after treatment to measure the reduction in the area

which is inflamed using a technique known in the art.

The term "cell" includes both prokaryotic and eukaryotic cells.

The term "animal" includes any organism with adenosine receptors or any organism susceptible to a compound of the invention. Examples of animals include yeast, mammals, reptiles and birds. It also includes transgenic animals.

The term "mammal" is known in the art and is intended to include an animal, more preferably a warm-blooded animal, more preferably cows, sheep, pigs, horses, dogs, cats, rats, mice and humans.

The term "modulating an adenosine receptor" is intended to include such instances where a compound interacts with an adenosine receptor(s) to cause increased, decreased or abnormal physiological activity associated with an adenosine receptor or subsequent cascade effects arising from the modulation of the adenosine receptor. Physiological activities associated with adenosine receptors include induction of anesthesia, vasodilation, lowering of heart rate and contractility, inhibition of platelet aggregation, stimulation of gluconeogenesis, inhibition of lipolysis, opening of potassium channels, reduction of current in calcium channels, etc.

The terms "modulate", "modulating" and "modulating" are intended to include preventing, removing or inhibiting the resulting increase in unwanted physiological activity associated with abnormal stimulation of an adenosine receptor, for example in the context of the therapeutic methods of the invention. In another embodiment, the term modulating antagonistic effects includes, for example, reducing the activity or production of mediators of allergy and allergic inflammation resulting from the over-stimulation of the adenosine receptor(s). For example, the therapeutic deazapurines according to the invention can interact with an adenosine receptor to inhibit, for example, adenylate cyclase activity.

The term "condition characterized by abnormal adenosine receptor activity" is intended to include those diseases, disorders or conditions that are associated with abnormal 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 , the disorder or condition. This stimulation of an adenosine receptor need not be the sole causative agent of the disease, disorder or condition, but only be responsible for causing some of the symptoms typically treated. The abnormal stimulation of the receptor may be the only factor, or at least another agent may be involved in the condition being treated. Examples of conditions include those disease states listed above, including inflammation, gastrointestinal disorders and the symptoms manifested by the presence of increased adenosine receptor activity. Preferred examples include such symptoms associated with asthma, allergic rhinitis, chronic obstructive pulmonary disease, emphysema, bronchitis, gastrointestinal diseases and cataracts.

The phrase "treating or treating a condition characterized by abnormal adenosine receptor activity" is intended to include the abrogation or reduction of at least one symptom typically associated with the condition. The treatment also includes the elimination or reduction of more than one symptom. Preferably, the treatment cures, eg, substantially removes, the symptoms associated with the condition.

The compounds according to the invention can be formed from water-soluble prodrugs which are described in WO 99/33815, International Application No. PCT/US98/04595, filed March 9, 1998 and published July 8, 1999. The water-soluble prodrugs are metabolized in vivo to an active drug, for example by esterase-catalyzed hydrolysis. Examples of potential prodrugs include deazapurines such as, for example, R2 as cycloalkyl substituted with -OC(0)(Z)NH2, where Z is a side chain of a naturally or unnaturally occurring amino acid or analog thereof, an a-, P~, y- or oo-amino acid or a dipeptide. Preferred amino acid side chains include those from glycine, alanine, valine, leucine, isoleucine, lysine, α-methylalanine, aminocyclopropane carboxylic acid, azetidine-2-carboxylic acid, β-alanine, γ-aminobutyric acid, alanine alanine or glycine alanine.

In a particularly preferred embodiment, the deazapurines according to the invention can be formed from water-soluble prodrugs which can be metabolized in vivo to an active drug, for example by esterase-catalyzed hydrolysis. Preferably, the prodrogen comprises an R2 group which is cycloalkyl substituted with -OC(0)(Z)NH2 where Z is a side chain of a naturally or unnaturally occurring amino acid, an analogue thereof, an a-, P~, y- or oo- amino acid or a dipeptide. Examples of preferred side chains include the side chains of glycine, alanine, valine, leucine, isoleucine, lysine, α-methylalanine, aminocyclopropane carboxylic acid, azetidine-2-carboxylic acid, β-alanine, γ-aminobutyric acid, alanine alanine or glycine alanine.

In another embodiment, the compound according to the invention is 4-(cis-3-hydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl-7ff-pyrrolo [2, 3d]pyrimidine,

4-(cis-3-(2-aminoacetoxy)cyclopentyl)amino-5,6-dimethyl-2-phenyl-7if-pyrrolo [2, 3d]pyrimidinetrifluoroacetic acid salt. 4-(3-acetamido)piperidinyl-5,6-dimethyl-2-phenyl-7if-pyrrolo[2,3d]pyrimidine.

4-(2-N'-methylureapropyl)amino-5,6-dimethyl-2-phenyl-7if-pyr-

rolo[2,3d]pyrimidine.

4-(2-acetamidobutyl)amino-5,6-dimethyl-2-phenyl-7ff-pyrrolo[2,3d]pyrimidine.

4-(2-N'-methylureabutyl)amino-5,6-dimethyl-2-phenyl-7if-pyrrolo[2,3d]pyrimidine.

4-(2-Aminocyclopropylacetamidoethyl)amino-2-phenyl-7ff-pyrrolo[2,3d]pyrimidine.

4-(trans-4-hydroxycyclohexyl)amino-2-(3-chlorophenyl)-1H-pyrrolo[2,3d]pyrimidine.

4-(trans-4-hydroxycyclohexyl)amino-2-(3-fluorophenyl)-1H-pyrrolo[2,3d]pyrimidine.

4-(trans-4-hydroxycyclohexyl)amino-2-(4-pyridyl)-7H-pyrrolo[2,3d]pyrimidine.

The compounds according to the invention can inhibit the activity of an adenosine receptor (for example Ai, A2a, A3b, or preferably A3) in a cell.

In one embodiment, the compound according to the invention can be 4-(2-acetylaminoethyl)amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine,

4-(2-acetylaminoethyl)amino-6-(4-fluorophenoxy)methyl-2-phenyl-7if-pyrrolo [2, 3d]pyrimidine,

4-(2-acetylaminoethyl)amino-6-(4-chlorophenoxy)methyl-2-phenyl-7if-pyrrolo [2, 3d]pyrimidine,

4-(2-acetylaminoethyl)amino-6-(4-methoxyphenoxy)methyl-2-phenyl-7if-pyrrolo[2,3d]pyrimidine,

4-(2-Acetylaminoethyl)amino-6-(2-pyridyloxy)methyl-2-phenyl-7ff-pyrrolo [2, 3d]pyrimidine,

4-(2-acetylaminoethyl)amino-6-(N-phenylamino)methyl-2-phenyl-7if-pyrrolo [2, 3d]pyrimidine,

4-(2-acetylaminoethyl)amino-6-(N-methyl-N-phenylamino)methyl-2-phenyl-7ff-pyrrolo [2, 3d]pyrimidine, or

4-(2-N'-methylureaethyl)amino-6-(4-phenoxymethyl)methyl-2-phenyl-7if-pyrrolo[2,3d]pyrimidine,

Compounds according to the invention can inhibit the activity of an adenosine receptor (for example an Aib adenosine receptor) in a cell if the cell is brought into contact with a compound according to the invention. Preferably, the compound is an antagonist of the receptor.

In another embodiment, the invention relates to a pharmaceutical composition containing a compound according to the invention as well as a pharmaceutically acceptable carrier.

The term "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. The term alkyl further includes alkyl groups which may further include oxygen, nitrogen, sulfur or phosphorus atoms by replacing one or more carbons of the hydrocarbon stem, for example oxygen, nitrogen, sulfur or phosphorus atoms. In preferred embodiments, a straight-chain or branched alkyl has 30 or fewer carbon atoms in its stem (for example C1-C30 for straight-chain, C3-C30 for branched) and more preferably 20 or fewer. Likewise, preferred cycloalkyls have from 4 - 10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.

Furthermore, the term alkyl, as used throughout the description and claims, is intended to include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl units that have substituents that replace a hydrogen on one or more carbons of the hydrocarbon stem. Such substituents may include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonate, phosphinate, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino ), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonate, sulfamoyl, sulfonamide, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic unit. It will be understood by those skilled in the art that the units substituted on the hydrocarbon chain may themselves be substituted if appropriate. Cycloalkyls can be further substituted, for example with the substituents described above. An "alkylaryl" moiety is an alkyl substituted with an aryl (for example, phenylmethyl (benzyl)). The term "alkyl" also includes unsaturated aliphatic groups which are analogous in length and possible substitution to the alkyls described above, but which contain at least one double or triple bond, respectively.

The term "aryl" as used herein refers to the radical of aryl groups including 5- and 6-membered single ring aromatic groups which may contain from 0 to 4 heteroatoms, for example benzene, pyrrole, furan, thiophene, imidazole, benzoxazole, benzothiazole, triazole, tetrazole , pyrazole, pyridine, pyrazine, pyridazine and pyrimidine and the like. Aryl groups also include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "arylheterocyclic compounds", "heteroaryls" or "heteroaromatic compounds". The aromatic ring may be substituted at one or more ring positions with such substituents as described above, such as, for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonate, phosphinate , cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonate, sulfamoyl, sulfonamide, nitro , trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl or an aromatic or heteroaromatic unit. Aryl groups may also be fused or bridged with alicyclic or heterocyclic rings that are not aromatic to form a polycyclic compound (eg tetralin).

The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups which are similar in length and possible substitution to the alkylenes described above, but which contain at least one double or triple bond, respectively. For example, the invention relates to cyano and propargyl groups.

Unless the number of carbons is indicated otherwise, "lower alkyl" as used herein means an alkyl group as defined above, but having from 1 to 10 carbons, more preferably from 1 to 6 carbon atoms in its stem structure, even more preferably 1 to 3 carbon atoms in its tribal structure. Likewise, "lower alkenyl" and lower alkynyl" have similar chain lengths.

The terms "Alkoxyalkyl", "Polyaminoalkyl" and "Thioalkoxyalkyl" refer to alkyl groups as described above, which further include oxygen, nitrogen or sulfur atoms replacing one or more carbons of the hydrocarbon chain, for example oxygen, nitrogen and sulfur atoms.

The terms "polycyclyl" or "polycyclic radical" refer to the radical of 2 or more cyclic rings (for example, cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) where 2 or more carbons are common to two adjacent rings, for example, the rings are " fused rings". Rings that are bonded together via non-adjacent atoms are termed "bridged" rings. Each of the rings of the polycyclic compound may be substituted with such substituents as described above, such as halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonate, phosphinate, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl or an aromatic or heteroaromatic moiety.

The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.

The term "amino acids" includes naturally and unnaturally occurring amino acids found in proteins such as glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine , tyrosine and tryptophan. Amino acid analogs include amino acids with extended or shortened side chains or variants of side chains with appropriate functional groups. Amino acids also include D and L stereoisomers of an amino acid when the structure of the amino acid allows for stereoisomeric forms. The term "dipeptide" includes 2 or more amino acids linked together. Preferably, dipeptides are two amino acids linked via a peptide bond. Particularly preferred dipeptides include, for example, alanine-alanine and glycine-alanine.

It will be noted that the structure of some of the compounds according to the present invention includes asymmetric carbon atoms and thus behaves as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. All such isomeric forms of these compounds are expressly included in the present invention. Each sterogenic carbon can be of the R or S configuration. Accordingly, it will be understood that the isomers resulting from such asymmetry (for example all enantiomers and diastereomers) are included in the scope of the present invention unless otherwise stated. Such isomers can be obtained mainly in pure form by classical separation techniques and by stereochemically controlled synthesis.

The invention further relates to pharmaceutical compositions for treating relevant disease states in a mammal, for example respiratory disorders, (for example asthma, bronchitis, chronic obstructive pulmonary disease and allergic rhinitis), kidney diseases, gastrointestinal disorders and eye disorders. The pharmaceutical composition comprises a therapeutically effective amount of a compound as described above, and a pharmaceutically acceptable carrier. It is understood that all the deazapurines described above are included for therapeutic treatment. It is further understood that deazapurines according to the invention can be used alone or in combination with other deazapurines according to the invention or in combination with further therapeutic compounds such as, for example, antibiotics, anti-inflammatory or anti-cancer agents.

The term "antibiotic" is recognized in the art and is intended to include such substances produced by growing microorganisms and synthetic derivatives thereof, which eliminate or inhibit the growth of pathogens and are selectively toxic to pathogens while producing minimal or no harmful effects on the infected host individual . Suitable examples of antibiotics include, but are not limited to, the major classes of aminoglycosides, cephalosporins, chloramphenicols, fusidic acids, macrolides, penicillins, polymyxins, tetracyclines and streptomycins.

The term "anti-inflammatory" is recognized in the art and is intended to include such agents that act on body mechanisms without directly antagonizing the causative agent of the inflammation such as glucocorticoids, aspirin, ibuprofen, NSAIDS, etc.

The term "anticancer agent" is recognized in the art, and is intended to include such agents which reduce, remove or prevent the growth of cancer cells without, preferably in an unfortunate way, affecting other physiological functions. Representative examples include cisplatin and cyclophosphamide.

When the compounds of the present invention are administered as pharmaceuticals to humans and animals, they may be administered as such 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.

The term "pharmaceutically acceptable carrier", as used herein, means a pharmaceutically acceptable material, composition or vehicle such as a liquid or a solid filler, diluent, excipient, solvent or encapsulating material involved in carrying or transporting a compound(s) of the present invention invention within or to the individual so that it can perform its intended function. Typically, such compounds are carried or transported from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense that it is compatible with the other components of the formulation and not harmful to the patient. Some examples of materials that may act 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; measured; gelatin; talc; excipients such as coconut butter and suppository wax; oils such as peanut oil, cottonseed oil, sunflower 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 physiological saline; Ringer's dissolution; ethyl alcohol; phosphate buffer solutions and other non-toxic compatible substances used in pharmaceutical formulations.

As noted above, certain embodiments of the present compounds may contain a basic functional group such as amino or alkylamino, and thus are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salts" in this context refers to the relatively non-toxic inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds according to the invention, or by separately reacting a purified compound according to the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate , glucoheptonate, lactobionate and lauryl sulfonate salts and the like (See, for example, Berge et al., (1977) "Pharmaceutical Salts", J.Pharm.Sei. 66:1-19).

In other cases, the compounds according to the present invention may contain one or more functional acid groups, and are thus able to form pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts", in these cases, refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts may also be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation with ammonia or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like. Representative organic amines useful for the preparation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

The term "pharmaceutically acceptable esters" refers to the relatively non-toxic esterified products of the compounds of the present invention. These esters can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form, or hydroxyl, with a suitable esterifying agent. Carboxylic acids can be converted to 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, for example alkyl esters, methyl, ethyl and propyl esters. (See for example Berge et al., supra..)

Prodrugs can be converted in vivo to the therapeutic compounds of the invention (See for example R.B. Silverman, 1992 "The Organic Chemistry of Drug Design and Drug Action", Academic Press, Chapter 8). Such prodrugs can be used to alter the biodistribution (eg, to allow compounds that would not typically enter the reactive site of the protease), or pharmacokinetics of the therapeutic compound. For example, a carboxylic acid group can be esterified, for example with a methyl group or an ethyl group to give an ester. When the ester is administered to a subject, the ester is cleaved, enzymatically or non-enzymatically, reductively or hydrolytically, to yield the anionic group. An anionic group can be esterified with units (eg acyloxymethyl esters) which are cleaved to give an intermediate compound which then decomposes to give the active compound. In another embodiment, the prodrug is a reduced form of a sulfate or sulfonate, such as a thiol, which is oxidized in vivo to the therapeutic compound. Furthermore, an anionic unit can be esterified to a group that is actively transported in vivo, or that is selectively taken up by measuring organs. The ester may be selected to allow specific targeting of the therapeutic entities to particular reactive sites as described below for carrier entities.

Humectants, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate as well as coloring agents, release agents, coating agents, sweeteners, flavoring and perfuming agents, preservatives and antioxidants may also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), 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.

Formulations according to the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form, and may be prepared by any method known in the pharmacological field. The amount of active ingredient which can be combined with a carrier material to form a single dosage form will generally be the amount of the compound which produces a therapeutic effect. In general, 100% of this amount will be from about 1% to about 99% of active ingredient, preferably about 5% to about 70%, most preferably from about 10% to about 30%.

Methods for preparing these formulations or compositions include the step of bringing into association a compound according to the present invention with carrier material and, optionally, one or more auxiliary ingredients. In general, the formulations are prepared by uniformly and intimately associating a compound of the present invention with liquid carrier materials or finely divided solid carrier materials or both, and then shaping the product if necessary.

Formulations according to the invention which are suitable for oral administration can be in the form of capsules, sachets, pills,

tablets, lozenges (which use a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in- oil liquid emulsion, or as an elixir or syrup, or as lozenges using an inert base such as gelatin and glycerin, or sucrose and acacia) and/or as mouth rinses and the like, each containing a predetermined amount of a compound according to the present invention as an active ingredient. A compound according to the present invention can also be administered as a bolus, laverge or paste.

In solid dosage forms according to the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), 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: filling materials or extenders such as starches, lactose, sucrose, glucose, mannitol and/or silicic acid; binders such as, for example, carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; humectants such as glycerol; disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; dissolution retarding agents such as paraffin; absorption accelerators such as quaternary ammonium compounds; humectants such as cetyl alcohol and glycerol monostearate; absorbents such as kaolin and bentonite clay; lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof and colorants. In the case of capsules, tablets and pills, the pharmaceutical compositions may also include buffering agents. Solid compositions of a similar type can also be used as fillers in soft and hard filled gelatin capsules using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

A tablet can be made by compression or molding, possibly with one or more auxiliary ingredients. Compressed tablets may be prepared using a binder (eg gelatin or hydroxypropylmethylcellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactant or dispersant. 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 according to the invention, such as lozenges, capsules, pills and granules, may optionally be added to a notch or prepared with coatings and shells such as enteric coatings and other coatings that are well known in the field of pharmaceutical formulation technology. They may also be formulated to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying amounts to provide the desired

release profile, other polymer matrices, liposomes and/or microspheres. They can be sterilized by, for example, filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient(s) alone or preferably in a certain part of the gastrointestinal tract, possibly in a delayed manner. Examples of inclusive compositions that may be used include polymeric substances and wax types. The active ingredient can also be in microencapsulated form, if appropriate with one or more of the excipients described above.

Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents that are usually used in the field such as water or other solvents, solubilizers and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, oils (especially cotton, peanut, corn, corn, olive, salmon and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof.

In addition to inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavourings, colourants, perfuming and preserving agents.

In addition to the active compounds, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum meta-hydroxide, bentonite, agar-agar and tragacanth, as well as mixtures thereof.

Formulations of the pharmaceutical compositions according to the invention for rectal or vaginal administration may be presented as a suppository which may be prepared by mixing one or more of the compounds according to the invention with one or more suitable non-irritating excipients or carriers comprising, for example, coconut butter , polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature but liquid at body temperature, and consequently will melt in the rectum or vagina and release the active compound.

Formulations according to the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be suitable.

Dosage forms for the topical or transdermal administration of a compound of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, plasters and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to an active compound according to the present invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicone acid , talc and zinc oxide or mixtures thereof.

Powders and sprays may contain, in addition to a compound according to the present invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder or mixtures of these substances. Sprays can also contain common propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

Transdermal patches have the further advantage of providing controlled delivery of a compound according to the present invention to the body. Such dosage forms may be prepared by dissolving or dispersing the compound in the appropriate medium. Absorption enhancing agents may also be used to increase the flux of the compound across the skin. The rate of such flow can be controlled by either providing a rate controlling membrane or dispersing the active compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like are also contemplated as being within the scope of the present invention. Preferably, the pharmaceutical preparation is an ophthalmic formulation (eg a periocular, retrobular or intraocular injection formulation, a systemic formulation or a surgical wetting formulation).

The ophthalmic formulations may include one or more deazapurines and a pharmaceutically acceptable vehicle. Different types of vehicles can be used. The vehicles will generally be watery in nature. Aqueous solutions are generally preferred based on considerations of formulation as well as a patient's ability to readily administer such compositions by instilling one or two drops of the solutions into the affected eye. However, the deazapurines according to the present invention can also be easily incorporated into other types of compositions such as suspensions, viscous or semi-viscous gels or other types of solid or semi-solid compositions. The ophthalmic compositions according to the present invention may also include various other ingredients such as buffers, preservatives, co-solutions and viscosity forming agents.

A suitable buffer system (eg sodium phosphate, sodium acetate or sodium borate) can be added to prevent pH drift under storage conditions.

Ophthalmic products are typically packaged in multidose form. Preservatives are thus necessary to prevent microbial contamination during use. Suitable preservatives include: benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenylethyl alcohol, disodium edetate, sorbic acid, polyquaternium-1 or other agents known to those skilled in the art. Such preservatives are typically used at a level of from 0.001 to 1.0% w/v ("% w/v").

When the compounds of the present invention are administered during intraocular surgical procedures such as via retrobulbar or periocular injection and intraocular perfusion or injection, the use of balanced wetting salt solutions as vehicles is preferred. BSS® Sterile Irrigating Solution and BSS Plus® Sterile Intra-ocular Irrigating Solution (Alcon Laboratories, Inc., Fort Worth, Texas, USA) are examples of physiologically balanced intraocular moisturizing solutions. The latter type of solution is described in US patent no. 4,550,022 (Garabedian, et al.,), the entire content of which is hereby incorporated into the present description by reference. Retrobulbar and periocular injection are known to those skilled in the art and are described in several publications, including, for example, Ophthalmic Surgery: Principles of Practice, Ed., G.L. Spaeth, W.B. Sanders Co., Philadelphia, Pa., USA, pp. 85-87 (1980).

As indicated above, the use of deazapurines to prevent or reduce damage to retinal and optic nerve head tissue at the cellular level is a particularly important aspect of one embodiment of the invention. Ophthalmic conditions that may be treated include, but are not limited to, retinopathies, macular degeneration, ocular ischemia, cataracts, and destruction associated with damage to ophthalmic tissue such as ischemia reperfusion injury, photochemical injury, and injury associated with ocular surgery, particularly injury to retina or the 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 or ophthalmic surgery. The compounds may be used for acute treatment of temporary conditions or may be administered chronically, particularly in the case of degenerative disease. The compounds may also be used prophylactically, particularly before ocular surgery or non-invasive ophthalmic procedures, or other types of surgery.

Pharmaceutical compositions according to the present invention, which are suitable for parenteral administration, comprise one or more compounds according to the invention, in combination with one or more pharmaceutically acceptable, sterile, isotonic, aqueous or non-aqueous 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, bacteriostatics, soluble materials which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions according to the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Appropriate fluidity can be maintained, for example, with the use of coating materials, such as lecithin, by maintaining the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Inhibition of the action of microorganisms can be ensured by including various anti-bacterial and anti-fungal 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, in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be provided by including agents that delay absorption, such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the drug, it is desirable to delay the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by the use of a liquid suspension of crystalline or amorphous material which has poor water solubility. The degree of absorption of the drug then depends on its degree of dissolution, which in turn may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are formed by forming micro-encapsulating matrices of the relevant compounds in biodegradable polymers, such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

The preparations according to the present invention can be given orally, parenterally, topically or rectally. They are of course given in forms that are suitable for each administration route. For example, they are administered in tablet or capsule form by injection, inhalation, eye lotion, ointment, suppository etc, administration by injection, infusion or inhalation, topically with lotion or ointment and rectally with suppositories. Oral administration is preferred.

The terms "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral and topical administration, usually by injection and include without limitation intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrasternal injection as well as infusion.

The terms "systemic administration", "administered systemically", "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system so that it penetrates the patient's system and is thus subject to metabolism and other similar processes, such as 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, rectal, intravaginally, parenterally, intracisternally, and topically, as by powders, ointments, or drops. , including buccal and sublingual.

Regardless of the route of administration chosen, the compounds according to the present invention, which can be used in a suitable hydrated form, and/or the pharmaceutical compositions according to the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to the person skilled in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied to maintain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without being toxic to the patient.

The selected dosage level will depend on a number of factors, including the activity of the particular compound of the present invention used, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of treatment, other drugs, compounds and/or materials used in combination with the particular compound used, age, sex, weight, condition, general health and previous medical history of the patient being treated and similar factors well known in the medical field.

A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the doctor or veterinarian could start doses of the compounds according to the invention, used in the pharmaceutical composition at levels that are lower than what is necessary to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In general, an appropriate daily dose of a compound according to the invention will be the amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above. In general, intravenous and subcutaneous doses of the compound of the present invention, for a patient when used for the indicated analgesic effect, will range from about 0.0001 to about 200 mg per kg of body weight per day, more preferably from about 0.01 to about 150 mg per kg per day, and even more preferably from about 0.2 to about 140 mg per kg per day.

If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more subdoses administered separately at appropriate intervals throughout the day, optionally in unit dose form.

Although it is possible for a compound of the present invention to be administered alone, it is preferred to administer the compound as a pharmaceutical composition.

The present invention also relates to packaged pharmaceutical compositions for treating relevant disease states, for example unwanted increased adenosine receptor activity in a mammal. The packaged pharmaceutical compositions include a container containing a therapeutically effective amount of at least one deazapurine as described above and instructions for using the deazapurine to treat the deazapurine-responsive condition in a mammal. The compounds according to the invention can be prepared using standard methods for organic synthesis. The compounds can be purified by reverse phase HPLC, chromatography, recrystallization etc. and their structure confirmed by mass spectral analysis, elemental analysis, IR and/or NMR spectroscopy.

Typically, synthesis of the intermediates as well as the deazapurines of the invention is carried out in solution. The additions and removal of one or more protecting groups are also typically carried out and are known to the person skilled in the art. Typical synthetic schemes for the production of the deaza-purine intermediates according to the invention are outlined below in Scheme 1.

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

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

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

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

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

In one embodiment of the pharmaceutical composition, said therapeutically effective amount is effective to treat a respiratory disorder or a digestive tract disorder.

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

In another embodiment of the pharmaceutical composition, said respiratory disorder is asthma, allergic rhinitis or chronic obstructive pulmonary disease.

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

In another embodiment of the pharmaceutical composition, the pharmaceutical composition is a periocular, retrobulbar or intraocular injection formulation.

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

In a further embodiment of the pharmaceutical preparation, said pharmaceutical composition is a surgical spray solution.

The present invention also provides a packaged pharmaceutical composition for treating a disease associated with adenosine Ai receptor in a subject comprising: (a) a container containing a therapeutically effective amount of an adenosine Ai-specific compound, and (b) instructions for using said compound to treat said disease in an individual.

As used herein, "a compound is Ai-selective" means that a compound has a binding constant to adenosine A1 receptor at least tenfold higher than that to adenosine A2a, A2b, or A3.

The invention is further illustrated by the following examples. It should be understood that the models used throughout the examples are accepted models and that the demonstration of effectiveness in these models is predictive of effectiveness in humans.

The present invention will be better understood from the experimental details that follow. However, the person skilled in the art will readily understand that the stated methods and results described are only illustrative of the invention.

EXPERIMENTAL DETAILS

The compounds according to the invention can be prepared using standard methods of organic synthesis. The compounds 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.

Typically, synthesis of the intermediates as well as the compounds of the invention is carried out in solution. The addition and removal of one or more protecting groups is also typical practice for the person skilled in the art.

In general, 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 pyrrolo[2,3d]pyrimidine-4 (3H)-one (Muller, CE. et al. J. Med. Chem. 40: 4396 (1997)). Removal of the pyrrolo-protecting group followed by treatment with a chlorinating agent, for example phosphorus oxychloride, gave substituted or unsubstituted 4-chloro-7H-pyrrolo[2,3d]pyrimidines. Treatment of the chloropyrimidine with amines gave 7-deazapurines.

For example, an N-(1-dl-phenylethyl)-2-amino-3-cyano-pyrrole was treated with an acyl halide in pyridine and dichloromethane. The resulting N-(1-dl-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]pyrimidin-4(3H)-one. Removal of the phenylethyl group by treatment of the pyrimidine with polyphosphoric acid (PPA) followed by POCl 3 gave a key intermediate, 4-chloro-7H-pyrrolo[2,3d]pyrimidine. Further treatment of the 4-chloro-7H-pyrrolo[2,3d]pyrimidine with various amines gives compounds listed in Table 1. Transesterification and alkylation of ethyl cyanoacetate with an α-haloketone gives a ketomethyl ester. Protection of the ketone followed by treatment with an amidine (eg, alkyl, aryl, or alkylaryl) hydrochloride gave the resulting ketal-protected pyrimidine. Removal of the protecting group followed by cyclization and treatment with phosphorus oxychloride gave the chloride intermediate which could be further treated with an amine to give an amine 6-substituted pyrrole. Furthermore, alkylation of the pyrrole nitrogen can be carried out under conditions known in the art.

Condensation of malononitrile and an excess of a ketone followed by bromination of the product gives a mixture of starting material, monobrominated and dibrominated products which are treated with an alkylamine, arylamine or alkylarylamine. The resulting amine product is acylated with an acid chloride and the monoacylated pyrrole is cyclized in the presence of acid to give the pyrimidine. The pyrrole protecting group is removed with polyphosphoric acid and treated with phosphorous oxychloride to give a chlorinated product. The chlorinated pyrrole can then be treated with an amine to give an amino 5-substituted pyrrole. Alkylation of the pyrrole nitrogen can be accomplished under conditions known in the art.

Special preparation of 6-methylpyrrolopyrimidines The key reaction towards 6-methylpyrrolopyrimidines was the cyclization of a cyanoacetate with benzamidine to a pyrimidine. It was hypothesized that methyl cyanoacetate would cyclize more efficiently with benzamidine to a pyrimidine than the corresponding ethyl ester. Consequently, when transesterification and alkylation of ethyl cyanoacetate in the presence of NaOMe and an excess of an α-haloacetyl unit, for example chloroacetone, the desired methyl ester in 79% yield. The ketoester was formed as the acetal in 81% yield. A new method of cyclization to the pyrimidine was achieved with an amidine hydrochloride, for example benzamidine hydrochloride, with 2 equivalents of DBU to give the compound in 54% isolated yield. This method improves the yield from 20% using the published conditions, which utilize NaOMe during the cyclization with guanidine. Cyclization to pyrrolepyrimidine was achieved via deblocking of the acetal in aqueous HCl in 78% yield. Reaction with phosphorus oxychloride at reflux temperature gave the corresponding 4-chloro derivative. Coupling with trans-4-aminocyclohexanol in dimethyl sulfoxide at 135 °C gave the product in 57%. The person skilled in the art will understand that the choice of reagents enables great flexibility in choosing the desired substituent R5.

Special preparation of 5-methylpyrrolopyrimidines

Knoevenage gel condensation of malononitrile as well as an excess of ketone, for example acetone by boiling under reflux in benzene gave 8 in 50% yield after distillation. Bromination of 8 with N-bromosuccinimide in the presence of benzoyl peroxide in chloroform gave a mixture of starting material, mono- and dibrominated products (5/90/5) after distillation (70%). The mixture was reacted with an α-methylalkylamine or α-methylarylamine, for example α-methylbenzylamine, to give the aminopyrrole. After passing through a short silica gel column, the partially purified amine (31% yield) was acylated with an acid chloride, for example benzoyl chloride, to give mono- and diacylated pyrroles, which were separated by flash chromatography. Acid hydrolysis of the disubstituted pyrrole gave a combined yield of 29% for the acyl pyrrole. Cyclization in the presence of concentrated sulfuric acid and DMF gave (23%), which was deblocked with polyphosphoric acid. Reaction with phosphorus oxychloride at reflux temperature gave the corresponding 4-chloro derivative. Coupling with trans-4-aminocyclohexanol in dimethylsulfoxide at 135 °C the relevant compound in 30%. The person skilled in the art will understand that the choice of reagents enables great flexibility.

Alternative synthesis route to 5-methylpyrrolopyrimidines

This alternative route to the relevant pyrroles, 5-methylpyrrolopyrimidines, involves transesterification and alkylation of ethyl cyanoacetate. Condensation of with benzamidine hydrochloride with 2 equivalents of DBU gives the pyrimidine. Cyclization to the pyrrolepyrimidine will be achieved via deblocking of the acetal in aqueous HCl. Reaction of the compound with phosphorus oxychloride at reflux temperature gave the corresponding 4-chloro derivative. Coupling with trans-4-aminocyclohexanol in dimethyl sulfoxide at 135 °C gives the final compound. This procedure reduces the number of synthesis reactions of the target compound from 9 to 4 steps. Furthermore, the yield is dramatically improved. Again, the person skilled in the art will appreciate that the choice of reagents enables great flexibility.

The invention is further illustrated by the following examples and it should be understood that the models used throughout the examples are accepted models and that the demonstration of effectiveness in these models is predictive of effectiveness in humans.

Exemplification

Preparation 1:

A modification of the alkylation method of Seela and Lupke was used.<1> To an ice-cold (0 °C) solution of ethyl cyanoacetate (6.58 g, 58.1 mmol) in MeOH (20 mL) was slowly added a solution of NaOMe (25% w/v; 58.1 mmol). After 10 min, chloroacetone (5 mL; 62.8 mmol) was slowly added. After 4 hours, the solvent was removed. The brown oil was diluted with EtOAc (100 mL) and washed with H 2 O (100 mL). The organic fraction was dried, filtered and concentrated to a brown oil (7.79 g; 79%). The oil (3)

(scheme IV) was a mixture of methyl/ethyl ester products (9/1), and was used without further purification. <1>H NMR (200 MHz, CDCl 3 ) 6_4.24 (q, J = 7.2 Hz, OCH 2 ), 3.91 (dd,

1H, J = 7.2, 7.0 Hz, CH) , 3.62 (s, 3H, OCH3) , 3.42 (dd, 1H, J = 15.0, 7.1 Hz, 1 x CH2) ; 3.02 (dd, 1H, J = 15.0, 7.0 Hz, 1 x CH 2 ); 2.44 (s, 3H, CH 3 ), 1.26 (t, J = 7.1 Hz, ester-CH 3 ).

^eela, F.; Lupke, U. Chem. Pray. 1977, 110, 1462-1469.

Preparation 2:

The procedure of Seela and Liipke was used.<1> Thus provided protection of the ketone; 5.0 g, 32.2 mmol) with ethylene glycol (4 mL, 64.4 mmol) in the presence of TsOH (100 mg) as an oil

(scheme IV; 5.2 g, 81.0 ) after flash chromatography (SiO 2 ; 3/7 EtOAc/hex, Rf 0.35). Still contains "5% ethyl ester: <1>H NMR (200 MHz, CDCl3) 5_4.24 (q, J = 7.2 Hz, 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-CH 3 ); MS (ES): 200.1 (M"+1).

<1> Seela, F.; Lupke, U. Chem. Pray. 1977, 110, 1462-1469.

Preparation 3:

A solution of acetal 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 hours. The mixture was diluted with CHCl 3 (30 mL) and washed with 0.5 N NaOH (10 mL) and H 2 O (20 mL). The organic fraction was dried, filtered and concentrated to a brown oil. Flash chromatography (SiO 2 ; 1/9 EtOAc/CH 2 Cl 2 , Rf 0.35) was attempted, but material crystallized on the column. The silica gel was washed with MeOH. Fractions containing the product (5) (Scheme IV) were concentrated and used without further purification (783 mg, 54.3%) : <1>R NMR (200 MHz, CDCl 3 ) 5 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"+1).

Preparation of the subject compound: A solution of acetal 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 mixture was diluted with 100 mL of CHCl 3 and washed with H 2 O (2 x 50 mL). The organic fraction was dried, filtered and concentrated to a dark brown oil. The dark brown oil was stirred in IN HCl (100 mL) for 2 h at room temperature. The resulting slurry was filtered to give the HCl salt of the title compound as a light brown solid (3.60 g, 70.6%); <X>H 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~+1).

Preparation 4:

A solution of the acetal (700 mg, 2.44 mmol) in 1N HCl (40 mL) was stirred for 2 h at RT. The resulting slurry was filtered to give the HCl salt of 2-phenyl-6-methyl-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one as a light brown solid (498 mg, 78.0%) : <1>H NMR (200 MHz, DMSO-d6) 11.78 (s, 1H), 8.05 (m, 2H, Ar-H), 7.45 (m, 3H, Ar-H), 6 .17 (s, 1H, pyrrole-H), 2.25 (s, 3H, CH 3 ); MS(ES):226, 1 (M"+1).

Preparation 5:

A modification of the Chen et al. crystallization method was used.<1> To an ice-cold (0 °C) solution of bromide; 20.0 g, 108 mmol; 90% pure) in isopropyl alcohol (60 mL) was slowly added to a solution of α-methylbenzylamine (12.5 mL, 97.3 mmol). The black solution was allowed to warm to RT and stirred for 15 h. The mixture was diluted with EtOAc (200 mL) and washed with 0.5 N NaOH (50 mL). The organic fraction was dried, filtered and concentrated to a black tar (19.2 g; 94%). The residue was partially purified by flash chromatography (SiO 2 ; 4/96 MeOH/CH 2 Cl 2 , Rf 0.35) to a black solid (6.38 g, 31%) as the compound dl-1-(1-phenyl-ethyl)- 2-Amino-3-cyano-4-methylpyrrole: MS(ES): 226.1 (M~+1). <1>Chen, 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.

Preparation 6:

To a solution dl-1-(1-phenylethyl)-2-amino-3-cyano-4,5-dimethylpyrrole<1>. (14.9 g, 62.5 mmol) and pyridine (10.0 mL) in dichloromethane (50.0 mL) was added benzoyl chloride (9.37 g, 66.7 mmol) at 0 °C. After stirring at 0 °C for 1 h, hexane (10.0 mL) was added to aid precipitation of the product. The solvent was removed in vacuo and the solid was recrystallized from EtOH/H 2 O to give 13.9 g (65%) of dl-1-(1-phenylethyl)-2-phenylcarbonylamino-3-cyano-4,5-dimethylpyrrole . m.p. 218-221 °C; <1>R NMR (200 MHz, CDCl 3 ) 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"+1).

1 Liebig's Ann. Chem. 1986, 1485-1505.

The following compounds were obtained in a similar manner.

Preparation 6A: dl-1-(1-phenylethyl)-2-(3-pyridyl)carbonylamino-3-cyano-4,5-dimethylpyrrole. <X>H NMR (200 MHz, CDCl 3 ) 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"+1). dl-1-(1-phenylethyl)-2-(2-furyl)carbonylamino-3-cyano-4,5-dimethylpyrrole. <X>H NMR ( 200 MHz, CDC13) 5 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 "+1), 230, 1. dl-1-(1-phenylethyl)-2-(3-furyl)carbonylamino-3-cyano-4,5-dimethylpyrrole. <X>H NMR (200 MHz, CDCl 3 ) δ 1.80 (d, J = 7 Hz, 3H), 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 (M"+1), 230.0. dl-1-(1-phenylethyl)-2-cyclophenylcarbonylamino-3-cyano-4,5-dimethylpyrrole. <X>H NMR (200 MHz, CDC13) 5 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, 1H), 5.43 (q, J = 7.4 Hz, 1H), 6.52 (s, 1H), 7.05-7.20 (m, 5H); MS (ES):336, 3 (M"+1). dl-1-(1-phenylethyl)-2-(2-thieyl)carbonylamino-3-cyano-4,5-dimethylpyrrole. <X>H NMR (200 MHz, CDCl 3 ) δ 1.82 (d, J = 6.8 Hz, 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"+1), 246.0. dl-1-(1-phenylethyl)-2-(3-thienyl)carbonylamino-3-cyano-4,5-dimethylpyrrole. <X >H NMR (200 MHz, CDCl 3 ) 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"+1), 246, 1. dl-1-(1-phenylethyl)-2-(4-fluorophenyl)carbonylamino-3-cyano-4,5-dimethylpyrrole. <X>H NMR (200 MHz, CDCl 3 ) 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"+1), 258, 1. dl-1-(1 -phenylethyl)-2-(3-fluorophenyl)carbonylamino-3-cyano-4,5-dimethylpyrrole <1>H NMR (200 MHz, CDCl3) 6 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"+1), 258.1. dl-1-(1-phenylethyl)-2-(2-fluorophenyl)carbonylamino-3-cyano-4,5-dimethylpyrrole. <1>R NMR (200 MHz, CDCl 3 ) 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. <X>H NMR (200 MHz, CDC13) 6 1.19 (d, J = 7.0 Hz, 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"+1) , 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-methylpyrrole were obtained. Monoacylated pyrrole: <1>R NMR (200 MHz, CDCl 3 ) 5_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-CH3), 1.85 (d, 3H, J = 7.2 Hz, CH-CH3); MS(ES):330.2 (M"+1); diacylated pyrrole: <1>R NMR (200 MHz, CDCl 3 ) 6 - 7.85 (d, 2H, J = 7.7 Hz, Ar-H), 7 .74 (d, 2H, J = 7.8 Hz, 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, 3H, J = 7.2 Hz, CH-CH3 ) ; 1.74 (s, 3H, pyrrole-CH 3 ); MS(ES): 434.1 (M"+1) .

Preparation 7:

To a solution dl-1-(1-phenylethyl)-2-phenylcarboxyamino-3-cyano-4,5-dimethylpyrrole<1>. (1.0 g, 2.92 mmol) in methanol (10.0 mL) was added concentrated sulfuric acid (1.0 mL) at 0 °C. The resulting mixture was refluxed for 15 hours and cooled to room temperature. The precipitate was filtered to give 0.48 g (48%) of dl-5,6-dimethyl-2-phenyl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3H)-one . <1>R NMR (200 MHz, CDCl 3 ) 6_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 (p, 1H); MS(ES): 344.2 (M"+1).

The following compounds were obtained in a similar manner to that in Preparation 7: dl-5,6-dimethyl-2-(3-pyridyl)-7H-7-(1-phenylethyl)pyrrolo-[2,3d]pyrimidine-4 (3H)-on. <1>R NMR (200 MHz, CDCl 3 ) 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"+1).

dl-5,6-dimethyl-2-(2-furyl)-7H-7-(1-phenylethyl)pyrrolo-[2,3d]pyrimidin-4(3H)-one. <X>H NMR (200 MHz, CDCl 3 ) δ 1.98

(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~+1).

dl-5,6-dimethyl-2-(3-furyl)-7H-7-(1-phenylethyl)pyrrolo-[2,3d]pyrimidin-4(3H)-one. <1>R NMR (200 MHz, CDCl 3 ) 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"+1).

dl-5,6-dimethyl-2-cyclopentyl-7H-7-(1-phenylethyl)pyrrolo-[2,3d]pyrimidin-4(3H)-one. <1>R NMR (200 MHz, CDCl 3 ) δ 1.95

(d, J= 7.4 Hz, 3H), 2.00 (s, 3H), 2.33 (s, 3H), 1.68-1.88 (m, 8H), 2.97 (m, 1H), 6.10 (q, J = 7.4 Hz, 1H), 7.16-7.30 (m, 5H), 9.29 (s, 1H); MS(ES):336, 3 (M"+1).

dl-5,6-dimethyl-2-(2-thienyl)-7H-7-(1-phenylethyl)pyrrolo-[2,3d]pyrimidin-4(3H)-one. <1>R NMR (200 MHz, CDCl 3 ) δ 2.02

(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" + 1).

dl-5,6-dimethyl-2-(3-thienyl)-7H-7-(1-phenylethyl)pyrrolo-[2,3d]pyrimidin-4(3H)-one. <1>R NMR (200 MHz, CDCl 3 ) δ 2.00

(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" + 1).

dl-5,6-dimethyl-2-(4-fluorophenyl)-7H-7-(1-phenylethyl)pyrrolo-[2,3d]pyrimidin-4(3H)-one. <1>R NMR (200 MHz, CDCl 3 ) δ 2.01

(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"+1).

dl-5,6-dimethyl-2-(3-fluorophenyl)-7H-7-(1-phenylethyl)pyrrolo-[2,3d]pyrimidin-4(3H)-one. <1>R NMR (200 MHz, CDCl 3 ) δ 2.02

(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"+1).

dl-5,6-dimethyl-2-(2-fluorophenyl)-7H-7-(1-phenylethyl)pyrrolo-[2,3d]pyrimidin-4(3H)-one. <1>R NMR (200 MHz, CDCl 3 ) δ 2.00

(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, 8H), 8.21 (m, 1H), 9.54 (s, 1H); MS(ES): 362.2 (M"+1).

dl-5,6-dimethyl-2-isopropyl-7H-7-(1-phenylethyl)pyrrolo-[2,3d]pyrimidin-4(3H)-one. <1>R NMR (200 MHz, CDCl 3 ) 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"+1).

Preparation 8:

A solution of dl-1-(1-phenylethyl)-benzoylamino-3-cyano-4-dimethylpyrrole (785 mg, 2.38 mmol) with concentrated H 2 SO 4 (1 mL) in DMF (13 mL) was stirred at 130 °C for 48 hours. The black mixture was diluted with CHCl 3 (100 mL) and washed with 1 N NaOH (30 mL), and brine (30 mL). The organic fraction was dried, filtered, and concentrated and purified by flash chromatography (SiO 2 ; 8/2 EtOAc/hex, Rf 0.35) to a brown solid (184 mg, 24%) as dl-5-methyl-2- phenyl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4-(3H)-one. <1>R NMR (200 MHz, CDCl 3 ) 5_8.18 (m, 2H, Ar-H), 7.62-7.44 (m, 3H, Ar-H), 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 -CH 3 ), 2.07 (d, 3H, J = 7.2 Hz, CH-CH 3 ); MS(ES): 330.2 (M"+1).

Preparation 9:

A mixture of dl-1-(1-phenylethyl)-2-amino-3-cyano-4,5-dimethylpyrrole (9.60 g, 40.0 mmol) and formic acid (50.0 mL, 98% ) was boiled under reflux for 5 hours. After cooling to room temperature and scraping the sides of the bottle, large amounts of precipitate formed and were filtered. The material was washed with water until washings showed neutral pH to give dl-5,6-dimethyl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3H)-one. <1>H NMR (200 MHz, CDCl 3 ) δ 1.96 (d, J = 7.4 hz, 3H), 2.00 (s, 3H), 2.38 (s, 3H), 6.21 ( q, 7 = 7.4 Hz, 1H), 7.11-7.35 (m, 5H), 7.81 (s, 1H), 11.71 (s, 1H);

MS (ES): 268.2 (M"+1).

Preparation 10: dl-5,6-dimethyl-2-phenyl-7H-7-(1-phenylethyl)pyrrolo[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 hours. The hot suspension was poured into ice water, stirred vigorously to disperse the 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-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. <X>H NMR (200 MHz, DMSO-de) 6_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" + 1) •

The following compounds were obtained in a similar manner to that in Preparation 10: 5-methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. MS(ES):226.0 (M"+1).

5,6-dimethyl-2-(3-pyridyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. MS (ES): 241.1 (M"+1).

5,6-dimethyl-2-(2-furyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. <X>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"+1).

5,6-dimethyl-2-(3-furyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. <1>H NMR (200 MHz, DMSO-d6) 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~+1).

5,6-dimethyl-2-cyclopentyl-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. <1>R NMR (200 MHz, DMSO-d6) δ 1.57-1.91 (m, 8H), 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"+1).

5,6-dimethyl-2-(2-thienyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. <X>H 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~ + 1) •

5,6-dimethyl-2-(3-thienyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. <1>R 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"+1).

5,6-dimethyl-2-(4-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. <1>R NMR (200 MHz, DMSO-d6) δ 2.17 (s, 3H) , 2.21

(s, 3H), 7.31 (m, 2H), 8.12 (m, 2H), 11.47 (m, 1H); MS(ES):258.2 (M"+1).

5,6-dimethyl-2-(3-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. <1>R 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"+1).

5,6-dimethyl-2-(2-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. <1>R 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"+1).

5,6-dimethyl-2-isopropyl-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. <1>R NMR (200 MHz, DMSO-d6) δ 1.17 (d, J = 6.6 Hz, 6H), 2.11 (s, 3H), 2.51 (s, 3H), 2, 81 (m, 1H), 11.20 (s, 1H), 11.39 (s, 1H); MS(ES):206, 1 (M"+1).

5,6-dimethyl-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. <1>H NMR (200 MHz, DMSO-d6) δ 2.13 (s, 3H), 2.17 (s, 3H), 7.65 (s, 1H); MS(ES): 164.0 (M"+1).

Production 11:

A solution of 5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one (1.0 g, 4.2 mmol) in phosphorus oxychloride (25.0 mL) was boiled under reflux for 6 hours and then concentrated in vacuo to dryness. Water was added to the residue to induce crystallization and the resulting solid was filtered and collected to give 0.90 g (83%) of 4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d ]pyrimidine. <1>H NMR (200 MHz, DMSO-d6) 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~ + 1) •

The following compounds were obtained in a similar manner to that of Preparation 11:

4-chloro-5-methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

MS (ES): 244.0 (M"+1).

4-chloro-6-methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS(ES):244.0 (M"+1).

4-chloro-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, DMSO-de) 8.35 (2, 2H) , 7.63 (br s, 1H) , 7.45 (m, 3H) , 6.47 (br s, 1H) ; MS (ES): 230.0 (M"+1).

4-chloro-5,6-dimethyl-2-(3-pyridyl)-7H-pyrrolo[2,3d]pyrimidine. MS(ES):259.0 (M"+1).

4-Chloro-5,6-dimethyl-2-(2-furyl)-7H-pyrrolo[2,3d]pyrimidine. <X>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~ + 1) •

4-Chloro-5,6-dimethyl-2-(3-furyl)-7H-pyrrolo[2,3d]pyrimidine. <X>H NMR (200 MHz, DMSO-d6) 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"+1).

4-chloro-5,6-dimethyl-2-cyclopentyl-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, DMSO-d6) δ 1.61-1.96 (m, 8H), 2.27 (s, 3H), 2.27 (s, 3H), 3.22 (m , 1H), 11.97 (s, 1H); MS (ES): 250.1 (M"+1).

4-chloro-5,6-dimethyl-2-(2-thienyl)-7H-pyrrolo[2,3d]pyrimidine. <X>H NMR (200 MHz, DMSO-d6) 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"+1).

4-chloro-5,6-dimethyl-2-(3-thienyl)-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, DMSO-d6) δ 2.32 (s, 3H) , 2.32 (s, 3H) , 7.62 (dd, J = 3.0 Hz, 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~+1).

4-Chloro-5,6-dimethyl-2-(4-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidine. <X>H NMR (200 MHz, DMSO-d6) δ 2.33 (s, 3H), 2.33 (s, 3H), 7.30 (m, 2H), 8.34 (m, 2H), 12.11 (p, 1H); MS (ES): 276.1 (M"+1).

4-chloro-5,6-dimethyl-2-(3-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidine. <X>H 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"+1).

4-Chloro-5,6-dimethyl-2-(2-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidine. <X>H 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) -. 216, 1 (M"+l) .

4-chloro-5,6-dimethyl-2-isopropyl-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, DMSO-d6) 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"+1).

4-chloro-5,6-dimethyl-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, DMSO-d6) δ 2.31 (s, 3H), 2.32 (s, 3H), 8.40 (s, 1H); MS(ES): 182.0 (M"+1). dl-4-chloro-5,6-dimethyl-2-phenyl-7H-7-(1-phenylethyl)pyrrolo-[2,3d]pyrimidine.

Production 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 (100.0 mL) and water (100.0 mL ) was added di-tert-dicarbonate (4.80 g, 22.0 mmol) at room temperature. The resulting mixture was stirred for 14 hours. 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. The filtrate was concentrated in vacuo to dryness to give a mixture of dl-1-amino-2-(1,1-dimethylethoxy)carbonylaminopropane and dl-2-amino-1-(1,1-dimethylethoxy)carbonylaminopropane which could not -separated with normal chromatography methods. The mixture was used for the reaction of Example 8.

Production 13:

To a solution of Fmoc-(3-Ala-OH (1.0 g, 3.212 mmol) and oxalyl chloride (0.428 g, 0.29 mL, 3.373 mmol) in dichloromethane (20.0 mL) was added a few drops of N ,N-dimethylformamide at 0° C. The mixture was stirred at room temperature for 1 h followed by the addition of cyclopropylmethylamine (0.229 g, 0.28 mL, 3.212 mmol) and triethylamine (0.65 g, 0.90 mL, 6.424 mmol) After 10 minutes, the mixture was treated with 1 M hydrochloride (10.0 mL) and the aqueous mixture was extracted with dichloromethane (3 x 30.0 mL). The organic solution was concentrated in vacuo to dryness. The residue was treated with a solution of 20% piperidine in N,N-dimethylformamide (20.0 mL) for half an hour. After removing the solvent in vacuo, the residue was treated with 1 M hydrochloride (20.0 mL) and ethyl acetate (20, 0 ml).The mixture was separated and the aqueous 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 with 20% pyridine to give 0.262 g (57%) of N-cyclopropylmethyl (3-alanine amide. <X>H NMR (200 MHz, CD3OD) 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"+1).

Preparation 14: N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine.

trans-1,4-cyclohexyldiamine (6.08 g, 53.2 mmol) was dissolved in dichloromethane (100 mL). A solution of di-t-butyl dicarbonate (2.32 g, 10.65 mmol in 40 mL dichloromethane) was added via cannula. After 20 hours, the reaction mixture was partitioned between CHCl 3 and water. The layers were separated and the aqueous layer was extracted with CHCl 3 (3x). The combined organic layers were dried over MgSO4, filtered and concentrated to give 1.20 g of a white solid (53%). <1>R NMR (200 MHz,

CDCl3) 5 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"+1).

4-(N-acetyl)-N-tert-butoxycarbonyl-trans-1,4-cyclohexyl-diamine.

N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine (530 mg, 2.47 mmol) was dissolved in dichloromethane (20 mL). Acetic anhydride (250 mg, 2.60 mmol) was added dropwise. After 16 hours, the reaction was diluted with water and CHCl 3 . The layers were separated and the aqueous layer was extracted with CHCl 3 (3x). The combined organic layers were dried over MgSO 4 , filtered and concentrated. Recrystallization (EtOH/H 2 O) gave 190 mg of white crystals (30%). <X>H NMR (200 MHz, CDCl 3 ) 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): 257.1 (M"+1), 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-cyclohexyl-diamine (190 mg, 0.74 mmol), was dissolved in dichloromethane (5 mL) and diluted with TFA (6 mL). After 16 hours, the reaction mixture was concentrated. The crude solid DMSO (2 mL), NaHCO 3 (200 mg, 2.2 mmol) and 4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (35 mg, 0.14 mmol) were combined in a bottle and heated to 130 °C. After 4.5 h, the reaction mixture was cooled to room temperature and diluted with EtOAc and water. The layers were separated and the aqueous layer was extracted with EtOAc (3x). The combined organic layers were dried over MgSO 4 , filtered and concentrated. Chromatography (preparative silica plate; 20:1 CHCl 3 :EtOH) gave 0.3 mg of a light brown solid (1% yield). MS(ES):378.2 (M"+1),

4-(N-methanesulfonyl)-N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine.

trans-1,4-cyclohexyldiamine (530 mg, 2.47 mmol) was dissolved in dichloromethane (20 mL) and diluted with pyridine (233 mg, 3.0 mmol). Methanesulfonyl chloride (300 mg, 2.60 mmol) was added dropwise. After 16 hours, the reaction mixture was diluted with water and CHCl 3 . The layers were separated and the aqueous layer was extracted with CHCl 3 (3x). The combined organic layers were dried over MgSO4, filtered and concentrated. Recrystallization (EtOH/H 2 O) gave 206 mg of white crystals (29%).

<1>R NMR (200 MHz, CDCl 3 ) 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"+1), 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 in dichloromethane (5 mL) and diluted with TFA (6 mL). After 16 hours, the reaction mixture was concentrated. The crude reaction mixture DMSO (2 mL), NaHCO 3 (100 mg, 1.1 mmol) and 1-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine were combined in a flask and heated to 130 °C. After 15 hours, the reaction mixture was cooled to room temperature and diluted with EtOAc (3x). The combined organic layers were dried over MgSO 0 , filtered and concentrated. Chromatography (preparative silica plate, 20:1 CHCl 3 /EtOH) gave 2.6 mg of a light brown solid (5% yield). MS(ES): 414.2 (M"+1).

Example 1:

A solution of 4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (0.50 g, 1.94 mmol) and 4-trans-hydroxy-cyclohexylamine (2, 23 g, 19.4 mmol) in methyl sulfoxide (10.0 mL) was heated to 130 °C for 5 h. After cooling to room temperature, water (10.0 mL) was added and the resulting aqueous solution was extracted into EtOAc (3 x 10.0 mL). The combined EtOAc solution was dried (MgSO 4 ) and filtered, the filtrate was concentrated in vacuo to dryness, the residue was chromatographed on silica gel to give 0.49 g (75%) of 4-(4-trans-hydroxycyclohexyl)amino-5 ,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. m.p. 197-199 °C; <X>H NMR (200 MHz, CDCl 3 ) 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"+1).

The following compounds were obtained in a similar manner to that of Example 1: 4-(4-trans-hydroxycyclohexyl)amino-6-methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <X>H NMR (200 MHz, CDCl 3 ) 6_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, CH 3 ) 1.68-1.20 (m, 4H); MS(ES):323.2 (M~ + 1) •

4-(4-trans-hydroxycyclohexyl)amino-5-methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) 5_11.37 (s, 1H, pyrrole-NH), 8.40 (m, 2H, Ar-H), 7.45 (m, 3H, Ar-H), 5.96 (s, 1H, pyrrole-H), 4.90 (br d, 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, CH 3 ) 1.68-1.20 (m, 4H); MS(ES):323.2 (M~ + 1) •

4-(4-trans-hydroxycyclohexyl)amino-2-2-phenyl-7H-pyrrolo-[2,3d]pyrimidine. m.p. 245.5-246.5 °C; <1>H NMR (200 MHz, CD3OD) δ 8.33 (m, 2H, Ar-H), 7.42 (m, 3H, Ar-H), 7.02 (d, 1H, J = 3, 6 Hz, pyrrole-H), 6.53 (d, 1H, J = 3.6 Hz, pyrrole-H), 4.26 (m, 1H, CH-O), 3.62 (m, 1H, CH -N), 2.30-2.12 (m, 2H), 2.12-1.96 (m, 2H), 1.64-1.34 (m, 4H); MS, M+1 = 309.3; Anal (Ci8H2oN40) C, H, N.

4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(3-pyridyl)-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) 6_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"+1).

4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(2-furyl)-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) 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"+1).

4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(3-furyl)-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) δ 1.25-1.63 (m, 8H), 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"+1).

4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-cyclopentyl-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) δ 1.26-2.04 (m, 16H), 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~+1).

4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(2-thienyl)-7H-pyrrolo[2,3d]pyrimidin-4-amine. <X>R NMR (200 MHz, CDCl 3 ) δ 1.28-1.59 (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~ +1) •

4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(3-thienyl)-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) 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), 11.23 (s, 1H); MS(ES): 343.2 (M"+1).

4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(4-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) 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"+1).

4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(3-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) 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"+1).

4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(2-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidine. <X>H NMR (200 MHz, CDCl 3 ) 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 (p, 1H) ; MS(ES):355, 3 (M"+1).

4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-isopropyl-7H-pyrrolo[2,3d]pyrimidine. <1>H NMR (200 MHz, CDCl 3 ) 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 (M"+1).

dl-4-(2-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-iso-propyl-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) δ 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 (m, 3H), 8.30 ( m, 2H), 10.08 (s, 1H); MS(ES):337.2 (M~ + 1) • 4-(3,4-trans-dihydroxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS(ES): 353.2 (M"+1). 4-(3,4-cis-dihydroxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS(ES): 353.2 (M"+1). 4-(2-acetylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo-[2,3d]pyrimidine. m.p. 196-199 °C; <1>R NMR (200 MHz, CDCl 3 ) 6_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, 2H), 10.76 ( s, 1H); MS(ES):324.5 (M"+1). dl-4-(2-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <1 > <1>R NMR (200 MHz, CDCl 3 ) 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, 1H), 7.41-7.49 (m, 3H), 8.22 (m, 2H), 10.51 ( s, 1H); MS(ES):323.2 (M"+1). <1> For the preparation of 2-trans-hydroxycyclopentylamine, see PCT 9417090.

dl-4-(3-trans-hydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.<1>

<X>H NMR (200 MHz, CDCl 3 ) δ_1.58-1.90 (br, 6H), 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 (M"+1). 1 For the preparation of 3-trans-hydroxycyclopentylamine, see EP-A-322242. dl-4-(3-cis-hydroxycyclopentyl)amino-5,6-dimethyl -2-phenyl-7H-pyrrolo[2,3d]pyrimidine 1 <1>R NMR (200 MHz, CDCl 3 ) 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"+1). <1> For the preparation of 3-cis-hydroxycyclopentylamine, see EP-A-322242. 4-(3,4-trans-dihydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.<1 > <1>H NMR (200 MHz, CDCl 3 ) 6_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, 1H), 8.38-8 .42 (m, 2H), 9.53 (s, 1H); MS(ES):339.2 (M"+1). 1 For the preparation of 3,4-trans-dihydroxycyclopentylamine, see PCT 9417090.

4-(3-amino-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <1>H NMR (200 MHz, CDCl 3 ) 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"+1).

4-(3-N-cyclopropylmethylamino-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <X>H 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"+1).

4-(2-amino-2-oxoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <1>H 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"+1).

4-(2-N-methylamino-2-oxoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <X>H NMR (200 MHz, CDCl 3 ) 6_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"+1).

4-(3-tert-butyloxyl-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <1>H NMR (200 MHz, CDCl 3 ) 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"+1).

4-(2-Hydroxyethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo-[2,3d]pyrimidine. <X>H NMR (200 MHz, CDCl 3 ) 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"+1).

4-(3-Hydroxypropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo-[2,3d]pyrimidine. <1>H NMR (200 MHz, CDCl 3 ) 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), 10.72 (s, 1H); MS (ES): 297.2 (M"+1).

4-(4-Hydroxybutyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo-[2,3d]pyrimidine. <1>H NMR (200 MHz, CDCl 3 ) 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"+1).

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.

4-(2-acetylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidine.

4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-(1-phenylethyl)pyrrolo[2,3d]pyrimidine.

4-(3-pyridylmethyl)amino-5,6-dimethyl-2-phenyl-7H-7-(1-phenyl-ethyl)pyrrolo[2,3d]pyrimidine.

4-(2-Methylpropyl)amino-5,6-dimethyl-2-phenyl-7H-7-(1-phenyl-ethyl)pyrrolo[2,3d]pyrimidine.

Example 2:

To a stirred suspension of triphenylphosphine (0.047 g, 0.179 mmol) and benzoic acid (0.022 g, 0.179 mmol) in THF (1.0 mL) cooled to 0 °C was added 4-(4-trans-hydroxycyclohexyl)amino -5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (0.05 g, 0.149 mmol) at 0 °C. Diethyl azodicarboxylate (0.028 mL, 0.179 mmol) was then added dropwise over 10 minutes. The reaction mixture was then allowed to warm to room temperature. After the reaction was complete in TCL, the reaction mixture was quenched with aqueous sodium bicarbonate (3.0 mL). The aqueous phase was separated and extracted with ether (2 x 5.0 mL). The organic extracts were combined, dried and concentrated in vacuo to dryness. Ether (2.0 ml) and hexane (5.0 ml) were added to the residue, whereupon most of the triphenylphosphine oxide was filtered off. Concentration of the filtrate gave a viscous oil which was purified by column chromatography (hexane:ethyl acetate = 4:1) to give 5.0 mg (7.6%) of 4-(4-cis-benzoyloxycyclohexyl)amino-5,6 -dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS (ES): 441.3 (M~+1). The reaction mixture also produced 50.0 mg (84%) of 4-(3-cyclohexenyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS (ES): 319.2 (M"+1).

Example 3:

To a solution of 4-(4-cis-benzoyloxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (5.0 mg, 0.0114 mmol) in ethanol ( 1.0 ml) 10 drops of 2M sodium hydroxide were added. After 1 hour, the reaction mixture was extracted with ethyl acetate (3 x 5.0 mL) and the organic layer was dried, filtered and concentrated in vacuo to dryness. The residue was subjected to column chromatography (hexane:ethyl acetate = 4:1) to give 3.6 mg (94%) of 4-(4-cis-hydroxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H- pyrrolo[2,3d]pyrimidine. MS (ES): 337.2 (M"+1).

The following compounds were obtained in a similar manner to that of Example 3: 4-(3-N,N-dimethyl-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. <1>H NMR (200 MHz, CDCl 3 ) 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"+1).

4-(2-Formylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) 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"+1).

4-(3-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS (ES): 338.2 (M"+1).

Example 4

4-(3-tert-butyloxy-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (70.0 mg, 0.191 mmol)) was dissolved in trifluoroacetic acid:dichloromethane (1:1, 5.0 ml). The resulting solution was stirred at room temperature for 1 hour and then refluxed for 2 hours. After cooling to room temperature, the mixture was concentrated in vacuo to dryness. The residue was subjected to preparative 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-7H-pyrrolo[2,3d]pyrimidine. <X>H NMR (200 MHz,CD2OD) 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+1).

The following compound was obtained in a similar manner to that of Example 4.

4-(3-aminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo-[2,3d]pyrimidine. MS (ES): 296.1 (M+1), 279.1 (M-NH 3 ).

Example 5

4-(3-Hydroxy-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (50.0 mg, 0.161 mmol) was dissolved in a mixture of N,N- dimethylformamide (0.50 ml), dioxane (0.50 ml) and water (0.25 ml). To this solution was added methylamine (0.02 ml, 40% by weight in water, 0.242 mmol). Triethylamine (0.085 mL) and N,N,N',N'-tetramethyl uronium tetrafluoroborate (61.2 mg, 0.203 mmol). After stirring at room temperature for 10 min, the solution was concentrated and the residue was subjected to preparative thin layer chromatography (EtOAc) to give 35.0 mg (67%) of 4-(3-N-methyl-3-oxopropyl)amino-5, 6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) 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 )ES): 311.1 (M+1).

The following compounds were prepared in a similar manner to that of Example 5.

4-(2-cyclopropanecarbonylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS (ES): 352.2 (M+1).

4-(3-propionylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) δ 1.00-1.08 (t, 3H), 1.71-2.03 (m, 4H), 2.08 (s, 3H), 2.37 (s, 3H), 3.26-3.40 (m, 2H), 3.79-3.96 (m, 2H), 5.43-5.62 (m, 1H), Jo,17-6 .33 (m, 1H), 7.33-7.57 (m, 3H), 8.31-8.39 (m, 2H), 9.69 (S, 1H); MS (ES): 352.2 (M+1).

4-(2-methylsulfonylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) δ 2.18 (s, 3H), 2.27 (s, 3H), 2.92 (s, 3H), 3.39-3.53 (m, 2H ), 2.71-3.88 (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+1).

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 (10.0 mL, 150 mmol) was refluxed under an inert atmosphere for 6 hours. Excess amine was removed in vacuo, the residue was washed sequentially with ether and hexane to give 0.75 g (98%) of 4-(2-aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[ 2,3d]pyrimidine. MS (ES); 282.2 (M+1), 265.1 (M-NH 3 ).

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, 0.274 mmol) at 0°C. After 1 hour, the mixture was concentrated in vacuo and the residue subjected to preparative thin layer chromatography (EtOAc) to give 22.0 mg (26%) of 4-(2-propionylaminoethyl)amino-5,6-dimethyl-2- phenyl-7H-pyrrolo[2,3d]-pyrimidine. MS (ES): 338.2 (M+1).

The following compounds were prepared in a similar manner to that of Example 7: 4-(2-N'-methylureaethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo-[2,3d]pyrimidine. <X>H NMR (200 MHz, CDCl 3 ) 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+1).

4-(2-N'-ethylureaethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo-[2,3d]pyrimidine. MS (ES): 353.2 (M+1).

Example 8:

To a solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (41.1 mg, 0.215 mmol), dimethylaminopyridine (2.4 mg, 0.020 mmol) and tartaric acid (18.9 mg, 0.015 mL) , 0.215 mmol) in dichloromethane (2.0 ml) was added 4-(2-aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]-pyrimidine (55.0 mg, 0.196 mmol). The mixture was stirred at room temperature for 4 hours. General workup and column chromatography (EtOAc) then gave 10.0 mg (15%) of 4-(2'-pyruvyl-amidoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine . MS (ES): 352.2 (M+1).

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) in dichloromethane (2.0 mL) was added N-trimethylsilyl isocyanate (43.3 mg, 0.051 mL, 0.320 mmol) was added. The mixture was stirred at room temperature for 3 hours followed by the addition of aqueous sodium bicarbonate. After filtration through a small amount of silica gel, the filtrate was concentrated in vacuo to 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+1).

The following compounds were prepared in a similar manner to that of Example 9: dl-4-(2-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) δ 1.28-1.32 (d, J=8 Hz, 3H), 1.66 (s, 3H), 1.96 (s, 3H), 2, 30 (s, 3H), 3.78-3.83 (m, 2H), 4.10-4.30 (m, 1H), 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+1).

(R)-4-(2-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) 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+1).

(R)-4-(1-methyl-2-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <1>H NMR (200 MHz, CDCl 3 ) 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+1).

(S)-4-(2-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) 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+1).

(R)-4-(1-methyl-2-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <1>H NMR (200 MHz, CDCl 3 ) δ 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, 1H), 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+1).

Example 10:

Reaction of 4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]-pyrimidine by the mixture of dl-1-amino-2-(1,1-dimethylethoxy)carbonylamino-propane and dl-2-amino-1-(1,1-dimethyl-thoxy)carbonylamino-propane was carried out in a manner similar to that 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 was separated by column chromatography (EtOAc:hexanes=1:3). The first fraction was dl-4-(1-methyl-2-(1,1-dimethylethoxy)carbonylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <X>H NMR (200 MHz, CDCl 3 ) δ 1.29-1.38 (m, 12H), 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+1). The second fraction was dl-4-(2-(1,1-dimethylethoxy)carbonylamino-propyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) δ 1.26-1.40 (m, 12H), 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+1).

The following compounds were prepared in a similar manner to that of Example 10: (S,S)-4-(2-acetylaminocyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine . <1>H NMR (200 MHz, CDCl 3 ) δ 1.43 (m, 4H), 1.60 (s, 3H), 1.83 (s, 2H), 2.18 (s, 3H), 2, 30 (m, 2H), 2.32 (s, 3H), 3.73 (br, 1H), 4.25 (br, 1H), 5.29 (d,lH), 7.43-7.48 (m,3H), 8.35-8.40 (m,2H), 9.05 (s,1H).

4-(2-Methyl-2-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) 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 (s, 1H), 8.39 (m, 2H), 10.07 (S,1H); MS (ES): 352.3 (M+1).

Example 11: dl-4-(1-methyl-2-(1,1-dimethylethoxy)carbonylaminoethyl)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 h. 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 was added at 0°C acetic anhydride (17.2 mg, 0.016, 0.169 mmol). The resulting mixture was stirred at room temperature for 48 hours and then concentrated in vacuo to dryness. The residue was subjected to preparative thin layer chromatography (EtOAc) to give 27.0 mg (52%) of dl-4-(1-methyl-2-acetylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[ 2,3d]pyrimidine. <X>H NMR (200 MHz, CDCl 3 ) δ 1.381.42 (d, J=8 Hz, 3H), 1.69 (s, 3H), 2.01 (s, 3H), 2.32 (s, 3H), 3.38-3.60(m, 2H), 4.65-4.80 (m, 1H), 5.43-5.26 (d, J=8 Hz, 1H), 7.40 -7.51 (m, 3H), 8.37-8.43 (m, 2H), 10.44 (S, 1H); MS (ES): 338.2 (M+1).

Example 12: (R,R)-4-(2-aminocyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine, prepared in a similar manner to that in Example 1 from 4- chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo-[2,3d]pyrimidine (0.15 g, 0.583 mmol) and (IR,2R)-(-)-1,2-diaminocyclohexane (0, 63 g, 5.517 mmol), was treated with triethylamine (0.726 g, 7.175 mmol) and acetic anhydride (0.325 g, 3.18 mmol) in N,N-dimethylformamide (10.0 mL) at room temperature for 2 hours. After removal of the solvent in vacuo, ethyl acetate (10.0 mL) and water (10.0 mL) were added to the residue. The mixture was separated and the aqueous layer was extracted with ethyl acetate (2 x 10.0 mL). The combined ethyl acetate solution was dried (MgSO 4 ) and filtered. The filtrate was concentrated in vacuo to dryness and the residue subjected to column chromatography (EtOAc; hexane=1:1) to give 57.0 mg (26%) of (R,R)-4-(2-acetylamino-cyclohexyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo-[2,3d]pyrimidine. <1>R NMR (200 MHz, CDCl 3 ) δ_1.43 (m, 4H), 1.60 (s, 3H), 1.84 (m, 2H), 2.22 (s, 3H), 2.30 (m, 2H), 2.33 (s, 3H), 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,1H); MS (ES): 378.3 (M+1).

Example 13:

To a solution of 4-(2-hydroxyethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (40.0 mg, 0.141 mmol) in pyridine (1.0 mL) was added acetic anhydride (0.108 g, 1.06 mmol) at 0°C was added. The mixture was stirred at room temperature for 4 hours and the solvent was removed in vacuo. The residue was subjected to preparative thin layer chromatography (EtOAc:hexane=1:1) to give 32.2 mg (71%) of 4-(2-acetyloxyethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo- [2,3d]pyrimidine. <1>H NMR (200 MHz, CDCl 3 ) 6_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-(3-Ala-OH (97.4 mg, 0.313 mmol) and oxalyl chloride (39.7 mg, 27.3 µl, 0.313 mmol) in dichloromethane (4.0 mL) with 1 drop of N, N-Dimethylformamide was stirred at 0°C for 1 hour followed by the addition of 4-(2-aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (80.0 mg, 0.285 mmol) and triethylamine (57.6 mg, 79.4 µl, 0.570 mmol) at 0° C. After 3 hours the mixture was concentrated in vacuo and the residue was treated with a solution of 20% piperidine in N,N-dimethylformamide (2.0 mL) for 0.5 h.After removal of the solvent in vacuo, the residue was washed with diethyl ether:hexane (1:5) to give 3.0 mg (3%) of 4-(6-amino -3-aza-4-oxohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine MS (ES): 353.2 (M+1).

Example 15:

A solution of 4-(2-aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (70.0 mg, 0.249 mmol) and succinic anhydride (27.0 mg, 0.274 mmol) in dichloromethane (4.0 mL) with a drop of N,N-dimethylformamide was stirred at room temperature for 4 h. The reaction mixture was extracted with 20% sodium hydroxide (3 x 5.0 mL). The aqueous solution was acidified with 3 M hydrochloric acid to give pH = 7.0. The whole mixture was extracted with ethyl acetate (3 x 10 mL). The combined organic solution was dried (MgSO 4 ) and filtered. The filtrate was concentrated in vacuo to dryness to give 15.0 mg (16%) of 4-(7-hydroxy-3-3aza-4,7-dioxoheptyl)amino-5,6-dimethyl-2-phenyl- 7H-pyrrolo[2,3d]pyrimidine. MS (ES: 382.2 (M+1).

Example 16:

To 10 ml of dimethylformamide (DMF) at room temperature was added 700 mg of 4-cis-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-dimethylamino-propyl)-3-ethylcarboimide hydrochloride (EDC1). The reaction mixture was kept under stirring overnight. The DMF was then removed under reduced pressure and the reaction mixture was partitioned between 20 mL ethyl acetate and 50 mL water. The aqueous portion was further extracted with 2 x 20 mL ethyl acetate and the combined organic portions were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. Purification on silica gel, elution with ethyl acetate/hexane gave 410 mg of the desired product: 4-(cis-2-(N-t-butoxycarbonyl-2-aminoacetoxy)cyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d]pyrimidine. MS (ES)

(M+1)=480.2. The ester was then treated with 5 ml of 20% trifluoroacetic acid in dichloromethane at room temperature, kept overnight and then concentrated. Trituration (pulverization) with ethyl acetate gave 300 mg of an off-white solid; 4-(cis-3-(2-aminoacetoxy)cyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine trifluoroacetic acid salt, MS (ES) (M+1)=380, l.

The person skilled in the art will appreciate that the following compounds can be synthesized by the methods described above:

4-(cis-3-Hydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine MS (ES) (M+1)=323.1. 4-(cis-3-(2(aminoacetoxy)cyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine trifluoroacetic acid salt, MS (ES) (M+1)=380, 1. 4-(3-acetamide)piperidinyl-5,6-dimethyl-2-phenyl-7H-pyrrolo-[2,3d]pyrimidine MS (ES) (M+1)=364.2. 4-2(2 -N'-methylureapropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine MS (ES) (M+1)=353.4 4-(2-acetamidebutyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo-[2,3d]pyrimidine MS (ES) (M+1)=352.4 4-(2-N'-methylureabutyl)amino-5, 6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine MS (ES) (M+1)=367.5 4-(2-aminocyclopropylacetamideethyl)amino-5,6-dimethyl-2- phenyl-7H-pyrrolo[2,3d]pyrimidine MS (ES) (M+1)=309.1 4-(trans-4-hydroxycyclohexyl)amino-2-(3-chlorophenyl)-7H-pyrrolo[ 2,3d]pyrimidine MS (ES) (M+1)=342.8 4-(trans-4-hydroxycyclohexyl)amino-2-(3-fluorophenyl)-7H-pyrrolo [2,3d]pyrimidine MS (ES ) (M+1)=327.2 4-(trans-4-hydroxycyclohexyl)amino-2-(4-pyridyl)-7H-pyrrolo[2,3d]pyrimidine MS (ES) (M+1) =310.2 Example 17

The pyrrole nitrogen of (7) (Scheme IX) was protected with di-t-butyl dicarbonate under basic conditions to give the corresponding carbamate (22). Radical bromination of (22) proceeded regioselectively to give bromide (23). In general, compound (23) acted as a key electrophilic intermediate for various nucleophilic coupling partners. Replacement of the alkyl bromide with sodium phenolate trihydrate gave compound (24). Subsequent replacement of the aryl chloride and removal of the t-butyl carbamate protecting group occurred in one step to give the desired compound (25).

Detailed synthesis of compound (22)-(25) according to Scheme IX

Di-t-butyl dicarbonate (5.37 g, 24.6 mmol) and dimethylaminopyridine (1.13 g, 9.2 mmol) were added to a solution containing (7) (1.50 g, 6.15 mmol ) and pyridine (30 mL). After 20 hours, the reaction mixture was concentrated and the residue was partitioned between CH 2 Cl 2 and water. The CH 2 Cl 2 layer was separated, dried over MgSO 4 , filtered and concentrated to give a black solid. Flash chromatography (SiO 2 ; 1/9 EtOAc/hexanes, Rf 0.40) gave 1.70 g (80%) of a white solid (22). <X>H NMR (200 MHz, CDCl 3 ) 5_8.50 (m, 2H, Ar-H), 7.45 (m, 3H, Ar-H), 6.39 (s, 1H, pyrrole-H), 2.66 (s, 3H, pyrrole-CH 3 ), 1.76 (s, 9H, carbamate-CH 3 ), MS, M+1=344.1; Mpt=175-177°C.

N-Bromosuccinimide (508 mg, 2.86 mmol) and AIBN (112 mg, 0.68 mmol) were added to a solution containing (22) (935 mg, 2.71 mmol) and CCl 4 (50 mL). The solution was heated to reflux. After 2 hours, the reaction mixture was cooled to room temperature and concentrated in vacuo to give a white solid. Flash chromatography (SiO 2 ; 1/1 CH 2 Cl 2 /hexanes, Rf 0.30) gave 960 mg (84%) of a white solid (23). <X>H NMR (200 MHz, CDCl 3 ) δ 8.52 (m, 2H, Ar-H), 7.48 (m, 3H, Ar-H), 6.76 (s, 1H, pyrrole-H), 4.93 (s, 2H, pyrrole-CH 2 Br), 1.79 (s, 9H, carbamate-CH 3 ), MS, M+1=423, 9; Mpt=155-157°C.

Sodium phenoxide trihydrate (173 mg, 1.02 mmol) was added in one portion to a solution of bromide (23) (410 mg, 0.97 mmol) dissolved in CH 2 Cl 2 (5 mL) and DMF (10 mL). After 2 hours, the reaction solution was partitioned between CH 2 Cl 2 and water. The aqueous layer was extracted with CH 2 Cl 2 . The combined CH 2 Cl 2 layers were washed with water, dried over MgSO 4 , filtered and concentrated to give a yellow solid. Flash chromatography (SiO 2 ; 1/6 EtOAc/hexanes, Rf 0.30) gave 210 mg (50%) of a white solid (21). <X>H NMR (200 MHz, CDCl 3 ) 5_8.53 (m, 2H, Ar-H), 7.48 (m, 3H, Ar-H), 7.34 (m, 2H, Ar-H), 7.03 (s, 3H, Ar-H), 6.83 (s, 1H, pyrrole-H), 5.45 (s, 2H, ArCH 2 O) , 1.76 (s, 9H, carbamate-CH 3 ), MS, M = 436.2.

A solution containing (25) (85 mg, 0.20 mmol), N-acetylethylenediamine (201 mg, 1.95 mmol) and DMSO (3 mL) was heated to 100°C. After 1 hour the temperature was raised to 130°C. After 3 hours, the reaction mixture was cooled to room temperature and partitioned between EtOAc and water. The aqueous layer was extracted with EtOAc (2x). The combined EtOAc layers are washed with water, dried over MgSO 4 , filtered and concentrated. Flash chromatography (SiO 2 ; 1/10 EtOH/CHCl 3 , Rf, 0.25) gave 73 mg (93%) of a white foamy solid (25). <1>R NMR (200 MHz, d6-DMSO) 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, ArCH2O) , 3.61 (m, 2H, NCH2) , 3.36 (m, 2H, NCH2) , 1.79 (s, 3H , COCH3) ; MS, M + 1 = 402, 6.

The following compounds were prepared in a manner similar to that of Example 17: 4-(2-acetylaminoethyl)amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. m.p. 196-197 °C; MS(ES): 401.6 (M"+1).

4-(2-acetylaminoethyl)amino-6-(4-fluorophenoxy)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS(ES): 420.1 (M++1).

4-(2-acetylaminoethyl)amino-6-(4-chlorophenoxy)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS(ES): 436, 1 (M++1).

4-(2-acetylaminoethyl)amino-6-(4-methoxyphenoxy)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS(ES): 432.1 (M<+>+1).

4-(2-Acetylaminoethyl)amino-6-(N-pyridin-2-one)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS(ES): 403.1 (M<+>+1).

4-(2-acetylaminoethyl)amino-6-(N-phenylamino)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS(ES): 400.9 (M"+1).

4-(2-acetylaminoethyl)amino-6-(N-methyl-N-phenylamino)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS(ES): 414.8 (M"+1).

4-(2-N'-methylureaethyl)amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS(ES): 416.9 (M"+1).

Example 18: Synthesis of Adenosine Ai Antagonists Compound 1319 and Compound 1320 (Table 13 below) can be synthesized by the general procedures herein.

Compound 1319 (81%) <1>H NMR (d 6 -DMSO) δ 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 = 8 Hz, 14 Hz), 8.03 (m, 1H), 8.18 (d, 1H, J = 8 Hz), 11.55 (brs, 1H) . MS(ES):327.0 (M"+1).

Compound 1320 (31%) MS(ES):343.1 (M"+1).

Example 19: Synthesis of Adenosine Ai Antagonist Compound 1321 (Table 13 below) can be synthesized by the general procedures given below.

Compound 28 (10.93 g, 50.76 mmol) was dissolved in DMF (67 mL). 4-Amidinopyridine hydrochloride (8.0 g, 50.76 mmol) and DBU (15.4 g, 101.5 mmol) were added sequentially and the reaction mixture was heated to 85 °C. After 22 hours, the reaction mixture was cooled to room temperature and the DMF was removed in vacuo. The dark oil was diluted with 2M HCl (80 mL). The reaction mixture was allowed to stand. After 2 hours, the solution was cooled to 10 °C and filtered. The solid was washed with cold water and dried to give 7.40 g of a yellow solid, compound 29 (69%). <X>H NMR (200 MHz, d6-DMSO) 6 6.58 (s, 1H), 7.27 (s, 1H), 8.53 (d, 2H, J = 5.6), 9.00 (d, 2H, J = 5.2 Hz), 12.53 (brs, 1H). MS (ES): 212.8 (M"+1).

Compound 29 (7.4 g, 29.8 mmol) was diluted with POCl 3 and heated to 105°. After 18 hours, the reaction mixture was cooled to room temperature and the POCl 3 material was removed in vacuo. The thick dark oil was diluted with MeOH (75 mL) followed by ether (120 mL). The amorphous red solid is filtered and washed with ether to give 3.82 g of a red solid. The impure solid is approximately 80% pure and is used without further purification in the next reaction. MS(ES):230.7 (M++1).

Compound 1321 <1>R NMR (15%) (200 MHz, d6-DMSO) δ 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, 4 Hz), 11.67 (brs, 1H). MS(ES):310.2 (M<+>+1).

Compound 1501 (Table 15 below) <1>H-NMR (70%) (200 MHz, CD3OD) 6 1.84 (s, 3H), 3.52 (t, 2H, J = 6.9 Hz), 3 .83 (t, 2H, J = 6.0 Hz), 6.51 (d, 1H, J = 3.4 Hz), 7.06 (d, 1H, J = 3.8 Hz), 7.42 (m, 3H), 8.36 (m, 2H). MS(ES):296.0 (M++1).

Compound 1502 (Table 15 below) MS(ES):345.0 (M~+1).

Compound 1500 (Table 15 below) <1>H-NMR (200 MHz, CDCl3) 6 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~+1).

Example 20: Synthesis of Adenosine Ai Antagonist Compound 1504 (Table 15 below) can be synthesized by the general procedures given below.

Compound 31 (200 mg, 0.47 mmol) was dissolved in DCM (4 mL). Triethylamine (51 mg, 0.5 mmol) and thiomorpholine (52 mg, 0.5 mmol) were added sequentially. The solution was mixed for several minutes and allowed to stand for 72 hours. The reaction mixture was diluted with DCM and H 2 O and the layers were separated. The aqueous layer was extracted with DCM. The combined DCM layers were dried over MgSO 4 , filtered and concentrated. Ethyl ether was added to the crude sample and the resulting solid was filtered to give 100 mg of a white solid, 32 (62%). <1>H-NMR (200 MHz, CDCl 3 ) δ 1.76 (s, 9H), 2.66 (brs, 2H), 2.79 (brs, 2H), 3.86 (s, 2H), 7 .46 (m, 3H) , 8.50 (m, 2H) .

Compound 32 was combined with DMSO (3 mL) and trans-4-aminocyclohexanol (144 mg, 1.25 mmol) and heated to 130 °C for 4 h. The reaction mixture was cooled to room temperature, and diluted with EtOAc and H 2 O. The layers were separated and the aqueous layer was extracted with EtOAc (2x). The combined organic layers were washed with H 2 O and brine, dried over MgSO 4 , filtered and concentrated. Chromatography (silica, 8:1 CHCl 3 /EtOH) gave 32 mg of a light brown oil. Ethyl ether was added and the resulting solid was filtered to give 5 mg of a white solid (9%). OSIC-148265: <1>H-NMR (200 MHz, CD3OD) : δ 1.44 (m, 6H) , 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) .

Example 21: Synthesis of adenosine Ai antagonist

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

The bromide, compound 31 (220 mg, 0.47 mmol) was dissolved in 1:1 DMF:dichloromethane (5 mL). To this was added K 2 CO 3 (71 mg, 0.52 mmol) and morpholine (0.047 mL, 0.47 mmol). The mixture was allowed to stir at room temperature overnight. Solvents were removed in vacuo and the residue was partitioned between H 2 O and dichloromethane. The organic layer was dried with MgSO 4 , filtered and concentrated to give an off-white solid which on trituration with ether/hexanes gave 175 mg of a white solid, 33 (84%). <1>H-NMR (200 MHz, CDCl 3 ) : δ 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 (2 mL). The resulting solution was sparged with N 2 and then heated to 100 °C in an oil bath and stirred overnight. The coarsening mixture was poured into water and extracted twice with ethyl acetate (50 mL). The combined organic layers were washed with H 2 O. After drying with MgSO 4 and filtration, the organic layer was concentrated in vacuo to give an orange solid, chromatography (silica, 10% CH 3 OH in CH 2 Cl 2 ) gave 15 mg (33%). <1>H-NMR (200 MHz, CDCl 3 ) : δ 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 using similar preparation steps from Example 20 by treating compound 32 with an appropriately substituted amine.

Yeast β-galactosidase reporter gene assays for human adenosine Ai and A2a receptor: Yeast strains (S. cerevisiae) were transformed with human adenosine Ai (AiR; CADUS strain CY12660) or human A2a (A2a; CAUDS strain CY8362) and the addition of a lacZ ((3-galactosidase) reporter gene to be used for a functional readout. A full description of the transformations is listed below (see yeast strains). NECA (5'-N-ethylcarboxamidoadenosine), which is a potent adenosine receptor agonist with similar affinity for A1 and A2a receptors, was used as a ligand for all assays. Test compounds were tested at 8 concentrations (0.1-10,000 nM) for the ability to inhibit NECA-induced (3-galactosidase activity of CY12660 or CY8362 .

Preparation of yeast strain cultures: Each of the respective yeast strains, CY12660 and CY8362, was streaked onto an LT-agar plate and incubated at 30°C until colonies were observed. Yeast from these colonies were added to LT medium (pH 6.8) and grown overnight at 30°C. Each yeast strain was then diluted to an OD600 = 1.0-2.0 (approximately 1-2 x 10<7 >cells/ml), as determined spectrophotometrically (Molecular Devices VMAX). For every 6 ml of liquid yeast culture, 4 ml of 40% glycerol (1:1.5 vol:vol) was added ("yeast/glycerol stock solution"). From this yeast/glycerol stock solution, ten 1 ml aliquots were prepared and stored at -80 °C until used for assay.

Yeast A]R and A2aR assay: One vial each of CY8362 and CY12660 yeast/glycerol stock solution was thawed and used to inoculate supplemented LT liquid medium, pH 6.8 (92 ml LT liquid, to which is added: 5 ml 40% glucose, 0.45 ml IM KOH and 2.5 ml Pipes, pH 6.8). Liquid cultures were grown for 16-18 hours (overnight) at 30°C. Aliquots from overnight cultures were then diluted in LT medium, containing 4U/ml adenosine deaminase (type VI or VII from calf intestinal mucosa, Sigma), to achieve OD60o = 0.15 (1.5 x 10<7 >cells/ml) for CY8362 (A2aR) and OD600 = 0.50 (5 x 10<6 >cells/ml) for CY12660 (AXR).

Assays were performed with a final volume of 100 µl in 96-well microtiter plates, so that a final concentration of 2% DMSO was achieved in all wells. For primary selection, 1-2 concentrations of test compounds were used (10 µl, 1 µM). For compound profiling, 8 concentrations were investigated (10,000, 1,000, 500, 100, 50, 10, 1 and 0.1 nM). To each microtiter plate, 10 µl of 20% DMSO was added to "control" and "total" wells while 10 µl of test compound (in 20% DMSO) was added to "unknown" wells. Then 10 µl NE CA (5 µM for AXR, 1 µM for A2aR) was added to "total" and "unknown" wells; 10 µl of PBS was added to the "control" wells. In the final addition, 80 µl of yeast strain CY8362 or CY12660 was added to all wells. All plates were then stirred briefly (LabLine orbital shaker 2-3 minutes) and allowed to incubate for 4 hours at 30 °C in a dry oven.

β-Galactosidase activity can be quantified using either colorimetric (eg ONPG, CPRG), luminescent (eg Galacton-Star) or fluorometric substrates (eg FDG, Resorufin). Currently, 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) which is a fluorescent β-galactosidase substrate was added to all wells at 20 µl/well (final concentration = 80 µM). Plates were shaken for 5-6 seconds (LabLine orbital shaker) and then incubated at 37°C for 90 minutes (95% O2/5% CO2 in-cubator). At the end of the 90 minute long incubation period β-galactosidase activity was stopped using 20 µl/well of IM Na 2 CO 3 and all plates were shaken for 5-6 seconds. Plates were then determined using a fluorometer (Tecan Spectrafluor; excitation = 485 nm, emission = 535 nm).

Calculations: Relative fluorescence values for "control" wells were interpreted as background and subtracted from the "total" and "unknown" values. Compound profiles were analyzed via logarithmic transformation (x-axis: compound concentration) followed by in situ competition curve fitting to calculate IC50 values (GraphPad Prism).

Yeast strains: Saccharomyces cerevisiae strain CY12660 [farl<*>1442 tbtl-1 fusl-HIS3 canl stel4::trpl::LYS2 ste3<*>1156 gpal(41)-Gai3 lys2 ura3 leu2 trpl: his3; LEU2 PGKp-MfalLeader-hAlR-PH05term 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 2muori REP3 Ampr] was developed.

LT medium: LT (Leu-Trp supplemented) medium consists of 100 g DIFCO yeast nitrogen base, supplemented with the following: 1.0 g valine, 1.0 g aspartic acid, 0.75 g phenylalanine, 0.9 g lysine, 0, 45 g tyrosine, 0.45 g isoleucine, 0.3 g methionine, 0.6 g adenine, 0.4 g uracil, 0.3 g serine, 0.3 g proline, 0.3 g cysteine, 0.3 g arginine, 0.9 g histidine and 1.0 g threonine.

Construction of Yeast Strains Expressing Human Ai Adenosine Receptor

In this example, the construction of yeast strains expressing a human Ai adenosine receptor functionally integrated into the yeast pheromone system pathway is described.

1. Expression vector construction

To construct a yeast expression vector for the human Ai adenosine receptor, the Ai adenosine receptor cDNA was prepared by reverse transcriptase PCR of human hippocampal mRNA using primers designed based on the published sequence of the human Ai adenosine receptor and standard techniques. The PCR product was subcloned into the NcoI and XbaI sites of the yeast expression plasmid pMP15.

The pMP15 plasmid was generated from pLPXt as follows: XbaI site of YEP51 (Broach, J.R. et al. (1983) "Vectors for high-level, inducible expression of cloned genes in yeast" pp. 83-117 in M. Inouye ( ed.), Experimental Manipulation of Gene Expression. Academic Press, New York) was removed by cleavage, end filling and religation to form Yep51NcoDXba. Another XbaI site was created at the BamHI site by digestion with BamHI, end filling, linker (New England Biolabs, #1081) ligation, XbaI cleavage and re-ligation to form YEP51NcoXt. This plasmid was cleaved with Esp31 and NcoI and ligated to Leu2 and PGKp fragments generated by PCR. The 2 kb Leu2 PCR product was generated by amplification from YEPS1Nco using primers containing Esp31 and Bgl11 sites. The 660 base pair PGKp PCR product was generated by amplification from pPGKas (Kang, Y.-

S. et al. (1990) Mol. Cell. Biol. 10: 2582-2590) with PCR primers containing Bglll and NcoI sites. The resulting plasmid is called plPXt. pLPXt was modified by inserting the coding region of the α-factor pre-pro leader into the NcoI site. The prepro leader was inserted such 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 cleavage of the plasmid with NcoI and XbaI. The resulting plasmid is called pMP15.

The pMP15 plasmid into which the human Ai adenosine receptor cDNA was inserted was designated p5095. In this vector, the receptor cDNA is fused to the 3' end of the yeast α-factor preproleader. During protein development, the prepro peptide sequences are cleaved to form the fully developed, full-length receptor. This occurs during processing of the receptor via the yeast secretory pathway. This plasmid is maintained by Leu selection (ie growth on medium lacking leucine). The sequence of the cloned region was determined and found to be equivalent to that in the published literature (GenBank accession numbers S45235 and S56143).

II. Yeast strain construction

To generate a yeast strain expressing the human Ai adenosine receptor, yeast strain CY7967 was used as the parental starting strain. The genotype of CY7967 is as follows: MATa gpaD1163 gpal(41)Gai3 farlD1442 tbt-1 FUS1-HIS3 canl stel4::trpl::LYS2 ste3D1156 lys2 ura3 leu2 trpl his3

An overview of the genetic markers is given below:

Two plasmids were transformed into strain CY7967 by electroporation: plasmid p5095 (encoding the human A1 adenosine receptor described above) and plasmid p1584, which is a FUS1-β-galactosidase reporter gene plasmid. Plasmid p1584 was derived from plasmid pRS426 (Christianson, T.W. et al.

(1992) Gene 110:119-1122). Plasmid pRS426 contains a polylinker site at nucleotides 2004-2016. A fusion between the FUS1 promoter and the β-galactosidase gene was inserted at the Eag1 and XhoI restriction sites to form plasmid p1584. The pl584 plasmid is maintained by Trp selection (ie growth on medium lacking leucine).

The resulting strain carrying p5095 and p1584 referred to as CY12660 expresses the human A1 adenosine receptor. To grow this strain in liquid or on agar plates, minimal medium lacking leucine and tryptophan was used. To perform a growth assay on plates (to examine FUS1-HIS3), the plates were maintained 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 check for specificity, a comparison with one or more other yeast-based seven transmembrane receptor assays was included in all experiments.

Construction of yeast strains expressing human A2a adenosine receptor

In this example, the construction of yeast strains expressing a human A2a adenosine receptor functionally integrated into the yeast pheromone system pathway is described.

I. Expression of vector construction

To construct a yeast expression vector for the human A2a adenosine receptor, the human A2a receptor cDNA was obtained from Dr. Phil Murphy (NIH). Upon receipt of this clone, the α2a receptor insert was sequenced and found to be identical to the published sequence (GenBank accession number S46950). Receptor cDNA was cleaved from the plasmid by PCR with VENT polymerase and cloned into the plasmid pLPBX, which drives receptor expression with a constitutive Phosphoglycerate Kinase (PGK) promoter in yeast. The sequence of the entire deposit was again sequenced and found to be identical to the published sequence. However, based on the cloning strategy used, there were three amino acids added to the carboxy terminus of the receptor, GlySerVal.

II. Yeast strain construction

To generate a yeast strain expressing the human A2a adenosine receptor, yeast strain CY8342 was used as the parental starting 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-rGasE10K (or gpalp-rGasD229S or gpalp-rGasE10K+D229S) .

The genetic markers are as described in Example 1, except for the G protein variation. For human A2a receptor expression, yeast strains were used in which the endogenous yeast G protein GPA1 had been removed and replaced with a mammalian Gas. Three Gas rat mutants were used. These variants contain one or two point mutations that convert them into proteins that bind efficiently to yeast Py. They are identified as Gas-E10K (where the glutamic acid at position ten is replaced by lysine), GasD229S (where the aspartic acid at position 229 is replaced by serine) and GasE10K+D229S (which contains both point mutations).

Strain CY8342 (carrying one of the three mutant rat Gas proteins) was transformed with either the parental vector pLPBX (Receptor") or with pLPBX-A2a (Receptor+). A plasmid with the FUS1 promoter fused to β-galactosidase coding sequences (described above) was added to confirm the degree of activation of the pheromone response pathway.

Functional assay using yeast strains expressing human Ai adenosine receptor

In this example, the development of a functional screening assay in yeast for modulators of the human Ai adenosine receptor is described.

I. Ligands used in the assay

Adenosine, which is a natural agonist for this receptor, as well as two other synthetic agonists, were used for the development of this assay. Adenosine was reported to have an EC50 of approximately 75 nM and (-)-N6-(2-phenylisopropyl)-adenosine (PIA) with a reported affinity of approximately 50 nM was used in a subset of experiments. 5'-N-ethylcarboxamidoadenosine (NECA) was used in all growth assays. To prevent signaling due to the presence of an adenosine in the growth medium, adenosine deaminase (4U/ml) was added to all assays.

II. Biological response in yeast

The ability of the Ai adenosine receptor to functionally couple in a heterologous yeast system was investigated by introducing the Ai receptor expression vector (p5095, described above) into a series of yeast strains expressing different G protein subunits. The majority of these transformants expressed Ga subunits of Gai or Gao subtypes. Additional Ga proteins were also examined for possible identification of promiscuous receptor-Ga protein coupling. In different strains, a STE18 or a chimeric STE18-Gy2 construct was integrated into the yeast genome. The yeast strains contained a defective HIS3 gene and an integrated copy of FUS1-HIS3 to thereby allow selection in selective medium containing 3-amino-1,2,4-triazole (examined at 0.2, 0.5 and 1, 0 mM) and without histidine. Transformants were isolated and monolayers were prepared on medium containing 3-amino-1,2,4-triazole, 4 U/ml adenosine deaminase and without histidine. Five microliters of different concentrations of ligand (eg, NECA at 0, 0.1, 1.0, and 10 mM) were applied. Growth was monitored for 2 days. Ligand-dependent growth responses were investigated in this way in different 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.

As indicated in Table 3, the strongest signaling was found to occur in a yeast strain expressing the GPAi (41)-Gai3 chimera fusion.

III fus1-LacZ assay

To more fully characterize activation of the pheromone response pathway, synthesis of β-galactosidase via fuslLacZ in response to agonist stimulation was measured. To perform the β-galatosidase assay, increasing concentrations of ligand were added to a mid-log culture of human Ai adenosine receptor expressed in a yeast strain co-expressing the Stel8-Gy2 chimeric compound and GPA4i-Gai3. Transformants were isolated and grown overnight in the presence of histidine and 4 u/ml adenosine deaminase. After 5 hours of incubation with 4 U/ml adenosine deaminase and ligand, induction of β-galactosidase was measured using CPRG as substrate for β-galactoside. 5 x 10<5> cells were used per assay.

The results obtained with NECA stimulation indicated that at a NECA concentration of 10<-8> M approximately 2-fold stimulation of β-galactosidase activity was obtained. Furthermore, a stimulation index of approximately 10-fold was observed at a NECA concentration of 10"<5> M.

The applicability of this assay was extended by validating the activity of antagonists on this strain. Two known adenosine antagonists, XAC and DPCPX, were examined for their ability to compete with NECA (at 5 mM) for activity in the β-galactosidase assay. In these assays, β-galactosidase induction was measured using FDG as substrate and 1.6 x 10<5> cells per assay. The results indicated that both XAC and DPCPX acted as potent antagonists for yeast expression A1 adenosine receptor with IC 50 values of 44 nM and 49 nM, respectively.

To determine whether this inhibitory effect was specific for the Ai subtype, a series of complementary experiments was performed with the yeast-based A2a receptor assay (described in Example 4). Results obtained with the A2a yeast-based assay indicated that XAC was a relatively effective A2a receptor antagonist, which is consistent with published reports. In contrast, DPCPX was relatively inert at this receptor as expected from published reports.

IV. Radioligand binding

The Ai adenosine receptor assay was further characterized by measuring the receptor's radioligand binding parameters. Displacement binding of [<3>H]CPX by several adenosine receptor reference compounds, XAC, DPCPX and CGS was analyzed using membranes prepared from yeast expressing the human Ai adenosine receptor. The results with yeast membranes expressing the human A1 adenosine receptor were compared with those from yeast membranes expressing the human A2a adenosine receptor or the human A3 receptor to examine binding specificity. To perform the assay, fifty mg of membranes were incubated with 0.4 nM [<3>H]CPX and increasing concentrations of adenosine receptor ligands. Incubation was in 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10 mM MgCl 2 , 0.25% BSA and 2 U/ml adenosine deaminase in the presence of protease inhibitors for 60 min at room temperature. Binding was terminated by addition of ice-cold 50 mM Tris-HCl, pH 7.4 plus 10 mM MgCl2, followed by rapid filtration over GF/B filters pre-saturated with 0.5% polyethyleneimine using a Packard 96-well harvester . Data were analyzed with a non-linear least square curve fitting procedure using Prism 2.01 software. The IC 50 values obtained in this experiment are summarized in Table 4 below.

These data indicate that the reference compounds have affinities consistent with those reported in the literature. The data further indicate that the yeast-based assays are of sufficient sensitivity to distinguish receptor type specificity.

Functional assay using yeast strains expressing human A2a adenosine receptor

In this example, the development of a functional screening assay in yeast for modulators of the human Ai adenosine receptor is described.

I. Ligands used in the assay

The natural ligand adenosine, as well as other thoroughly characterized and commercially available ligands, were used to study the human A2a receptor functionally expressed in yeast. Three ligands have been used in the establishment of this assay. They include:

To prevent signaling due to the presence of adenosine in the growth medium, adenosine deaminase (4U/ml) was added to all assays.

II. Biological response in yeast

A2a receptor agonists were examined for their capacity to stimulate the pheromone response pathway in yeast transformed with A2a receptor expression plasmid and expressing either GasE10K, GasD229S or GasElOK+D229S. The ability of the ligand to stimulate the pheromone response pathway in a receptor-dependent manner was indicated by a change in the yeast phenotype. Receptor activation modified the phenotype from histidine auxotrophy to histidine prototrophy (activation of fusl-HIS3). Three independent transformants were isolated and grown overnight in the presence of histidine. Cells were washed to remove histidine and diluted to 2 x 10<6> cells/ml. 5 µl of each transformant was spotted on non-selective medium (including histidine) or selective medium (1 mM AT) in the absence or presence of 4 U/ml adenosine deaminase. Plates were grown at 30°C for 24 hours. In the presence of histidine, both receptor"1"

(R<+>) and receptor (R~) strains capable of growth. However, in the absence of histidine only R<+-> cells grow. Since no ligand had been added to these plates, two explanations were possible for this result. One possible interpretation was that the receptor-bearing yeast had a growth advantage due to ligand-independent receptor-mediated activation (LIRMA). Alternatively, the yeast could have synthesized the ligand adenosine. To distinguish between these two possibilities, an enzyme that cleaves the ligand adenosine deaminase (ADA) was added to the growing yeast and plates. In the presence of adenosine deaminase, R<+-> cells no longer grew in the absence of histidine, indicating that the yeast organisms actually synthesized the ligand.

This interpretation was confirmed with an A2a growth assay in liquid. In this experiment, R<+->yeast (a GasE10K strain expressing the A2a receptor) was inoculated at three densities (1 x 10<6 >cell/ml; 3 x 10<5> cells/ml or 1 x 10< 5> cell/ml) in the presence or absence of adenosine deaminase (4 U/ml). The stringency of the assay was increased with increasing concentrations (0, 0.1, 0.2 or 0.4 mM) of 3-amino-1,2,4-triazole (AT), which is a competitive antagonist of imidazoleglycerol- P dehydrogenase which is the protein product of the HIS3 gene. In the presence of adenosine deaminase and 3-amino-1,2,4-triazole, yeast grows less vigorously. However, in the absence of 3-amino-1,2,4-triazole, adenosine deaminase had little effect. Thus, adenosine deaminase itself had no direct effect on the pheromone response pathway.

An alternative way to measure growth and one that can be miniaturized for high throughput research is an A2a receptor ligand spot assay. A GasE10K strain expressing the A2a receptor (A2aR+) or lacking the receptor (R-) was grown overnight in the presence of histidine and 4 U/ml adenosine deaminase. Cells were washed to remove histidine and diluted to 5 x 10<6> cells/ml. 1 x 10<6> cells were spread on selective plates containing 4 U/ml adenosine deaminase and 0.5 or 1.0 mM 3-amino-1,2,4-triazole (AT) and allowed to dry for 1 hour. 5 µl of the following reagents were applied to the monolayer: 10 mM adenosine, 38.7 mM histidine, dimethyl sulfoxide (DMSO), 10 mM PIA, or 10 mM NECA. Cells were grown for 24 h at 30 °C. The results showed that cells without the receptor could only grow when histidine was added to the medium. In contrast, R<+> cells only grew in areas where the A2a receptor ligands PIA and NECA had been applied. Since the plates contained adenosine deaminase, the lack of growth where adenosine had been applied confirmed that adenosine deaminase was active.

III. fusion LacZ assay

To quantify activation of the yeast mating pathway, synthesis of β-galactosidase via fuslLacZ was measured. Yeast strains expressing GasE10K, GasD229S or GasE10K+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<7> cells were diluted to 1 x 10<6> cells/ml and exposed to increasing concentrations of NECA for 4 hours followed by determination of β-galactosidase activity in the cells. The results showed that essentially no β-galactosidase activity was detected in R- strains, while increasing amounts of β-galactosidase activity were detected in R+ strains expressing either GasE10K, GasD229S or GasE10K+D229S as the concentration of NECA increased, indicating a dose-dependent increase in units of β-galactosidase detected in response to exposure to increased ligand concentration. This dose dependence was only observed in cells expressing the A2a receptor. Furthermore, the most potent Gas construct was for the A2a receptor GasE10K. The GasD229S construct was the second most potent Gas construct for the A2a receptor, while the GasE10K+D229S construct was the least potent of the three Gas constructs examined although even the GasElOK+D229S construct readily stimulated detectable amounts of β-galactosidase activity .

For a further description of the identified assays, see U.S. application, serial no. 09/088985, with the title "Functional Expression of Adenosine Receptors in Yeast", filed on 2 June 1998 (Authorized file no. CPI-093) the entire contents of which are incorporated here by reference.

Pharmacological characterization of the human adenosine receptor subtypes

Material and methods

Materials [<3>H]-DPCPX [Cyclopentyl-1,3-dipropylxanthine, 8-[dipropyl-2,3-3H(N) ] (120.0 Ci/mmol) [<3>H]-CGS 21680, [carboxyethyl-<3>H (N) ] (30 Ci/mmol) and [125<1>]-AB-MECA ([<125>I]-4-aminobenzyl-5'-N-methylcarboxamidoadenosine) ( 2,200 Ci/mmol) was purchased from New England Nuclear (Boston MA). XAC (Xanthine amine congeners), NECA (5'-N-ethylcarboxamidoadenosine) and IB-MECA from Research Biochemicals International (RBI, Natick, MA). The adenosine deaminase and complete protease inhibitor combination tablets were purchased from Boehringer Mannheim Corp. (Indianapolis, IN). Membranes from HEK-293 cells stably expressing the human adenosine 2a [RB-HA2a], adenosine 2b [RB-HA2b], or adenosine 3 [RB-HA3] receptor subtypes were purchased from Receptor Biology (Beltsville, MD), respectively. Cell culture reagents were from Life Technologies (Grand Island, NY) except for serum which was from Hyclone (Logan, UT).

Yeast strains: Saccharomyces cerevisiae strain CY12660 [farl<*>1442 tbtl-1 fusl-HIS3 canl stel4::trpl::LYS2 ste3<*>1156 gpal (41)-Gai3 lys2 ura3 leu2 trpl: his3; LEU2 PGKp-MfalLeader-halR-PH05term 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] was developed as described above.

Yeast culture: Transformed yeast were grown in Leu-Trp (LT) medium (pH 5.4) supplemented with 2% glucose. For the production of membranes, 250 ml of LT medium was inoculated with a starting titer of 1-2 x 10<6> cells/ml from a 30 ml overnight culture and incubated at 30 °C under permanent oxygenation by rotation. After 16 hours of growth, the cells were harvested by centrifugation and membranes were prepared as described below.

Mammalian tissue culture: HEK-293 cells stably expressing the human adenosine 2a receptor subtype (Cadus clone # 5) were grown in Dulbeco's minimal essential medium (DMEM) supplemented with 10% fetal bovine serum and IX penicillin/streptomycin under selective pressure by use 500 mg/ml G418 antibiotic at 37 °C in a humidified 5% C02 atmosphere.

Yeast cell membrane preparations: 250 ml cultures were harvested after overnight incubation by centrifugation at 2000 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 of ice-cold lysis buffer [5 mM Tris-HCl, pH 7.5, 5 mM EDTA and 5 mM EGTA] supplemented with Protease inhibitor mix tablets (1 tablet per 25 ml buffer). Glass beads (17 g, Mesh 400-600, Sigma) were added to the suspension and the cells were disrupted by vigorous vortexing at 4°C for 5 minutes. The homogenate was diluted with an additional 30 ml of lysis buffer plus protease inhibitors and centrifuged at 3000 x g for 5 minutes. The membranes were then pelleted at 36,000 x g (Sorvall RC5B, type SS34 rotor) for 45 minutes. The resulting membrane pellet was resuspended in 5 ml membrane buffer [50 mM Tris-HCl, pH 7.5, 0.6 mM EDTA and 5 mM MgCl2] supplemented with protease inhibitor mixture tablets (1 tablet per 50 ml buffer) and stored at -80° C for further attempts.

Mammalian cell membrane preparations: HEK-293 cell membranes were prepared as described previously (Duzic E et al.: J. Biol. Chem., 267, 9844-9851, 1992). Briefly, cells were washed with PBS and harvested with a rubber mallet. Cells were pelleted at 4 °C 200 x g in a Sorvall RT6000 centrifuge. The pellet was resuspended in 5 ml/well of lysis buffer at 4 °C (5 mM Tris-HCl, pH 7.5, 5 mM EDTA, 5 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 10 mg/ml pepstatin A and 10 mg/ml ml aprotinin) and homogenized in a Dounce homogenizer. The cell lysate was then centrifuged at 36,000 x g (Sorvall RC5B type SS34 rotor) for 45 minutes and the pellet was resuspended in 5 ml of membrane buffer [50 mM Tris-HCl, pH 7.5, 0.6 mM EDTA, 5 mM MgCl2, 0.1 mM phenylmethylsulfonyl fluoride, 10 mg/ml pepstatin A and 10 mg/ml aprotinin] and stored at -80 °C for further experiments.

Bio-Rad protein assay kit, based on the Bradford color binding procedure (Bradford, M.: Anal. Biochem. 72:248

(1976)) was used to determine total protein concentration in yeast and mammalian membranes.

Adenosine 1 receptor subtype saturation and radioligand competition binding: Saturation and competition binding on membranes from yeast cells transformed with human Ai receptor subtype was performed using antagonist [<3>H] DPCPX as a radioactive ligand. Membranes were 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 inhibitor mixture tablet/50 ml] at concentrations of 1.0 mg/ml.

In saturation binding, membranes (50 µg/well) were incubated with increasing concentrations of [<3>H] DPCPX (0.05 - 25 nM) in a final volume of 100 µl of binding buffer at 25 °C for 1 h in the absence and presence of 10 µM unlabeled XAC in a 96-well microtiter plate.

In competition binding, membranes (50 µg/well) were incubated with [<3>H]DPCPX (1.0 nM) in a final volume of 100 ml binding buffer at 25 °C for one hour in the absence and presence of 10 µM unlabeled XAC or increasing concentrations of competing compounds in a 96-well microtiter plate.

Adenosine 2a receptor subtype competitive radioligand binding: Competitive binding on membranes from HE293 cells stably expressing the human A2a receptor subtype was performed using agonist [<3>H] CGS-21680 as a radioligand. Membranes were 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 inhibitor mixture tablet/50 ml] at concentrations of 0.2 mg/ml. Membranes (10 µg/well) were incubated with [<3>H] CGS-21680 (100 nM) in a final volume of 100 ml binding buffer at 25 °C for 1 h in the absence and presence of 50 µM unlabeled NECA or increasing concentrations of competing compounds

1 a 96-well microtiter plate.

Adenosine 3 receptor competitive radioligand binding: Competitive binding on membranes from HEK293 cells stably expressing the human A3 receptor subtype was performed using agonist [12<5>I] AB-MECA as a radioactive ligand. Membranes were 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 inhibitor mixture tablet/50 ml] at concentrations of 0.2 mg/ml. Membranes (10 µg/well) were incubated with [<125>I] AB-MECA (0.75 nm) in a final volume of 100 µl binding buffer at 25 °C for 1 h in the absence and presence of 10 µM unlabeled IB- MECA or increasing concentrations of competing compounds in a 96-well microtiter plate.

At the end of incubation, Ai, A2a and A3 receptor subtype radioligand binding assays were terminated by addition of ice-cold 50 mM Tris-HCl (pH 7.4) buffer supplemented with 10 mM MgCl2, followed by rapid filtration over glass fiber filters (96-well GF/B UniFilters , Packard pre-saturated in 0.5% polyethyleneimine in a Filtermate 196 cell harvester (Packard). Filter plates were dried coated with 50 µl/well scintillation fluid (MicroScint-20, Packard) and counted in a TopCount (Packard). Assays were performed in triplicate Nonspecific binding was 5.6 ± 0.5%, 10.8 ± 1.4%, and 15.1 ± 2.6% of total binding in an AIR, A2aR, and A3R binding assay, respectively.

Adenosine 2b receptor subtype competitive radioligand binding: Competitive binding on membranes from HEK293 cells stably expressing the human A2b receptor subtype was performed using the Ai receptor antagonist [<3>H] DPCPX as a radioactive ligand. Membranes were diluted in binding buffer

[10 mu 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 µg/well) were incubated with [<3>H] DPCPX (15 nM) in a final volume of 100 µl binding buffer at 25 °C for 1 h in the absence and presence of 10 µM unlabeled XAC or increasing concentrations of competitor compounds in a 96-well microtiter plate. At the end of incubation, the assay was terminated by the addition of ice-cold 10 mM Hepes-KOH (pH 7.4) buffer followed by rapid filtration over glass fiber filters (96-well GF/C UniFilters, Packard) pre-saturated in 0.5% polyethyleneimine in a Filtermate 196 cell harvester (Packard). The filter plates were dried coated with 50 µl/well scintillation fluid (MicroScint-20, Packard) and counted in a TopCount (Packard). Assays were performed in triplicate. Non-specific binding was 14.3 +2.3% of the total binding.

Specific binding of [<3>H] DPCPX, [<3>H] CGS-21680 and [125I] AB-MECA was defined as the difference between the total binding and non-specific binding. Percentage inhibition of the compounds was calculated against total binding. Competition data were analyzed with iterative curve fitting to a single-seat model and Ki values were calculated from ICso values

(Cheng and Prusof, Biochem. Pharmacol. 22, 3099-3109, 1973)

using the GraphPad Prizm 2.01 program.

Results

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 in yeast. Rough membranes prepared from Saccharomyces cerevisiae transformed with the human Adenosine 1 receptor subtype construct showed specific saturable binding of [<3>H] DPCPX with a KD of 4.0 + 0.19 nM. The KD and Bmax value were calculated from the saturation isotherm and the Scatchard transformation of the data indicated a simple class of binding sites. The densities of adenosine binding sites in the yeast membrane preparations were calculated to be 716.8 ± 4 3.4 fmol/mg membrane protein.

The pharmacological subtype characteristics of the recombinant yeast cells transformed with the human Ai receptor subtype were investigated with subtype-selective adenosine ligands (XAC, DPCPX, CGS-15943, compound 600, compound 1002, NECA, (R)-PIA, IB-MECA and Alloxazin) that competed with [<3>H] DPCPX in the expected ranking order. Replacement curves recorded with these compounds show the typical steepness with all ligands and the data for each of the ligands could be modulated by single-site fitting. The observed dissociation constants calculated for the individual compounds from the curves (table 5) are consistent with the value published for the receptor obtained from other sources.

Tables 6 to 12 show the effectiveness and structure-activity profiles of deazapurines according to the invention. Tables 13 and 14 show that selectivity can be achieved for human adenosine receptor sites by modulating the functionality around the deazapurine structure. Table 14 also shows the surprising discovery that the compounds disclosed herein have subnanomolar activity and higher selectivity for the A2b receptor compared to the compounds in Table 13.

Compounds specific for the Aia receptor Summary.

The present invention is also based on compounds that selectively bind to the adenosine A2a receptor in order to thereby treat a disease associated with the A2a adenosine receptor in an individual. The disease to be treated is associated with, for example, a disorder of the central nervous system, a cardiovascular disorder, a kidney disorder, an inflammatory disorder, a gastrointestinal disorder, an eye disorder, an allergic disorder or a respiratory disorder in order to inhibit the activity of an A2a adenosine receptor in a cell which comprises contacting said cell with the above-mentioned compounds.

Said A2a adenosine receptor may be associated with Parkinson's disease and diseases associated with movement activity, vasodilation, platelet inhibition, neutrophil superoxide formation, cognitive disorders or senile dementia.

Disorders associated with adenosine A1, A2a, A2b and A3 receptors are described 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 invention is further illustrated by the following examples. It should be understood that the models used throughout the examples are accepted models and that demonstration of efficacy in these models is predictive of efficacy in humans.

The present invention will be better understood from the experimental details that follow. However, those skilled in the art will readily appreciate that the particular methods and results discussed are merely illustrative of the invention. Example 22: Synthesis of adenosine A2a antagonists, compounds 1601, 1602 and 1603.

Compound 26 (10.93 g, 50.76 mmol) was dissolved in DMF (67 mL). 4-Amidinopyridine hydrochloride (8.0 g, 50.76 mmol) and DBU (15.4 g, 101.5 mmol) were added sequentially and the reaction mixture was heated to 85 °C. After 22 hours, the reaction mixture was cooled to room temperature and the DMF was removed in vacuo. The dark oil was diluted with 2M HCl (80 mL). The reaction mixture was allowed to stand. After 2 hours, the solution was cooled to 10 °C and filtered. The solid was washed with cold water and dried to give 7.40 g of a yellow solid, compound 27 (69%). <X>H NMR (200 MHz, d6-DMSO) δ 6.58 (s, 1H), 7.27 (s, 1H), 8.53 (d, 2H, J = 5.6), 9.00 (d, 2H, J = 5.2 Hz), 12.35 (brs, 1H). MS (ES): 212.8 (M"+1).

Compound 27 (7.4 g, 29.8 mmol) was diluted with POCl 3 and heated to 105 °C. After 18 hours, the reaction mixture was cooled to room temperature and the POCl 3 was removed in vacuo. The thick dark oil was diluted with MeOH (75 mL) followed by ether (120 mL). The amorphous red solid is filtered and washed with ether to give 3.82 g of a red solid. The impure solid, compound 28 is approximately 80% pure and is used without further purification in the next reaction. <1>R NMR (200 MHz, d6-DMSO) δ 6.58 (s, 1H) , 7.27

(s, 1H) , 8.53 (d, 2H, J = 5.6), 9.00 (d, 2H, J = 5.2 Hz), 12.35 (brs, 1H). MS (ES): 212.8 (M"+1).

Compound 1601: DMSO (5 mL) and D-prolinol (500 mg, 4.94 mmol) were added to compound 28 (500 mg, 2.17 mmol). The reaction mixture was heated to 120 °C. After 18 h, the reaction mixture was cooled to room temperature and diluted with EtOAc and H 2 O. The layers were separated and the aqueous layer was extracted with EtOAc (2x). The combined organic layers were washed with H 2 O (2x), brine, dried over MgSO 4 , filtered and concentrated to give 200 mg of a light brown solid. The solid was recrystallized from EtOAc to give 82 mg of a light brown solid (13%). <X>H NMR (200 MHz, d6-DMSO) δ 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"+1), mp = 210-220 °C (decomp.).

Compound 1602: Chromatography (silica, 9:1 CHCl 3 /MeOH) gave 10 mg of a light brown solid (2%). <X>H NMR (d6-DMSO) δ 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"+1).

Compound 1603: Chromatography (silica, 20:1 hexanes/EtOAc) afforded 135 mg of a light brown solid (53%). <X>H NMR (d 6 -DMSO) δ 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"+1).

Compound 1605: In 50 ml of RBF, 60 mg of 2-(4'-pyridyl)-4-chloropyrimidinopyrrole HCl salt was dissolved in 2 ml of anhydrous DMSO. 3-(R)-hydroxy-(D)-prolinol TFA salt (380 mg) and 500 mg of sodium bicarbonate were added thereto. The mixture was then sparged with nitrogen gas for 5 minutes and heated to 130°C. After 2 hours, the reaction mixture was cooled to room temperature and the DMSO was removed in vacuo. The residue was partitioned between EtOAc (15 mL) and saturated aqueous sodium bicarbonate solution (15 mL). The organic layer was separated and washed with brine (15 mL) and dried over Na 2 SO 4 . After removal of the solvent, the crude product was purified by preparative TLC (CH 2 Cl 2 /MeOH = 95/5) to give 35 mg (50%). <1>H NMR (200 MHz, CDCl 3 ) 2.3-2.5 (1H) , 3.4-3.8 (3H) , 4.4-4.6 (2H), 6.4 (1H) ; 7.1 (1H); 8.2 (d, 1H); 8.7 (d, 2H); 11.0 (1H). MS(ES):312 (M"+1).

Example 23: Synthesis of adenosine A2a antagonist, compound 1606

Compound 28 (200 mg) was treated with DMF (30 mL), (-dimethylglycine methyl ester) (73 mg HCl salt in 2 mL water) and 500 mg sodium bicarbonate. After 18 hours, DMF was removed in vacuo. The residue was partitioned between EtOAc (30 mL) and saturated aqueous sodium bicarbonate solution (15 mL). The organic layer was washed with brine (15 mL), dried over sodium sulfate, filtered and concentrated. Chromatography (silica, 10:4 hexanes/EtOAc) gave 150 mg of pure product, compound 29 (69%). <X>H NMR (200 MHz, CDCl 3 ), 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 (p, 1H).

Compound 1606:

Procedure is the same as compound 1605 (72%). <1>H NMR (200 MHz, CDCl 3 ), 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"+1).

The following compounds can be synthesized in the same way.

Compound 1600: (51%) MS(ES):326.0 (M"+1).

Compound 1607: <X>H NMR (200 MHz, CDCl 3 ), 1.40-1.80 (m, 5H), 2.80-3.50 (m, 3H), 4.60-4.80 (m , 3H), 6.66 (d, 1H, J = 6.2 Hz), 7.26 (m, 1H), 8.21 (d, 2H, J = 6.3 Hz), 8.65 (d , 2H, J= 5.8 Hz), 11.90 (s, 1H). MS (ES): 310.1 (M"+1).

Compound 1608: (64%) . <X>H NMR (200 MHz, 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 = 6.0 Hz), 8.63 (d, 2H, J = 5, 4 Hz), 8.82 (brs, 1H), 11.56 (brs, 1H). MS(ES): 444.0 (M"+1).

Compound 1604: <X>H NMR (200 MHz, 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).

Compounds specific to the A3 receptor Summary

The present invention is also based on compounds that selectively bind to the adenosine A3 receptor, thereby treating a disorder associated with the A3 adenosine receptor in an individual by administering to the individual a therapeutically effective amount of such compounds. The disorder to be treated is associated with, for example, asthma, hypersensitivity, rhinitis, hay fever, serum sickness, allergic vasculitis, atopic dermanitis, dermanitis, psoriasis, eczema, idiopathic pulmonary fibrosis, eosinophilic chlorcystitis, chronic airway inflammation, hypereosinophilic syndromes, eosinophilic gastroenteritis , oedema, urticaria, eosinophilic myocardial disease, episodic angioedema with eosinophilia, inflammatory bowel disease, ulcerative colitis, allergic granulomatosis, carcinomatosis, eosinophilic granuloma, familial histiocytosis, hypertension, mast cell degranulation, cancer, cardiac hypoxia, cerebral ischaemia, diuresis , renal failure, neurological disorder, mental disorder, cognitive disorder, myocardial ischemia, bronchoconstriction, arthritis, autoimmune disease, Crohn's disease, Graves' disease, diabetic, multiple sclerosis, anemia, psoriasis, fertility disorders, lupus erythematosus, reperfusion injury, arteriole diameter in brain, release of allergic mediators, scleroderma, stroke, global ischaemia , central nervous system disorder, cardiovascular disease, kidney disease, inflammatory disease, gastrointestinal disease, eye disease, allergic disease, respiratory disease, or immunological disease.

Compounds of the invention are active against A3 adenosine receptor associated with central nervous system disorder, a cardiovascular disorder, asthma, hypersensitivity, rhinitis, hay fever, serum sickness, allergic vasculitis, atopic dermantitis, dermantitis, psoriasis, eczema, idiopathic pulmonary fibrosis, eosinophilic chlorcystitis, chronic airway inflammation, hypereosinophilic syndromes, eosinophilic gastroenteritis, oedema, urticaria, eosinophilic myocardial disease, episodic angioedema with eosinophilia, inflammatory bowel disease, ulcerative colitis, allergic granulomatosis, carcinomatosis, eosinophilic granuloma, familial histiocytosis, hypertension, mast cell degranulation, cancer, cardiac hypoxia, cerebral ischemia, diuresis, renal failure, neurological disorder, mental disorder, cognitive disorder, myocardial ischemia, bronchoconstriction, arthritis, autoimmune disease, Crohn's disease, Graves' disease, diabetic, multiple sclerosis, anemia, psoriasis, fertility disorders, lupus erythematosus, reperfusion injury, arteriole dia meters in brain, release of allergic mediators, scleroderma, stroke, global ischemia, central nervous system disorder, cardiovascular disorder, kidney disorder, inflammatory disorder, gastrointestinal disorder, eye disorder, allergic disorder, respiratory disease, or immunological disease.

Diseases associated with adenosine A1, A2a, A2b and A3 receptors are described in WO 99/06053 and WO-09822465, WO-09705138, WO-09511681, WO-09733879, JP-09291089, PCT/US98/16053 and US patent no. .5,516,894.

As used herein, "a compound is A3 selective" means that a compound has a binding constant to the adenosine A3 receptor at least tenfold higher than that of adenosine A1, A2a, or A2b.

The invention is further illustrated by the following examples. It should be understood that the models used throughout the examples are accepted models and that the demonstration of efficacy in these models is predictive of efficacy in humans.

The person skilled in the art will know that metabolism of the compounds described herein in an individual forms certain biologically active metabolites that can act as drugs.

The present invention will be better understood from the experimental details that follow. However, the person skilled in the art will readily understand that the particular methods and results discussed are only illustrative of the invention.

Example 24: Adenosine A3 antagonist test

Compound 1700 (Table 17 below): MS (ES): 336.1 (M"+1).

Compound 1710 (Table 17 below): MS (ES): 381.1 (M"+1).

Compound 1316 (Table 17 below): MS (ES): 353.2 (M"+1).

Compound 1703 (Table 17 below): MS (ES): 357.1 (M"+1).

Compound 1719 (Table 17 below): <1>H-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 (m, 1H), 6.55 (s, 1H) , 7.15 (s, 1H), 7.49 (m, 2H), 8.32 (m, 2H).

Compound 1704 (Table 17 below): MS (ES): 367.0 (m~+1).

Compound 1706 (Table 17 below): <1>H-NMR (200MHz, CDcl3) 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++1).

Compound 1708 (Table 17 below): MS (ES) 399.0 (M"+1).

Compound 1709 (Table 17 below): MS (ES) 385.9 (M"+1).

Compound 1710 (Table 17 below): MS (ES) 434.0

(M~+l) .

Compound 1711 (Table 17 below): <1>H-NMR (200 MHz, CD3OD) d 3.95 (d, 2H, J = 5.8 Hz), 4.23-4.31 (m, 2H), 4.53 (t, 2H, J = 8.8 Hz), 6.30 (d, 1H, J = 3.0 Hz), 6.98 (d, 1H, J = 3.0 Hz), 7, 45-7.48 (m, 3H), 7.83-8.42 (m, 2H), 9.70 (brs, 1H). MS (ES): 281.1 (M<+>+1).

OSIC-148313 <X>H-NMR (200 MHz, CD3OD) d 3.02 (m, 2H), 3.92

(m, 2H) , 5.09 (2, 2H) , 6.53 (s, 1H), 6.90-7.04 (br s, 1H), 6.92 (m, 2H), 7.02 (m, 1H), 7.21 (dd, 1H, J = 8.2 Hz), 7.40 (m, 3H), 7.50-7.80 (br s, 1H), 8.33 (m , 2H), MS (ES): 445.1 (M<+>+1) .

Compound 1713 (Table 17 below): <1>H-NMR (200 MHz, CDCl 3 ) 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 = 14 Hz), 3.53-3.60 (m, 2H) , 3.84-3.92 (m, 2H), 3.97 (d, 1H, J = 14 Hz), 5.55 (t, 1H, J = 5.8 Hz), 6.17 (s, 1H), 6.55-6.59 (m, 2H), 6.64-6.71 (m , 1H), 7.11-7.19 (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++1).

Compound 1715 (Table 17 below): MS (ES): 505.0 (M++1).

Compound 1716 (Table 17 below): <X>H-NMR (200 MHz, CD3OD) d 1.65 (m, 1H) , 2.18 (m, 1H) , 2.49 (br, 2H, J= 6 ,2 Hz), 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 = 8.1 Hz), 7.39 (m, 3H), 8.32 (m, 2H).

MS (ES): 447.1 (M"+1).

Compound 1717 (Table 17 below): <1>H-NMR (200 MHz, CD30D) d 1.69 (m, 1H) , 2.26 (m, 1H) , 2.42 (d, 2H, J = 7 .4 Hz), 2.72 (m, 1H), 3.53 (m, 1H), 3.83 (m, 1H), 4.02 (m, 1H), 4.14 (dd, 1H, J = 10.6, 7.0 Hz), 5.14 (2, 2H), 6.69 (s, 1H), 6.96 (m, 2H), 7.06 (m, 1H), 7.25 (dd, 1H, J = 8.0 Hz), 7.39 (m, 3H), 8.35 (m, 2H).

MS (ES): 462.2 (M"+1).

Compound 1718 (Table 17 below): <1>H-NMR (200 MHz, CD3OD) d 1.40-2.00 (m, 5H), 3.52 (d, 2H, 7.6 Hz), 3, 80-4.00 (m, 1H), 4.00-4.20 (m, 3H), 4.50 (m, 2H), 6.36-6.50 (m, 2H), 6.54 ( p. 1H), 6.84-6.92 (m, 1H), 7.05 (t, 1H, J = 8.2 Hz), 7.30 -7.45 (m, 3H), 8.24 (d, 2H, J = 9.8 Hz). MS (ES): 449.0 (M"+1).

Table 17. Adenosine A3 receptor-selective compounds

<*> at least 10 times more selective than other three subtypes

The present invention provides a compound with the structure:

The present invention provides a compound with the structure:

The present invention provides a compound with the structure:

The present invention also provides a connection with the structure:

The present invention further provides a connection with the structure:

The present invention also provides a connection with the structure:

The present invention also provides a connection with the structure:

The present invention provides a further connection with the structure:

The present invention also provides a connection with the structure:

The present invention provides a further connection with the structure:

The present invention provides a further connection with the structure:

The present invention also provides a connection with the structure:

The present invention provides a further connection with the structure:

The present invention also provides a connection with the structure:

The present invention provides a further connection with the structure:

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

In a further embodiment, the invention provides the above-mentioned pharmaceutical composition(s), where said pharmaceutical composition is a periocular, retrobulbar or intraocular injection formulation.

In a further embodiment, the invention provides the above-mentioned pharmaceutical composition(s), where said pharmaceutical composition is a systemic formulation.

In a further embodiment, the invention provides the above-mentioned pharmaceutical composition(s), where said pharmaceutical composition is a surgical spray solution.

In a further embodiment, the invention provides a packaged pharmaceutical composition for treating a disease associated with Ai adenosine receptor in an individual comprising: (a) a container containing a therapeutically effective amount of compounds 1505, 1506, 1507, 1508, 1509, 1510, 1511 , 1512, 1513, 1514, 1516, 1517, 1518, 1519 or 1520, and (b) instructions for using said compound to treat said disease in an individual.

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

Exemplification

Example 21: Synthesis of 1-[6-(4-hydroxy-4-phenyl-piperidin-1-yl-methyl)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-pyrro -lidin-2-carboxylic acid amide.

Compound 1505 was synthesized in a manner similar to that of Example 17 using Synthetic Scheme IX with L-prolinamide and 4-phenyl-piperidin-4-ol to obtain:

<1>H-NMR (d 6 -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 = 6.8 Hz), 4.73 (s, 1H), 6.44 (s, 1H), 6.94 (s, 1H), 7.12-7.50 (m, 10H), 8, 35 (m, 2H), 11.6 (brs, 1H); MS (ES): 305.1 (M"+1), mp = 234-235 °C.

Example 22: Synthesis of [N-(2-phenyl-7H-pyrrolo[2,3-3]pyrimidin-4-yl)(L)-prolinamide (1506).

Compound 1506 was synthesized using Synthesis Scheme VII with L-prolineamide to obtain:

<1>H-NMR (DMSO-d6) d 2.05 (m, 4H) , 3.85 (m, 1H) , 4.05 (m, 1H), 4.70 (d, 1H, J= 8 ,0 Hz), 6.58 (brs, 1H), 6.95 (brs, 1H), 7.15 (d, 1H, J= 3.4 Hz), 7.40 (m, 3H), 7, 50 (brs, 1H), 8.40 (m, 2H), 11.6 (brs, 1H); MS (ES): 308.3 (M~+1). mp = 236-238 °C.

Example 23: Synthesis of [N-(2-phenyl-6-methoxymethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-(L)-prolinamide (1507).

Compound 1507 was synthesized using precursor compound 23 of Synthesis Scheme IX to obtain:

Bromide 23 (4.23 g, 10 mmol) is dissolved in anhydrous methanol (60 mL) and DCM (120 mL) and treated with AgO 2 CCF 3 under N 2 at room temperature for 1 hour. The solid is removed by filtration and washed with DCM (2x20 mL). The filtrate is concentrated in vacuo. The residue is redissolved in DCM (80 mL). The resulting solution is then washed with saturated NaHCO 3 solution and brine, dried over MgSO 4 , filtered and concentrated to give 3.71 g (4.99%) off-white solid. <1>H-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 (2.448 g, 6.55 mmol), DMSO (15 mL), L-prolinamide (4.0 g, 35.0 mmol) and NaHCO 3 (2.9 g) are combined and heated to 120 °C under nitrogen. After 4 hours, the reaction mixture is cooled to room temperature and diluted with water (60 ml). The resulting slurry is extracted with DMC (10x). The combined organic layers are washed with saturated NaHCO 3 solution and brine, dried over MgSO 4 , filtered and concentrated to give 2.48 g of brown solid. Pure product (1.86 g, 81%) is obtained after flash column chromatography as a white solid. White crystals are obtained from THF/hexane. Melting point = 213-215 °C. <1>H-NMR (CDCl 3 ) 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 = 8.2 Hz), 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<+>+1).

Example 24: Synthesis of 4-hydroxy-1-(2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-pyrrolidine-2-carboxylic acid amide

(1508).

Compound 1508 was obtained by Synthesis Scheme VII using cis-hydroxy prolinamide to form:

<1>H-NMR (d6-DMSO) d 1.90 (m, 1H), 3.85 (d, 1H, J = 9.2 Hz), 4.08 (m, 1H), 4.37 8s , 1H), 4.67 (dd, 1H, J = 8.8, 4.0 Hz), 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"+1); mp = 268-271 °C.

Example 25: Synthesis of 3-[4-((S)-2-carbamoyl-pyrrolodin-1-yl)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl]-propionic acid

(1509).

Compound 1509 was obtained using precursor compound 23 from Synthesis Scheme IX to obtain:

The tert-butoxycarbonyl-protected aryl bromide 23 (4.0 g, 9.5 mmol), dry DMSO (25 mL), NaH 2 PO 4 (454 mg, 3.79 mmol) and Na 2 HPO 4 (1.62 g, 11.4 mmol) were combined and heated to 50 °C under argon for about 3.5 hours. The mixture was then poured into water (200 mL) and extracted with three 100 mL portions of EtOAc. The combined organic layers were thoroughly washed with water, brine, dried over MgSO 4 , filtered and concentrated to give a yellow solid which was purified by trituration with ethanol to give 1.55 g of a bright yellow solid (7 ). The mother liquor was purified by flash chromatography (10% EtOAc in hexane) to give an additional 454 mg (60%). <1>H-NMR (CDCL3) d 1.77 (s, 9H), 7.25 (s, 1H), 7.48 8m, 3H), 8.52 (m, 2H), 10.39 (s , 1H); Melting point = 156 °C (dec) .

Aldehyde 7 (600 mg, 1.7 mmol) was dissolved in dry THF (20 mL) and cooled to 0 °C under argon. To this was added a 0 °C solution of (tert-butoxycarbonylmethylene)-triphenylphosphorane (694 mg, 1.8 mmol) in 10 mL of dry THF dropwise through a cannula. After 3 h, the mixture was concentrated and purified by trituration with ethanol to give 565 mg (73%) of a white solid (8). <1>HNMR (CDCl 3 ) 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).

A solution of compound 8 (565 mg 1.2 mmol) in 5 mL THF was diluted to 100 mL with EtOAc. After addition of 600 mg of catalyst (5 wt% Pd, 50% H 2 O) and purging with argon, the mixture was hydrogenated under atmospheric pressure. After 8 h, the mixture was filtered, concentrated and purified by flash chromatography (10% EtOAc in hexane) to isolate 200 mg (35%) of 9 as a clear oil which crystallized on storage. <1>HNMR (CDCl 6 ) d 1.42 (s, 9H) , 1.75 (s, 9H) , 2.65 (t, 2H), 3.32 (t, 2H) , 6.41 (s, 1H), 7.45 (m, 3H), 8.51 (m, 2H).

Aryl chloride 9 (200 mg, 0.44 mmol), DMSO (10 mL) and L-prolinamide (440 mg, 4.4 mmol) were combined and heated to 85 °C under argon. After 14 hours, the mixture was cooled to room temperature and partitioned between water and ethyl acetate. The layers were separated and the aqueous layer washed with EtOAc (3x). The combined organic layers were thoroughly washed with water (3x), brine, dried over MgSO 4 , filtered and concentrated to give 10 as a yellow film which was purified by flash chromatography (2.5% MeOH in CH 2 Cl 2 ). 185 mg (97%) . MS (ES): 435.8 (M"+1).

Ester 10 (30 mg, mmol) in 5 mL dioxane was hydrolyzed by adding 0.5 mL concentrated HCl. After 3 h, the mixture was concentrated in vacuo and recrystallized from EtOH/EtOAc to give 1509 as a white solid (20 mg, 61%). MS (ES): 380 (M"+1).

Example 26: Synthesis of [N-(2-phenyl-6-aminocarbonyl methoxymethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-(L)-prolinamide

(1510).

Compound 1510 was obtained using precursor compound 23 from Synthesis Scheme IX to form:

Bromide 23 (1.27 g, 3 mmol) and molecular sieve (5 g) are stirred in anhydrous methyl glycolate (5.8 g, 60 mmol) and DCM (40 mL). The solution is treated with AgOTf under N 2 and allowed to stir for 3 hours. The solid is removed by filtration and washed with DCM (2x20 mL). The filtrate is concentrated in vacuo. The residue is redissolved in DCM (80 mL). The resulting solution is then washed with water, saturated NaHCO 3 solution, brine, dried over MgSO 4 , filtered and concentrated to give 1.35 g (99%) of an off-white solid (12). <X>H-NMR (CDC13 d 175 (s, 9H) , 3.80 (s, 3H), 5.0 (s, 2H), 6.78 (s, 1H), 7.47 (m, 3H ), 8.52 (m, 2H).

Aryl chloride 12 (177 mg, 0.41 mmol), DMSO (10 mL), L-prolinamide (466 mg, 4 mmol) and NaHCO 3 (500 mg) are combined and heated to 120 °C under nitrogen. After 4 hours, the mixture is cooled to room temperature and diluted with water (60 ml). The resulting slurry is extracted with DCM (5x30 mL). The combined organic layers are washed with saturated NaHCO 3 solution and brine, dried over MgSO 4 , filtered and concentrated to give a brown solid. Pure product (154 mg, 92%) is obtained after flash column as a white solid (13). <X>H-NMR (CDCl 3 ) 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"+1).

Methyl ester 13 (124 mg, 0.3 mmol) is dissolved in HOCH3 (15 mL). Ammonia is bubbled through the solution for half an hour. The reaction mixture is then stirred for a further 3 hours at room temperature. After removal of the solvent, 111 mg of a white solid (1510, 93%) is formed. <1>H-NMR (CDCl 3 ) d 1.82 (m, 3H) , 2.20 (m, 1H) , 2.80 (m, 1H) , 3.10 (m, 1H) , 3.63 ( dd, 2H, Jx = 13.8 Hz, J2 = 19.4 Hz), 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"+1).

Example 27: Synthesis of [4-(2-carbamoylpyrrolidin-1-yl)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid] (1511).

Compound 1511 was synthesized using precursor compound 15 from Synthesis Scheme VII to form:

To a suspension of sodium hydride (780 mg of a 60% oil suspension, 19.5 mmol) in dry DMF (20 mL), cooled with an ice/water bath under nitrogen is added a solution of pyrrolopyrimidine 15 (2.00 g, 7.52 mmol) in DMF (10 mL) over 5 min. After 15 minutes, benzenesulfonyl chloride (1.2 mL, 9.40 mmol) is added, then the cooling bath is removed. After 4 hours, the reaction mixture is poured into a mixture of ice and saturated NaHCO 3 solution, the precipitated solid is filtered off and triturated with acetone (3) and methanol (2) to give 2.37 g of a beige solid. This solid (16) contains about 10 mol% DMF (based on this 83% yield) and can be used in the next step. A pure sample can be prepared by chromatography on silica gel using acetone as eluent. <1>H-NMR (CDCl3) : d 6.70 (d, J = 4.2 Hz, 1H), 7.47-7.68 (m, 6H), 7.76 (d, J = 4, 2 Hz, 1H), 8.24-8.32 (m, 2H), 8.48-8.56 (m, 2H); IR (fixed): n = 3146 cm"<2>, 1585, 1539, 1506, 1450, 1417, 1386, 1370, 1186, 1176, 1154, lill, 1015, 919, 726, 683, 616, 607; MS ( ES): 372/370 MH") ; melting point = 226-227 °C.

To a solution of the N-sulfonyl compound 16 (337 mg, 0.911 mmol) in dry THF (34 mL), cooled with dry ice/acetone is added LDA-THF (1.0 mL, 1.5 M solution in cyclohexane, 1.5 mmol). After 45 minutes, carbon dioxide is bubbled into the solution for 5 minutes, then the cooling bath is removed. When the solution has reached room temperature, the solvents are evaporated, giving 398 mg of the salt 17, containing 0.5 equivalents of (iPr)2NC02Li as a yellow solid. The salt is used without purification in the next step. <1>H-NMR (Di-DMSO): d= 6.44 (s, 1H), 7.50-7.75 (m, 6H), 8.33-8.40 (m, 2H), 8 .53 (dd, J = 8.0, 1.6 Hz, 2H).

A solution of the lithium salt 17 (50 mg) and L-prolinamide (122 mg, 1.07 mmol) in DMSO (1.5 mL) is heated under nitrogen to 80 °C for 15.5 h. 4% aqueous acetic acid (10 mL) is added to the cooled solution and the mixture is extracted with EtOAc (5x10 mL). The combined organic layers are washed with 4% aqueous acetic acid (10 mL), water (10 mL) and brine (10 mL) and dried over MgSO 4 . Filtration and concentration gives 40 mg of 18 as a yellowish solid which is used without purification in the next step. <1>H-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).

A solution of sodium hydroxide in methanol (1.5 mL, 5M, 7.5 mmol) is added to a solution of pyrrolopyrimidine 18 (40 mg, 0.081 mmol) in methanol (2 mL). After 2 hours the pH is adjusted to 5, most of the methanol is evaporated, the mixture is extracted with EtOAc (5x10 mL), the combined organic layers are washed with brine and dried over MgSO 4 . Filtration and concentration gives 24 mg of a light yellow solid which is triturated with toluene/EtOAc/MeOH to give 15.6 mg (55%) of the acid 1511 as a light yellowish solid. <1>H-NMR (CD3OD) : d = 2.05-2.20 (m, 4H), 3.95-4.10 (m, 1H), 4.15-4.25 (M, lh) , 4.85 (BRS, lh), 7.14 (S, lh) , 7.35-7.42 (M, 3h), 8.38-8.45 (M, 2h); IR (solid): n = 3192 cm"<1>, 2964, 2923, 2877, 1682, 1614, 1567, 1531, 1454, 1374, 1352, 1295, 1262, 1190, 974, 754, 700; MS (ES ): 352 (M"+1); melting point = 220 °C (decomp.).

Example 28: Synthesis of 1-(6-methyl-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-(S)-pyrrolidine-2-carboxylic acid amide

(1512).

Compound 1512 was synthesized by the following steps:

Aryl chloride 20 (3 g, 10.7 mmol), DMSO (50 mL) and ( S )-prolinamide were combined and heated to 85 °C under argon. After stirring overnight (14 hours), the mixture was cooled to room temperature and poured into 800 ml of water. This was extracted with three 200 mL portions of EtOAc. The combined organic layers were thoroughly washed with water (3 x 300 m), brine, dried over MgSO 4 , filtered and concentrated to give a dark brown solid. The solid was recrystallized twice from EtOAc to give 1.95 g (57%) of a light brown solid (1512). "HNMR (DMSO-d6) d 1.82-2.2 (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"+1).

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

Compound 1513 was synthesized in a similar manner to that of Example 17 using Synthetic Scheme IX with L-prolinamide and ethane-1,2-diol to form:

MS (ES): 382 (M<+>+1).

Example 30: Synthesis of 4-(6-imidazol-1-ylmethyl-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-cyclohexanol (1514).

Compound 1514 was synthesized in a similar manner to that of Example 17 using Synthesis Scheme IX with N-6 amino cyclohexanol and imidazole to form:

MS (ES): 389 (M"+1).

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 similar manner to that of Example 27 using Synthetic Scheme IX with N-6 aminocyclohexanol to form:

MS (ES): 353 (M"+1).

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 similar manner to that of compound 1513 using Synthetic Scheme IX with N-6 aminocyclohexanol to form:

MS (ES) : 383 (rrf+1) .

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

(1517).

A solution of the lithium salt 17 (0.13 mmol) in dry DMF (4 mL) is stirred with methyl iodide (0.1 mL, 1.6 mmol) at 20 °C under argon for 3 h. DMF is evaporated and aqueous ammonium chloride solution is added (15 mL). The mixture is extracted with EtOAc (3x15 mL), the combined organic layers are washed with water (2x10 mL) and brine (10 mL) and dried over MgSO 4 . Filtration and concentration yield 21 mg (38%) of methyl ester 22.

A solution of the methyl ester 22 (24.5 mg, 0.057 mmol) and 4-trans-aminocyclohexanol (66 mg, 0.57 mmol) in DMSO (1.5 mL) is heated under nitrogen to 80 °C for 5 h, then stopped the heating and stirring at 20 °C continues for 13.5 hours. 4% aqueous acetic acid (10 mL) is added to the cooled solution and the mixture is extracted with EtOAc (3x10 mL). The combined organic layers are washed with 4% aqueous acetic acid (10 mL), water (10 mL), 2N NaOH (10 mL), water (10 mL), and brine (10 mL) and dried over MgSO 4 . To a solution of the crude material obtained after filtration and concentration (1H NMR indicates about 50% removal of the benzenesulfonyl group) in THF (2 mL) is added a solution of NaOH in MeOH (0.5 mL 5M solution, 2.5 mmol) at room temperature. After 20 minutes, water and saturated NaHCO 3 ~ solution (5 mL each) are added and the mixture is extracted with EtOAc (4x15 mL). The combined organic layers are washed with 2N NaOH

(10 mL), water (10 mL) and brine (10 mL) and dried over MgSO 4 . Chromatography of the crude material obtained after filtration and concentration on silica gel, eluting with hexanes/EtOAc 1:1 ® 1:2 gives 8.6 mg (41%) of 1517 as a white solid, mp 225-227 ° C. <1>H-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, lh), 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, 1165, 1074, 938, 756, 705 ; MS (ES) : 367 (MH") .

Example 34: Synthesis of [4-(2-carbamoyl-pyrrolidin-1-yl)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-6-ylmethoxy]-acetic acid methyl ester (1518).

Compound 1518 was synthesized in a similar manner to Example 2 6 using precursor compound 12 to form:

MS (ES): 410 (m<+>+1).

Example 35: Synthesis of [4-(2-carbamoyl-pyrrolidin-1-yl)-2-phenyl-7H-pyrrolo[2,3-d]pyrimidin-6-ylmethoxy]-acetic acid

(1519).

Compound 1519 was synthesized in a similar manner to compound 1515 where the methyl ester group was hydrolyzed with a base to form:

MS: 396 (M"+1).

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

(1520).

Gaseous ammonia is condensed into a solution of pyrrolopyrimidine 23 (7.8 mg, 0.021 mmol) in methanol (6 mL), cooled with dry ice/acetone until a total volume of 12 mL is reached. After stirring for 10 days at 20 °C, the solvents are evaporated and the residue is purified by preparative TLC on silica gel by elution with 5% MeOH in CH 2 Cl 2 . The material thus formed is triturated with ether to give 6.5 mg (88%) of the amide 1520 as a white solid, mp 210-220 °C (decomp.). <1>H-NMR (CD3OD) : d = 1.40-1.60 (m, 4H) , 2.00-2.15 (nm 2G(m 2ml5.2m25 /nm 2G(m 3m55.3m69 /nm lG(m 4m29.4m35 /nm lG(m 6ml5 /sm lG(m 6m35.6m46 /nm 3G(m 7m34.7m49 /nm 2G(M UR (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") .

Activity of compounds

Adenosine 1 (Ai) receptor subtype saturation and competitive radioligand binding were performed for compounds 1505, 1506,

1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1516, 1517, 1518, 1519 and 1520 as described herein and i.a. on pages 152-153 of the present description. All of the compounds listed above equal or exceeded the Ai receptor binding affinity of reference compounds 1318 or 1319 as described herein, and inter alia in table 13.

The water solubilities of the above compounds listed in Table 18 are expected to be better than reference compounds 1318 or 1319 due to their cLogP values which were calculated using the computer program CS ChemDraw, ChemDraw Ultra ver. 6.0 ©1999 as provided by CambridgeSoft corporation, 100 Cambridge Park Drive, Cambridge, MA 02140.

The compounds specific for the Ai receptor listed in Table 18 had lower cLogP values between about 1.5 to about 3.4, compared to reference compounds 1318 or 1319 with a cLogP value of about 3.8. It was not predicted that the more polar A1 receptor compounds listed in Table 18 that have lower cLogP values than reference compounds 1318 or 1319 would still maintain efficacy and A1 receptor binding selectivity relative to these reference compounds.

Additional compounds that are specific to the A2a receptor

The present invention provides a compound having the structure:

The present invention also provides a compound having the structure:

The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of compound 1609 or 1610 and a pharmaceutically acceptable carrier.

The invention also provides the above pharmaceutical composition wherein the therapeutic amount is effective to treat Parkinson's disease and diseases associated with locomotor activity, vasodilation, platelet inhibition, neutrophil superoxide formation, cognitive disorder or senile dementia.

The invention also provides the above-mentioned pharmaceutical composition where the pharmaceutical composition is an ophthalmic formulation.

The invention also provides the above-mentioned pharmaceutical composition where the pharmaceutical composition is a periocular, retrobulbar or intraocular injection formulation.

The invention also provides the above-mentioned pharmaceutical composition where the pharmaceutical composition is a systemic formulation.

The invention also provides the above-mentioned pharmaceutical composition where the pharmaceutical composition is a surgical spray solution.

The invention also provides a packaged pharmaceutical composition for treating a disease associated with A2a adenosine receptor in a subject, comprising: (a) a container containing a therapeutically effective amount of compound 1609 or 1610, and (b) instructions for using the compound to treat said disease in an individual.

Exemplification

Example 41: Synthesis of 1-(6-phenyl-2-pyridin-4-yl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-pyrrolidine-2-carboxylic acid amide

(1609).

Compound 1609 was synthesized by reacting L-prolinamide with the appropriate chloride intermediate described in Synthesis Scheme II on page 82 to form:

<1>H-NMR (de-DMSO) 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 = 7.0 Hz), 7.44 (t, 2H, J = 7.0 Hz ), 7.59 (s, 1H), 7.92 (brs, 2H), 8.26 (d, 2H, J = 6.2 Hz), 8.65 (d, 2H, J = 6.2 Hz ); MS (ES): 384.9 (M"+1); Melting point = 280-316 °C (decomp.).

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

Compound 1610 was synthesized by reacting L-prolinamide with the appropriate chloride intermediate described in Synthesis Scheme II on page 82 to form:

<1>H-NMR(d6-DMSO) d 2.07 (m, 4H), 3.85 (s, 3H), 4.02 (m, 1H), 4.17 (M, 1H), 4, 75 (m, 1H), 6.89 (m, 1H), 7.00 (s, 1H), 7.23 (s, 1H), 7.35 (t, 1H, J = 8.2 Hz), 7.53 (s, 2H), 7.60 (s, 1H), 8.28 (d, 2H, J = 5.8 Hz), 8.67 (d, 2H, J = 5.8 Hz), 12.37 (s, 1H); MS (ES): 415.0 (M<+>+1).

Activity of compounds

Adenosine 2a (A2a) receptor subtype competitive radioligand binding was performed for compounds 1609 and 1610 as described herein. Compounds 1609 and 1610 were found to have A2a receptor binding affinity and selectivity.

Pages 294-300 concern further compounds that are specific to the A3 receptor

The present invention also provides a compound having the structure:

The compound can be used to inhibit the activity of an A3 adenosine receptor in a cell where the cell is contacted with the compound 1720.

In a further embodiment, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of compound 1720 and a pharmaceutically acceptable carrier.

In a further embodiment, the invention provides a packaged pharmaceutical composition for treating a disease associated with A3 adenosine receptor in an individual, comprising: (a) a container containing a therapeutically effective amount of compound 1720, and (b) instructions for using said compound to treat said disease in an individual.

In a further embodiment, the invention provides a method for preparing a composition comprising compound 1720, where the method comprises mixing compound 1702 with a suitable carrier material.

In a further embodiment, the invention provides a pharmaceutically acceptable salt of compound 1720 wherein the pharmaceutically acceptable salt contains an anion selected from the group consisting of maleic, fumaric, tartaric, acetate, phosphate and mesylate.

Exemplification

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

Compound 1720 was synthesized using precursor compound 1 from Synthesis Scheme VII to form:

Aryl chloride 1 (400 mg, 1.50 mmol), DMSO (10 mL) and histamine (1.67 g, 15.0 mmol) are combined and heated to 120 °C under nitrogen. After 6.5 h, the reaction mixture is cooled to room temperature and partitioned between EtOAc and water. The layers are separated and the aqueous layer is extracted with EtOAc (3x). The combined organic layers are washed with brine (2x), dried over MgSO 4 , filtered and concentrated to give 494 mg of a brown solid. The solid is washed with cold MeOH and recrystallized from MeOH to give 197 mg (43%) of an off-white solid (1720). <1>H-NMR (CD30D) d 3.05 (t, 2H, J = 7.0 Hz), 3.94 (t, 2H, J = 7.0 Hz), 6.50 (d, 1H, J = 3.5 Hz), 6.88 (brs, 1H), 7.04 (d, 1H, J = 3.5 Hz), 7.42 (m, 3H), 7.57 (s, 1H) , 8.34 (m, 2H); MS (ES): 305.1 (M"+1); Melting point = 234-235 °C.

Activity of compounds

Adenosine 3 (A3) receptor competitive radioligand binding was performed for compound 1720 as described herein. Compound 1720 was found to have an A3 receptor binding affinity greater than 10 times that of reference compound 1308 as described herein and inter alia. in table 13.

The present invention further provides compounds having the formula:

where RiNR2 together form a ring having the structure: or Ri is H and R2 is:

aR;;;;ryrr subsutured — —— alkyl or

Claims (64)

1. Compound having the structure: in which R1NR2 together form a ring having the structure: or R 1 is H and R 2 is: and, when RiNR2 together is R5 is H, -CH2 (NC5H8) (OH) (C6H5) , -CH2OCH3, - (CH2) 2C (0) OH, - CH2OCH2C (0)NH2, -C(0)OH, - CH3, -CH20(CH2)20H, -CH2OCH2(0)OCH3 or -CH2OCH2C(0)OH, or when R1 is H and R2 is is R5 -CH2 (N2C3H3), -C(0)OH, CH2O (CH2)20H, -C(0)0CH3 or -C(0)NH2 or a pharmaceutically acceptable salt thereof. 2. Compound according to claim 1 which has the structure: 3. Compound according to claim 1 which has the structure: 4. Compound according to claim 1 which has the structure: 5. Compound according to claim 1 which has the structure: 6. Compound according to claim 1 which has the structure: 7. Compound according to claim 1 which has the structure: 8. Compound according to claim 1 which has the structure: 9. Compound according to claim 1 which has the structure: 10. Compound according to claim 1 which has the structure: 11. Compound according to claim 1 which has the structure: 12. Compound according to claim 1 which has the structure: 13. Compound according to claim 1 which has the structure: 14. Compound according to claim 1 which has the structure: 15. Compound according to claim 1 which has the structure: 16. Compound according to claim 1 which has the structure: 17. Compound according to claim 1 which has the structure: 18. Use of a compound according to claim 1 in the preparation of a medicament which is useful for treating a disease associated with Ai adenosine receptor in an individual. 19. Use according to claim 18 where said Ai adenosine receptor is associated with cognitive disease, kidney failure, cardiac arrhythmia, respiratory epithelium, transmitter release, anaesthesia, vasoconstriction, bradycardia, negative cardiac inotropy and dromotropic, bronchoconstriction, neutrophil chemotaxis, acid reflux or peptic ulcer conditions. 20. Use of a compound according to claim 1 in the preparation of a drug which is useful for inhibiting the activity of an Ai adenosine receptor in a cell. 21. Use according to claim 18 where said disease is asthma, chronic obstructive pulmonary disorder, allergic rhinitis or a disorder in the upper respiratory tract. 22. Pharmaceutical composition comprising the compound according to claim 1, a pharmaceutically acceptable carrier and at least one of a steroid, β2 agonist, glucocorticoid, leukotriene antagonist or an anticholinergic agonist. 23. Pharmaceutical composition comprising a therapeutically effective amount of the compound according to claim 1 and a pharmaceutically acceptable carrier. 24. Use according to claim 18 where the disease is asthma, allergic rhinitis or chronic obstructive pulmonary disease. 25. A packaged pharmaceutical composition for treating a disease associated with Ai adenosine receptor in a subject comprising: (a) a container containing a therapeutically effective amount of the compound of claim 1, and (b) instructions for using said compound to treat said disease in an individual. 26. Pharmaceutically acceptable salt of the compound according to claim 6, 8, 12, 15 or 16 wherein the salt contains a cation selected from the group consisting of sodium, calcium and ammonium. 27. Use according to claim 18, where the Ai adenosine receptor is associated with congestive heart failure. 28. Compound having the structure: 29. Compound having the structure: 30. Use of a compound according to claim 28 or 29 in the preparation of a drug which is useful in the treatment of a disease associated with A2a-adenosine receptor in an individual. 31. Application according to claim 18 or 30 where the individual is a human. 32. Use according to claim 31, where said A2a adenosine receptor is associated with movement activity, vasodilation, platelet inhibition, neutrophil superoxide formation, cognitive disorder, senile dementia or Parkinson's disease. 33. Use according to claim 30, wherein the compound treats said diseases by stimulating adenylate cyclase. 34. Use of a compound according to claim 28 or 29 for the preparation of a drug to inhibit the activity of an A2a adenosine receptor in a cell. 35. Pharmaceutical composition comprising a therapeutically effective amount of the compound according to claim 28 or 29 and a pharmaceutically acceptable carrier. 36. Pharmaceutical composition according to claim 35, wherein said therapeutically effective amount is effective to treat Parkinson's disease and diseases associated with locomotor activity, vasodilation, platelet inhibition, neutrophil superoxide formation, cognitive disorder or senile dementia. 37. Pharmaceutical composition according to claim 35, wherein said pharmaceutical composition is an ophthalmic formulation. 38. Pharmaceutical composition according to claim 23 or 35, wherein said pharmaceutical composition is a periocular, retrobular or intraocular injection formulation. 39. Pharmaceutical composition according to claim 23 or 35, wherein said pharmaceutical composition is a systemic formulation. 40. Pharmaceutical composition according to claim 23 or 35, wherein said pharmaceutical composition is a surgical injection solution. 41. A pharmaceutical composition comprising the compounds of claim 28 or 29, a pharmaceutically acceptable carrier and any dopamine enhancing material. 42. A packaged pharmaceutical composition for the treatment of a disease associated with A2a adenosine receptor in an individual, comprising: a) a container containing a therapeutically effective amount of the compound according to claim 28 or 29; and b) instructions for using said compound to treat said disease in an individual. 43. Pharmaceutically acceptable salt of the compound according to claim 28 or 29. 44. Compound having the structure: or a pharmaceutically acceptable salt thereof. 45. Use of a compound according to claim 44 in the preparation of a drug which is useful for inhibiting the activity of an A3-adenosine receptor in a cell. 46. Use according to claim 20, 34 or 45, where the cell is a human cell. 47. Use of a compound according to claim 44 in the preparation of a drug which is useful for the treatment of damage to the eye of an individual. 48. Use according to claim 47, where the damage comprises retinal or optic nerve head damage. 49. Use of a compound according to claim 44 in the preparation of a drug which is useful in the treatment of cataracts in an individual. 50. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to claim 46 and a pharmaceutically acceptable carrier. 51. A packaged pharmaceutical composition for treating a disease associated with an A3 adenosine receptor in an individual, comprising: a) a container containing a therapeutically effective amount of the compound of claim 49; and b) instructions for using said compound to treat said disease in an individual. 52. Method for producing a composition comprising the compound according to claim 44, wherein the method comprises mixing the compound with a suitable carrier. 53. A pharmaceutically acceptable salt of the compound according to claim 28, 29 or 44, wherein the pharmaceutically acceptable salt contains an anion selected from the group consisting of maleic, fumaric, tartaric, acetate, phosphate and mesylate. 54. Use of a compound according to claim 44 in the preparation of a drug which is useful for the treatment of a disease associated with an A3 adenosine receptor in an individual in need of such treatment where the disease associated with the A3 adrenosine receptor is asthma, bronchitis, chronic obstructive pulmonary disease or bronchoconstriction . 55. Process for the preparation of a compound according to claim 44, comprising the steps: (a) reacting the compound with histamine in the presence of dimethyl sulfoxide: (b) heating the reaction mixture under an inert atmosphere; (c) cooling the reaction mixture and separating the organic layer from the aqueous layer; (d) washing the organic layer with brine, drying and filtering to give the compound. 56. Method according to claim 55 where in step (b) the reaction mixture is heated under a nitrogen atmosphere. 57. Method according to claim 55 where in step (d) the organic layer is dried with MgSO4. 58. Use of a compound according to claim 1 in the preparation of a drug that is useful for the treatment of a disease associated with an Ai adenosine receptor in an individual in need of such treatment where the disease is antidiuresis, bradycardia, bronchitis, bronchoconstriction, Alzheimer's disease, cardiac arrhythmia , cardiac hypoxia, hypertension, inflammation, negative cardiac inotropy and dromotropic, renal failure, anesthesia or is associated with transmitter release, respiratory epithelium, contraction of smooth muscles underlying the respiratory epithelium, vasoconstriction or mast cell degranulation. 59. Use of a compound according to claim 1 for the preparation of a drug for increasing the memory of an individual. 60. Use of a compound according to claim 28 or 29 in the preparation of a drug which is applicable for the treatment of a disease associated with an A2a adenosine receptor in an individual in need of such treatment where the disease associated with an A2a adenosine receptor is Parkinson's disease or glaucoma. 61. Use of a compound according to claim 44 in the preparation of a drug which is applicable for the treatment of a disease associated with an A3 adenosine receptor in an individual in need of such treatment where the disease associated with an A3 adenosine receptor is myocardial ischaemia, bronchitis or bronchoconstriction. 62. Use of a compound according to claim 44 for the preparation of a drug which is useful for treating inflammation in the eye associated with an A3 adenosine receptor in an individual. 63. Use of a compound according to claim 44 for the preparation of a drug which is useful for the treatment of a disease associated with an A3 adenosine receptor in an individual in need of such treatment where the disease associated with the A3 adenosine receptor is associated with mast cell granulation. 64. Use of a compound according to claim 44 in the preparation of a drug which is applicable for the treatment of a disease associated with an A3 adenosine receptor in an individual in need of such treatment where the disease associated with the A3 adenosine receptor is asthma, glaucoma, retinopathy, ocular ischemia or macular degeneration.