EA007254B1 - Compounds specific to adenosine a, aand areceptor and uses thereof - Google Patents

Abstract

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

Description

This application is a partial continuation, and it claims priority on the basis of applications for US patent No. 09/728316, filed December 1, 2000, 09/728607, filed December 1, 2000, and 09/728616, filed December 1, 2000 ., each of which is incorporated into this description by reference in its entirety.

This application refers to compounds that specifically bind to ί) A ₁ adenosine receptors (such as, but not limited to, pages 4-76, 130-175 and 257-287);

ίί) A _2a adenosine receptors (such as, but not limited to, p.176-201 and p. 288-293) and A ₃ adenosine receptors (such as, but not limited to, p. 202-256 and 294-300).

BACKGROUND OF THE INVENTION

Adenosine is a widespread modulator of numerous types of physiological activity, in particular in the cardiovascular and nervous systems. It turns out that the action of adenosine is mediated by specific receptor proteins on the cell surface. Adenosine modulates various physiological functions, including the induction of sedation, vasodilation, suppression of heart rate and cardiac contractility, inhibition of platelet aggregation, stimulation of gluconeogenesis and inhibition of lipolysis. It has been shown that in addition to its effect on adenylate cyclase, adenosine opens potassium channels, reduces the current through calcium channels and inhibits or stimulates phosphoinositide turnover using receptor-mediated mechanisms (see, for example, S.E. Mi. and apb B.81esh Abepokshe Keser1og Ap1adoshk1k: ByisShtek apb Ro1epya1 Tyegareijs Arriesayopk, Siggep! Ryattaseijsa1 Oek1dp 2: 501 (1996) and S.E. Mi11eg Agabepokshe Residuyid Ayidopyk, Exp.

Adenosine receptors belong to the superfamily of purine receptors, which are currently divided into P ₁ (adenosine) and P ₂ (ATP, ADP and other nucleotides) receptors. To date, four subtypes of receptors for adenosine nucleoside have been cloned from various species, including humans. Two receptor subtypes (A ₁ and A _2a ) exhibit affinity for adenosine in the nanomolar range, while two other known subtypes of A _2b and A ₃ are low affinity receptors for adenosine in the lower micromolar range. Activation of A ₁ and A ₃ adenosine receptors can lead to inhibition of adenylate cyclase activity, while activation of A _2a and A _2b causes adenylate cyclase to be stimulated.

Several A1 antagonists have been developed to treat cognitive impairment, renal failure, and cardiac arrhythmias. It has been suggested that A2a antagonists may be beneficial for patients suffering from parkinsonism (Parkinson's disease). In particular, given the ability to deliver locally, adenosine receptor antagonists may be valuable in the treatment of allergic inflammation and asthma. Available information (for example, Yuse & Mehdeg ΌΝΑ apykepka Tjetaru £ from Akbppa sh ap Ashsha1 Mobe1, No. 1 Shge (1997) 385: 721-5) indicates that, in this pathophysiological context, A ₁ antagonists can block the contraction of smooth muscles lying under the respiratory epithelium whereas A _2b or A ₃ receptor antagonists can block mast cell degranulation, decreasing the release of histamine and other inflammatory mediators. And _2b receptors were found in the gastrointestinal tract, especially in the epithelium of the rectum and small intestine. It has been suggested that A _2b receptors mediate a cAMP response (81 echel1 e1 a1., EByu.Syesh. (1995) 270: 2387-94).

It has also been shown that adenosine receptors exist on the retina of various mammalian species, including cattle, pig, monkey, rat, guinea pig, mouse, rabbit, and humans (see Baxophellus e1a1., 0 | tashshayap Keipa, 1oit1 о £ №igosyet1k11u, uuppe 54, Radek 648-655 (1990); Хуобк e1 a1., SyagasUpkhiop o £ Abepokshe ₁ Resergy bshbd Beyek sh Vogte Re1a1 Metepecke33, 33 (1991); and Vgaak e1 a1., Epbodepoik Abepokshe Apb Abepokshe Geser Yugk 1osheb 1o dapdnop s11k o £ 111e teipa, Rtoseybshdk about £ 111e No. Shop1 Asabetu about £ BOens, woopte 84, Radek 3906-3910 (1987)). Recently, the results of observations of adenosine transport sites in a cultured human retinal cell line have been reported (Sjyub e1 a1., Shskoybe TgapkroP Beyek sha Siyigek Nitap Keipa se11 Leke Eklabekeb uyu 8U-40 T Aptsdep depe, Sztep! Eyeu radieke -118 (1994)).

Compounds that regulate adenosine uptake have previously been proposed as potential therapeutic agents for treating retinal and optic nerve damage. In US Pat. No. 5,780,450 to Biabe, the author discusses the use of adenosine absorption inhibitors for the treatment of eye diseases. Byabe does not describe the use of specific inhibitors of the A ₃ receptor. The full contents of US patent No. 5780450 included in the present description by reference.

As pharmacological tools, additional antagonists of adenosine receptors are needed, and they are of significant interest as drugs for the above-mentioned disease states and / or diseases.

Summary of the invention

The present invention relates to compounds that selectively bind to the A ₁ adenosine receptor, thereby treating a disease associated with the A1 adenosine receptor in

- 1 007254 of the subject when a therapeutically effective amount of such compounds is administered to the subject. The disease being treated is associated with impaired cognitive ability, renal failure, cardiac arrhythmia, respiratory epithelium, release of transmitters, sedation, constriction of blood vessels, bradycardia, negative cardiac inotropy and dromotropia, bronchostenosis, neutrophilic chemotrexemia, and chemotaxis.

The present invention is based, at least in part, on the fact that some N-6 substituted 7-desazapurines described below can be used to treat a disease condition that responds to N-6 substituted 7-desazapurin. Examples of such conditions include those in which adenosine receptor activity is increased, for example, bronchitis, gastrointestinal diseases, or asthma. Such conditions can be characterized by the fact that activation of the adenosine receptor can lead to inhibition or stimulation of adenylate cyclase activity. Compositions and methods of the invention include enantiomerically or diastereomerically pure N-6 substituted 7-desazapurines. Preferred N-6 substituted 7-desazapurines include those containing fragments of acetamide, carboxamide, substituted cyclohexyl, for example cyclohexanol, or urea, attached to N-6 nitrogen through an alkylene chain.

The present invention relates to methods for modulating the adenosine receptor (s) in a mammal by administering to the mammal a therapeutically effective amount of N-6 substituted 7-deazapurin so that adenosine receptor activity is modulated. Suitable adenosine receptors include the A _b A ₂ and A ₃ family. In a preferred embodiment, N-6 substituted 7-desazapurin is an adenosine receptor antagonist.

The invention further relates to methods for treating diseases that respond to N-6 substituted 7-desazapurines, for example, asthma, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease, kidney disease, gastrointestinal diseases and eye diseases in a mammal by administering to the mammal a therapeutically effective amount of N-6 substituted 7-desazapurin, so that the disease is treated in the mammal. Suitable N-6 substituted 7-desazapurines include those illustrated by general formula I:

(I) and their pharmaceutically acceptable salts. Each of Κι and K ₂ independently represents a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl fragment, or together they form a substituted or unsubstituted heterocyclic ring. K ₃ represents a substituted or unsubstituted alkyl, aryl or alkylaryl moiety. K ₄ represents a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety. Each of K ₅ and Κ ₆ , independently, represents a halogen atom, for example, chlorine, fluorine or bromine, a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety, or K ₅ represents a carboxyl, carboxyl esters or carboxamides, or K ₄ and Κ ₅ , or K ₅ and K ₆ together form a substituted or unsubstituted heterocyclic or carbocyclic ring.

In some embodiments, each of Κι and Κ ₂ , independently, may be substituted or unsubstituted cycloalkyl or heteroarylalkyl moieties. In other embodiments, K ₃ is a hydrogen atom or a substituted or unsubstituted heteroaryl moiety. In still other embodiments, each of K ₄ , K _5, and K, independently, may be a heteroaryl moiety. In a preferred embodiment, Κι represents a hydrogen atom, K ₂ represents cyclohexanol, for example, trans-cyclohexanol, K ₃ represents phenyl, K ₄ represents a hydrogen atom, K ₅ represents a methyl group and K <5 represents a methyl group. In another embodiment, K represents a hydrogen atom,

K ₂ represents

K ₃ represents phenyl, K ₄ represents a hydrogen atom, K ₅ and K represent methyl groups.

- 2 007254

The invention further relates to pharmaceutical compositions for treating conditions that respond to N-6 substituted 7-desazapurines in mammals, for example, asthma, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease, kidney disease, gastrointestinal diseases and eye diseases. The pharmaceutical composition comprises a therapeutically effective amount of N-6 substituted 7-desazapurin and a pharmaceutically acceptable carrier.

The present invention also relates to packaged pharmaceutical compositions for treating conditions that respond to N-6 substituted 7-deazapurins in mammals. Packed pharmaceutical compositions include a container containing a therapeutically effective amount of at least one N-6 substituted 7-desazapurine, and instructions for using N-6 substituted 7-desazapurin for treating conditions that respond to N-6 substituted 7-desazapurines, mammals.

The invention further relates to compounds of formula I, wherein

Κ ₁ represents hydrogen;

K ₂ represents a substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkyl, or Κ ₁ and K ₂ together form a substituted or unsubstituted heterocyclic ring;

K ₃ represents unsubstituted or substituted aryl;

K ₄ represents hydrogen; and each of K ₅ and Κ5 independently represents hydrogen or alkyl, and their pharmaceutically acceptable salts. The desazapurins of this embodiment can advantageously be selective A ₁ receptor antagonists. These compounds may be useful for many therapeutic applications, such as, for example, the treatment of asthma, renal failure associated with heart failure, and glaucoma. In a particularly preferred embodiment, desazapurin is a water-soluble prodrug that is capable of metabolizing ίη νίνο into an active drug, for example, by hydrolysis catalyzed by esterase.

In yet another embodiment, the invention relates to a method for inhibiting the activity of an adenosine receptor (e.g., A ₃ ) in a cell by contacting the cell with an N-6 substituted 7-dezazapurin (e.g., preferably an adenosine receptor antagonist).

In another aspect, the invention relates to a method for treating eye damage in a mammal (e.g., a human) by administering to the animal an effective amount of N-6 substituted 7-desazapurin of Formula I. Preferably, N-6 substituted 7-desazapurin is an antagonist of A ₃ adenosine receptors in mammalian cells. Damage refers to the retina or optic head nerve and can be acute or chronic. Damage may result from, for example, glaucoma, edema, ischemia, hypoxia, or trauma.

The invention also relates to a pharmaceutical composition comprising an N-6 substituted compound of formula I. Preferably, the pharmaceutical preparation is an ophthalmic composition (for example, a composition for periocular, retrobulbar or intraocular injection, a systemic composition and a surgical irrigation solution).

where X is N or CB ^ each of B! and K ₂ independently represents hydrogen or substituted or unsubstituted alkoxy, aminoalkyl, alkyl, aryl or alkylaryl, or together they form a substituted or unsubstituted heterocyclic ring, provided that both Κ ₁ and K ₂ are not hydrogen; K ₃ represents substituted or unsubstituted alkyl, arylalkyl or aryl; K ^L represents hydrogen or substituted or unsubstituted C _T C ₆ alkyl; B represents hydrogen, substituted or unsubstituted alkyl, or B ₄ and B together form a substituted or unsubstituted heterocyclic or carbocyclic ring; Κ6 represents hydrogen, substituted or unsubstituted alkyl, or halogen; Ω represents CH ₂ , O, 8 or IV ₇ , where K ₇ represents hydrogen or substituted or unsubstituted C ₁ -C ₆ alkyl; and is unsubstituted or substituted alkyl, cycloalkyl, aryl, arylalkyl, biaryl, heteroaryl, substituted carbonyl, substituted thiocarbonyl or substituted sulfonyl; provided that if Κ3 is pyrrolidino, then Κ4 is not methyl. The invention also includes pharmaceutically acceptable salts and prodrugs of the compounds of the invention.

- 3 007254

In an advantageous embodiment, X is SC ₆ , and P is CH ₂ , O, 8 or ΝΗ in formula II, wherein K ₆ is as defined above.

In another embodiment of Formula II, X is Ν.

The invention further relates to a method of inhibiting the activity of an adenosine receptor (e.g., A _2b -adenozinovogo receptor) in a cell when the cell is contacted with a compound of the invention. Preferably, the compound is a receptor antagonist.

The invention also relates to a method for treating a gastrointestinal disease (e.g., diarrhea) or a respiratory disease (e.g., allergic rhinitis, chronic obstructive pulmonary disease) in an animal by administering to the animal an effective amount of a compound of formula II (e.g., L _2b antagonist). Preferably, the animal is a human.

A distinctive feature of the invention is also a compound having the structure:

N

IV where K ₁ is trans-4-hydroxycyclohexyl, 2-methylaminocarbonylaminocyclohexyl, 2-methylaminocarbonylaminocyclohexyl, acetamidoethyl or methylaminocarbonylaminoethyl;

where K ₃ represents a substituted or unsubstituted four-six membered ring;

where K ₅ represents H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, where said substituted alkyl is —C (K ₇ ) (Κ ₈ ) ΧΚ ₉ , where X is O, 8 or ΝΚ ₁₀ where each of K ₇ and K ₈ independently represents H or alkyl, where each of K ₉ and K ₁₀ independently represents alkyl or cycloalkyl, or K ₉ , K ₁₀ and nitrogen together form a substituted or unsubstituted ring containing 4-7 members;

where K ₆ represents H, alkyl, substituted alkyl, cycloalkyl; or a pharmaceutically acceptable salt or prodrug derivative or biologically active metabolite; provided that when K ₁ is acetylaminoethyl, K _{3 is} not 4-pyridyl.

In one embodiment, Compound K ₃ is phenyl, pyrrole, thiophene, furan, thiazole, imidazole, pyrazole, 1,2,4-triazole, pyridine, 2 (1H) -pyridone, 4 (1H) -pyridone, pyrazine, pyrimidine , pyridazine, isothiazole, isoxazole, oxazole, tetrazole, naphthalene, tetraline, naphthyridine, benzofuran, benzothiophene, indole, 2,3-dihydroindole, 1H-indole, indoline, benzopyrazole, 1,3-benzodioxole, benzoxazole, purine, coumarin , quinoline, tetrahydroquinoline, isoquinoline, benzimidazole, quinazoline, pyrido [2,3b] pyrazine, pyrido [3,4-b] pyrazine, pyrido [3,2-c] pyridazine, pyrido [3,4-b] pyridine , 1H-pyrazole [3,2-6] pyrimidine, pteridine, 2 (1H) -quinolone, 1 (2H) -isoquinolone, 1,4-benzisoxazine, benzothiazole, quinoxaline, quinoline-nooxide, isoquinoline-no-oxide, quinoxaline-Noxide, quinazoline ^ -oxide, benzoxazine, phthalazine, cinnolin or has the structure:

where Υ represents carbon or nitrogen;

where K ₂ and K ₂ 'independently represent H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, halogen, methoxy, methylamino or methylthio.

This invention also relates to a compound having the structure:

V where K ₃ represents aryl, substituted aryl or heteroaryl;

- 4 007254 where Kb represents H, alkyl, substituted alkyl or cycloalkyl; where B ₅ represents H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, wherein said substituted alkyl is —C (K ₇ ) (Κ ₈ ) ΝΚ ₉ Κ ₁₀ , where each of K ₇ and K ₅ represents H or alkyl, where each of K. ₉ and K ₁₀ represents alkyl or cycloalkyl, or K ₉ , K ₁₀ and nitrogen together form a ring system containing 4-7 members.

The present invention also relates to a method for inhibiting the activity of an L ₁ adenosine receptor in a cell, comprising contacting said cell with the above compounds.

Detailed description

Distinctive features and other details of the invention will now be described more specifically and are reflected in the claims. It should be understood that specific embodiments of the invention are provided by way of illustration and not limitation of the invention. The principal distinguishing features of this invention can be used in various embodiments without departing from the scope of the invention.

The present invention relates to methods for treating conditions that respond to Ν-

6-substituted 7-desazapurin in a mammal. The methods include administering to the mammal a therapeutically effective amount of β-substituted 7-desazapurine as described below, such that a condition is treated in the mammal that responds to β-substituted 7-desazapurin.

The expression “condition that responds to Ν-b-substituted 7-desazapurine" is intended to indicate a disease state or disease characterized by a response to treatment with Ν-b-substituted 7-desazapurin according to the invention, as described below, for example, treatment includes significant the reduction of at least one symptom or the consequences of the disease state achieved using the Ν-b substituted 7-desazapurin according to the invention. Typically, such conditions are associated with an increase in the amount of adenosine in the host, so that the host often experiences physiological symptoms, which include, but are not limited to, the release of toxins, inflammation, coma, water retention, weight gain or weight loss, pancreatitis, emphysema, rheumatoid arthritis, osteoarthritis, multiple organ damage, pediatric or adult respiratory distress syndrome, allergic rhinitis, chronic obstructive pulmonary disease, eye diseases, gastrointestinal diseases, stimulated skin lesions, immunodeficiency, and asthma (see, for example, S.E. Mi11eg apb B. 81st. .. a | -Lbspo5ts VessrYug Lp1adosh515 Ehr.Ort.Tyst Ra1sp15 7 (5): 419 (1997), and I. Esok1Yous V. Ro1o§a, 8.T. No1da1s and I. V1addyush Abspots L _2b gessrYugh and pous1 Shegarsibs 1agds1 w Lkshta T1P8 19: 148 (1998) .Activities often associated with such symptoms include, but are not limited to, fever, lack of breathing, nausea, diarrhea, weakness, headache, and even death. In one embodiment, the disease state responding to β-substituted 7-desazapurin includes those diseases that are mediated by stimulation of adenosine receptors, for example A !, L _2a , L _2b , A ₃ , etc., such in which the concentration of calcium in the cells and / or the activation of PBC (phospholipase C) is modulated. In a preferred embodiment, the disease state responding to β-substituted

7-desazapurin is associated with adenosine receptor (s), for example, β-substituted 7-desazapurin acts as an antagonist. Examples of suitable responsive conditions that can be treated with the compounds of the invention, for example, subtypes of adenosine receptors that mediate a biological effect, include central nervous system (CNS) action, cardiovascular effect, renal effect, respiratory effect, immunological effect gastrointestinal effects and metabolic effects. The relative amount of adenosine in a subject may be related to the following action; that is, elevated adenosine levels can trigger an action, for example, an undesirable physiological response, for example, an asthma attack.

CNS effects include decreased transmitter release (L!), Sedative effect (A!), Decreased locomotor activity (L _2a ), anticonvulsant activity, chemoreceptor stimulation (L ₂ ), and hyperalgesia. The therapeutic use of the compounds of the invention includes the treatment of dementia, Alzheimer's disease and memory improvement.

Cardiovascular effects include vasodilation (L _2a ), (L _2b ) and (A ₃ ), vasoconstriction (L ₁ ), bradycardia (L ₁ ), platelet inhibition (L _2a ), negative cardiac inotropy and dromotropia, arrhythmia, tachycardia and angiogenesis. The therapeutic use of the compounds of the invention includes, for example, prophylaxis of ischemia-induced damage to the heart and stimulation of the activity of the heart, protection of myocardial tissue and restoration of heart function.

Renal effects include reduced glomerular filtration rate (L1), mesangial cell contraction (L ₁ ), antidiuresis (L ₁ ), and inhibition of renin release (L ₁ ). Suitable therapeutic uses of the compounds of the invention include the use of the compounds of the invention as diuretics, natriuretics, preparations for potassium deficiency, for the protection of

- 5,007,254 check / prophylaxis of acute renal failure, antihypertensive, anti-edematous and anti-nephritic agents.

Respiratory effects include bronchodilation (A ₂ ), narrowing of the bronchi (Αι), chronic obstructive pulmonary disease, allergic rhinitis, mucous discharge and respiratory depression (A ₂ ). Suitable therapeutic uses of the compounds of the invention include anti-asthma, treatment of lung disease after transplantation and respiratory diseases.

Immunological effects include immune suppression (A ₂ ), neutrophilic chemotaxis (A ₁ ), neutrophilic superoxide generation (A _2a ) and mast cell degranulation (A _2b and A ₃ ). The therapeutic use of antagonists includes allergic and non-allergic inflammation, for example, the release of histamine and other inflammatory mediators.

Gastrointestinal effects include inhibition of acid release (A ₁ ); therapeutic use may include reflux and peptic ulcer disease. The gastrointestinal effect also includes diseases of the rectum, small intestine and diarrhea, for example, diarrhea associated with inflammation of the intestine (A _2b ).

Ophthalmic diseases include diseases associated with damage and trauma to the retina and the optic nerve (A ₃ ). In a preferred embodiment, the ocular disease is glaucoma.

Other therapeutic uses of the compounds of the invention include the treatment of obesity (lipolytic properties), high blood pressure, the treatment of depression, a sedative, antipsychotic effect, and action as anti-leprosy and laxatives, for example, affecting mobility but not causing diarrhea.

The term “disease state” is intended to include those conditions that are caused or associated with undesirable levels of adenosine, adenylyl cyclase activity, increased physiological activity associated with abnormal stimulation of adenosine receptors and / or increased cAMP. In one embodiment, the disease state is, for example, asthma, chronic obstructive pulmonary disease, allergic rhinitis, bronchitis, kidney disease, gastrointestinal disease, or eye disease. Additional examples include chronic bronchitis and cystic fibrosis. Suitable examples of inflammatory diseases include non-lymphocytic leukemia, myocardial ischemia, angina pectoris, heart attack, cerebrovascular ischemia, intermittent claudication, critical limb anemia, venous hypertension, varicose veins, peptic ulcer disease and arteriosclerosis. Deteriorated reperfusion conditions include, for example, any post-surgical trauma, such as during reconstructive surgery, thrombolysis and angioplasty.

The term “treating a condition responding to N-6 substituted 7-desazapurine” or “treating a condition responding to N-6 substituted 7-desazapurin” is intended to include such changes in the disease or condition, as described above, that physiological symptoms in a mammal can be significantly reduced or minimized. The expression also includes controlling, preventing or inhibiting physiological symptoms or actions associated with abnormal amounts of adenosine. In one preferred embodiment, controlling the disease or disease state is to eradicate the disease or disease state. In another preferred embodiment, the control is selective such that abnormal levels of adenosine receptor activity are controlled, while other physiological systems and parameters are not affected.

The term "N-6 substituted 7-desazapurines" is understood to be generally accepted in the art and includes compounds that have the formula I:

“Ν-substituted 7-desazapurin” includes pharmaceutically acceptable salts thereof, and in one embodiment also includes some Ν-6 substituted purines described herein.

In some embodiments, Ν-6 substituted 7-desazapurin is not Ν-6 benzyl or Ν-6 phenylethyl substituted. In other embodiments, K4 is not benzyl or phenylethyl substituted. In preferred embodiments, both K ₁ and K ₂ are not hydrogen atoms. In still other preferred embodiments, K _{3 is} not a hydrogen atom.

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The expression “therapeutically effective amount” of N-6 substituted 7-desazapurine, described below, is that amount of a therapeutic compound that is necessary or sufficient to fulfill its intended function in a mammal, for example, to treat a condition that responds to # 6- substituted 7-desazapurin, or diseases in a mammal. The effective amount of a therapeutic compound may vary in accordance with factors such as the amount of the causative agent already present in the mammal, the age, gender and weight of the mammal and the ability of the therapeutic compounds of the present invention to affect a condition that responds to N-6 substituted 7-desazapurin, a mammal.

An ordinary person skilled in the art will be able to study the above factors and determine the effective amount of a therapeutic compound without undue experimentation. An ίη νίίτο or ίη νίνο analysis can also be used to determine the “effective amount” of the therapeutic compounds described above. One of ordinary skill in the art will select the appropriate amount of therapeutic compound for use in the above analysis or as a therapeutic treatment.

The therapeutically effective amount preferably reduces at least one symptom or effect associated with the condition responding to N-6 substituted 7-dezapapurin, or with the disease being treated by at least about 20% (more preferably at least about 40 %, even more preferably at least about 60%, and even more preferably at least about 80%) compared with the untreated subject. Assays can be developed by one skilled in the art to measure a reduction in such symptoms and / or actions. It is understood that any analysis known in the art capable of measuring such parameters is included as part of this invention. For example, if asthma is a condition being treated, then the volume of air consumed by the lungs of the subject can be measured before and after treatment to measure the increase in volume using known techniques in the art. Similarly, if inflammation is a condition to be treated, then the area that is inflamed can be measured before and after treatment to measure a decrease in the area of inflammation using known techniques in the art.

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

The term “animal” includes any organism containing adenosine receptors, or any organism susceptible to a condition that responds to N-6 substituted 7-desazapurin. Examples of animals include yeast, mammals, reptiles and birds. The term also includes transgenic animals.

The term “mammal” is generally accepted in the art, and is intended to include an animal, more preferably a warm-blooded animal, most preferably cattle, sheep, pigs, horses, dogs, cats, rats, mice and humans. Mammals susceptible to a condition responding to N-6 substituted 7-desazapurin, for example, inflammation, emphysema, asthma, central nervous system disease or acute respiratory distress syndrome, are included as part of this invention.

In another aspect, the present invention encompasses methods for modulating the adenosine receptor (s) in a mammal by administering to the mammal a therapeutically effective amount of N-6 substituted 7-deazapurin, such that modulation of the adenosine receptor in the mammal occurs. Suitable adenosine receptors include the A1, A2, or A3 family. In a preferred embodiment, N-6 substituted 7-desazapurin is an adenosine receptor antagonist.

The term “modulation of the adenosine receptor” is meant to include those cases where the compound interacts with the adenosine receptor (s), causing increased, decreased or abnormal physiological activity associated with the adenosine receptor or subsequent cascading effects resulting from modulation of the adenosine receptor.

Physiological activity associated with adenosine receptors includes inducing sedation, vasodilation, suppressing heart rate, inhibiting platelet aggregation, stimulating gluconeogenesis, inhibiting lipolysis, opening calcium channels, decreasing the flow through calcium channels, etc.

The terms “modulate,” “modulate,” and “modulate” are meant to prevent, correct, or inhibit the resulting increase in undesired physiological activity associated with abnormal stimulation of the adenosine receptor, for example, in the context of therapeutic methods of the invention. In another embodiment, the term “modulation” includes antagonistic effects, for example, a decrease in the activity or production of allergic mediators and allergic inflammation that occurs when the adenosine receptor (s) are overstimulated. For example, the therapeutic desazapurins of the invention can interact with an adenosine receptor to inhibit, for example, adenylate cyclase activity.

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The term “condition characterized by abnormal activity of the receptor” is intended to include those diseases, disorders or disease states that are associated with abnormal stimulation of the adenosine receptor, in which stimulation of the receptor causes a biochemical or physiological chain of events that are directly or indirectly associated with the disease, disorder or painful condition. Such stimulation of the adenosine receptor should not be the sole cause of the disease, disorder or disease state, but is only responsible for causing some symptoms typically associated with the disease, disorder or disease state being treated. Abnormal receptor stimulation may be the only factor, or at least one other agent may be included in the condition being treated. Examples of conditions include those diseases that are listed previously, including inflammation, gastrointestinal diseases, and those symptoms that are manifested due to increased activity of the adenosine receptor. Preferred examples include those associated with asthma, allergic rhinitis, chronic obstructive pulmonary disease, emphysema, bronchitis, gastrointestinal diseases, and glaucoma.

The term “treatment or treatment of a condition characterized by abnormal activity of the adenosine receptor” is intended to mean a decrease or decrease in the manifestation of at least one symptom associated with the disease. Treatment also includes alleviating or reducing the manifestation of more than one symptom. Preferably, the treatment cures, for example, substantially eliminates the symptoms associated with the disease.

The present invention relates to compounds N-6 substituted with 7-deazapurines having the formula I:

where each of Κ1 and Κ2 independently represents a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety, or together they form a substituted or unsubstituted heterocyclic ring; Κ3 represents a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety; Κ4 represents a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety. Each of Κ5 and Κ6, independently, represents a halogen atom, for example, chlorine, fluorine or bromine, a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety, or Bd and B ₅ , or Κ5 and Κ6 together form a substituted or unsubstituted heterocyclic or carbocyclic ring. Also included are pharmaceutically acceptable salts of N-6 substituted 7-deazapurines.

In some embodiments, each of B ₁ and B ₂ , independently, may be a substituted or unsubstituted cycloalkyl or heteroarylalkyl moiety. In another embodiment, B ₃ represents a hydrogen atom or a substituted or unsubstituted heteroaryl moiety. In still other embodiments, each of Vd, B _5, and Vb may independently be a heteroaryl moiety.

In one embodiment, B1 represents a hydrogen atom, B2 represents a substituted or unsubstituted cyclohexane, cyclopentyl, cyclobutyl or cyclopropane fragment, B3 represents a substituted or unsubstituted phenyl fragment, B4 represents a hydrogen atom, and both B5 and B6 are methyl.

In another embodiment, B2 is cyclohexanol, cyclohexanediol, cyclohexyl sulfonamide, cyclohexanamide, cyclohexyl ester, cyclohexene, cyclopentanol or cyclopentanediol, and B3 is a phenyl moiety.

In yet another embodiment, B1 represents a hydrogen atom, B2 represents cyclohexanol, B3 represents a substituted or unsubstituted phenyl, pyridine, furan, cyclopentane or thiophene fragment, B4 represents a hydrogen atom, a substituted alkyl, aryl or arylalkyl fragment, each of B5 and B6, independently, represents a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety.

In yet another embodiment, B1 represents a hydrogen atom, B2 represents substituted or unsubstituted alkylamine, arylamine or alkylarylamine, substituted or unsubstituted alkylamide, arylamide or alkylarylamide, substituted or unsubstituted alkylsulfonamide, arylsulfonamide or alkylaryl urea, substituted unsubstituted aryl, substituted substituted or unsubstituted alkyl carbamate, aryl carbamate or alkyl aryl carbamate, substituted or unsubstituted alkyl carboxylic acid, aryl Coupons acid

- 8 007254 or alkylaryl carboxylic acid, K ₃ represents a phenyl moiety, K ₄ represents a hydrogen atom, and K ₅ and K ₆ represent methyl groups.

In a further embodiment, K ₂ is guanidine, modified guanidine, cyanoguanidine, thiourea, thioamide or amidine.

In one embodiment, K ₂ may be

where K _2a -K _2c each independently represents a hydrogen atom or a saturated or unsaturated alkyl, aryl or alkylaryl fragment, and Κ _2ά represents a hydrogen atom or a saturated or unsaturated alkyl, aryl or alkylaryl fragment, ΝΚ _2ε Κ _{2 £} or OK _2d , where K _2e —K _2d each independently represents a hydrogen atom or a saturated or unsaturated alkyl, aryl or alkylaryl moiety. Alternatively, K _2a and K _2b may together form a carbocyclic or heterocyclic ring having a ring size of 3 to 6 members, for example, cyclopropyl, cyclopentyl, cyclohexyl groups.

In one aspect of the invention, both K ₅ and K ₆ are not methyl groups, preferably one of K ₅ and K ₆ is an alkyl group, for example, a methyl group, and the other is a hydrogen atom.

In another aspect of the invention, when K ₄ is 1-phenylethyl and Κι is a hydrogen atom, then K _{3 is} not phenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 3,4-dichlorophenyl, 3-methoxyphenyl or 4- methoxyphenyl, or when K ₄ and Κ ₁ are ₁ phenylethyl, then K _{3 is} not a hydrogen atom, or when K ₄ is a hydrogen atom and K ₃ is phenyl, then Κ _{1 is} not phenylethyl.

In another aspect of the invention, when K ₅ and K ₆ together form a carbocyclic ring, for example,

or pyrimido [4,5-6] indole, then K _{3 is} not phenyl when K ₄ is 1- (4-methylphenyl) ethyl, phenylisopropyl, phenyl or 1-phenylethyl, or K _{3 is} not a hydrogen atom when K ₄ represents 1-phenylethyl. The carbocyclic ring formed by K ₅ and Κ ₆ may be either aromatic or aliphatic and may contain from 4 to 12 carbon atoms, for example, naphthyl, phenylcyclohexyl, etc., preferably from 5 to 7 carbon atoms, for example, cyclopentyl or cyclohexyl. Alternatively, K ₅ and K ₆ together may form a heterocyclic ring, such as those discussed below. Typical heterocyclic rings include from 4 to 12 carbon atoms, preferably from 5 to 7 carbon atoms, and can be either aromatic or aliphatic. The heterocyclic ring may be further substituted, including the substitution at one or more carbon atoms of a ring structure containing one or more heteroatoms.

In another aspect of the invention, Κ ₁ and K ₂ form a heterocyclic ring. Illustrative examples include, but are not limited to, the following heterocyclic rings, such as morpholino, piperazine and the like, for example, 4-hydroxypiperidines, 4-aminopiperidines. When Κ ₁ and K ₂ together form a piperazino group, “g

then K ₇ may be a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety.

In another aspect of the invention, K ₄ and K ₅ together can form a heterocyclic ring, for example,

- 9 007254 where the heterocyclic ring can be either aromatic or aliphatic and can form a ring containing from 4 to 12 carbon atoms, for example, naphthyl, phenylcyclohexyl, etc. and can be either aromatic or aliphatic, for example, cyclohexyl, cyclopentyl.

The heterocyclic ring may be further substituted, including the substitution of carbon atoms of the ring structure containing one or more heteroatoms. Alternatively, K4 and K ₅ together may form a heterocyclic ring, such as those discussed below.

In some embodiments, N-6 substituted 7-desazapurin is not # 6-benzyl or # 6-phenylethyl substituted. In other embodiments, K _{4 is} not benzyl or phenylethyl substituted. In preferred embodiments, both K1 and K2 are not hydrogen atoms. In still other preferred embodiments, K _{3 is} not N.

Compounds of the invention may include water-soluble prodrugs as described in ν099 / 33815, International Patent Application No. PCT / I8 98/04595, filed March 9, 1998 and published July 8, 1999. The full contents of XV0 99/33815 are specifically included in this description by reference. Water-soluble prodrugs metabolize ίη νίνο to the active drug, for example, using esterase-catalyzed hydrolysis. Examples of potential prodrugs include desazapurines, in which, for example, K2 is cycloalkyl substituted with -C (O) ^) H ₂ , where Ζ is the side chain of a natural or synthetic amino acid or analogue thereof, α-, β-, γ- or ω-amino acids or dipeptide. Preferred side chains include the side chains of glycine, alanine, valine, leucine, isoleucine, lysine, α-methylalanine, aminocyclopropanecarboxylic acid, azetidine-2-carboxylic acid, β-alanine, γ-aminobutanoic acid, alanine-alanine, or glycine-alanine.

In a further embodiment, the invention relates to desazapurines of formula (I), wherein K ₁ is hydrogen; K ₂ represents a substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkyl, or K1 and K2 together form a substituted or unsubstituted heterocyclic ring; K3 is unsubstituted or substituted aryl; K4 is hydrogen, and each of K5 and K6, independently, is hydrogen or alkyl, and their pharmaceutically acceptable salts. The desazapurins of this embodiment can potentially be selective A3 receptor antagonists.

In one embodiment, K ₂ is substituted (e.g., hydroxy-substituted) or unsubstituted cycloalkyl. In an advantageous embodiment, K ₁ and K ₄ are hydrogen, K ₃ is unsubstituted or substituted phenyl, and each of K ₅ and K6 is alkyl. Preferably, K2 is mono-hydroxycyclopentyl or mono-hydroxycyclohexyl. K ₂ may also be substituted with a group -NH-C (0) E, where E is a substituted or unsubstituted C1-C4 alkyl (e.g., alkylamine, e.g. ethylamine).

Also, K1 and K2 together can form a substituted or unsubstituted heterocyclic ring, which may be substituted by an amine or acetamido group.

In another aspect, K ₂ may be -A-NOH-C (0) B, where A is unsubstituted C1-C4 alkyl (e.g., ethyl, propyl, butyl), and B is substituted or unsubstituted C1-C4 alkyl (e.g. methyl, aminoalkyl, e.g. aminomethyl or aminoethyl, alkylamino, e.g. methylamino, ethylamino), preferably when K ₁ and K ₄ are hydrogen, K ₃ is unsubstituted or substituted phenyl, and each of K ₅ and K ₆ is alkyl. B may be substituted or unsubstituted cycloalkyl, for example cyclopropyl or 1-aminocyclopropyl.

In another embodiment, K ₃ may be substituted or unsubstituted phenyl, preferably when each of K5 and K6 is alkyl. Preferably K3 may contain one or more substituents (for example, o-, m- or p-chlorophenyl, o-, m- or p-fluorophenyl).

Advantageously, K3 may be substituted or unsubstituted heteroaryl, preferably when each of K5 and K6 is alkyl. Examples of heteroaryl groups include pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, pyrrolyl, triazolyl, thioazolyl, oxazolyl, oxadiazolyl, furanyl, methylenedioxyphenyl and thiophenyl. Preferably K ₃ is 2 pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl or 3-pyrimidyl.

Preferably, in one embodiment, each of K ₅ and K ₆ is hydrogen. In another embodiment, each of K ₅ and K ₆ is methyl.

In a particularly preferred embodiment, the desazapurins of the invention are water-soluble prodrugs that can metabolize ίη νίνο to an active drug, for example, by esterase-catalyzed hydrolysis. Preferably, the prodrug includes a K ₂ group which is substituted on cycloalkyl-OOC (O) ^) OH ₂ , where Ζ represents the side chain of a natural or synthetic amino acid, its analogue, α-, β-, γ- or ω-amino acid or dipeptide. Examples of preferred side chains include the side chains of glycine, alanine, valine, leucine, isoleucine, lysine, α-methylalanine, aminocyclopropanecarboxylic acid, azetidine-2-carboxylic acid, β-alanine, γ-aminobutanoic acid, alanine-alanine or glycine-alanine.

- 10 007254

In a particularly preferred embodiment, Ζ is a glycine side chain, K ₂ is cyclohexyl, K ₃ is phenyl, and K ₅ and Kb are methyl.

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

In another embodiment, desazapurin is a trifluoroacetic acid salt

4- (cis-3- (2-aminoacetoxy) cyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine.

In another embodiment, desazapurin is 4- (3-acetamido) piperidinyl-

5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine.

In another embodiment, desazapurin is 4- (2-Y′-methylureapropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine.

In another embodiment, desazapurin is 4- (2-acetamidobutyl) amino-5,6dimethyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine.

In another embodiment, desazapurin is 4- (2-Y′-methylureabutyl) amino

5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine.

In another embodiment, desazapurin is 4- (2-aminocyclopropylacetamidoethyl) amino-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine.

In another embodiment, desazapurin is 4- (trans-4-hydroxycyclohexyl) amino-2- (3-chlorophenyl) -7H-pyrrolo [2,3-6] pyrimidine.

In another embodiment, desazapurin is 4- (trans-4-hydroxycyclohexyl) amino-2- (3-fluorophenyl) -7H-pyrrolo [2,3-6] pyrimidine.

In another embodiment, desazapurin is 4- (trans-4-hydroxycyclohexyl) amino-2- (4-pyridyl) -7H-pyrrolo [2,3-6] pyrimidine.

In yet another embodiment, the invention relates to a method for inhibiting the activity of an adenosine receptor (for example, A ₁ , A _2a , A _2b, or preferably A ₃ ) in a cell by contacting the cell with a β-6 substituted 7-deazapurin (for example, preferably with an adenosine receptor antagonist )

In another aspect, the invention relates to a method for treating eye damage in an animal (eg, a human) by administering to the animal an effective amount of Ν-6 substituted 7-desazapurin. Preferably, Ν-6 substituted 7-desazapurin is an antagonist of A ₃ adenosine receptors in animal cells. Damage refers to the retina or head optic nerve and can be acute or chronic. Damage may result from, for example, glaucoma, edema, ischemia, hypoxia, or trauma.

In a preferred embodiment, the invention relates to desazapurin having the formula II, see above, where X represents Ν or SK ₆ ; each of K ₁ and K ₂ independently represents hydrogen or a substituted or unsubstituted alkoxy, aminoalkyl, alkyl, aryl or alkylaryl, or together they form a substituted or unsubstituted heterocyclic ring, provided that both K ₁ and K ₂ are not hydrogen; K ₃ represents substituted or unsubstituted alkyl, arylalkyl or aryl; K ₄ represents hydrogen or substituted or unsubstituted C ₁ -Cb alkyl; B represents hydrogen, substituted or unsubstituted alkyl, or K ₄ and b together form a substituted or unsubstituted heterocyclic or carbocyclic ring; K ₆ represents hydrogen, substituted or unsubstituted alkyl, or halogen; O represents CH ₂ , O, 8 or ΝΚ ₇ , where K ₇ represents hydrogen or substituted or unsubstituted C ₁ -C ₆ alkyl; and represents unsubstituted or substituted alkyl, cycloalkyl, alkynyl, aryl, arylalkyl, biaryl, heteroaryl, substituted carbonyl, substituted thiocarbonyl or substituted sulfonyl, provided that if K ₃ is pyrrolidino, then K _{4 is} not methyl.

In one embodiment, the compounds of formula II, X represents SC ₆ , and O represents CH ₂ , O, 8 or ΝΗ. In another embodiment, X represents Ν.

In a further embodiment of the compounds of formula II, is a substituted or unsubstituted aryl, 5- or 6-membered heteroaryl or biaryl. may be substituted by one or more substituents. Examples of substituents include: halogen, hydroxy, alkoxy, amino, aminoalkyl, aminocarboxyamide, CH CE ₃ , CO ₂ K ₈ , ί.ΌΝΗΗ _χ . SOK ₈ K ₉ , ₈ OK ₈ and 8Ο ₂ ΝΚ ₈ Κ ₉ , where each of K ₈ and K ₉ independently represents hydrogen or substituted or unsubstituted alkyl, cycloalkyl, aryl or arylalkyl. Preferably, it may be substituted or unsubstituted phenyl, for example methylenedioxyphenyl. may also be a substituted or unsubstituted 5-membered heteroaryl ring, for example, pyrrole, pyrazole, oxazole, imidazole, triazole, tetrazole, furan, thiophene, thiazole and oxadiazole. It may preferably be a 6 membered heteroaryl ring, for example pyridyl, pyrimidyl, pyridazinyl, pyrazinyl and thiophenyl. In a preferred embodiment, is 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl or

5-pyrimidyl.

- 11 007254

In one advantageous embodiment of the compounds of formula II, O is ΝΗ, and represents a 3-pyrazole ring, which is unsubstituted or Ν-substituted or unsubstituted by alkyl, cycloalkyl, aryl or arylalkyl.

In another embodiment of the compounds of formula II, O is oxygen, and is a 2-thiazole ring, which is unsubstituted or substituted or unsubstituted by alkyl, cycloalkyl, aryl or arylalkyl.

In another embodiment, the compounds of formula II are substituted or unsubstituted alkyl, cycloalkyl, for example cyclopentyl or arylalkyl. Examples of substituents include halogen, hydroxy, substituted or unsubstituted alkyl, cycloalkyl, aryl, arylalkyl or ΝΗΚ ₁₀ , where K ₁₀ represents hydrogen or substituted or unsubstituted alkyl, cycloalkyl, aryl or arylalkyl. In yet another embodiment, the invention relates to a desazapurin of formula II, wherein is - (CH ₂ ) _a -C (= O ^ or - (CH ₂ ) _a ^- C (= 8 ^, and a is an integer from 0 to 3, Υ represents aryl, alkyl, arylalkyl, cycloalkyl, heteroaryl, alkynyl, ΝΗΚ ₁₁ Κ ₁₂ , or, provided that O represents ΝΗ, OK ₁₃ , where each of K ₁₁ , K ₁₂ and K ₁₃ independently represents hydrogen or unsubstituted or substituted alkyl, aryl, arylalkyl or cycloalkyl Preferably, Υ represents a 5- or 6-membered heteroaryl ring.

In addition, it can be - (CH ₂ ) _b -8 (= O ^, where 1 or 2, b is 0, 1, 2 or 3, Υ is aryl, alkyl, arylalkyl, cycloalkyl, alkynyl, heteroaryl, ΝΗΚ ₁₄ Κ ₁₅ or, provided that when b is 1, O represents C1E, and where each of K ₁₄ , K ₁₅ and K ₁₆ independently represents hydrogen or unsubstituted or substituted alkyl, aryl, arylalkyl or cycloalkyl.

In another embodiment, Κ3 is selected from the group consisting of substituted or unsubstituted phenyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinal, pyrrolyl, triazolyl, thiazolyl, oxazolyl, oxadiazolyl, pyrazolyl, furanyl, methylenedioxyphenyl and thiophenyl. When Κ3 is phenyl, it may be substituted, for example, with hydroxyl, alkoxy (e.g. methoxy), alkyl (e.g. tolyl) and halogen (e.g. o-, m- or p-fluorophenyl or o-, m- or p chlorophenyl). Advantageously, K ₃ may be 2-, 3- or 4-pyridyl or 2- or 3-pyrimidyl.

The invention also includes desazapurin, wherein K ₆ is hydrogen or C ₁ -C ₃ alkyl. Preferably, Κ ₆ is hydrogen.

The invention also includes desazapurines in which Κ1 is hydrogen and Κ2 is substituted or unsubstituted alkyl or alkoxy, substituted or unsubstituted alkylamine, arylamine or alkylarylamine, substituted or unsubstituted aminoalkyl, aminoaryl or aminoalkylaryl, substituted or unsubstituted alkylamide, arylamide or alkyl substituted or unsubstituted alkylsulfonamide, arylsulfonamide or alkylarylsulfonamide, substituted or unsubstituted alkyl urea, arylurea or alkylarylurea, substituted whether unsubstituted alkyl carbamate, aryl carbamate or alkilarilkarbamat or a substituted or unsubstituted alkyl carboxylic acid, aryl carboxylic acid or alkilarilkarbonovuyu acid.

Preferably, Κ ₂ is substituted or unsubstituted cycloalkyl, for example, mono- or dihydroxy substituted cyclohexyl or cyclopentyl (preferably monohydroxy substituted cyclohexyl or monohydroxy substituted cyclopentyl).

Advantageously, K ₂ may have one of the following formulas:

Oh Oh

or

B ^ 17 where A represents C ₁ -C ₆ alkyl, C ₃ -C ₇ cycloalkyl, a chain of one to seven atoms or a ring of three to seven atoms, optionally substituted with C ₁ -C ₆ alkyl, halogens, hydroxyl, carboxyl, thiol or amino groups; where B is methyl, Ν (Μθ) ₂ , Ν (Εΐ) ₂ , ΝΗΜθ, ΝΗΕΐ, (ΕΗ ₂ ) ₂ ΝΗ ₃ ⁺ , ΝΗ (^ 2 ^ Η3, (^ 2ΕΝΗ2, (^ / ΝΗΜθ, ( OSH UCHSCH number Κ18Κ19 or

CHMeOH, where g is an integer from 0 to 2, t is 1 or 2, K ₁₈ represents alkyl, K ₁₉ represents ΝΗ ₃ ⁺ or CO ₂ H, or K ₁₈ and K ₁₉ together represent:

- CH - ΝΗ \ / (CH ₂ ) p where p is 2 or 3; and K ₁₇ is C ₁ -C ₆ alkyl, C ₃ -C ₇ cycloalkyl, a chain of one to seven atoms, or a ring of three to seven atoms, optionally substituted with C ₁ -C ₆ alkyl, halogens, hydroxyl, carboxyl, thiol, or amino groups.

Preferably, L is unsubstituted or substituted C ₁ -C ₆ alkyl. B may be unsubstituted or substituted C ₁ -C ₆ alkyl.

In a preferred embodiment, K ₂ has the formula -L-GCFO ^. In a particularly advantageous embodiment, L is —CH ₂ CH ₂ -, and B is methyl.

- 12 007254

The compounds of the invention may include water-soluble prodrugs that metabolize ίη νίνο into an active drug, for example, by esterase-catalyzed hydrolysis. Examples of potential prodrugs include desazapurines, in which, for example, K ₂ is in the form of cycloalkyl substituted with -Θί. '(ΫχΖ) ΝΗ ₂ . where Ζ represents the side chain of a naturally occurring or non-naturally occurring amino acid or its analogue, α-, β-, γ- or ω-amino acid or dipeptide. Preferred amino acid side chains include glycine, alanine, valine, leucine, isoleucine, lysine, α-methylalanine, aminocyclopropanecarboxylic acid, azetidine-2-carboxylic acid, β-alanine, γ-aminobutyric acid, alanine-alanine or glycine alanine chains.

In another embodiment, Κι and K ₂ together represent

where η is 1 or 2, and where the ring may optionally be substituted with one or more hydroxyl, amino, thiol, carboxyl, halogen, CH ₂ OH, CΗ ₂ NΗC (= Ο) alkyl or ΟΗ ₂ ΝΗΟ (= Ο) ΝΗ alkyl groups . Preferably, η is 1 or 2, and said ring is substituted with ΝΗί. '(= Ο) alkyl.

In one advantageous embodiment, Κ ₁ is hydrogen, K ₂ is substituted or unsubstituted C ₁ -C ₆ alkyl, K ₃ is substituted or unsubstituted phenyl, Κ 4 is hydrogen, b is hydrogen or substituted or unsubstituted C ₁ - C ₆ alkyl represents Θ, 8 or ΝΚ ₇ , where K ₇ represents hydrogen or substituted or unsubstituted C ₁ -C ₆ alkyl, and represents substituted or unsubstituted aryl.

Preferably K ₂ is —Α-ΝΗΠ (= Ο) Β, where each of A and B independently is unsubstituted or substituted C ₁ -C ₄ alkyl. For example, A may be CH ₂ CH ₂ . B may be, for example, alkyl (e.g. methyl) or aminoalkyl (e.g. aminomethyl). Preferably, K ₃ is unsubstituted phenyl, and b is hydrogen. Kb may be methyl or, preferably, hydrogen. Preferably, it is Θ, 8 or ΝΚ ₇ , where K ₇ is hydrogen or substituted or unsubstituted C ₁ C ₆ alkyl, for example methyl. represents unsubstituted or substituted phenyl (for example, alkoxy, halogen-substituted). Preferably, it is p-fluorophenyl, p-chlorophenyl or p-methoxyphenyl. may also be heteroaryl, for example, 2-pyridyl.

In a particularly preferred embodiment, desazapurin is 4- (2-acetylaminoethyl) amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine.

In a particularly preferred embodiment, desazapurin is 4- (2-acetylaminoethyl) amino-6- (4-fluorophenoxy) methyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine.

In a particularly preferred embodiment, desazapurin is 4- (2acetylaminoethyl) amino-6- (4-chlorophenoxy) methyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine.

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

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

In a particularly preferred embodiment, desazapurin is 4- (2-acetylaminoethyl) amino-6- (Y-phenylamino) methyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine.

In a particularly preferred embodiment, desazapurin is 4- (2-acetylaminoethyl) amino-6- (K-methyl-M-phenylamino) methyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine.

In a particularly preferred embodiment, desazapurin is 4- (2-Ν'methylureaethyl) amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine.

The invention further relates to a method for inhibiting the activity of an adenosine receptor (eg, A ₂ adenosine receptor) in a cell by contacting the cell with a compound of the invention. Preferably, the compound is an adenosine receptor antagonist.

The invention also relates to a method for treating a gastrointestinal disease (e.g., diarrhea) in an animal by administering to the animal an effective amount of a compound of the invention (e.g., A ₂ antagonist). Preferably, the animal is a human.

In another embodiment, the invention relates to a pharmaceutical composition comprising Ν-6 substituted 7-desazapurin according to the invention and a pharmaceutically acceptable carrier.

The invention also relates to a method for treating a disease state that responds to a Ν-6 substituted 7-desazapurin in an animal by administering to a mammal a therapeutically effective amount of desazapurin according to the invention, such that a condition that responds to a Ν-6 substituted 7- desazapurin in an animal. Mostly for

- 13 007254 ailment may be a disorder mediated by adenosine. Examples of preferred disease states include: central nervous system disorders, cardiovascular diseases, kidney diseases, inflammatory diseases, allergic diseases, gastrointestinal diseases, eye diseases and respiratory diseases.

The term “alkyl” refers to a radical of saturated aliphatic groups, including linear alkyl groups, branched 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 that replace one or more carbons in the main hydrocarbon chain, for example, oxygen, nitrogen, sulfur or phosphorus atoms. In preferred embodiments, the linear or branched chain alkyl contains 30 or less carbon atoms in the main chain (for example, C ₁ -C ₃₀ for the linear chain, C ₃ -C ₃₀ for the branched chain) and, more preferably, 20 or less. Similarly, preferred cycloalkyls have 4-10 carbon atoms in their ring structure and, more preferably, have 5, 6 or 7 carbons in the ring structure.

Moreover, it is understood that the term alkyl, as used in the description and claims, includes both “unsubstituted alkyls” and “substituted alkyls,” the latter referring to alkyl moieties having substituents that replace hydrogen on one or more carbons in the base hydrocarbon chain. Such substituents may include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato-aminoamino-aminoamino-aminoamino-aminoamino-aminoamino-aminoamino-aminoamino-aminoamino-aminoamino-aminoamino-aminoamino-aminoamino-aminoamino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-amino-... alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl or an aromatic or heteroaromatic moiety. Specialists in this field should understand that the fragments located as substituents in the hydrocarbon chain themselves can be substituted, if possible. Cycloalkyls may additionally be substituted, for example, with the substituents described above. An “alkylaryl moiety” is an alkyl substituted with an aryl (eg, phenylmethyl (benzyl)). The term “alkyl” also includes unsaturated aliphatic groups, similar in length and possible substitution to the above alkyls, but also containing at least one double or triple bond, respectively.

The term “aryl,” as used herein, refers to an aryl radical, including 5- and 6-membered aromatic groups, consisting of one ring, which may include from zero to four 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 that have heteroatoms in the ring structure may also be referred to as “aryl heterocycles”, “heteroaryls” or “heteroaromatic” groups. The aromatic ring may be substituted at one or more ring positions by such substituents as described above, such as, for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthionocarbonato, phosphato, , cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, s lfgidril, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl groups can also be fused or linked by bridges to alicyclic or heterocyclic rings that are not aromatic, such that they form a polycycle (e.g. tetralin).

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groups, similar in length and possible substitution to the above alkyls, but also containing at least one double or triple bond, respectively. For example, the invention contemplates cyano and propargyl groups.

Unless the number of carbon atoms is indicated otherwise, “lower alkyl,” as used herein, means an alkyl group as defined above but containing from one to ten carbons, more preferably from one to six carbons in the backbone structure, even more preferably from one to three carbon atoms in the structure of its main chain. Similarly, "lower alkenyl" and "lower alkynyl" have a similar chain length.

The terms “alkoxyalkyl”, “polyaminoalkyl” and “thioalkoxyalkyl” refer to alkyl groups as described above, which further include oxygen, nitrogen or sulfur atoms that replace one or more carbons in the hydrocarbon backbone, for example, oxygen, nitrogen or sulfur atoms.

- 14 007254

The terms "polycyclyl" or "polycyclic radicals" refer to radicals consisting of two or more cyclic rings (eg, cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and / or heterocyclyls) in which two or more carbons are common to two adjacent rings, for example , rings are "fused rings." Rings that are joined by non-adjacent atoms are called "bridged" rings. Each of the polycyclic rings may be substituted with substituents as described above, such as, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phospho, alkoxy , amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, iokarboksilat, sulfate, 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 natural and synthetic 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 elongated or shortened side chains or differing side chains with suitable functional groups. Amino acids also include the Ό and b stereoisomers of amino acids when the structure of the amino acid allows for the existence of stereoisomeric forms. The term "dipeptide" includes two or more amino acids linked together. Preferably, dipeptides are two amino acids linked by a peptide bond. Particularly preferred dipeptides include, for example, alanine-alanine or glycine-alanine.

It should be noted that the structure of some compounds of this invention includes asymmetric carbon atoms, and thus they exist in the form of racemates and racemic mixtures, individual enantiomers, diastereomeric mixtures and individual diastereomers. All such isomeric forms of these compounds are included in this invention. Each stereogenic carbon may have a Κ or 8 configuration. Accordingly, it should be understood that isomers arising from such an asymmetry (for example, all enantiomers and diastereomers) are included in the scope of this invention, unless otherwise indicated. Such isomers can be obtained essentially in pure form using classical separation methods and by stereochemically selective synthesis.

The invention further relates to pharmaceutical compositions for treating conditions that respond to Ν-6 substituted 7-deazapurins in mammals, for example, respiratory diseases (e.g. asthma, bronchitis, chronic obstructive pulmonary disease and allergic rhinitis), renal diseases, gastrointestinal diseases and eye diseases. The pharmaceutical composition comprises a therapeutically effective amount of Ν-6 substituted 7-dezazapurin as described above and a pharmaceutically acceptable carrier. It should be understood that all of the above desazapurins are included for therapeutic treatment. Additionally, it should be understood that the desazapurins of the invention can be used individually or in combination with other desazapurins of the invention, or in combination with additional therapeutic compounds, such as, for example, antibiotics, anti-inflammatory or anti-cancer agents.

The term “antibiotic” is generally accepted in the art and is intended to include those produced by growing microorganisms and their synthetic derivatives that exclude or inhibit the growth of pathogens and are selectively toxic to pathogens, with minimal or no adverse effect on infected host subject. Suitable examples of antibiotics include, but are not limited to, the principal classes of aminoglycosides, cephalosporins, chloramphenicol, fuscinic acids, macrolides, penicillins, polymyxins, tetracyclines and streptomycin.

The term “anti-inflammatory” is generally accepted in the art and is intended to include those that act on the mechanisms of the body without directly antagonizing the agent causing the inflammation, such as glucocorticoids, aspirin, ibuprofen, NSAIDs (non-steroidal anti-inflammatory drugs), and t . d.

The term “anti-cancer agent” is generally accepted in the art and is intended to include those that reduce, eradicate or prevent the growth of cancer cells, preferably without adversely affecting other physiological functions. Illustrative examples include cisplatin and cyclophosphamide.

When the compounds of the present invention are administered in the form of drugs to humans and mammals, they can be given in pure form or in the form of a pharmaceutical composition containing

- 15 007254, for example, from 0.1 to 99.5% (more preferably from 0.5 to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

The term “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable substance, composition or carrier, such as a liquid or solid excipient, diluent, excipient, solvent or encapsulating substance, included in the delivery or transportation of the compound (s) according to the invention inside the subject or to the subject in such a way that the connection performs the intended function. Typically, such compounds are delivered or transported from an organ or body part to another organ or body part. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not harm the patient. Some examples of substances that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered gums; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; 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 glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; phosphate buffers and other non-toxic compatible substances used in pharmaceutical preparations.

As indicated above, some embodiments of the present compounds may contain basic functional groups, such as amino or alkylamino, and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salts" in this regard refers to the relatively non-toxic acid addition salts of the compounds of the present invention with inorganic or organic acids. These salts can be obtained by ίΐη δίΐιι during the final isolation and purification of the compounds of the invention or by separately reacting the purified compound of the invention in the form of its free base with a suitable organic or inorganic acid and isolating the salt thus formed. Illustrative 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, mesylate , dactobionate and lauryl sulfonate salts and the like (see, for example, Vegde s1 a1. (1977) Rattasei11ca1 8a118, 1. PHAGt. 8c1., 66: 1-19).

In other cases, the compounds of the present invention may contain one or more acidic functional groups and are thus capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in such cases refers to the relatively non-toxic base addition salts of the compounds of the present invention with inorganic or organic bases. Such salts can be prepared in a similar manner to ίΐη δίΐιι during the final isolation and purification of the compounds, or by separately reacting the purified compound in the form of its free acid with a suitable base, such as a pharmaceutically acceptable metal cation hydroxide, carbonate or bicarbonate, with ammonia or with pharmaceutically acceptable organic primary, secondary or tertiary amines. Illustrative alkaline or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like. Illustrative organic amines useful for the formation 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 obtained ίη δίΐιι during the final isolation and purification of the compounds or by separately reacting the purified compound in the form of its free acid or hydroxyl with a suitable esterifying reagent. Carboxylic acids can be converted to esters by treatment with alcohol in the presence of a catalyst. Hydroxyl-containing derivatives can be converted to esters by treatment with an esterifying agent such as alkanoyl halides. In addition, the term is intended to include lower hydrocarbon groups capable of solvation under physiological conditions, for example, alkyl esters, methyl, ethyl and propyl esters (see, for example, Vegde e1 a1., See above).

The invention further contemplates the use of prodrugs that are converted ίη νίνο into the therapeutic compounds of the invention (see, for example, KVEZhettasch 1992, WHERE YOU WILL NOT GET ABOUT Aegid Oeyod aob Egid LsOoi. Lsabetu Rgeck, SNarGeg 8). Such prodrugs can be used to alter biodistribution (for example, to make possible compounds that would not normally enter into the reactive protease sites) or for the pharmacokinetics of a therapeutic compound. For example, a carboxylic acid group may be esterified to

- 16 007254 example, methyl group or ethyl group to obtain an ester. When an ester is administered to a subject, the ester is cleaved by enzymes or non-enzymatically, reducingly or hydrolytically, releasing the anionic group. The anionic group can be esterified with fragments (for example, acyloxymethyl esters) that cleave to liberate the intermediate, which subsequently decomposes, to give the active compound. In another embodiment, the prodrug is a reduced form of a sulfate or sulfonate, for example, a thiol, which is oxidized to a therapeutic compound. In addition, the anionic fragment can be esterified to a group that is actively transported by ίπ у1уо, or which is selectively absorbed by the target organs. The ester can be selected so as to enable specific therapeutic fragments to be targeted to specific reactive sites, as described below for carrier fragments.

Wetting agents, emulsifiers and lubricating agents, such as sodium lauryl sulfate and magnesium stearate, as well as dyes, releasing agents, coating agents, sweeteners, flavoring agents and fragrances, 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, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

The compositions of the present invention include formulations suitable for oral, nasal, topical, transdermal, buccal, sublingual, rectal, vaginal and / or parenteral administration. The preparations may conveniently exist as unit dosage forms and may be prepared by any means well known in the pharmaceutical art. The amount of active ingredient that can be combined with a carrier to form a unit dosage form will usually be that amount of a compound that exerts a therapeutic effect. Usually, excluding one hundred percent, this amount will range from about 1% to about ninety-nine percent of the active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%.

Methods for preparing these compositions or compositions include the step of combining a compound of the present invention with a carrier and, optionally, one or more accessory ingredients. Typically, compositions are prepared by uniformly and thoroughly mixing a compound of the present invention with liquid carriers or finely divided solid carriers, or both, and then, if necessary, molding the product.

Compositions of the invention suitable for oral administration can be presented as capsules, wafers, pills, tablets, lozenges (using a flavoring base, usually sucrose and gum arabic or tragacanth), powders, granules, or as a solution or suspension in 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 gum arabic), and / or in the form of mouth rinses and so on each of which contains a predetermined amount of a compound of the present invention as an active ingredient. The compound of the present invention can also be administered as a bolus, electuary or paste.

In the solid oral dosage formulations of the invention (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 agents: fillers or fillers, such as starches, lactose, sucrose, glucose, mannitol and / or silicic acid; binders, such as, for example, carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and / or gum arabic; humectants, such as glycerin; disintegrating agents such as agar agar, calcium carbonate, potato or maize starch, alginic acid, certain silicates and sodium carbonate; dissolution retarding agents such as paraffin; absorption accelerators such as quaternary ammonium compounds; wetting agents, such as, for example, 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 dyes. In the case of capsules, tablets and pills, pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type can also be used as filler in soft and hard filled gelatin capsules using excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be prepared by compression or molding, optionally using one or more additional ingredients. Compressed tablets may be prepared with

- 17 007254 using a binder (e.g. gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, preservative, disintegrant (e.g. sodium starch glycolate or crosslinked sodium carboxymethyl cellulose), a surfactant or dispersant. Molded tablets may be prepared by molding in a suitable device a mixture of the powdered compound moistened with an inert liquid diluent.

Tablets and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be notched or prepared using coatings or coatings, such as enteric coatings and other coatings well known in the art. obtaining pharmaceutical formulations. They can also be formulated to provide slow or controlled release of the active ingredient from them using, for example, hydroxypropyl methyl cellulose in various proportions to create the desired release profile, other polymer matrices, liposomes and / or microspheres. They can be sterilized, for example, 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 turbidity agents and may be a composition that releases the active ingredient (s) only or preferably in a certain part of the gastrointestinal tract, optionally, in a delayed manner. Examples of implantable compositions that can be used include polymeric substances and waxes. The active ingredient may 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 commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular cottonseed, peanut, corn, seedling oil, olive, castor and sesame oils), glycerin, tetrahydrofuryl alcohol, poly ilenglikoli and sorbitan esters of fatty acids and mixtures thereof.

In addition to inert diluents, oral compositions may also include auxiliary agents, such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, coloring agents, flavoring agents and preservatives.

Suspensions, in addition to the active compounds, may contain suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar agar and tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which can be prepared by mixing one or more of the compounds of the invention with one or more suitable non-irritant excipients or carriers, including, for example, cocoa butter, polyethylene glycol, suppository wax or salicylate, which are solid at room temperature but liquid at body temperature and therefore will melt into the rectum e or vaginal cavity and release the active compound.

Formulations of the present invention that are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foamy or nebulized preparations containing such carriers that are known in the art as suitable for this application.

Formulations for topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, plasters and inhalers. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and with any desired preservative, buffer or propellant.

Ointments, pastes, creams and gels may contain, in addition to the active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays may contain, in addition to the compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain conventional propellants, such as chlorofluorocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

- 18 007254

Transdermal patches have the additional advantage of providing controlled delivery of the compounds of the present invention to the body. Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable medium. To increase the penetration of the substance through the skin, absorption enhancers can be added. The speed of such a flow through the skin can be controlled either by using a speed-controlling membrane, or by dispersing the active compound in a polymer matrix or gel.

Ophthalmic preparations, ophthalmic ointments, powders, solutions and the like are also contemplated as being within the scope of this invention. Preferably, the pharmaceutical preparation is an ophthalmic composition (for example, a preparation for periocular, retrobulbar or intraocular injection, a systemic preparation or a surgical irrigation solution).

Ophthalmic preparations of the present invention may include one or more desazapurines and a pharmaceutically acceptable carrier. You can use various types of media. The carrier will usually be aqueous in nature. Aqueous solutions are usually preferred based on the type of composition, as well as the patient's ability to easily administer such compositions by instilling one or two drops of the solution into the affected eye. However, the desazapurines of the present invention can also easily be incorporated into other types of compositions, such as suspensions, viscous or semi-viscous gels, or other types of solid or semi-solid compositions. Ophthalmic compositions of the present invention may also include various types of ingredients, such as buffers, preservatives, cosolvents and viscosity-generating agents.

A suitable buffer system (e.g., sodium phosphate, sodium acetate or sodium borate) may be added to prevent pH changes during storage.

Ophthalmic products are usually packaged in a variety of dosage forms. Thus, preservatives are required to prevent microbial contamination during use. Suitable preservatives include: benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben, 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% weight / volume (% weight / volume).

When the desazapurins of the present invention are administered during an intraocular surgical procedure, for example by retrobulbar or periocular injection and intraocular injection or injection, it is most preferable to use balanced saline irrigating solutions as carriers. Examples of physiologically balanced intraocular irrigation solutions are B88® sterile irrigation solution and B88 P1i§® Sterile intraocular irrigation solution (A1cop Laboguepec, Sch., Eoy \ Uog111. Techak, I8A). The latter type of solution is described in US Pat. Retrobulbar and periocular injections are well known to those skilled in the art and are described in numerous publications, including, for example, ORYMME 8: Prophylactic Surgery: Ophthalmologic Surgery: Principles of Practice) Eb. 6.b.8pae1y, U.V. 8apbeg 5 Co. ., P1labe1aRa Ra., I8A, pages 85-87 (1990).

As indicated above, the use of desazapurines to prevent or reduce tissue damage to the retina or brain optic nerve at the cellular level is a particularly important aspect of one embodiment of the invention. Ophthalmic diseases that can be treated include, but are not limited to, retinopathy, macular degeneration, ischemia of the eye, glaucoma, and damage associated with damage to eye tissue, such as ischemic reperfusion damage, photochemical damage, and damage associated with eye surgery, in features of damage to the retina or head optic nerve caused by exposure to light or surgical instruments. The compounds can also be used as concomitant treatment in ophthalmic surgery, for example, as an injection into the retina or supraconjunctival injection after ophthalmic surgery. The compounds can be used for the short-term treatment of temporarily occurring diseases, or they can be administered continuously, especially in the case of a degenerative disease. The compounds can also be used prophylactically, especially before eye surgery or non-invasive ophthalmic procedures, or other types of surgery.

Pharmaceutical compositions of this invention suitable for parenteral administration include one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or in the form of sterile powders, the moisture content of which is restored immediately before use in sterile injectable solutions or dispersions, which may contain antioxidants, buffers, bacteriostats chemical agents, soluble substances conferring isotonic properties to the formulation with the blood of the intended recipient, or suspending or thickening agents.

- 19 007254

Examples of suitable aqueous and non-aqueous vehicles that can be used in the pharmaceutical compositions of the invention include water, ethanol, polyhydric alcohols (such as glycerin, propylene glycol, polyethylene glycol and the like) and their suitable mixtures, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by using coating materials such as lecithin, maintaining the desired particle size in the case of dispersions, and using surfactants.

These compositions may also contain auxiliary agents, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be guaranteed by the inclusion of various antimicrobial and antifungal agents, for example, paraben, chlorobutanol, phenolsorbic 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 achieved by the administration of agents that delay absorption, such as aluminum monostearate and gelatin.

In some cases, to achieve the long-term effect of the drug, it is desirable to slow the absorption of the drug by subcutaneous or intramuscular injection. This can be achieved through the use of a liquid suspension of a crystalline or amorphous substance having a low solubility in water. The absorption rate of the drug then depends on its dissolution rate, which, in turn, may depend on the size of the crystal and crystalline form. Alternatively, delayed absorption of a parenterally administered formulation is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are obtained by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of release of the drug can be controlled. Examples of other biodegradable polymers include poly (ortho esters) and poly (anhydrides). Injectable depoforms are also obtained by capturing the drug with liposomes or microemulsions that are compatible with body tissues.

The preparations of the present invention can be administered orally, parenterally, locally or rectally. Of course, they are administered in the form of forms suitable for each route of administration. For example, they are administered in the form of tablets or capsules, by injection, inhalation, in the form of an eye lotion, ointment, suppository, etc., they are administered by injection, infusion or inhalation; topically using lotion or ointment; and rectally using suppositories. Oral administration is preferred.

The expressions “parenteral administration” and “administered parenterally”, as used herein, mean methods of administration other than enteral and local administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraocular , intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrathoracic injection and infusion.

The terms “systemic administration” and “systemically administered”, “peripheral administration” and “peripherally administered”, as used herein, mean administration of a compound, drug or other substance in a different way than direct administration to the central nervous system, so it enters the patient’s system and thus undergoes metabolism and other similar processes, for example, by subcutaneous administration.

These compounds can be administered to humans and other animals for treatment using any suitable route of administration, including oral, nasal, for example, using a spray, rectal, intravaginal, parenteral, intracisternal and topical administration, for example, using powders, ointments and drops, including buccal and sublingual introduction.

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

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and route of administration without being toxic to the patient.

The dosage levels chosen will depend on many factors, including the activity of the particular compound of the present invention or its ester, salt or amide, route of administration, time of administration, rate of excretion of the particular compound used, duration of treatment, other drugs agents, compounds and / or substances used in combination with the particular compound used, age, gender, weight, disease, general health and previous medical history of the patient the substance being treated, and similar factors well known in medicine.

An attending physician or veterinarian who is an ordinary specialist in this field can easily determine and prescribe an effective amount of the required pharmaceutical composition. For example, the attending physician or veterinarian may begin by applying doses of the compounds used in the pharmaceutical composition at levels lower than those required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

Typically, a suitable daily dose of a compound of the invention will be that amount of a compound that is the smallest dose effective to provide a therapeutic effect. Such an effective dose will usually depend on the factors described above. Typically, intravenous and subcutaneous doses of the compounds of this invention to a patient, when used to provide the indicated analgesic effect, will range from about 0.0001 to about 200 mg per kilogram of body weight per day, more preferably from about 0.01 to about 150 mg per kg per day, and even more preferably from about 0.2 to about 140 mg per kg per day.

If desired, an effective daily dose of the active compound can be administered in the form of two, three, four, five, six or more sub-doses, administered individually at suitable intervals throughout the day, optionally in unit dosage forms.

Although the compound of the present invention can be administered on its own, it is preferable to administer the compound in the form of a pharmaceutical composition.

The present invention also relates to packaged pharmaceutical compositions for treating painful conditions that respond to a Ν-6 substituted 7-desazapurin, for example, with an undesirable increased activity of adenosine receptor in a mammal. Packed pharmaceutical compositions include a container containing a therapeutically effective amount of at least one desazapurin as described above, and instructions for using desazapurin to treat a condition that responds to desazapurin.

The desazapurines of this invention can be obtained using standard methods of organic synthesis. Desazapurines can be purified by reverse phase HPLC, chromatography, recrystallization, etc. and their structures are confirmed by mass spectral analysis, elemental analysis, IR and / or NMR spectroscopy.

Typically, the synthesis of intermediates as well as the desazapurines of the invention is carried out in solution. The attachment and removal of one or more protecting groups is also common in practice and is known to those skilled in the art.

Typical synthesis schemes for the intermediates for preparing the desazapurines of the invention are presented below in Scheme I.

This invention further relates to a compound having structure (IV):

where Κ ₁ is trans-4-hydroxycyclohexyl, 2-methylaminocarbonylaminocyclohexyl, acetylaminoethyl or methylaminocarbonylaminoethyl;

where K ₃ represents a substituted or unsubstituted four-six membered ring; phenyl, pyrrole, thiophene, furan, thiazole, imidazole, pyrazole, 1,2,4-triazole, pyridine,

2 (1 H) -pyridone, 4 (1 H) -pyridone, pyrazine, pyrimidine, pyridazine, isothiazole, isoxazole, oxazole, tetrazole, naphthalene, tetraline, naphthyridine, benzofuran, benzothiophene, indole, 2,3-dihydroindole, 1 Nindol indoline, benzopyrazole, 1,3-benzodioxole, benzoxazole, purine, coumarin, chromon, quinoline, tetrahydroquinoline, isoquinoline, benzimidazole, quinazoline, pyrido [2,3-b] pyrazine, pyrido [3,4-b] pyrazine, pyrido [ 3,2-c] pyridazine, pyrido [3,4-b] pyridine, 1H-pyrazole [3,4-b] pyrimidine, pteridine, 2 (1H) -quinolone, 1 (2H) -isoquinolone, 1,4- benzisoxazine, benzothiazole, quinoxaline, quinoline - ^ oxide, isoquinoline - ^ oxy , ^ Quinoxalin-oxide, ^ quinazoline-oxide, benzoxazine, phthalazine, cinnoline, or having a structure:

where Υ represents carbon or nitrogen;

- 21 007254 where K ₂ and K ₂ ', independently, are H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, halogen, methoxy, methylamino or methylthio; where K ₅ represents H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, wherein said substituted alkyl is —C (K ₇ ) (K ₈ ) XK ₉ , where X is O, 8 or ΝΚ ₁₀ where each of K ₇ and K ₈ independently represents H or alkyl, where each of K ₉ and K ₁₀ independently represents alkyl or cycloalkyl, or ΝΚ ₉ Κι ₀ represents a substituted or unsubstituted ring containing from 4 up to 7 members;

where K ₆ represents H, alkyl, substituted alkyl, cycloalkyl; or a pharmaceutically acceptable salt thereof, a prodrug derivative, or a biologically active metabolite, provided that when Κι is acetylaminoethyl, K _{3 is} not 4-pyridyl.

In one embodiment of the compound having structure IV, ΝΚ ₉ Κι ₀ is a substituted or unsubstituted ring containing from 4 to 7 members, which is selected from the group consisting of:

where m is 0, 1 or 2,

where η is 0, 1, 2 or 3; where K ₈ 'represents hydrogen, —OH, —CH ₂ OH, —C (= O) ΝΚ ₉ 0ι ₀ , ΝΗΚ _π ; where Kp represents —C (═O) CH ₃ or —8O ₂ Me, or

where K represents H, alkyl or aryl.

In another embodiment of the compound having structure IV, K ₃ has the structure

where Υ represents carbon or nitrogen; where K ₂ represents H or halogen, an alkyl group, an amino group or a sulfide group;

where K5 represents H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, where said substituted alkyl is —C (K ₇ ) (Κ ₈ ) Κ ₉ Κι ₀ , where each of K ₇ and K ₈ , independently represents H or alkyl, where each of K ₉ and K ₁₀ independently is alkyl or cycloalkyl, or K ₉ , K ₁₀ and nitrogen together form a substituted or unsubstituted ring of 4-7 members.

In another embodiment, compound Υ is carbon.

In another embodiment, compound K ₂ is hydrogen.

In another embodiment, compound K ₆ is hydrogen.

In another embodiment, compound K ₅ is hydrogen.

In another embodiment of the compound, each of K ₅ and K ₆ is methyl.

In another embodiment, the compound K ₅ is —C (K ₇ ) (Κ ₈ ) ΝΚ ₉ Κι ₀ , where each of K ₇ and K ₈ , independently, is H or alkyl, where each of K ₉ and K ₁₀ , independently, is alkyl or cycloalkyl, or K9, K10 and nitrogen together form a substituted or unsubstituted ring of 4-7 members.

In another embodiment, the compound K ₂ is halogen.

In another embodiment, compound Υ is nitrogen.

In yet another embodiment, the compound K ₂ is hydrogen.

In a further embodiment of the compound, each of K ₅ and K ₆ is hydrogen.

This invention also relates to a compound having structure (V):

- 22 007254

where K ₃ represents aryl, substituted aryl or heteroaryl;

where K ₆ represents H, alkyl, substituted alkyl or cycloalkyl; where K ₅ represents H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, where said substituted alkyl is —C (K ₇ ) (Κ ₈ ) ΝΚ ₉ Κ ₁₀ , where each of K ₇ and K ₈ independently represents H or alkyl, where each of K ₉ and K ₁₀ represents alkyl or cycloalkyl, or K ₉ , K ₁₀ and nitrogen together form a ring system consisting of 4-7 members.

In one embodiment of a compound having structure V, each of K ₇ and K ₈ is H; where K ₉ represents H, and K ₁₀ represents -K ₁₂ C (= 0) K ₁₃ .

In another embodiment of a compound having structure V, each of K ₇ and K ₈ is H; where the ring system is morpholino, thiomorpholino, Ν-4-substituted piperazino, 2-substituted piperizine or K ₈ 'substituted pyrrolidino, piperidine, where K ₈ ' represents H, OH, CH ₂ 0H, -C (= Ο) ΝΚ ₉ Κ ₁₀ , ΝΚ ₁₁ , where K ₁₁ represents -C (= 0) CH ₃ , -80 ₂ Me.

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

(Compound 706)

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

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

(Compound 1318-a)

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

(Compound 1318-b)

- 23 007254

(Compound 1319)

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

(Compound 1320)

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

(Compound 1321)

The compound has the structure

where K ₃ represents a 5-membered aromatic ring; where K ₅ and K _b independently represent H or alkyl.

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

(Compound 1500)

- 24 007254

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

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

In another embodiment of compound 1500, the compound has the structure:

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

This invention also relates to a compound having the structure:

where K ₃ represents a 5-6 membered aromatic ring; where K ₅ and Kb independently represent H or alkyl; provided that K _{3 is} not 4-pyridyl.

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

- 25 007254

(Compound 1501)

This invention further relates to a compound having the structure:

where P ₃ represents a substituted 5-6 membered aromatic ring; where P ₅ and B ₆ independently represent H or alkyl.

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

(Compound 1520)

This invention further relates to a compound having the structure:

where P ₃ represents a 5-6 membered aromatic ring; where X represents oxygen or sulfur.

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

"TO

(Compound 1503)

This invention further relates to a compound having the structure:

- 26 007254 where K ₃ represents a 5-6-membered aromatic ring; where X represents oxygen or sulfur.

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

(Compound 1504)

The invention also relates to a method for treating a disease associated with an A ₁ adenosine receptor in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula IV, V, VI, VI, VII, VIII, IX or X.

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

In a further embodiment of the method, A ₁ adenosine receptor is associated with cognitive impairment, renal failure, cardiac arrhythmia, epithelium of the respiratory tract, sedation, constriction of blood vessels, bradycardia, negative cardiac inotropy and dromotropia, bronchostenosis, neutrophilic reflux or peptic ulcer.

This invention also relates to combination therapy for asthma, including compounds IV and V, and a steroid, β2 agonist, glucocorticoid, lucotriene antagonist or anticholinergic agonist. Diseases associated with A _b A _{2 a} , A ₂ b and A ₃ adenosine receptors are described in No. 0 99/06053 and No. 0-09822465, No. 0-09705138, No. 0-09511681, No. 0-09733879, 1P-09291089, PCT / I8 98/16053 and US Pat. No. 5,516,894, the entire contents of which are incorporated by reference in their entirety.

The present invention also relates to a water-soluble prodrug of compounds having the structure IV, V, VI, VI, VII, VIII, IX or X, wherein said water-soluble prodrug metabolizes ίη νίνο to an active drug that selectively inhibits A! adenosine receptor.

In one embodiment of the prodrug, said prodrug metabolizes ίη νίνο by esterase-catalyzed hydrolysis.

This invention also relates to a pharmaceutical composition comprising a prodrug and a pharmaceutically acceptable carrier.

The present invention further relates to a method for inhibiting A1 activity of an adenosine receptor in a cell, which comprises contacting said cell with a compound having a structure of IV, V, VI, VI, VII, VIII, IX or X.

In one embodiment of the method, the compound is an antagonist of said A ₁ adenosine receptor.

The present invention also relates to a method for treating a gastrointestinal disease in a subject, comprising administering a therapeutically effective amount of a compound having a structure of IV, V, VI, VI, VII, VIII, IX or X.

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

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

In another embodiment of the method, the compound is an A ₁ antagonist _of adenosine receptors.

The invention also relates to a method for treating a respiratory tract disease in a subject, comprising administering to the subject an effective amount of a compound having a structure of IV, V, VI, VI, VII, VIII, IX or X.

In one embodiment of the method, said disease is asthma, chronic obstructive pulmonary disease, allergic rhinitis, or upper respiratory tract disease.

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

In another embodiment of the method, said compound is an antagonist of A! adenosine receptors.

The present invention also relates to a method for treating eye damage in a subject, comprising administering to said subject an effective amount of a compound having a structure of IV, V, VI, VI, VII, VIII, IX or X.

In one embodiment of the method, said damage includes damage to the retina or optic head nerve.

- 27 007254

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

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

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

In another embodiment of the method, the compound is an antagonist of Αι adenosine receptors.

The invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound having the structure IV, V, VI, VI, VII, VIII, IX or X, and a pharmaceutically acceptable carrier.

In another embodiment of the pharmaceutical composition, said therapeutically effective amount is effective for treating respiratory disease or gastrointestinal disease.

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

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

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

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

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

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

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

As used herein, the expression “compound is selective” means that the compound has a binding constant with A _{g of the} adenosine receptor at least ten times higher than that of A _{2 a} , A _{2 b} or A ₃ adenosine.

This invention also relates to a method for producing a compound having structure (IV), comprising the steps of:

a) interactions

P to obtain

P where P is a removable protecting group;

B) processing the product of stage a) under cyclization to obtain

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

- 28 007254

ά) processing the chlorinated product of step c) ΝΗ ₂ Κ ₁ to obtain

where Kd is trans-4-hydroxycyclohexyl, 2-methylaminocarbonylaminocyclohexyl, acetylaminoethyl or methylaminocarbonylaminoethyl;

where K ₃ represents a substituted or unsubstituted four-six membered ring;

where K ₅ represents H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, wherein said substituted alkyl is —C (K ₇ ) (K ₈ ) XK ₉ , where X is O, 8 or ΝΚ ₁₀ where each of K ₇ and K ₈ independently is H or alkyl, where each of K ₉ and K ₁₀ independently is alkyl or cycloalkyl, or ΝΚ ₉ Κ ₃₀ is a substituted or unsubstituted ring containing from 4 up to 7 members;

where K ₆ represents H, alkyl, substituted alkyl, cycloalkyl; or a pharmaceutically acceptable salt, prodrug derivative or biologically active metabolite thereof; with the proviso that when Kd is acetylaminoethyl, K _{3 is} not 4-pyridyl.

The present invention also relates to a method for producing a compound having a structure V, comprising the steps of a) reacting

obtaining where P represents a removable protecting group; B) processing the product of stage a) under cyclization to obtain

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

- 29 007254 where K ₃ represents aryl, substituted aryl, heteroaryl;

where In _b represents H, alkyl, substituted alkyl or cycloalkyl; where B ₅ represents H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, where said substituted alkyl is —C (B ₇ ) (Β ₈ ) ΝΒ ₉ Β ₁₀ , where each of B ₇ and K ₈ represents represents H or alkyl, where each of B ₉ and B ₁₀ represents alkyl or cycloalkyl, or ΝΒ ₉ Β ₁₀ represents a ring system containing from 4 to 7 members.

Compounds represented by formulas VI, VII and VIII can be synthesized according to any of the schemes ΕνΗ! The compounds represented by formula IX and X can be obtained according to scheme IX.

The invention is further illustrated by the following examples, which should in no way be construed as an additional limitation. The contents of all references pending patent applications and published patent applications cited in this application, including those cited in the "Background of the invention", are incorporated into this description by reference. It should be understood that the models used in the examples are generally accepted models, and demonstrating performance on these models predicts performance in humans.

The invention will be better understood from the following experimental details. However, one skilled in the art will readily understand that the specific methods and results discussed are merely illustrative of the invention, as described in more detail in the following claims.

Experimental details

The desazapurines of the invention can be prepared by standard methods of organic synthesis. Desazapurines can be purified by reverse phase HPLC, chromatography, recrystallization, etc., and their structures are confirmed by mass spectral analysis, elemental analysis, IR and / or NMR spectroscopy.

Typically, the synthesis of intermediates as well as the desazapurines of the invention is carried out in solution. The addition and removal of one or more protective groups is also typical for practical implementation and is known to specialists in this field. Typical synthesis schemes for the preparation of the intermediate products of the desazapurines of the invention are presented below in Scheme I.

where K ₃ , B ₅ and B _b are as defined above.

In general, protected 2-amino-3-cyano-pyrrole can be treated with an acyl halide to form carboxamido-3-cyanopyrrole, which can be treated with acidified methanol to close the ring in pyrrolo [2,3b] pyrimidin-4- (3H) - he (Mi11sg S.E. s! a1., I. Furniture. SNST. 40: 439b (1997)). Removal of the pyrrole protecting group followed by treatment with a chlorinating reagent, for example phosphorus oxychloride, results in substituted or unsubstituted 4-chloro-7Hpyrrolo [2,3b] pyrimidines. Treatment of chloropyrimidine with amines afforded 7-deazapurines.

For example, as shown in Scheme I, ^ (1-b1-phenylethyl) -2-amino-3-cyanopyrrole was treated with an acyl halide in pyridine and dichloromethane. The resulting No. (1-b1-phenylethyl) -2-phenylcarboxamido-3 cyanopyrrole was treated with a 10: 1 methanol / sulfuric acid mixture to close the ring, resulting in b1-7H-7- (1-phenylethyl) pyrrolo [2,3- b] pyrimidin-4 (3H) -one. Removal of the phenylethylene group by treatment of pyrimidine with polyphosphoric acid (PPA) followed by treatment with POC13 gave the key intermediate 4-chloro-7H-pyrrolo [2,3-b] pyrimidine.

Subsequent treatment of 4-chloro-7H-pyrrolo [2,3-b] pyrimidine with various amines listed in Table 1 gives the compounds of formula (I) and (II).

- 30 007254

Table 1

TO M '+ n TO M '+ n Le 343.2 about D / H / D 351.27 343.18 wsl 430.35 337.21 359.44 BUT, 364.19 XX .. 404.32 + Ό 330.18 '* M 330.45 V ^m alaa 347.22 * y | from 339.47 1> ζνν> * one 350.28 La / h. 9 ”” * '*** ^$ \ 1 353.41

- 31 007254

A / M ” and 344.19 0 ____ Υ ι 1 n 324.45 394.16 359.38 CO ~ “ ΝΗ --- 371.12 Al x ^Α Υ A 379.40 ‘AE it 359.39 0Υ + 387.41 n ^ Υν Ζχ \ ι 1 403.33 344.48 σΖ 351.49 hl 337.53 Cg ^A 330.37 Αγ 295.2 n 407.23 n 321.2 CO— * 355.45 Υ ^ζ 0Η 337.53

- 32 007254

AAO 441.33 413.24 L / AC / O N N 372.48

, X) * n 350,2 X1 343.2 n 373.2 BUT 307.2

A general approach to the preparation of 6-substituted pyrroles is depicted in the following scheme (Scheme II).

Scheme II

- 33 007254 where Κι-Κ ₅ are as defined above.

Transesterification and alkylation of ethyl cyanoacetate with α-halogen ketone lead to a ketomethyl ester. The protection of the ketone followed by treatment with amidine hydrochloride (e.g., alkyl, aryl or alkylaryl) leads to the final pyrimidine protected with ketal. Removal of the protecting group followed by cyclization and treatment with phosphorus oxychloride leads to a chlorine-containing intermediate, which can then be treated with an amine to give amine-6-substituted pyrrole. Additionally, pyrrole nitrogen may be alkylated under conditions known in the art.

A general approach to the synthesis of 5-substituted pyrroles is depicted in the following scheme (Scheme III).

where K ₁ -K ₆ are as defined above, and P represents a removable protecting group.

Condensation of malononitrile and excess ketone followed by bromination of the product leads to a mixture of the starting material, monobrominated and dibrominated products that have been treated with alkylamine, arylamine or alkylarylamine.

The resulting amine product was acylated with acid chloride and the mono-acylated pyrrole was cyclized in the presence of acid to give the corresponding pyrimidine. The pyrrole protecting group was removed with polyphosphoric acid and treated with phosphorus oxychloride to give a chlorinated product. Chlorinated pyrrole could subsequently be treated with an amine to give an amino-5-substituted pyrrole. Alkylation of pyrrole nitrogen can be carried out under conditions known in the art.

Schemes IV and V depict methods for the preparation of desazapurines 1 and 2 according to the invention.

2

- 34 007254 where B ₅ and B ₆ are as described above, for example, CH ₃ .

Specific preparation of 6-methylpyrrolopyrimidines

The key reaction to obtain 6-methylpyrrolopyrimidines (1) [B ₅ = CH ₃ ] was the cyclization of cyanoacetate with benzamidine to pyrimidine.

It was suggested that methyl cyanoacetate would cyclize with benzamidine to pyrimidine more efficiently than the corresponding ethyl ester. Therefore, the transesterification and alkylation of ethyl cyanoacetate in the presence of NOOMe and an excess of the α-haloacetyl moiety, for example, chloroacetone, gave the desired methyl ester (3) in 79% yield (Scheme IV). The keto ester (3) was protected as acetal (4) in 81% yield. A new method of cyclization to pyrimidine (5) was carried out using amidine hydrochloride, for example, benzamidine hydrochloride, using 2 equivalents of DBU, obtaining 5 with a yield of 54% of the isolated product. This method allows to improve the yield from 20% in accordance with the published conditions, where, during cyclization with guanidine, No. OMe is used. Cyclization to pyrrole-pyrimidine (6) was carried out by removal of acetal protection in aqueous HC1 with 78% yield. The interaction of (6) with phosphorus oxychloride at the boil under reflux gave the corresponding 4-chloro derivative (7). The combination with trans-4-aminocyclohexanol in dimethyl sulfoxide at 135 ° C gave (1) with a yield of 57% from (7). It will be apparent to those skilled in the art that the choice of reagents allows for greater flexibility in selecting the desired substituent B5.

Scheme IV

Specific preparation of 5-methylpyrrolopyrimidines

The condensation of Knevenagel malonitrile and an excess of ketone, for example, acetone in boiling benzene, led to 8 with a yield of 50% after distillation. The bromination with 8 X-bromosuccinimide in the presence of benzoyl peroxide in chloroform gave a mixture of the starting material, mono (9), and dibrominated products (5/90/5) after distillation (70%). The mixture was reacted with α-methylalkylamine or α-methylarylamine, e.g., α-methylbenzylamine, to obtain aminopyrrole (10). 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 (11) and diacylated (12) pyrroles, which were separated by flash chromatography. Acid hydrolysis of disubstituted pyrrole (12) gave a total yield of 29% for acylpyrrole (11). Cyclization in the presence of concentrated sulfuric acid

- 35 007254 and DMF gave (13) (23%), in which the protection was removed with polyphosphoric acid, obtaining (14). The interaction of (14) with phosphorus oxychloride upon boiling under reflux gave the corresponding 4-chloro derivative (15). The combination with trans-4-aminocyclohexanol in dimethyl sulfoxide at 135 ° C gave (2) [K ₆ = CH ₃ ] with a yield of 30% from (14) (see Scheme V). It will be apparent to those skilled in the art that the choice of reagents allows greater flexibility in the selection of the desired substituent K ₆ .

Scheme V

nine

An alternative way of synthesis of K_b-substituted pyrroles, for example, 5-methylpyrrolopyrimidines

This alternative route to K ₆ -substituted pyrroles, e.g., 5-methylpyrrolopyrimidines, involves transesterification and alkylation of ethyl cyanoacetate to form (16) (Scheme VI). Condensation (16) with benzamidine hydrochloride using 2 equivalents of DBU gave pyrimidine (17). Cyclization to pyrrole-pyrimidine (14) will be carried out by removing acetal protection in aqueous HC1. The interaction of (14) with phosphorus oxychloride upon boiling under reflux gave the corresponding 4-chloro derivative (15). Combination with trans-4-aminocyclohexanol in dimethyl sulfoxide at 135 ° C gave (2). According to this technique, the number of synthetic reactions for the target compound (2) is reduced from 9 to 4 stages. In addition, the output improves dramatically. Again, it will be apparent to a person skilled in the art that the choice of reagents allows for greater flexibility in choosing the desired substituent K ₆ .

- 36 007254

A general approach to the production of des-methylpyrrole is depicted in the following diagram (Scheme VII).

where Κι-Κ ₃ are as defined above.

- 37 007254

Alkylation of the alkyl cyanoacetate with diethyl acetal in the presence of a base gave cyanodiethyl acetal, which was treated with an amidine salt to give the methyl pyrrolopyrimidine precursor.

The precursor was chlorinated and treated with amine to form the desired des-methylpyrrolopyrimidine as shown above.

For example, Scheme VIII shows the synthesis of compound (18).

Scheme VIII

Commercially available methyl cyanoacetate was alkylated with bromoacetaldehyde diethylacetal in the presence of potassium carbonate and railway preparation (19). Cyclization to pyrimidine (20) was carried out in two stages. Initially, pyrimidine-acetal was formed by reaction (19) with benzamidine hydrochloride using 2 equivalents of DBU. In the resulting pyrimidine-acetal without purification, the protective group of aqueous 1N was removed. HCl and the resulting aldehyde were cyclized to pyrrolopyrimidine (20), which was isolated by filtration. The interaction of (20) with phosphorus oxychloride upon boiling under reflux led to the corresponding 4-chloro derivative (21). The binding of the chloro derivative to trans-4-aminocyclohexanol in DMSO at 135 ° C gave compound (18) from compound (21).

It has been shown in the P ^ W schemes that the 5- and 6-position of the pyrrolopyrimidine ring can be functionalized. Using various starting reagents and minor modifications of the above reaction schemes, various functional groups can be introduced at the 5- and 6-positions in the formula (I) and (II). Some examples are illustrated in table. 2.

- 38 007254

It is known to those skilled in the art that the metabolism of the compounds described herein in a subject will lead to some biologically active metabolites that serve as medicaments.

The invention is further illustrated by the following examples, which should in no way be construed as an additional limitation. The contents of all references pending patent applications and published patent applications cited in this application, including those cited in the "Background of the invention", are incorporated into this description by reference. It should be understood that the models used in the examples are generally accepted models, and demonstrating performance on these models predicts performance in humans.

Illustrative examples

Getting 1. Used a modification of the alkylation method 8ee1a and Birke ¹ . To an ice-cooled (0 ° C) solution of ethyl cyanoacetate (6.58 g, 58.1 mmol) in MeOH was slowly added a solution of # YUMe (25% w / v; 58.1 mmol). After 10 minutes, chloroacetone (5 ml; 62.8 mmol) was slowly added. After 4 hours, the solvent was removed. The brown oil was diluted with EuAc (100 ml) and washed with H2O (100 ml). The organic fraction was dried, filtered and concentrated to give a brown oil (7.79 g; 79%). 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, ί.Όί.Ί3) δ 4.24 (q, 1 = 7.2 Hz, OCH ₂ ), 3.91 (dd, 1H, 1 = 7.2, 7.0 Hz , CH), 3.62 (s, 3H, OCH ₃ ), 3.42 (dd, 1H, 1 = 15.0, 7.1 Hz, 1 x CH ₂ ); 3.02 (dd, 1H, 1 = 15.0, 7.0 Hz, 1 x CH ₂ ); 2.44 (s, 3H, CH ₃ ), 1.26 (t, 1 = 7, 1 Hz, ester-CH ₃ ).

¹ 8ee1a K; Tag and. Sat. Veg. 1977, 110, 1462-1469.

Getting 2. Used method 8e1a and Birke ¹ . Thus, ketone protection (3) (Scheme IV; 5.0 g,

32.2 mmol) with ethylene glycol (4 ml, 64.4 mmol) in the presence of TUN (100 mg) gave 4 in the form of an oil (Scheme IV; 5.2 g, 81.0) after flash chromatography (8U ₂ ; 3 / 7 EJAc / Hexanes, K, 0.35). The product still contains 5% ethyl ester: ¹ H-NMR (200 MHz, SBS1 ₃ ) δ 4.24 (q, 1 = 7.2 Hz, OCH ₂ ), 3, 98 (s, 4H, 2 x acetal -CH ₂ ), 3.79 (s, 3H, OCH ₃ ), 3.62 (dd, 1H, 1 = 7.2, 7.0 Hz, CH), 2.48 (dd, 1H, 1 = 15 0, 7.1 Hz, 1 x CH ₂ ), 2.32 (dd, 1H, 1 = 15.0, 7.0 Hz, 1 x CH ₂ ); 1.35 (s, 3H, CH ₃ ), 1.26 (t, 1 = 7.1 Hz, ester-CH ₃ ); mass spectrum (E8 electrospray): 200.1 (M ⁺ +1).

¹ 8ee1a K; Tag and. Sat. Veg. 1977, 110, 1462-1469.

Preparation 3. Acetal solution (4) (Scheme IV; 1.0 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 at 85 ° C for 15 hours. The mixture was diluted with CHCl ₃ (30 ml) and washed with 0.5N. No. JUN (10 ml) and H ₂ O (20 ml). The organic fraction was dried, filtered and concentrated to give a brown oil. An attempt was made to flash chromatography (81O ₂ ; 1/9 EJAc / CH ₂ Cl ₂ , K 0.35), but the substance crystallized on a column. Silica gel was washed with MeOH. Fractions containing product (5) (Scheme IV) were concentrated and used without further purification (783 mg, 54.3%): ¹ H-NMR (200 MHz, SOSB) δ 8.24 (m, 2H, Ar-H) 7.45 (m, 3H, Ar-H), 5.24 (br s, 2H, ΝΉ ₂ ), 3.98 (s, 4H, 2 x acetal-CH ₂ ), 3.60-3, 15 (m, 2H, CH ₂ ), 1.38 (s, 3H, CH ₃ ); mass spectrum (E8): 288.1 (M ⁺ +1).

Preparation of compound (20) (Scheme VIII): Acetal solution (19) (4.43 g, 20.6 mmol) 1, benzamidine hydrochloride (3.22 g, 20.6 mmol) and DBU (6.15 ml, 41 , 2 mmol) in anhydrous DMF (20 ml) was heated at 85 ° C for fifteen hours. The mixture was diluted with CHCl ₃ and washed with H ₂ O (2x50 ml). The organic fraction was dried, filtered and concentrated to give a dark brown oil. Dark brown oil was stirred in 1N. HCl (100 ml) for 2 hours at room temperature. The resulting suspension was filtered to give the HCl salt of compound (20) as a golden brown solid (3.60 g, 70.6%); ¹ H-NMR (200 MHz, DMSO-4 ₆ ) 11.92 (s 1H), 8.05 (m, 2H, Ar-H), 7.45 (m, 3H, Ar-H), 7.05 (s, 1H, pyrrole-H); mass spectrum (E8): 212.1 (M ⁺ +1).

Preparation 4. A solution of acetal (5) (700 mg, 2.44 mmol) in 1N HCl (40 ml) was stirred for 2 hours at room temperature. The resulting suspension was filtered to obtain the HC1 salt of 2-phenyl-6-methyl7H-pyrrolo [2,3-4] pyrimidin-4 (3H) -one as a golden brown solid (498 mg, 78.0%); ¹ H-NMR (200 MHz, DMSO-4 ₆ ) δ 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 ); mass spectrum (E8): 226.1 (M + 1).

Getting 5. Used a modification of the method of cyclization SBep e! a1. ¹ . To an ice-cooled (0 ° C) solution of bromide (9) (Scheme V; 20.0 g, 108 mmol, 90% purity) in isopropyl alcohol (60 ml), a solution of α-methylbenzylamine (12.5 ml, 97, 3 mmol). The black solution was allowed to warm to room temperature and was stirred for 15 hours. The mixture was diluted with EuAc (200 ml) and washed with 0.5N. No. JUN (50 ml). The organic fraction was dried, filtered and concentrated to give a black gum (19.2 g; 94%). The residue was partially purified by flash chromatography (occupies 8 _2; 4/96 MeOH / CH ₂ C1 _2, R, 0.35) to give a black solid (6.38 g, 31%), constituting 41-1- (1feniletil) -2-amino-3-cyano-4-methylpyrrole: Mass spectrum (E8): 226.1 (M ⁺ +1).

¹ Sat ΥΤ .; Mapkas K.8 .; \ Ut1eg 8.M .; Vgook E .; Sashpk 1 .; Sogtap M.B .; ΟυηηίκΚίκ A.K .; Eagas1 No. 8 .; SaIaksBip K..B; 8cbt141 A .; Zsik Ό.№. BMe4.Sbet., 1997, 40, 1749-1754.

- 39 007254

Preparation 6. To a solution of 61-1- (1-phenylethyl) -2-amino-3-cyano-4,5-dimethylpyrrole (14.9 g, 62.5 mmol) and pyridine (10.0 ml) in dichloromethane ( 50.0 ml) benzoyl chloride (9.37 g, 66.7 mmol) was added at 0 ° C. After stirring at 0 ° C for 1 h, hexane was added to facilitate crystallization of the product. The solvent was removed in vacuo and the solid was recrystallized from a mixture of E1OH / H ₂ O to give 13.9 g (65%) 61-1- (1-phenylethyl) -2-phenylcarbonylamino-3-cyano-4,5-dimethylpyrrole, t .pl. 218-221 ° C; 'H-NMR (200 MHz, ATP1 ₃ ) δ 1.72 (s, 3H), 1.76 (d, 1 = 7.3 Hz, 3H), 1.98 (s, 3H),

5.52 (q, 1 = 7.3 Hz, 1H), 7.14-7.54 (m, 9H), 7.68-7.72 (dd, 1 = 1.4 Hz, 6.9 Hz, 2H ), 10.73 (s, 1H); mass spectrum (E8): 344.4 (M ⁺ +1).

¹ Exile App. Syet., 1986, 1485-1505.

The following compounds were similarly prepared.

Getting 6A. 61-1- (1-Phenylethyl) -2- (3-pyridyl) carbonylamino-3-cyano-4,5-dimethylpyrrole. 'NMR (200 MHz, ATP1 ₃ ) δ 1.83 (d, 1 = 6.8 Hz, 3H), 2.02 (s, 3H), 2.12 (s, 3H), 5.50 (q, 1 = 6.8 Hz, 1H), 7.14-7.42 (m, 5H), 8.08 (m, 2H), 8.75 (m, 3H); mass spectrum (E8): 345.2 (M ⁺ +1).

61-1- (1-Phenylethyl) -2- (2-furyl) carbonylamino-3-cyano-4,5-dimethylpyrrole. 'H-NMR (200 MHz, ATP1 ₃ ) δ 1.84 (d, 1 = 7.4 Hz, 3H), 1.92 (s, 3H), 2.09 (s, 3H), 5, 49 ( q, 1 = 7.4 Hz, 1H), 6.54 (dd, 1 = 1.8 Hz, 3.6 Hz, 1H), 7.12-7.47 (m, 7H); mass spectrum (E8): 334.2 (M ⁺ +1), 230.1.

61-1- (1-Phenylethyl) -2- (3-furyl) carbonylamino-3-cyano-4,5-dimethylpyrrole. 'H-NMR (200 MHz, ATP1 ₃ ) δ 1.80 (d, 1 = 7 Hz, 3H), 1.89 (s, 3H), 2.05 (s, 3H), 5.48 (q, 1 = 7 Hz, 1H), 6.59 (s, 1H), 7.12-7.40 (m, 6H), 7.93 (s, 1H); mass spectrum (E8): 334.1 (M ⁺ +1), 230.0.

61-1- (1-Phenylethyl) -2-cyclopentylcarbonylamino-3-cyano-4,5-dimethylpyrrole. 'H-NMR (200 MHz, ATP1 ₃ ) δ 1.82 (d, 1 = 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, 1 = 7.4 Hz, 1H), 6.52 (s, 1H), 7.05-7.20 (m, 5H); mass spectrum (E8): 336.3 (M + 1).

61-1- (1-Phenylethyl) -2- (2-thienyl) carbonylamino-3-cyano-4,5-dimethylpyrrole. 'H-NMR (200 MHz, ATP1 ₃ ) δ 1.82 (d, 1 = 6.8 Hz, 3H), 1.96 (s, 3H), 2.09 (s, 3H), 5.49 ( q, 1 = 6.8 Hz, 1H), 7.05-7.55 (m, 8H); mass spectrum (E8): 350.1 (M ⁺ +1), 246.0.

61-1- (1-Phenylethyl) -2- (3-thienyl) carbonylamino-3-cyano-4,5-dimethylpyrrole. 'H-NMR (200 MHz, ATP1 ₃ ) δ 1.83 (d, 1 = 7.0 Hz, 3H), 1.99 (s, 3H), 2.12 (s, 3H), 5.49 ( q, 1 = 7.0 Hz, 1H), 6.90 (m, 1H), 7.18-7.36 (m, 6H), 7.79 (m, 1H); mass spectrum (E8): 350.2 (M ⁺ +1), 246.1.

61-1- (1-Phenylethyl) -2- (4-fluorophenyl) carbonylamino-3-cyano-4,5-dimethylpyrrole. 'H-NMR (200 MHz, ATP1 ₃ ) δ 1.83 (d, 1 = 7.4 Hz, 3H), 1.96 (s, 3H), 2.08 (s, 3H), 5, 51 ( q, 1 = 7.4 Hz, 1H), 7.16-7.55 (m, 9H); mass spectrum (E8): 362.2 (M ⁺ +1), 258.1.

61-1- (1-Phenylethyl) -2- (3-fluorophenyl) carbonylamino-3-cyano-4,5-dimethylpyrrole. 'H-NMR (200 MHz, ATP1 ₃ ) δ 1.83 (d, 1 = 7.4 Hz 3H), 1.97 (s, 3H), 2.10 (s, 3H), 5.50 (q , 1 = 7.4 Hz, 1H), 7.05-7.38 (m, 7H), 7.67-7.74 (m, 2H); mass spectrum (E8): 362.2 (M ⁺ +1), 258.1.

61-1- (1-Phenylethyl) -2- (2-fluorophenyl) carbonylamino-3-cyano ~ 4,5-dimethylpyrrole. ’H-NMR (200

MHz, ATP1 ₃ ) δ 1.85 (d, 1 = 7.2 Hz, 3H), 1.94 (s, 3H), 2.11 (s, 3H), 5.50 (q, 1 = 7, 2 Hz, 1H), 7.12-7.35 (m, 6H),

7.53 (m, 1H), 7.77 (m, 1H), 8.13 (m, 1H); mass spectrum (E8): 362.2 (M ⁺ +1), 258.0.

61-1- (1-Phenylethyl) -2-isopropylcarbonylamino-3-cyano-4,5-dimethylpyrrole. 'H-NMR (200 MHz, ATP1 ₃ ) δ 1.19 (d, 1 = 7.0 Hz, 6H), 1.82 (d, 1 = 7.2 Hz, 3H), 1.88 (s, 3H), 2.06 (s, 3H), 2.46 (m, 1H), 5.39 (m, 1 = 7.2 Hz, 1H), 6.64 (s, 1H), 7.11- 7.36 (m, 5H); mass spectrum (E8): 310.2 (M ⁺ +1), 206.1.

By acylating 61-1- (1-phenylethyl) -2-amino-3-cyano-4-methylpyrrole, mono-acylated 61-1- (1-phenylethyl) -2-benzoylamino-3-cyano-4-dimethylpyrrole and diacylated pyrrole 61 were obtained -1 (1-phenylethyl) -2-dibenzoylamino-3-cyano-4-methylpyrrole. Mono-acylated pyrrole: 'H-NMR (200 MHz, ATP1 ₃ ) δ 7.69 (d, 2H, 1 = 7.8 Hz, Ar-H), 7.58-7.12 (m, 8H, Ar-H ), 6.18 (s, 1H, pyrrole-H), 5.52 (q, 1H, 1 = 7.2 Hz, CH-CH ₃ ), 2.05 (s, 3H, pyrrole-CH ₃ ), 1.85 (d, 3H, 1 = 7.2 Hz, CH-CH ₃ ); mass spectrum (E8): 330.2 (M ⁺ +1); Diacylated pyrrole: 'H-NMR (200 MHz, SES1 ₃ ) δ 7.85 (d, 2H, 1 = 7.7 Hz, Ar-H), 7.74 (d, 2H, 1 = 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, 1 = 7.2 Hz, CH-CH ₃ ), 1.77 (d, 3H, 1 = 7.2 Hz, CH-CH ₃ ), 1.74 (s, 3H, pyrrole-CH ₃ ); mass spectrum (E8): 434.1 (M ⁺ +1).

Preparation 7. To a solution of 61-1- (1-phenylethyl) -2-phenylcarboxyamino-3-cyano-4,5-dimethylpyrrole (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 heated at the boil under reflux for 15 hours and cooled to room temperature. The precipitate was filtered off, yielding 0.48 g (48%) of 61-5,6-dimethyl-2-phenyl-7H-7 (1-phenylethyl) pyrrolo [2,3-6] pyrimidin-4 (3H) -one. 'H-NMR (200 MHz, SESE) δ 2.02 (d, 1 = 7.4 Hz, 3H), 2.04 (s, 3H), 2.41 (s, 3H), 6.25 (q , 1 = 7.4 Hz, 1H), 7.22-7.50 (m, 9H), 8.07-8.12 (dd, 1 = 3.4 Hz, 6.8 Hz, 2H), 10 51 (s, 1H); mass spectrum (E8): 344.2 (M ⁺ +1). The following compounds were obtained by a method similar to that described in obtaining 7.

61-5,6-Dimethyl-2- (3-pyridyl) -7H-7- (1-phenylethyl) pyrrolo [2,3-6] pyrimidin-4 (3H) -one. 'H-NMR (200 MHz, ATP1 ₃ ) δ 2.03 (d, 1 = 7.2 Hz, 3H), 2.08 (s, 3H), 2.42 (s, 3H), 6.24 ( q, 1 = 7.2 Hz, 1H), 7.09-7.42 (m, 5H), 8.48 (m, 2H), 8.70 (m, 3H); mass spectrum (E8): 345.1 (M + 1).

- 40 007254 b1-5,6-Dimethyl-2- (2-furyl) -7H-7- (1-phenylethyl) pyrrolo [2,3-b] pyrimidin-4 (3H) -one. ¹ H-NMR (200 MHz, SIS) δ 1.98 (d, 1 = 7, 8 Hz, 3H), 1.99 (s, 3H), 2.37 (s, 3H), 6.12 (q , 1 = 7.8 Hz, 1H), 6.48 (dd, 1 = 1.8 Hz, 3.6 Hz, 1H), 7.17-7.55 (m, 7H), 9.6 (s , 1H); mass spectrum (E8): 334.1 (M + 1).

b1-5,6-dimethyl-2- (3-furyl) -7H-7- (1-phenylethyl) pyrrolo [2,3-b] pyrimidin-4 (3H) -one. ¹ H-NMR (200 MHz, SIS) δ 1.99 (d, 1 = 7 Hz, 3H), 2.02 (s, 3H), 2.42 (s, 3H), 6.24 (q, 1 = 7 Hz, 1H), 7.09 (s, 1H), 7.18-7.32 (m, 5H), 7.48 (s, 1H), 8.51 (s, 1H); mass spectrum (E8): 334.2 (M + 1).

b1-5,6-dimethyl-2-cyclopentyl-7H-7- (1-phenylethyl) pyrrolo [2,3-b] pyrimidin-4 (3H) -one. ¹ H-NMR (200 MHz, SIS) δ 1.95 (d, 1 = 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, 1 = 7.4 Hz, 1H), 7.16-7.30 (m, 5H), 9.29 ( s, 1H); mass spectrum (E8): 336.3 (M ⁺ +1).

b1-5,6-dimethyl-2- (2-thienyl) -7H-7- (1-phenylethyl) pyrrolo [2,3-b] pyramidin-4 (3H) -one. ¹ H-NMR (200 MHz, C1) C1 _; ) δ 2.02 (d, 1 = 7.2 Hz, 3H), 2.06 (s, 3H), 2.41 (s, 3H), 6.13 (q, 1 = 7.2 Hz, 1H ), 7.12 (dd, 1 = 4.8, 2.8 Hz, 1H), 7.26-7.32 (m, 5H), 7.44 (d, 1 = 4.8 Hz, 1H) 8.01 (d, 1 = 2.8 Hz, 1H); 11.25 (s, 1H); mass spectrum (E8):

350.2 (M ⁺ +1).

b1-5,6-dimethyl-2- (3-thienyl) -7H-7- (1-phenylethyl) pyrrolo [2,3-b] pyrimidin-4 (3H) -one. ¹ H-NMR (200 MHz, SIS) δ 2.00 (d, 1 = 7.4 Hz, 3H), 2.05 (s, 3H), 2.43 (s, 3H), 6.24 (q , 1 = 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); mass spectrum (E8): 350.2 (M ⁺ +1).

b1-5,6-dimethyl-2- (4-fluorophenyl) -7H-7- (1-phenylethyl) pyrrolo [2,3-b] pyrimidin-4 (3H) -one. ¹ H-NMR (200 MHz, SIS) δ 2.01 (d, 1 = 6.8 Hz, 3H), 2.05 (s, 3H), 2.42 (s, 3H), 6.26 (q , 1 = 6.8 Hz, 1H), 7.12-7.36 (m, 7H), 8.23-8.30 (m, 2H), 11.82 (s, 1H); mass spectrum (E8): 362.3 (M ⁺ +1).

b1-5,6-dimethyl-2- (3-fluorophenyl) -7H-7- (1-phenylethyl) pyrrolo [2,3-b] pyrimidin-4 (3H) -one. 'H-NMR (200 MHz, SIS) δ 2.02 (d, 1 = 7.4 Hz, 3H), 2.06 (s, 3H), 2.44 (s, 3H), 6.29 (q , 1 = 7.4 Hz, 1H), 7.13-7.51 (m, 7H), 8.00-8.04 (m, 2H), 11.72 (s, 1H); mass spectrum (E8): 362.2 (M ⁺ +1).

b1-5,6-dimethyl-2- (2-fluorophenyl) -7H-7- (1-phenylethyl) pyrrolo [2,3-b] pyrimidin-4 (3H) -one. ¹ H-NMR (200 MHz, SIS) δ 2.00 (d, 1 = 7.2 Hz, 3H), 2.05 (s, 3H), 2.38 (s, 3H), 6.24 (q , 1 = 7.2 Hz, 1H), 7.18-7.45 (m, 8H), 8.21 (m, 1H), 9.54 (s, 1H); mass spectrum (E8): 362.2 (M ⁺ +1).

b1-5,6-dimethyl-2-isopropyl-7H-7- (1-phenylethyl) pyrrolo [2,3-b] pyrimidin-4 (3H) -one. ¹ H-NMR (200 MHz, SIS) δ 1.30 (d, 1 = 6.8 Hz, 3H), 1.32 (d, 1 = 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) ; mass spectrum (E8): 310.2 (M ⁺ +1).

Preparation 8. A solution of b1-1- (1-phenylethyl) -2-benzoylamino-3-cyano-4-methylpyrrole (785 mg, 2.38 mmol) with concentrated H ₂ 8O ₄ (1 ml) in DMF (13 ml) stirred at 130 ° C for 48 hours. The black solution was diluted with CHCl ₃ (100 ml) and washed with 1N. No. 1OH (30 ml) and brine (30 ml). The organic fraction was dried, filtered, concentrated and purified by flash chromatography (81O ₂ ; 8/2 EULc / hexanes, K _£ 0.35) to give a brown solid (6.38 g, 31%), which was b1-5- methyl-2-phenyl-7H-7- (1-phenylethyl) pyrrolo [2,3-ά] pyrimidin-4 (3H) -one. ¹ NMR (200 MHz, SIS) δ 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, 1 = 7.2 Hz, CH-CH ₃ ), 2.18 (s, 3H, pyrrole - CH ₃ ), 2.07 (d, 3H, 1 = 7.2 Hz, CH-CH ₃ ); mass spectrum (E8): 330.2 (M ⁺ +1).

Preparation 9. Mixture of b1-1- (1-phenylethyl) -2-amino-3-cyano-4,5-dimethylpyrrole (9.60 g, 40.0 mmol) and formic acid (50.0 ml, 98%) heated at the boil under reflux for 5 hours. After cooling to room temperature and rubbing the sides of the flask, an abundant precipitate formed, which was filtered off. The substance was washed with water until the washings showed a neutral pH, obtaining b1-5,6-dimethyl-7H-7- (1-phenylethyl) pyrrolo [2,3-b] pyrimidin-4 (3H) -one . ^! H-NMR (200 MHz, SIS) δ 1.96 (d, 1 = 7.4 Hz, 3H), 2.00 (s, 3H), 2.38 (s, 3H), 6.21 (q, 1 = 7.4 Hz, 1H), 7.11-7.35 (m, 5H), 7.81 (s, 1H), 11.71 (s, 1H); mass spectrum (E8): 268.2 (M ⁺ +1).

Preparation 10. b1-5,6-Dimethyl-2-phenyl-7H-7- (1-phenylethyl) pyrrolo [2,3-b] pyrimidin-4 (3H) -one (1.0 g, 2.91 mmol ) 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 made basic with solid KOH to pH 6. The resulting solid was filtered and isolated to obtain 0.49 g (69%) of 5.6 dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidin-4 (3H) -one. ¹ H-NMR (200 MHz, DMSO-b ₆ ) δ 2.17 (s, 3H), 2.22 (s, 3H), 7.45 (br., 3H), 8.07 (br., 2H ,), 11.49 (s, 1H), 11.82 (s, 1H); mass spectrum (E8): 344.2 (M ⁺ +1).

The following compounds were prepared in a similar manner to that described in Preparation 10: 5-methyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidin-4 (3H) -one. Mass spectrum (E8): 226.0 (M ^ + 1);

5.6-dimethyl-2- (3-pyridyl) -7H-pyrrolo [2,3-b] pyrimidin-4 (3H) -one. Mass spectrum (E8): 241.1 (M ⁺ +1);

5.6-dimethyl-2- (2-furyl) -7H-pyrrolo [2,3-b] pyrimidin-4 (3H) -one. ¹ H-NMR (200 MHz, DMSO-b ₆ ) δ

2.13 (s, 3H), 2.18 (s, 3H), 6.39 (dd, 1 = 1.8, 3.6 Hz, 1H), 6.65 (dd, 1 = 1.8 Hz, 3 6 Hz, 1H), 7.85 (dd, 1 = 1.8, 3.6 Hz, 1H,), 11.45 (s, 1H), 11.60 (s, 1H); mass spectrum (E8): 230.1 (M ⁺ +1);

5.6-dimethyl-2- (3-furyl) -7H-pyrrolo [2,3-b] pyrimidin-4 (3H) -one. ¹ H-NMR (200 MHz, DMSO-b ₆ ) 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); mass spectrum (E8): 230.1 (M ⁺ +1);

- 41 007254

5.6-dimethyl-2-cyclopentyl-7H-pyrrolo [2,3-a] pyrimidin-4 (3H) -one. ¹ H-NMR (200 MHz, DMSO-ώ ·,) δ 1.57-1.91 (m, 8 N), 2.12 (s, 3H), 2.16 (s, 3H), 2.99 (m, 1H), 11.24 (s, 1H), 11.38 (s, 1H); mass spectrum (E8):

232.2 (M ⁺ +1);

5.6-dimethyl-2- (2-thienyl) -7H-pyrrolo [2,3-th] pyrimidin-4 (3H) -one. ¹ H-NMR (200 MHz, DMSO-0 ₆ ) δ

2.14 (s, 3H), 2.19 (s, 3H), 7.14 (dd, 1 = 3.0, 5.2 Hz, 1H), 7.70 (d, 1 = 5.2 Hz , 1H), 8.10 (d, 1 = 3.0 Hz, 1H), 11.50 (s, 1H); mass spectrum (E8): 246.1 (M ⁺ +1);

5.6-dimethyl-2- (3-thienyl) -7H-pyrrolo [2,3-th] pyrimidin-4 (3H) -one. ¹ H-NMR (200 MHz, DMS0-b ₆ ) 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); mass spectrum (E8): 246.1 (M ⁺ +1);

5.6-dimethyl-2- (4-fluorophenyl) -7H-pyrrolo [2,3-th] pyrimidin-4 (3H) -one. ¹ H-NMR (200 MHz, DMSO- ₆ ) δ 2.17 (s, 3H), 2.21 (s, 3H), 7.31 (m, 2H), 8.12 (m, 2H), 11.47 (s, 1H); mass spectrum (E8): 258.2 (M ⁺ +1);

5.6-dimethyl-2- (3-fluorophenyl) -7H-pyrrolo [2,3-th] pyrimidin-4 (3H) -one. ¹ H-NMR (200 MHz, DMSO- ₆ ) 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); mass spectrum (E8): 258.1 (M ⁺ +1);

5.6-dimethyl-2- (2-fluorophenyl) -7H-pyrrolo [2,3-b] pyrimidin-4 (3H) -one. ¹ H-NMR (200 MHz, DMS0-b ₆ ) 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); mass spectrum (E8): 258.1 (M ⁺ +1);

5.6-dimethyl-2-isopropyl-7H-pyrrolo [2,3-th] pyrimidin-4 (3H) -one. ¹ H-NMR (200 MHz, DMS0-b ₆ ) δ

1.17 (d, 1 = 6.6 Hz, 6H), 2.11 (s, 3H), 2.15 (s, 3H), 2.81 (m, 1H), 11.20 (s, 1H), 11.39 (s, 1H); mass spectrum (E8): 206.1 (M ⁺ +1);

5.6-dimethyl-7H-pyrrolo [2,3-b] pyrimidin-4 (3H) -one. ¹ H-NMR (200 MHz, DMSO-th ₆ ) δ 2.13 (s, 3H),

2.17 (s, 3H); 7.65 (s, 1H); mass spectrum (E8): 164.0 (M ⁺ +1).

Preparation 11. A solution of 5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-th] pyrimidin-4 (3H) -one (1.0 g, 4.2 mmol) in phosphorus oxychloride (25.0 ml) was heated at the boil under reflux for 6 h, and then concentrated in vacuo to dryness. Water was added to the residue to induce crystallization, and the resulting solid was filtered and collected, yielding 0.9 g (83%) of 4-chloro-5,6dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. ¹ H-NMR (200 MHz, DMSO- ₆ ) 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); mass spectrum (E8): 258.1 (M ⁺ +1).

The following compounds were obtained by a method similar to that described in obtaining 11:

4-chloro-5-methyl-2-phenyl-7H-pyrrolo [2,3-th] pyrimidine, mass spectrum (E8): 244.0 (M ⁺ +1); 4-chloro-6-methyl-2-phenyl-7H-pyrrolo [2,3-th] pyrimidine, mass spectrum (E8): 244.0 (M ⁺ +1);

4-chloro-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. ¹ H-NMR (200 MHz, DMSO-b ₆ ) δ 8.35 (2.2H), 7.63 (br s, 1H), 7.45 (m, 3H), 6.47 (br s , 1H); mass spectrum (E8): 230.0 (M ⁺ +1);

4-chloro-5,6-dimethyl-2- (3-pyridyl) -7H-pyrrolo [2,3-th] pyrimidine, mass spectrum (E8): 259.0 (M ⁺ +1);

4-chloro-5,6-dimethyl-2- (2-furyl) -7H-pyrrolo [2,3-th] pyrimidine, ¹ H-NMR (200 MHz, DMSO-th ₆ ) δ 2.35 (s, 3H), 2.35 (s, 3H), 6.68 (dd, 1 = 1.8, 3.6 Hz, 1H), 7.34 (dd, 1 = 1.8 Hz, 3.6 Hz, 1H), 7.89 (dd, 1 = 1.8, 3.6 Hz, 1H); mass spectrum (E8): 248.0 (M ⁺ +1).

4-chloro-5,6-dimethyl-2- (3-furyl) -7H-pyrrolo [2,3-th] pyrimidine. ¹ H-NMR (200 MHz, DMSO- ₆ ) 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); mass spectrum (E8): 248.1 (M ⁺ +1).

4-chloro-5,6-dimethyl-2-cyclopentyl-7H-pyrrolo [2,3-b] pyrimidine. ¹ H-NMR (200 MHz, DMSO-b ₆ ) δ 1.61-1.96 (m, 8H), 2.27 (s, 3H), 2.27 (s, 3H), 3.22 (m , 1H); 11.97 (s, 1H); mass spectrum (E8): 250.1 (M ⁺ +1).

4-chloro-5,6-dimethyl-2- (2-thienyl) -7H-pyrrolo [2,3-th] pyrimidine.

Я-NMR (200 MHz, DMSO-a ₆ ) δ 2.29 (s, 3H), 2.31 (s, 3H), 7.14 (dd, 1 = 3.1 Hz, 4.0 Hz, 1H ), 7.33 (d, 1 = 4.9 Hz, 1H), 7.82 (d, 1 = 3.1 Hz, 1H), 12.19 (s, 1H); mass spectrum (E8): 264.1 (M ⁺ +1).

4-chloro-5,6-dimethyl-2- (3-thienyl) -7H-pyrrolo [2,3-th] pyrimidine. Ή-NMR (200 MHz, DMSO- ₆ ) δ 2.32 (s, 3H), 2.32 (s, 3H), 7.62 (dd, 1 = 3.0, 5.2 Hz, 1H) 7.75 (d, 1 = 5.2 Hz, 1H); 8.20 (d, 1 = 3.0 Hz, 1H); mass spectrum (E8): 264.0 (M ⁺ +1).

4-chloro-5,6-dimethyl-2- (4-fluorophenyl) -7H-pyrrolo [2,3-th] pyrimidine. Я-NMR (200 MHz, DMSO-0 ₆ ) δ 2.33 (s, 3H), 2.33 (s, 3H), 7.30 (m, 2H), 8.34 (m, 2H), 12 11 (s, 1H); mass spectrum (E8): 276.1 (M ⁺ +1).

4-chloro-5,6-dimethyl-2- (3-fluorophenyl) -7H-pyrrolo [2,3-th] pyrimidine. Я-NMR (200 MHz, DMSO-0 ₆ ) 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); mass spectrum (E8): 276.1 (M ⁺ +1).

4-chloro-5,6-dimethyl-2- (2-fluorophenyl) -7H-pyrrolo [2,3-th] pyrimidine. Я-NMR (200 MHz, DMSO-0 ₆ ) 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); mass spectrum (E8): 276.1 (M ⁺ +1).

4-chloro-5,6-dimethyl-2-isopropyl-7H-pyrrolo [2,3-th] pyrimidine. ¹ H-NMR (200 MHz, DMSO-ώ ·,) δ 1.24 (d, 1 = 6.6 Hz, 6H), 2.28 (s, 3H), 2.28 (s, 3H), 3 08 (q, 1 = 6.6 Hz, 1H); 11.95 (s, 1H); mass spectrum (E8): 224.0 (M ⁺ +1).

4-chloro-5,6-dimethyl-7H-pyrrolo [2,3-th] pyrimidine. ¹ H-NMR (200 MHz, DMSO-ώ ·) δ 2.31 (s, 3H), 2.32 (s, 3H), 8.40 (s, 1H); mass spectrum (E8): 182.0 (M ⁺ +1).

- 42 007254 ά1-4-chloro-5,6-dimethyl-2-phenyl-7H-7- (1-phenylethyl) pyrrolo [2,3-ά] pyrimidine.

Preparation 12. To a solution of 41-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) were added di-tert-butyl 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 E10Ac and then filtered. The filtrate was concentrated in vacuo to dryness to give a mixture of 41-1-amino-2 (1,1-dimethylethoxy) carbonylaminopropane and 41-2-amino-1- (1,1-dimethylethoxy) carbonylaminopropane, which could not be separated by conventional chromatography methods. The mixture was used for the reaction in example 8.

Preparation 13. To a solution of Btoc-β-A1a-ΟH (1.0 g, 3.212 mmol) and oxalyl chloride (0.428 g, 0.29 ml, 3.373 mmol) in dichloromethane (20.0 ml) were added a few drops of 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 min, the mixture was treated with 1M hydrochloride (10.0 ml) and the aqueous mixture was extracted with dichloromethane (3x30.0 ml). The organic solution was concentrated in vacuo to dryness. The residue was triturated with a 20% solution of piperidine in ^^ dimethylformamide (20.0 ml) for 0.5 h. After removal of the solvent in vacuo, the residue was triturated with 1M hydrogen chloride (20.0 ml) and ethyl acetate (20.0 ml ) The mixture was separated and the aqueous layer was made basic with solid sodium hydroxide to pH 8.

The precipitate was removed by filtration, and the aqueous layer was subjected to ion exchange column chromatography eluting with 20% pyridine to obtain 0.262 g (57%) of N-cyclopropylmethyl-β-alanine amide. ¹ H-NMR (200 MHz, (Ό _; 0Ι)) 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); mass spectrum (E8): 143.1 (M ⁺ +1).

Getting 14. No. tert-Butoxycarbonyl-trans-1,4-diaminocyclohexane. Trans-1,4-diaminocyclohexane (6.08 g, 53.2 mmol) was dissolved in dichloromethane (100 ml). A solution of ditret-butyl dicarbonate (2.32 g, 10.65 mmol in 40 ml of 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 MD80 ₄ , filtered and concentrated to give 1.20 g of a white solid (53%). 'H-NMR (200 MHz, C1) (b) δ 1.0-1.3 (m, 4H), 1.44 (s, 9H), 1.8-2.1 (m, 4H), 2 62 (broad s, 1H); 3.40 (broad s, 1H); 4.37 (broad s, 1 HO); mass spectrum (E8): 215.2 (M + 1).

4- (No. Acetyl) -H-tert-butoxycarbonyl-trans-1,4-diaminocyclohexane.

No. tert-Butoxycarbonyl-trans-1,4-diaminocyclohexane (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 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 MD80 ₄ , filtered and concentrated. Recrystallization (E10N / H ₂ 0) gave 190 mg of white crystals (30%). 'H-NMR (200 MHz, COC1 _; ) δ 0.9-1.30 (m, 4H), 1.43 (s, 9H), 1.96-2.10 (m, 7H), 3.40 (broad s, 1H), 3.70 (broad s, 1H), 4.40 (broad s, 1H), 4, 40 (broad s, 1H); mass spectrum (E8): 257.2 (M ⁺ + 1), 242.1 (M ⁺ -15), 201.1 (M ⁺ -56).

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

4- (No. Acetyl) -H-tert-butoxycarbonyl-trans-1,4-diaminocyclohexane (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-2phenyl-7H-pyrrolo [2,3-4] pyrimidine (35 mg, 0 , 14 mmol) were combined in a flask and heated to 130 ° C. After 4.5 hours, the reaction mixture was cooled to room temperature and diluted with E10Ac and water. The layers were separated and the aqueous layer was extracted with E10Ac (3x). The combined organic layers were dried over MD80 ₄ , filtered and concentrated. Chromatography (preparative plate with silica gel; 20: 1, CHCl ₃ : E10H) afforded 0.3 mg of a golden brown solid (1% yield). Mass spectrum (E8):

378.2 (M ⁺ +1).

4- (No. Methanesulfonyl) -I-tert-butoxycarbonyl-trans-1,4-diaminocyclohexane.

Trans-1,4-diaminocyclohexane (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 CHC13. The layers were separated and the aqueous layer was extracted with CHCl ₃ (3x). The combined organic layers were dried over MD80 ₄ , filtered and concentrated. Recrystallization (E10N / H ₂ 0) gave 206 mg of white crystals (29%). 'H-NMR (200 MHz, SES1 ₃ ) δ 1,101.40 (m, 4H), 1.45 (s, 9H), 2.00-2.20 (m, 4H), 2.98 (s, 3H) ), 3.20-3.50 (br s, 2H), 4.37 (br s, 1H); mass spectrum (E8): 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 [2,3-ά] pyrimidine.

4- (No. Sulfonyl) -H-tert-butoxycarbonyl-trans-1,4-diaminocyclohexane (206 mg, 0.71 mmol) (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 4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3

- 43 007254

b] pyrimidine (35 mg, 0.14 mmol) was combined in a flask and heated to 130 ° C. After 15 hours, the reaction mixture was cooled to room temperature and diluted with EAAc (3x). The combined organic layers were dried over Md8O _4, filtered and concentrated. Chromatography (preparative plate with silica gel; 20: 1, CHC1 ₃ : JUN) gave 2.6 mg of a golden brown solid (5% yield). Mass spectrum (E8): 414.2 (M ⁺ +1).

Example 1. A solution of 4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine (0.50 g, 1.94 mmol) and 4-trans-hydroxycyclohexylamine (2.23 g, 19.4 mmol) in methyl sulfoxide (10.0 ml) was heated at 130 ° C for 5 hours. After cooling to room temperature, water (10.0 ml) was added and the resulting aqueous solution was extracted with EyAc (3x10.0 ml) . EYuAs The combined solution was dried (Md8O _4), filtered and the filtrate concentrated in vacuo to dryness and the residue chromatographed on silica gel to give 0.49 g (75%) of 4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- phenyl-7H-pyrrolo [2,3-b] pyrimidine. Mp 197-199 ° C; ^! H-NMR (200 MHz, ATP1 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, 1 = 8 Hz, 1H), 7.26-7.49 (m, 3H), 8.40-8 44 (dd, 1 = 2.2, 8 Hz, 2H); 10.60 (s, 1H); mass spectrum (E8): 337.2 (M ⁺ +1).

The following compounds were obtained by a method similar to that described in example 1:

4- (4-trans-hydroxycyclohexyl) amino-6-methyl-2-phenyl-7H-pyrrolo [2,3-ά] pyrimidine. ¹ H-NMR (200 MHz, ATP1 ₃ ) δ 11.37 (s, 1H, pyrrole-PN), 8.45 (m, 2H, Ar-H), 7.55 (m, 3H, Ar-H) 6.17 (s, 1H, pyrrole H), 4.90 (broad d, 1H, ΝΉ), 4.18 (m, 1H, CH-O), 3.69 (m, 1H, CH-Ν) 2.40-2.20 (m, 2H), 2.19-1.98 (m, 2H), 2.25 (s, 3H, CH ₃ ), 1.68-1.20 (m, 4H) ); mass spectrum (E8): 323.2 (M ⁺ +1);

4- (4-trans-hydroxycyclohexyl) amino-5-methyl-2-phenyl-7H-pyrrolo [2,3-ά] pyrimidine. ¹ H-NMR (200 MHz, ATP1 ₃ ) δ 11.37 (s, 1H, pyrrole-ΝΉ), 8.40 (m, 2H, Ar-H), 7.45 (m, 3H, Ar-H) 5.96 (s, 1H, pyrroleH), 4.90 (broad d, 1H, N4), 4.18 (m, 1H, CH-O), 3.69 (m, 1H, CH-Ν) 2.38-2.20 (m, 2H), 2.18-1.98 (m, 2H) 2.00 (s, 3H, CH ₃ ), 1.68-1.20 (m, 4H) ; mass spectrum (E8): 323.1 (M ⁺ +1);

4- (4-trans-hydroxycyclohexyl) amino-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. Mp 245.5246.5 ° C; ^! H-NMR (200 MHz, SP ₃ 0P) δ 8.33 (m, 2H, Ar-H), 7.42 (m, 3H, Ar-H), 7.02 (d, 1H, 1 = 3, 6 Hz, pyrrole-H), 6.53 (d, 1H, 1 = 3.6 Hz, pyrrole-H), 4.26 (m, 1H, CH-O), 3.62 (m, 1H, CH -Ν), 2.30-2.12 (m, 2H), 2.121.96 (m, 2H), 1.64-1.34 (m, 4H); mass spectrum M + 1 = 309.3; Elemental analysis (C ^ gCO) C, H, Ν;

4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- (3-pyridyl) -7H-pyrrolo [2,3-b] pyrimidine. ^! H-NMR (200 MHz, SES1 ₃ ) δ 1.21-1.54 (m, 8H); 2.28 (s, 3H); 2.33 (s, 3H); 3.70 (m, 1H), 4.31 (m, 1H), 4.89 (d, 1H), 7.40 (m, 1H), 8.61 (m, 2H), 9.64 (m , 1H); mass spectrum (E8): 338.2 (M ⁺ +1);

4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- (2-furyl) -7H-pyrrolo [2,3-b] pyrimidine. ¹ NNMR (200 MHz, SES1 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); mass spectrum (E8): 327.2 (M ⁺ +1);

4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- (3-furyl) -7H-pyrrolo [2,3-b] pyrimidine. ¹ NNMR (200 MHz, SES1 ₃ ) δ 1.25-1.63 (m, 8 N), 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); mass spectrum (E8): 327.2 (M + 1);

4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2-cyclopentyl-7H-pyrrolo [2,3-b] pyrimidine. ^! H-NMR (200 MHz, SES1 ₃ ) δ 1.26-2.04 (m, 16 N), 2.26 (s, 3H), 2.27 (s, 3H), 3.15 (m, 1H ), 3.70 (m, 1H), 4.12 (m, 1H), 4.75 (d, 1H); mass spectrum (E8): 329.2 (M + 1);

4- (4-trans-hydroxycyclohexyl) amino-5, 6-dimethyl-2- (2-thienyl) -7H-pyrrolo [2,3-b] pyrimidin-4amine. ^! H-NMR (200 MHz, SES1 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); mass spectrum (E8): 343.2 (M ⁺ +1);

4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- (3-thienyl) -7H-pyrrolo [2,3-b] pyrimidine. ¹ H-NMR (200 MHz, SES1 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); mass spectrum (E8): 343.2 (M ⁺ +1);

4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- (4-fluorophenyl) -7H-pyrrolo [2,3-b] pyrimidine. ^! H-NMR (200 MHz, SES1 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); mass spectrum (E8): 355.2 (M ⁺ +1);

4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- (3-fluorophenyl) -7H-pyrrolo [2,3-b] pyrimidine. '11-NMR (200 MHz, SES1 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); mass spectrum (E8): 355.2 (M ⁺ +1);

4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2- (2-fluorophenyl) -7H-pyrrolo [2,3-b] pyrimidine. Я-NMR (200 MHz, SES1 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 ( s, 1H); mass spectrum (E8): 355.3 (M ⁺ +1);

4- (4-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2-isopropyl-7H-pyrrolo [2,3-b] pyrimidine. ^! H-NMR (200 MHz, SES1 ₃ ) δ 1.31 (d, 1 = 7.0 Hz, 6H), 1.30-1.65 (m, 8H), 2.27 (s, 3H), 2 28 (s, 3H), 3.01 (m, 1 = 7.0 Hz, 1H), 3.71 (m, 1H), 4.14 (m, 1H), 4.78 (d, 1H) ; mass spectrum (E8): 303.2 (M + 1);

b1-4- (2-trans-hydroxycyclohexyl) amino-5,6-dimethyl-2-isopropyl-7H-pyrrolo [2,3-b] pyrimidine.

¹ H-NMR (200 MHz, SES1 ₃ ) δ 1.31-1.42 (br. 4H), 1.75-1.82 (br. 4H), 2.02 (C, 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); mass spectrum (E8): 337.2

- 44 007254 (M ⁺ +1). 4- (3,4-trans-dihydroxycyclohexyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3SH] pyrimidine. Mass spectrum (E8): 353.2 (M ⁺ +1);

4- (3,4-cis-dihydroxycyclohexyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-th] pyrimidine. Mass spectrum (E8): 353.2 (M ⁺ +1);

4- (2-acetylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-ά] pyrimidine. Mp 196-199 ° C; ¹ H-NMR (200 MHz, COC1 _; ) δ 1.72 (s, 3H), 1.97 (s, 3H), 2.31 (s, 3H), 3.59 (m, 2H), 3, 96 (m, 2H), 5.63 (br., 1H), 7.44-7.47 (m, 3H), 8.36-8.43 (dd, 1 = 1 Hz, 7 Hz, 2H) 10.76 (s, 1H); mass spectrum (E8): 324.5 (M ⁺ +1);

y1-4- (2-trans-hydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-ά] pyrimidine. ¹ H-NMR (200 MHz, C1) C1 ₃ ) δ 1.62 (m, 2H), 1.79 (broad, 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); mass spectrum (E8): 323.2 (M ⁺ +1).

For 2-trans-hydroxycyclopentylamine, see PCT 9417090. 01-4- (3-transTydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-0] pyrimidine.

Ή-NMR (200 MHz, C1) C1 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); mass spectrum (E8): 323.2 (M ⁺ +1).

For 3-trans-hydroxycyclopentylamine, see EP-A-322242. 01-4- (3-cisTidroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-0] pyrimidine.

1-NMR (200 MHz, C1) C1 ₃ ) δ 1.82-2.28 (Lg, 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); mass spectrum (E8): 323.2 (M ⁺ +1).

For 3-cis-hydroxycyclopentylamine, see EP-A-322242. 4- (3,4-trans-dihydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-0] pyrimidine. Я-NMR (200 MHz, COC1 _; ) 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, 3H), 8, 38-8.42 (m, 2H); 9.53 (s, 1H); mass spectrum (E8):

339.2 (M ⁺ +1).

For 3,4-trans-dihydroxycyclopentylamine, see PCT 9417090.

4- (3-amino-3-oxopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-0] pyrimidine. ¹ H-NMR (200 MHz, C1) C1 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), 8.378.41 (m, 2H), 10.42 (s, 1H); mass spectrum (E8): 310.1 (M ⁺ +1).

4- (3-Y-Cyclopropylmethylamino-3-oxopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3SH] pyrimidine. ¹ H-NMR (200 MHz, C1) C1 ₃ ) δ 0.51 (q, 2H), 0.40 (q, 2H), 1.79-1.95 (broad, 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); mass spectrum (E8): 364.2 (M ⁺ +1).

4- (2-amino-2-oxoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-0] pyrimidine. Ή-NMR (200 MHz, C1) C1 ₃ ) δ 2.31 (s, 3H), 2.38 (s, 3H), 4.26 (s, 2H), 7.36 (m, 3H), 8 33 (m, 2H); mass spectrum (E8): 396.1 (M ⁺ +1).

4- (2-Y-Methylamino-2-oxoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3SH] pyrimidine. Ή-NMR (200 MHz, C1) C1 ₃ ) δ 1.99 (s, 3H), 2.17, (s, 3H), 2.82 (d, 3H), 4.39 (d, 2H), 5, 76 (t, 1H), 6.71 (broad, 1H),

7.41-7.48 (m, 3H), 8.40 (m, -4H), 1-0.66 (s, 1H); mass spectrum (E8): 310.1 (M ⁺ +1).

4- (3-tert-Butyloxy-3-oxopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3SH] pyrimidine. Ή-NMR (200 MHz, C1) C1 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); mass spectrum (E8): 367.2 (M ⁺ +1).

4- (2-Hydroxyethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-ά] pyrimidine. Я-NMR (200 MHz, С0С1 _; ) 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); mass spectrum (E8): 283.1 (M ⁺ +1).

4- (3-Hydroxypropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3SH] pyrimidine. Я-NMR (200 MHz, С0С1 _; ) δ 1.84 (m, 2H), 1.99 (s, 3H), 2.32 (s, 3H), 3.62 (t, 2H), 3.96 (m, 2H), 3.35 (t, 1H), 7.39-7.48 (m, 3H), 8.36 (m, 2H), 10.27 (s, 1H); mass spectrum (E8): 297.2 (M ⁺ +1).

4- (2-hydroxybutyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3SH] pyrimidine. Ή-NMR (200 MHz, C1X'1 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); mass spectrum (E8): 311.2 (M ⁺ +1).

4- (4-trans-Acetylamino-cyclohexyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3SH] pyrimidine.

4- (4-trans-methylsulfonylaminocyclohexyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3SH] pyrimidine.

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

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

4- (3-Pyridylmethyl) amino-5,6-dimethyl-2-phenyl-7H-7- (1-phenylethyl) pyrrolo [2,3SH] pyrimidine. 4- (2-Methylpropyl) amino-5,6-dimethyl-2-phenyl-7H-7- (1-phenylethyl) pyrrolo [2,3SH] 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., 4- (4-trans-hydroxycyclohexyl) amino was added -5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3SH] pyrimidine (0.05 g, 0.149 mmol) at 0 ° C. Then, diethyl azodicarboxylate (0.028 ml, 0.179 mmol) was added dropwise over 10 minutes. Reaction

The 45 007254 mixture was allowed to warm to room temperature. After completion of the reaction by TLC, the reaction mixture was quenched with an aqueous solution of sodium bicarbonate (3.0 ml). The aqueous phase was separated and extracted with ether (2x5.0 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, after which a bulk precipitate of 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 obtain 5.0 mg (7.6%) of 4- (4-cis-benzoyloxycyclohexyl) amino-5,6-dimethyl-2- phenyl-7H-pyrrolo [2,3-b] pyrimidine. Mass spectrum (EB): 441.3 (M ⁺ +1). The reaction also yielded 50.0 mg (84%) of 4- (3-cyclohexenyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. Mass spectrum (EB): 319.2 (M ⁺ +1).

Example 3. To a solution of 4- (4-cis-benzoyloxycyclohexyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine (5.0 mg, 0.0114 mmol) in ethanol (1.0 ml) was added 10 drops of 2M sodium hydroxide. After 1 h, 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 obtain 3.6 mg (94%) of 4- (4-cis-hydroxycyclohexyl) amino-5,6-dimethyl-2-phenyl7H-pyrrolo [2,3- b] pyrimidine. Mass spectrum (EB): 337.2 (M ⁺ +1).

The following compounds were prepared by a method similar to that described in example 3: 4- (3- ^ dimethyl-3-oxopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine.

¹ H-NMR (200 MHz, CHC1 _; ) 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); mass spectrum (EB): 338.2 (M ⁺ +1).

4- (2-Formylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. ¹ H-NMR (200 MHz, SIS1 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); mass spectrum (EB): 310.1 (M ⁺ +1).

4- (3-Acetylaminopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. Mass spectrum (EB): 338.2 (M ⁺ +1).

Example 4. 4- (3-tert-Butyloxy-3-oxopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine (70.0 mg, 0.191 mmol) was dissolved in mixtures of trifluoroacetic acid: dichloromethane (1: 1, 5.0 ml). The resulting solution was stirred at room temperature for 1 h, and then heated at the boil under reflux for 2 h. After cooling to room temperature, the mixture was concentrated in vacuo to dryness. The residue was subjected to preparative thin layer chromatography (EULAc: hexane: AcOH = 7: 2.5: 0.5) to obtain 40.0 mg (68%) of 4- (3-hydroxy-3-oxopropyl) amino-5,6-dimethyl2 -phenyl-7H-pyrrolo [2,3-b] pyrimidine. ¹ H-NMR (200 MHz, CO ₃ OE) 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); mass spectrum (EB): 311.1 (M ⁺ +1).

The following compound was obtained by a method similar to that described in example 4: 4- (3-aminopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-3] pyrimidine. Mass spectrum (EB): 296.1 (M ⁺ +1), 279, 1 (^ -N43).

Example 5. 4- (3-Hydroxy-3-oxopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine (50.0 mg, 0.161 mmol) was dissolved in a mixture of Ν , Ν-dimethylformamide (0.50 ml), dioxane (0.50 ml) and water (0.25 ml). Methylamine (0.02 ml, 40 wt.% In water, 0.242 mmol), triethylamine (0.085 ml) and раф, Ν, Ν ', Ν'-tetramethyluronium tetrafluoroborate (61.2 mg, 0.203 mmol) were added to this solution. After stirring at room temperature for 10 minutes, the solution was concentrated, and the residue was subjected to preparative thin layer chromatography (EuAc) to obtain 35.0 mg (67%) of 4- (3-methyl-3-oxopropyl) amino

5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. ¹ H-NMR (200 MHz, SEZOE) 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); mass spectrum (EB): 311.1 (M ⁺ +1).

The following compounds were prepared by a method similar to that described in example 5: 4- (2-cyclopropanecarbonylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. Mass spectrum (EB): 350.2 (M + 1);

4- (2-isobutyrylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. Mass spectrum (EB): 352.2 (M + 1);

4- (3-propionylaminopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. ¹ H-NMR (200 MHz, CHC1 _; ) 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), 3.793.96 (m, 2H), 5.53-5.62 (m, 1H), 6.17-6.33 (m , 1H), 7.33-7.57 (m, 3H), 8.31-8.39 (m, 2H), 9.69 (s, 1H); mass spectrum (EB): 352.2 (M + 1);

4- (2-methylsulfonylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. ¹ H NMR (200 MHz, CH _; C1) δ 2.18 (s, 3H), 2.27 (s, 3H), 2.92 (s, 3H), 3.39-3.53 (m, 2H ), 3.71-3.88 (m, 2H), 5.315.39 (m, 1H), 6.17-6.33 (m, 1H), 7.36-7.43 (m, 3H), 8.20-8.25 (m, 2H); 9.52 (s, 1H); mass spectrum (EB): 360.2 (M ⁺ +1).

Example 6. A mixture of 4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine (0.70 g, 2.72 mmol) and 1,2-diaminoethane (10.0 ml, 150 mmol) was heated at the boil under reflux in an inert atmosphere for 6 hours. The excess amine was removed in vacuum, the residue was washed successively with ether

46 007254 and hexane to give 0.75 g (98%) of 4- (2-aminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. Mass spectrum (E8): 265.1 (M'-HH ₃ ).

Example 7. To a solution of 4- (2-aminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] 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 h, the mixture was concentrated in vacuo and the residue was subjected to preparative thin layer chromatography (E! OAc) to obtain 2.0 mg (26%) of 4- (2propionylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2 , 3-b] pyrimidine. Mass spectrum (E8):

338.2 (M ⁺ +1).

The following compounds were obtained by a method similar to that described in example 7:

4- (2-No.-methylureaethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. ¹ H-NMR (200 MHz, COC1 _; ) 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); mass spectrum (E8): 339.3 (M ⁺ +1);

4- (2-No-ethylurea-ethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. Mass spectrum (E8):

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 pyruvic 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-7Npyrrolo [2,3-b] pyrimidine (55.0 mg, 0.196 mmol). The mixture was stirred at room temperature for 4 hours. Conventional work-up and column chromatography (E! OAc) then gave 10.0 mg (15%) of 4- (2'-pyruvylamidoethyl) amino-5,6-dimethyl-2-phenyl-7H pyrrolo [2,3-b] pyrimidine. Mass spectrum (E8): 352.2 (M ⁺ +1).

Example 9. To a solution of 4- (2-aminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine (60.0 mg, 0.213 mmol) in dichloromethane (2.0 ml) was added No.trimethylsilyl isocyanate (43.3 mg, 0.051 ml, 0.32 mmol). The mixture was stirred at room temperature for 3 hours, followed by the addition of an aqueous solution of sodium bicarbonate. After filtering 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-dimethyl2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. Mass spectrum (E8): 325.2 (M ⁺ +1).

The following compounds were obtained by a method similar to that described in example 9:

b1-4- (2-acetylaminopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. ¹ H-NMR (200 MHz, COC1 _; ) δ 1.28-1.32 (d, 1 = 8 Hz, 3 H), 1.66 (s, 3H), 1.96 (s, 3H), 2 30 (s, 3H) 3.76-3.83 (m, 2H), 4.104.30 (m, 1H), 5.60-5.66 (t, 1 = 6 Hz, 1H), 7.40 -7.51 (m, 3H), 8.36-8.43 (m, 2H), 10.83 (s, 1H); mass spectrum (E8):

338.2 (M ⁺ +1).

(B) -4- (2-acetylaminopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. ¹ H-NMR (200 MHz, COC1 _; ) 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); mass spectrum (E8): 338.2 (M ⁺ +1).

(B) -4- (1-methyl-2-acetylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. ¹ NNMR (200 MHz, СОС1 _; ) δ 1.41 (d, 3Н), 1.68 (s, 3Н), 2.21 (s, 3Н), 2.34 (s, 3Н), 3.46- 3.52 (broad 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); mass spectrum (E8): 338.2 (M ⁺ +1).

(8) -4- (2-acetylaminopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. ¹ H-NMR (200 MHz, COC1 _; ) 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); mass spectrum (E8): 338.2 (M ⁺ +1).

(8) -4- (1-methyl-2-acetylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. ¹ NMR (200 MHz, COC1 _; ) 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); mass spectrum (E8): 338.2 (M ⁺ +1).

Example 10. The interaction of 4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine with a mixture of b1-1-amino-2- (1,1-dimethylethoxy) carbonylaminopropane and b1- 2-amino-1 - (1,1-dimethylethoxy) carbonylaminopropane was carried out in a manner similar to that described in example 1. The reaction gave a mixture of b1-4- (1methyl-2- (1,1-dimethylethoxy) carbonylamino) ethylamino-5,6- dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine and b1-4- (2-methyl-2- (1,1-dimethylethoxy) carbonylamino) ethylamino-5,6-dimethyl-2-phenyl- 7H-pyrrolo [2,3-b] pyrimidine, which were separated by column chromatography (E! OAc: hexanes = 1: 3). The first fraction was b1-4- (1-methyl-2- (1,1-dimethylethoxy) carbonylaminoethyl) amino-5,6-dimethyl-2phenyl-7H-pyrrolo [2,3-b] pyrimidine. 'H-NMR (200 MHz, COC1 _; ) 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, 1 = 8 Hz, 1H), 5.53 (br., 1H), 7.37- 7.49 (m, 3H), 8.378.44 (m, 2H), 10.75 (s, 1H). Mass spectrum (E8): 396.3 (M ⁺ +1). The second fraction was b1-4- (2 (1,1-dimethylethoxy) carbonylaminopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidine. ¹ H-NMR (200 MHz, CHC1 _; ) δ 1.26-1.40 (m, 12 N), 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, 1 = 6.0 Hz, 1H), 5.65 (br., 1H), 7 40-7.46 (m, 3H), 8.37-8.44 (m, 2H), 10.89 (s, 1H); mass spectrum (E8): 396.2 (M ⁺ +1).

The following compounds were obtained by a method similar to that described in example 10:

- 47 007254 (8.8) -4- (2-acetylaminocyclohexyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine. ¹ NNMR (200 MHz, SECE) δ 1.43 (m, 4H), 1.60 (s, 3Η), 1.83 (m, 2H), 2.18 (s, 3Η), 2.30 (m , 2H), 2.32 (s, 3Η), 3.73 (br., 1H), 4.25 (br., 1H), 5.29 (d, 1H), 7.43-7.48 ( m, 3Η), 8.35-8.40 (m, 2H), 9.05 (s, 1H).

4- (2-methyl-2-acetylaminopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine. ¹ NNMR (200 MHz, СЕС1 _; ) δ 1.51 (s, 6Н), 1.56 (s, 3Η), 2.07 (s, 3Η), 2.36 (s, 3Η), 3.76 ( d, 2H), 5.78 (t, 1H), 7.417.48 (m, 3Η), 7.93 (s, 1H), 8.39 (m, 2H), 10.07 (s, 1H); mass spectrum (E8): 352.3 (M + 1).

Example 11. 61-4- (1-Methyl-2- (1,1-dimethylethoxy) carbonylaminoethyl) amino-5,6-dimethyl-2-phenyl7H-pyrrolo [2,3-6] pyrimidine (60.6 mg, 0.153 mmol) was treated with trifluoroacetic acid (0.5 ml) in dichloromethane (2.0 ml) for 14 hours. The organic solvent was removed in vacuo to dryness. The residue was dissolved in Ν, Ν-dimethylformamide (2.0 ml) and triethylamine (2.0 ml). Acetic anhydride (17.2 mg, 0.169 mmol) was added to the solution at 0 ° C. 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 (EULAc) to obtain 27.0 mg (52%) of 61-4- (1-methyl-2-acetylaminoethyl) amino-5,6dimethyl-2-phenyl-7H-pyrrolo [2,3-6 ] pyrimidine. ’H-NMR (200 MHz, CECE) δ 1.38-1.42 (d, ί = 8 Hz, 3Η),

1.69 (s, 3H), 2.01 (s, 3H), 2.32 (s, 3H) 3.38-3.60 (m, 2H), 4.65-4.80 (m, 1H ), 5.23-5.26 (d, 1 = 6 Hz, 1H), 7.407.51 (m, 3H), 8.37-8.43 (m, 2H), 10.44 (s, 1H) ; mass spectrum (E8): 338.2 (M ⁺ +1).

Example 12. (K, K) -4- (2-Aminocyclohexyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine obtained by a method similar to that described in example 1, from 4-chloro-5,6-dimethyl-2-phenyl-7Npyrrolo [2,3-6] pyrimidine (0.15 g, 0.583 mmol) and (1K, 2K) - (-) - 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 Ν, д-dimethylformamide (10.0 ml) at room temperature for 2 hours. After removing 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 (2x10.0 ml). The combined ethyl acetate solution was dried (Md8O ₄₎ and filtered. The filtrate was concentrated in vacuo to dryness and the residue was subjected to column chromatography (EULAc: hexane = 1: 1) to obtain 57.0 mg (26%) (K, K) -4- (2-acetylaminocyclohexyl) amino

5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine. 'H-NMR (200 MHz, SESE) 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 (broad, 1H), 4.24 (broad, 1H), 5.29 (d, 1H), 7.43-7, 48 (m, 3H), 8.35-8.39 (m, 2H), 8.83 (s, 1 H); mass spectrum (E8): 378.3 (M ⁺ +1).

Example 13. To a solution of 4- (2-hydroxyethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-6] 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. 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 (EULAc: hexane = 1: 1) to obtain 32.3 mg (71%) of 4- (2-acetyloxyethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3 -6] pyrimidine. 'H-NMR (200 MHz, CEC1 _; ) δ 1.90 (s, 3H), 2.08 (s, 3H), 2.31 (s, 3H), 4.05 (m, 2H), 4, 45 (t, 2H), 5.42 (m, 1H), 7.41-7.49 (m, 3H), 8.42 (m, 2H), 11.23 (s, 1H).

Example 14. A solution of Yeshos-P-A1a-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 one drop of Ν, Ν -dimethylformamide was stirred at 0 ° C for 1 h, followed by the addition of 4- (2-aminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-6] 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 triturated with 20% piperidine in Ν, dimethylformamide (2.0 ml) for 0.5 hours. After removal of the solvent in vacuum, the residue was washed with diethyl ether: hexane (1: 5 ), yielding 3.0 mg (93%) of 4- (6-amino-3-aza-4-oxohexyl) amino-5,6dimethyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine. Mass spectrum (E8): 353.2 (M ⁺ +1).

Example 15. A solution of 4- (2-aminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine (70.0 mg, 0.249 mmol) and succinic anhydride (27.0 mg, 0.274 mmol) in dichloromethane (4.0 ml) with 1 drop of Ν, Ν-dimethylformamide was stirred at room temperature for 4 hours. The reaction mixture was extracted with 20% sodium hydroxide (3x5.0 ml). The aqueous solution was acidified with 3M hydrogen chloride to pH 7.0. The whole mixture was extracted with ethyl acetate (3x10 ml). The combined organic solution was dried (Md8O ₄₎ and filtered. The filtrate was concentrated in vacuo to dryness to give 15.0 mg (16%) of 4- (7-hydroxy-3-aza-4,7-dioxoheptyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2, 36] pyrimidine. Mass spectrum (E8): 382.2 (M ⁺ +1).

Example 16. To 10 ml of dimethylformamide (DMF), 700 mg of 4 (cis-3-hydroxycyclopentyl) amino-2-phenyl-5,6-dimethyl-7H-pyrrolo [2,3-6] pyrimidine was added at room temperature, followed by the addition of pyrimidine 455 mg of Ν-Boc glycine, 20 mg of Ν, Ν-dimethylaminopyridine (OMAP), 293 mg of hydroxybenzotriazole (HOBT) and 622 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EES1). The reaction mixture was allowed to stir overnight. DMF was then removed under reduced pressure and the reaction mixture was partitioned between 20 ml of ethyl acetate and 50 ml of water. The aqueous portion was extracted with an additional 2x20 ml of ethyl acetate and the combined organics were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. Purification on silica gel, eluting with ethyl acetate / hexane, afforded 410 mg of the expected product: 4 (cis-3- (N-tert-butoxycarbonyl-2-aminoacetoxy) cyclopentyl) amino-2-phenyl-5,6-dimethyl-7H-pyrrolo

- 48 007254 [2,3-d] pyrimidine. Mass spectrum (E8): (M ⁺ +1) = 480.2. The ester was then treated with 5 ml of 20% trifluoroacetic acid in dichloromethane at room temperature, left overnight and then concentrated. Rubbing with ethyl acetate gave 300 mg of an off-white solid; salts of trifluoroacetic acid 4- (cis-3- (2-aminoacetoxy) cyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-d] pyrimidine. Mass spectrum (E8): (M ⁺ +1) = 380.1.

One skilled in the art will recognize that the following compounds can be synthesized using the methods described above.

4- (cis-3-hydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-d] pyrimidine. Mass spectrum (E8): (M ⁺ +1) = 323.1.

Salt of trifluoroacetic acid 4- (cis-3- (2-aminoacetoxy) cyclopentyl) amino-5,6-dimethyl-2phenyl-7H-pyrrolo [2,3-d] pyrimidine. Mass spectrum (E8): (M ⁺ +1) = 380.1.

4- (3-Acetamido) piperidinyl-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-d] pyrimidine. Mass spectrum (E8): (M ⁺ +1) = 364.2.

4- (2-No.-Methylureapropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-d] pyrimidine. Mass spectrum (E8): (M ⁺ +1) = 353.4.

4- (2-Acetamidobutyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-3] pyrimidine. Mass spectrum (E8): (M ⁺ +1) = 352.4.

4- (2-No.-Methylureabutyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3-d] pyrimidine. Mass spectrum (E8): (M ⁺ +1) = 367.5.

4- (2-aminocyclopropylacetamidoethyl) amino-2-phenyl-7H-pyrrolo [2,3-d] pyrimidine. Mass spectrum (E8): (M ⁺ +1) = 309.1.

4- (trans-4-hydroxycyclohexyl) amino-2- (3-chlorophenyl) -7H-pyrrolo [2,3-d] pyrimidine. Mass spectrum (E8): (M ⁺ +1) = 342.8.

4- (trans-4-hydroxycyclohexyl) amino-2- (3-fluorophenyl) -7H-pyrrolo [2,3-d] pyrimidine. Mass spectrum (E8): (M ⁺ +1) = 327.2.

4- (trans-4-hydroxycyclohexyl) amino-2- (4-pyridyl) -7H-pyrrolo [2,3-d] pyrimidine. Mass spectrum (E8): (M ⁺ +1) = 310.2.

Example 17

Scheme IX

Pyrrole nitrogen (7) (Scheme IX) was protected with di-tert-butyl dicarbonate in an alkaline medium to obtain the corresponding carbamate (22). Radical bromination (22) was regioselective to give bromide (23). Typically, compound (23) serves as a key electrophilic intermediate for various nucleophilic partners in coupling reactions. Substitution of the alkyl bromide with sodium phenolate trihydrate afforded compound (24). Subsequent substitution of the aryl chloride and removal of the tert-butyl carbamate protecting group occurred in one step, giving the target compound (25).

Detailed synthesis of compounds (22) - (25) in accordance with scheme IX.

- 49 007254

Di-tert-butyl dicarbonate (5.37 g, 24, b mmol) and dimethylaminopyridine (1.13 g, 9.2 mmol) were added to a solution containing (7) (1.50 g, b.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 layer containing CH ₂ Cl ₂ was separated, dried over MD8O ₄ , filtered and concentrated to give a black solid. Flash chromatography (81O ₂ ; 1/9 UES / hexanes, K _£ 0.40) gave 1.70 g (80%) of a white solid. ¹ H-NMR (200 MHz, SBS ^) b 8.50 (m, 2H, Lg-H), 7.45 (m, 3H, Lg-H), b, 39 (s, 1H, pyrrole-H) 2, bb (s, 3H, pyrroleCH ₃ ); 1.7b (s, 9H, carbamate-CH ₃ ); mass spectrum, M + 1 = 344.1; Mp = 175-177 ° C.

©

Ν-bromosuccinimide (508 mg, 2.8 b mmol) and AIΒN (azobisisobutyronitrile) (112 mg, 0, b8 mmol) were added to a solution containing (22) (935 mg, 2.71 mmol) and CC14 (50 ml) . The solution was heated at the boil under reflux. After 2 hours, the reaction mixture was cooled to room temperature and concentrated in vacuo to give a white solid. Flash chromatography (occupies 8 _2; 1/1 CH ₂ C1 ₂ / Hexanes, R _£ 0,30) afforded 9b0 mg (84%) of a white solid (23). ¹ H-NMR (200 MHz, SBS1 ₃ ) δ 8.52 (m, 2H, Lg-H), 7.48 (m, 3H, Lg-H), b, 7b (s, 1H, pyrrole-H) 4.93 (s, 2H, pyrrole-CH2Bg), 1.79 (s, 9H, car-

Sodium phenoxide trihydrate (173 mg, 1.02 mmol) was added in one go to a solution of bromide (23) (410 mg, 0.97 mmol) dissolved in CH2C12 (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 MD8O ₄ , filtered and concentrated to give a yellow solid. Flash chromatography ₍₂ occupies 8); 1 / b

ELS / Hexanes, K _£ 0.30) gave 210 mg (50%) of a white solid (24). ¹ H-NMR (200 MHz, SBS13) δ 8.53 (m, 2H, Lg-H), 7.48 (m, 3H, Lg-H), 7.34 (m, 2H, Lg-H), 7.03 (m, 3H, Lg-H), b, 83 (s, 1H, pyrroleH), 5, 45 (s, 2H, AGSN2O), 1.7b (s, 9H, carbamate-CH); mass spectrum, M ⁺ = 43b, 2.

A solution containing (24) (85 mg, 0.20 mmol), Ν-acetylethylenediamine (201 mg, 1.95 mmol) and DMSO (3 ml) was heated at 100 ° C. After 1 h, the temperature was raised to 130 ° C. After 3 hours, the reaction mixture was cooled to room temperature and partitioned between E! OLs and water. The aqueous layer was extracted with EULS (2x). The combined ELSs layers were washed with water, dried over MgSO ₄ , filtered and concentrated. Flash chromatography (occupies 8 _2; 1/10 EtOH / CHC1 _3, R _£ 0,25) afforded 73 mg (93%) of a white foamy solid (25). ¹ H-NMR (200 MHz, DMSO-b _b ) δ 11.81 (br s, 1H, Ν-H), 8.39 (m, 2H, LH), 8.03 (br t, 1H, Ν-H), 7.57 (broad t, 1H, Ν-H), 7.20-7.50 (m, 5H, Lg-H), b, 89-7.09 (m, 3H, Lg -H), b, 59 (s, 1H, pyrrole-H), 5.12 (s, 2H, LVSN2O), 3, b1 (m, 2H, ΝΜ), 3.3b (m, 2H, ΝΜ), 1.79 (s, 3H, COCH3); mass spectrum, M + 1 = 402, b

The following compounds were obtained by a method similar to that described in example 17.

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4- (2-Lactylaminoethyl) amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine. Mp. 196197 ° C. Mass spectrum (E8): 401.6 (M ⁺ +1).

4- (2-Lactylaminoethyl) amino-6- (4-fluorophenoxy) methyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine. Mass spectrum (E8): 420.1 (M ⁺ +1).

4- (2-Lactylaminoethyl) amino-6- (4-chlorophenoxy) methyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine. Mass spectrum (E8): 436.1 (M ⁺ +1).

4- (2-Lactylaminoethyl) amino-6- (4-methoxyphenoxy) methyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine. Mass spectrum (E8): 432.1 (M ⁺ +1).

4- (2-Lactylaminoethyl) amino-6- (No. pyridin-2-one) methyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine. Mass spectrum (E8): 403.1 (M ⁺ +1).

4- (2-Lactylaminoethyl) amino-6- (No. phenylamino) methyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine. Mass spectrum (E8): 400.9 (M ⁺ +1).

4- (2-Lactylaminoethyl) amino-6- (No. methyl-No. phenylamino) methyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine. Mass spectrum (E8): 414.9 (M ⁺ +1).

4- (2-X'-methylureaethyl) amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3-6] pyrimidine. Mass spectrum (E8): 416.9 (M ⁺ +1).

Example 18. Synthesis of A ₁ adenosine antagonists.

Compound 1319 and compound 1320 (table 13 below) can be synthesized using the general methods described herein.

C1

Compound 26 X = P

Compound 27 X = C1

Compound 1319

Compound 1320

Compound 1319 (81%). ¹ H-NMR (DMSO-6 ₆ ) δ 1.37 (m, 4H), 1.93 (m, 2H), 2.01 (m, 2H), 4.11 (br s, 1H), 4 61 (d, 1H, 1 = 4.4 Hz), 6.59 (m, 1H), 7.09 (m, 1H), 7.21 (m, 2H), 7.49 (dd, 1H, > 8Hz, 14Hz), 8.03 (m, 1H), 8.18 (d, 1H, 1 = 8 Hz), 11.55 (broad s, 1H). Mass spectrum (E8): 327.0 (M ⁺ +1).

Compound 1320 (31%). Mass spectrum (E8): 343.1 (M ⁺ +1).

Example 19. The synthesis of the antagonist L ₁ adenosine.

Compound 1321 (table 13, below) can be synthesized using the following general methods.

Compound 1321

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 successively added and the reaction mixture was heated to 85 ° C. After 22 hours, the reaction mixture was cooled to room temperature and DMF was removed in vacuo. The dark oil was diluted with 2M 11C1 (80 ml). The reaction mixture was left. After 2 hours, the solution was cooled to 10 ° C and filtered. The solid was washed with cold water and dried, yielding 7.40 g of a yellow solid. Compound 29 (69%). ¹ H-NMR (200 MHz, DMSO-f) δ 6.58 (s, 1H), 7.27 (s, 1H), 8.53 (d, 2H, 1 = 5.6), 9.00 ( d, 2H, 1 = 5.2 Hz), 12.35 (br s, 1H). Mass spectrum (E8): 212.8 (M ⁺ +1).

Compound 29 (7.4 mmol, 29.8 mmol) was diluted with POC1 ₃ and heated to 105 ° C. After 18 hours, the reaction mixture was cooled to room temperature and POC1 ₃ was removed in vacuo. The thick dark oil was diluted with MeOH (75 ml) followed by ether (120 ml). The lmorphic red solid was filtered and washed with ether to give 3.82 g of a red solid. The crude solid is approximately 80% pure and is used without further purification in the next reaction. Mass spectrum (E8): 230.7 (M ⁺ +1).

Compound 1321. ¹ H-NMR (15%) (200 MHz, DMSO-6 ₆ ) δ 1.38 (m, 4H), 1.92 (broad s, 2H), 2.02 (broad s, 2H ), 3.44 (broad s, 1H), 4.14 (broad s, 1H), 4.56 (d, 1H, 1 = 4 Hz), 6.63 (m, 1H), 7.15 (m, 1H), 7.32 (d, 1H, 1 = 6.2 Hz), 8.20 (d, 2H, 1 = 4.4 Hz), 8.65 (d, 2H, 1 = 4, 4Hz), 11.67 (br s, 1H). Mass spectrum (E8): 310.2 (M ⁺ +1).

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Compound 1501 (table 15 below). ¹ H-NMR (70%) (20 MHz, SOYU) δ 1.84 (s, 3Η), 3.52 (t, 2H, 1 = 6.0 Hz), 3.83 (t, 2H, 1 = 6 Hz), 6.51 (d, 1H, 1 = 3.4 Hz), 7.06 (d, 1H, 1 = 3.8 Hz), 7.42 (m, 3Η), 8.36 ( m, 2H). Mass spectrum (E8): 296.0 (M ⁺ +1).

Compound 1502 (table 15 below). Mass spectrum (E8): 345.0 (M ⁺ +1).

Compound 1500 (table 15 below). ¹ Η-NMR (70%) (200 MHz, ATP1 ₃ ) δ 1.40-1.80 (m, 6H), 1.85-2.10 (m, 2H), 2.18 (s, 3Η) 2.33 (s, 3Η), 2.50 (d, 3Η), 3.90-4.10 (m, 2H), 4.76 (m, 1H), 5.50 (d, 1H), 6.03 (m, 1H), 7.40 (m, 3Η), 8.37 (m, 2H), 9.15 (br s, 1H). Mass spectrum (E8): 393.3 (M ⁺ +1).

Example 20. The synthesis of the antagonist of Αχ adenosine.

Compound 1504 (table 15 below) can be synthesized according to the General methods described 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 stirred for several minutes, then left for 72 hours. The reaction mixture was diluted with DCM and Η ₂ Ο and the layers were separated. The aqueous layer was extracted with DCM. The combined DCM layers were dried over Μ§8Ο ₄ , filtered and concentrated. Diethyl ether was added to the crude sample and the resulting solid was filtered off to obtain 100 mg of a white solid 32 (62%). ¹ H-NMR (200 MHz, SES1 ₃ ) δ 1.76 (s, 9H), 2.66 (broad s, 2H), 2.79 (broad s, 2H), 3.86 (s, 2H) ), 7.46 (m, 3Η), 8.50 (m, 2H).

Compound 32 was combined with DMSO (3 ml) and trans-4-aminocyclohexanol (144 mg, 1.25 mmol) and heated at 130 ° C. for 4 hours. The reaction mixture was cooled to room temperature and diluted with ΕΐΟΑο and Η ₂ Ο. The layers were separated and the aqueous layer was extracted with ΕΐΟΑο (2x). The combined organic layers were washed with Η ₂ Ο and brine, dried over Μ§8Ο ₃ , filtered and concentrated. Chromatography (silica, 8: 1 CΗC1 ₃ / EΐOH) gave 32 mg of a golden brown oil. Diethyl ether was added and the resulting solid was filtered to give 5 mg of a white solid (9%). Ο8Κ'-148265: Ή-NMR (200 MHz, SE ^ E) δ 1.44 (broad.m, 4H), 2.03 (broad m, 2H), 2.21 (broad m, 2H), 2.70 (broad m, 8H), 3.63 (m, 4H), 3.92 (m, 1H), 4.26 (broad s, 1H), 6.42 (s, 1H), 7 42 (m, 3Η), 8.33 (m, 2H).

Example 21. The synthesis of antagonist Α1 adenosine.

Compound 1503 (table 15 below) can be synthesized according to the General methods below.

C1 JOS * C1 ^{H0 /} a ~ La (V- = o 31 33 Compound 1503

Bromide compound 31 (220 mg, 0.47 mmol) was dissolved in a 1: 1 mixture of DMF: dichloromethane (5 ml). To this solution were 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. The solvents were removed in vacuo and the residue was partitioned between Η ₂ Ο and dichloromethane. The organic layer was dried over Μ§8Ο ₄ , filtered and concentrated to give an off-white solid, which when triturated with an ether / hexanes mixture gave 175 mg of a white solid 33 (84%). Я-NMR (200 MHz, SES1 ₃ ) δ 1.9 (9H, s), 2.54 (4H, s), 3.65 (4H, s), 3.85 (1H, s), 6.59 (1H, s), 7.45 (3Η, m), 8.5 (2H, m).

Compound 33 (50 mg, 0.11 mmol) and trans-4-aminocyclohexanol (105 mg, 0.91 mmol) were placed in DMSO (2 ml). The resulting solution was purged with Ν ₂ , and then heated to 100 ° C in an oil bath and stirred overnight. The crude reaction mixture was poured into water and extracted twice with ethyl acetate (50 ml). The combined organic layers were washed with Η ₂ Ο. After drying Μ§8Ο ₄ and filtering, the organic layer was concentrated in vacuo to give an orange solid. Chromatography (silica, 10% CH ₃ ΟH in SCN1 ₂ ) gave 15 mg (33%). ¹ Η-NMR (200 MHz, SES1 ₃ ) δ 1,241.62 (4Н, m), 1.85 (2Н, m), 2.10 (2Н, m), 2.26 (4Н, m), 3, 53 (4H, m), 4.22 (1H, m), 4.73 (1Η, m), 5.85 (1Η, d), 6.15 (1Η, s), 7.25 (3Η, m) ), 8.42 (2H, M), 10.0 (1H, s). Mass spectrum (E8): 408.0 (M ⁺ +1).

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Compounds 1500, 1501, and 1502 can be synthesized using the same steps as in Example 20 by treating compound 32 with a suitable substituted amine.

Analysis of Αι and A _{2a of} the human adenosine receptor using the yeast β-galactosidase reporter gene

Yeast strains (8.ssgsuyas) were transformed with human adenosine L ₁ (L ₁ B; SLEI8 strain SU12 bb0) or human adenosine A _2a (A _2a ; SLPI8 strain SU83b2) and were supplemented with 1ac2 (β-galactosidase) reporter gene for use as a functional data output. A complete description of the transformations is listed below (see Yeast Strains). As a ligand for all analyzes, IHD was used (5'-Y-ethylcarboxamidoadenosine), which is an effective agonist of the adenosine receptor with similar affinity for L1 and A2a receptors. The tested compounds were studied in 8 concentrations (0.1-10000 nM) on their ability to inhibit the HESL-induced activity of β-galactosidase SU12bb0 or SU83b2.

Preparation of initial yeast cultures: each of the corresponding yeast strains, SU12bb0 or SU83b2, was applied in stripes on agar plates and incubated at 30 ° C until colonies were observed. The yeast of these colonies was added to LF liquid (pH b, 8) SU83b2 and grown overnight at 30 ° C. Each yeast strain was then diluted to an OD of _b0 about = 1.0-2.0 (approximately 1-2x10 ⁷ cells / ml), as determined spectrophotometrically (Mo1cci1a Esuvus VMAX). Based on each b ml of liquid yeast culture, 4 ml of 40% glycerol (1: 1.5 v / v) (“yeast / glycerol starting preparation”) was added. Ten aliquots of 1 ml each were prepared from this yeast / glycerin starting preparation and stored at -80 ° C until analysis.

Yeast L ₁ ° C and L _2a analysis: thawed one ampoule of each of the СУ83б2 and СУ12бб0 yeast / glycerin starting preparation and used to inoculate the supplemented LТ liquid medium, pH b, 8 (92 ml liquid L to which was added: 5 ml 40% glucose, 0.45 ml of 1M KOH and 2.5 ml of P1pc8, pH b, 8). Liquid cultures were grown for 1b-18h (overnight) at 30 ° C. Aliquots of the cultures obtained overnight were then diluted in LB medium containing 4ME / ml adenosine deaminase (type VI or VII from bovine intestinal mucosa, 81 dtA) to achieve an OD of _b00 = 0.15 (1.5x10 ^b cells / ml) for SU83b2 (L2aV) and OPb00 = 0.50 (5x10 ^b cells / ml) for SU12bb (L1B).

Assays were performed at a final volume of 100 μl in 9b-well microtiter plates at a final DMSO concentration of 2%, achieved in all wells. For primary screening, 1-2 concentrations of test compounds (10 μM, 1 μM) were used. Eight concentrations (10,000, 1,000, 500, 50, 10, 1, and 0.1 nM) were studied for profiling the compound. In each microtiter plate, 10 μl of 20% DMSO was added to the “control” and “common” wells, while 10 μl of the test compound (in 20% DMSO) was added to the “unknown” wells. Subsequently, 10 μl of HESL (5 μl for HPL, 1 μl for L _2a B) was added to the “control” and “general” wells; 10 μl of PB8 (phosphate buffered saline) was added to the “control” wells. Upon final addition, 80 μl of yeast strain, SU83b2 or SU12bb0 was added to all wells. Then, all plates were shaken for a short period of time (Ebbc orbital shaker, 2-3 min) and allowed to incubate for 4 hours at 30 ° C in an oven.

The activity of β-galactosidase can be quantified using either colorimetric (e.g., ООС, СРВС), luminescent (e.g., 6a1ac1op-81ag) or fluorimetric substrates (e.g., EES, KezogiGt). Currently, fluorescence recording is preferred based on a better signal to noise ratio, relative freedom from interference, and low cost. Fluorescein digalactopyranoside (EEC, MoCl2, PrgoBc3, or Magx2 Oscope Tespepoi fluorescent β-galactosidase substrate, was added to all wells at a concentration of 20 μl / well (final concentration = 80 μM).

The plates were shaken for 5 s (Lbbc orbital shaker) and then incubated at 37 ° C for 90 min (95% O ₂ /5% CO ₂ incubator). At the end of the 90-minute incubation period, β-galactosidase activity was stopped using 20 μl / well of 1M NbCO ₃ and all plates were shaken for 5-sec. The plates were then shaken for 6 seconds and the relative fluorescence intensity was determined using a fluorimeter (Tssap 8pcc1ga £ 1; excitation = 485 nm, emission = 535 nm).

Calculations: Relative fluorescence values for the “control” wells were interpreted as background and subtracted from the “general” and “unknown” values. The compound profiles were analyzed using a logarithmic transformation (x-axis: compound concentration) followed by a single-site competition curve corresponding to the calculation of Κ. ' ₅₀ (SgarNRab Rpzt).

Yeast strains: strains of 8assyagotuss5 ssgsu181ac SU 12bb0 [£ a1 * 1442 1U1-1 £ s1Sh83 sap1 81s14 :: 1gr1 :: LU82 81c3 * 115b dra1 (41) -6αί3 1uz2 yga3 1si2 1gr1: Sz3; LIE2 RSKr-MEaKsabegiL1V-RNO51st 2t-ogsh KEP3 Ltrg] and SU83b2 [dra1r-g6oE10K £ ar1 * 1442 1Y-1 £ i81-Sh83 sap1 81s14 :: 1gr1: LU82 81s3 * 115b 1s2y2a3 LIE2 R6Kr-yL ₂ aK 2ti-og1 KEP3 Ltr].

LB medium: LB (Lc-Tgr supplemented) medium consists of 100 g of EXO of a yeast nitrogen base, supplemented with the following: 1.0 g of valine, 1.0 g of aspartic acid, 0.75 g of phenylalanine, 0.9 g

- 53 007254 lysine, 0.4 5 g of tyrosine, 0.45 g of isoleucine, 0.3 g of methionine, 0.6 g of adenine, 0.4 g of uracil, 0.3 g of serine, 0.3 g of proline, 0.3 g cysteine, 0.3 g of arginine, 0.9 g of histidine and 1.0 g of threonine.

Construction of yeast strains expressing A! human adenosine receptor

This example describes the construction of yeast strains expressing the A ₁ human adenosine receptor functionally integrated into the pathway of the yeast pheromone system.

The construction of the espression vector

To construct a yeast expression vector for the human Adenosine A ₁ receptor A1 receptor cDNA, the A ₁ Adenosine receptor cDNA was obtained using human hippocampal mRNA reverse transcriptase PCR using primers developed based on the published human Adenosine receptor A ₁ sequence and standard techniques. The PCR product was subcloned into the XCOT and XM sites of the yeast plasmid pMP15 expression.

The PMP15 plasmid was constructed from ρΚΡΧΐ as follows: XM site UEP51 (Bgosy Ι.Β. e! A1., (1983) Ues! Ogg Gd Ydy-1eue1, tdis1b1e exrgyeop OG c1oped depes ΐη Uea§1 p.83-117e in MTn (unit) Exreptiopalis Mashri1a1yup oGe exrgesiop. Asadesh M Prgesus, Να Wagk) was eliminated by splitting, filling the end and re-ligation to create Epeploxe. Another XM site was created on your WG site by burning with WGW, filling the end, linker (Еα Epd1apd Vy1aB§, # 1081) ligation, XBaT cleavage and re-ligation to generate Vep5ShCOX1. This plasmid was digested with Esp31 and Xcot and ligated to the Le2 and PCB fragments generated by PCR. The PCR product 1, ei2, with a size of 2 thousand bases, was generated by amplification from Uep5ShCO using primers containing the sites Ezp31 and Vd1P. The PCR product of RSCr from 660 base pairs was generated by amplification from rRSCase (Kapd Υ.-8. E! A1., (1990), Mo1.Ce11.Vu1. 10: 25822590) using PCR primers containing the Bd1P and XcOR sites. The resulting plasmid is called pbx p1.px! modified with the insertion of the coding region of the p-leader of the α-factor into the ΝαΜ site. The insertion of the cge-leader of the α-factor was carried out so that the проводилиα) 1 cloning site was maintained at the 3 'end of the leader, but was not regenerated at the 5'end. Thus, the receptors can be cloned by plasmid digestion with α4 and XbT. The resulting plasmid is called pMP15, the leader factor factor leader.

The pMP15 plasmid into which the human adenosine A ₁ receptor cDNA was inserted was designated p5095. In this vector, the cDNA of the receptor is fused to the 3 'end of the pro-yeast α-factor leader leader. During the maturation of the protein rgo-rgo, the peptide sequences are cleaved, generating a mature receptor with a full length. This occurs during processing of the receptor on the secretory pathway of yeast. This plasmid is supported by Rey selection (i.e., growth on a medium lacking leucine). The sequence of the cloned coding region was determined and it was found to be equivalent to that published in the literature (assigned numbers in SepVapk 845235 and 856143).

II. Construction of a yeast strain.

To create a yeast strain expressing the A1 human adenosine receptor, the SU7967 yeast strain was used as the initial parent strain. The genotype SU7967 is as follows:

MATA dray Ό1163 dray1 (41) Oa13 1agY1442 1Y-1 81υ81-ΗI83 sap 1 §1е14 :: 1gr1 :: LU82 §1e3E1156 1y2 yoke3 1ey2 1gr1 §§3

Genetic markers are discussed below ._______________________

MAT ' ^: '

Mating type a

Endogenous yeast C-protein CPA-1 was removed from the nucleus! (41) Sag3 nucleus! (41) -Ca13

This chimeric was integrated into the yeast genome.

£ 'ayu1442

Bjc-1

Eu31-H133 glanders!

zbe14 :: bgr1 :: bY32 amino acids

Ca protein consists of the first 41 endogenous subunits of CPA1 fused to mammals, in which yeast

C-protein originating Ν-terminal amino acids removed

Sa

Sagz total were

EAK. 1 gene (responsible for stopping the cell cycle) was removed (thus preventing the cell cycle from stopping when the response pathway to the pheromone strain was activated with high transformation efficiency by electroporation fusion between the Eu31 promoter and the ΗΙ33 coding region (thus creating an inducible ΗΙ33 pheromone gene) arginine / canavinin permease gene destruction ZTE14,

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C-farnesylmethyl transferase (thus lowering the main signal through the pheromone pathway ZEZO1156 endogenous yeast ZTH, pheromone receptor factor (ZTEZ) was destroyed 1uz2 2-aminoadipat reductase defect, yeast lysine is needed for growth igaz defect orotidine-5'-phosphate decarboxylase, yeast needs uracil for growth 1ei2 defect b-isopropyl malate dehydrogenase, yeast needs leucine for growth Bgr1 defect phosphoribosylanthranilate, yeast tryptophan is needed for growth MZZ imidazole glycerol phosphate dehydrogenase defect, yeast needs histidine for growth

Two plasmids were transformed into strain SU7967 by electroporation: plasmid p5095 (encoding A ₁ human adenosine receptor; described above) and plasmid p1584, which is an EIB1-p-galactosidase reporter gene plasmid. Plasmid p1584 was obtained from plasmid pRB426 (Snipkapkop T. ^. E! A1. 91992) Oepe 110: 119-1122. Plasmid rRB426 contains a polylinker site on nucleotides 2004-2016. The fusion of the EIB1 promoter and β-galactosidase gene was inserted into the restriction sites Ead! and X1y1 to create plasmid p1584. Plasmid z1584 was maintained by Tg selection (i.e., growth on a medium lacking leucine).

The resulting strain bearing p5095 and p 1584, referred to as SU12 660, expresses the human A ₁ adenosine receptor. To grow this strain in a liquid or on agar plates, a minimal medium with a lack of leucine and tryptophan was used. To perform growth analysis on tablets (analysis Γϋδ 1 ^ 83), the tablets were at pH 6.8 and contained 0.5–2.5 mM 3 amino-1,2,4-triazole and lack of leucine, tryptophan and histidine. As a control of specificity, all experiments included comparisons with one or more other seven yeast-based transmembrane receptor selections.

Construction of yeast strains expressing A _2a human adenosine receptor

This example describes the construction of yeast strains expressing the human A _2a adenosine receptor functionally integrated into the pathway of the yeast pheromone system.

Construction of the expression vector

To construct the yeast expression vector A2a of the human adenosine receptor, adenosine receptor cDNA A ₂ from Όγ was obtained. РЫ1 МигрКу (ΝΜ). Upon receipt of this clone, the A _2a receptor insert was sequenced, and it was found to be identical to the published sequence (assigned number in OepWapk # B46950). The receptor cDNA was excised from the PCR plasmid using νΡ ^ polymerase and cloned into the rBVC plasmid, which stimulated the expression of the receptor using the phosphoglycerate kinase (POK) compound promoter in yeast. Once again, the amino acid sequence of the whole insert was determined and found to be identical to the published sequence. However, by virtue of the cloning strategy used here, three amino acids were added to the carboxyl end of the receptor, O1uBe ^ a1.

Construction of a yeast strain

To create a yeast strain expressing A _2a human adenosine receptor, the SU8342 yeast strain was used as the initial parent strain. The genotype SU8342 is as follows:

MATa 1agY1442 1Y1-1 1uk2 yy3 1ei2 1gr1 Yk3 GikEN ^ sap1 k! E3E1156 §raV1163 k1e14 :: 1gr1 :: BUB2 dra1r-go _ak E10K (or dra1r-go _Ak O229B or dra1r-go _Ak 9E10K +22)

Genetic markers are as described above, with the exception of the marker for varying the O-protein. For expression of the human A _2a receptor used strains of yeast, which was deleted by the endogenous yeast G-protein ORA1 and replaced by O _ak mammal. Three rat O _ak mutants were used. Such variants contain one or two mutation points that turn them into proteins that crosslink rationally with βγ-yeast. They were identified as O _ak E10K (in which glutamic acid in the tenth position is replaced by lysine), O _ak O229B (in which aspartic acid at position 229 is replaced by a series) and O _ak E10K + O229B (which contains both mutation points).

Strain SU8342 (carrying one of the three mutant rat _Oak proteins) was transformed using either the parent vector rBVX (Receptor ^- ) or using rBVX-A _2a (Receptor ⁺ ). A plasmid with an EIB1 promoter fused to β-galactosidase coding sequences (described above) was added to evaluate the activation value of the pheromone response pathway.

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Functional analysis using yeast expressing strains

Αχ adenosine receptor

This example describes the development of functional screening analysis of modulators of A _x human adenosine receptor in yeast

I. Ligands used in the analysis.

To develop this analysis, adenosine, a natural agonist of this receptor, and two other synthetic agonists were used. A subset of the experiments used adenosine having, according to published data, an EC _{50 of} approximately 75 nM, and (-) - Ν6- (2-phenylisopropyl) adenosine (LA) with a published affinity of approximately 50 nM. All growth assays used 5'-X-ethylcarboxamidoadenosine (HESA). To prevent signal transmission due to the presence of adenosine in the growth medium, adenosine deaminase (4 IU / ml) was added in all assays.

II. Biological response in yeast.

The ability of the A ₁ adenosine receptor to bind functionally to the heterologous yeast system was evaluated by introducing the expression vector of the A _x receptor (p5095, described above) into a series of yeast strains that express various subunits of protein O. Most of these transformants expressed O _a subunits of O _a ; or o _ao subtype. Additional O _a proteins have also been tested for possible identification of the heterogeneity of their binding to receptor-Οα protein. In various strains, the 8TE18 or chimeric 8TE18-Oy construct was integrated into the yeast genome.

Yeast strains concealed the defective J83 gene and the integrated copy of 81υ81-HI83, thus giving the opportunity for selection in a selective medium containing 3-amino-1,2,4-triazole (tested at 0.2, 0.5 and 1.0 mmol ) and not having histidine. Transformants were isolated and monolayers prepared on a medium containing 3-amino-1,2,4-triazole, 4 IU / ml adenosine deaminase and not containing histidine. Five microliters of various ligand concentrations were used (for example, NECA at a concentration of 0, 0.1, 1.0, and 10 mM). Growth was monitored for 2 days. Thus, a ligand-dependent growth response was investigated in various yeast strains. The results are summarized in Table 1 below. The symbol (-) indicates that ligand-dependent receptor activation has not been determined, while (+) indicates a ligand-dependent response. The term "BSMA" refers to ligand-independent receptor-mediated activation.

Table 3

Yeast strain C _a subunit Soo slbby pin that Options strain Result SU1316 CPA1 5TE18 - CPA41-Ssch1 + CPA41-C _a12 + ΟΡΑ41-Ο _α ΐ3 + 6PA41-Ο _α ί2-Ο _α οΒ YKMA ΘΡΑ41- (3 _α 5Εΐοκ - CPA41 ~ S _a zo229z - SU7967 CPA41-Ca1 integrated 5TE18 +++ SU2120 With RA! 5TE18 33Ε2Δ + SRA41-6 _au + CPA41-C _a 1 ₂ +

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СРА41-С _а 1з СРА41- <З _а 12-Сссов СРА41-САЗЕЮК СРА4 1 ~ С _а 302293 + YKMA SU9438 SRA1 SRA41-C _and C and SRA41 _and SRA41-12 Sa1z SRA41 ^_ With _a 12-Saov ZTE18-Su2 + + + YKMA <ZRA41- (3 _α 3Εΐοκ - CPA41- <З _а зо229з - SU10560 CPA1- integrated 5TE18-Su2 33Γ2Δ ++

As indicated in the table. 3, it was found that the strongest signal transmission occurred in a yeast strain expressing OPA '(41) -O _a ; ₃ chimeric protein.

III. 1 of 1 Gus-Ζ analysis.

In order to more fully characterize the activation of the pheromone response pathway, β-galactosidase synthesis was measured by ί and 81 ^ acΖ in response to agonistic stimulation. To carry out β-galactosidase analysis, increased ligand concentrations were added to the T16-1 ° culture of human A 'adenosine receptor expressed in a yeast strain co-expressing 8 (E18-Cy2 chimera and OPA' (41) -O _a ; _3. Transformants were isolated and grown overnight in the presence of histidine and 4 IU / ml adenosine deaminase After five hours of incubation with 4 IU / ml adenosine deaminase and ligand, β-galactosidase induction was measured using CPCO as a substrate for β-galactosidase. 5x10 ⁵ cells were used for analysis.

The results obtained with NEC stimulation indicated that approximately 2-fold stimulation of β-galactosidase activity is achieved at a concentration of NECΑ 10 ^-8 M. In addition, an approximately 10-fold stimulation index was observed at a NECΑ concentration of 10 ^-5 M.

The use of this analysis was expanded to evaluate the activity of antagonists in this strain. Two known adenosine antagonists, HAC and ^ PCPX, were tested for their ability to compete with NECΑ (at 5 mM) for activity in the analysis of β-galactosidase. In these assays, β-galactosidase induction was measured using EES as a substrate and 1.6 × 10 ⁵ cells per analysis. The results show that both XAC and ^ RSRX are potent antagonists of yeast-expressed A 'adenosine receptor at values of 44 nm, ₅₀ HS and 49 nM, respectively.

To determine whether this inhibitory effect is specific for the A 'subtype, a series of complementary experiments were performed using an analysis of the A _2a receptor based on yeast. Results from a yeast-based A _2a receptor assay indicated that HAC was a relatively effective antagonist of the A _2a receptor, consistent with published data. In contrast, ^ PCPX was relatively inert for this receptor, as expected from published reports.

IV. Radioligand binding.

Analysis of the A 'adenosine receptor was further characterized by measuring the parameters of the radioligand binding of the receptor. Substitutional binding of [ ³ H] CPX to several reference compounds of the adenosine receptor, HAC, ^ PCPX, and CO8 was analyzed using membranes derived from yeast expressing human A1 adenosine receptor. Results for yeast membranes expressing human A1 adenosine receptor were compared with results for yeast membranes expressing human A2a adenosine receptor or human A3 receptor to evaluate binding specificity. For this analysis, fifty milligrams of membranes were incubated with 0.4 nM [ ³ N] CPX and increasing concentrations of adenosine receptor ligands. Incubation was carried out in 50 mM TPZ-HCl, pH 7.4, 1 mM EETA, 10 mM MdC12, 0.25% BSA and 2 IU / ml adenosine deaminase in the presence of protease inhibitors for 60 min at room temperature. Binding was stopped by the addition of ice-cold 50 mM TPZ-HC1, pH 7.4,

- 57 007254 plus 10 mM MDS1 ₂ , followed by rapid filtration through OE / B, filters pre-washed with 0.5% polyethyleneimine using a 96-well Raskagb harvester. The data were analyzed by the method of selecting a non-linear curve by the least squares method using the software Рпзт2.02. The IC5 ₀ values obtained in this experiment are summarized below in Table. 4:

Table 4

1C ₅₀ (nM) Compound EA1K EA2aK KAZN XAS b, b 11.7 53.1 RRPC 8.5 326, 4 1307.0 SS5-15943 13, 1 15.8 55.5 ΝΕΟΑ 215.5 294.9 34.9 Κ-ΡΙΑ 67 b 678.1 23, 6 1B MESA 727.7 859.4 3,1 Alloxosin 1072.0 1934.0 8216,0

These data indicate that the linking compounds had an affinity corresponding to that published in the literature. The data further indicates that yeast-based assays have sufficient sensitivity to recognize the specificity of the receptor subtype.

Functional analysis using yeast expressing strains

A2a adenosine receptor

This example describes the development of functional screening analysis of modulators A! human adenosine receptor in yeast

I. Ligands used in the analysis.

The natural adenosine ligand, as well as other carefully characterized and commercially available ligands, have been used to study human A2a receptor functionally expressed by yeast. When creating this analysis used three ligands. These include:___________

Ligand Accounted for Κι | Function Adenosine 500 nM agonist 5 '-Ν- ethylcarboxamidoadenosine (ΝΕΟΑ) 10-15 nm agonist (-) - Ν6- (2 phenylisopropyladenosine (ΡΙΑ) 100-125 nM agonist

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

II. Biological response in yeast.

A2a receptor agonists were tested for their ability to stimulate the pheromone response pathway in yeast transformed with the A2a receptor expression plasmid and expressing either O _a8 E10K, O _a8 E2298, or O _a8 E10K + E2298. The ability of the ligand to stimulate the pathway of the pheromone response in a receptor-dependent manner was indicated by a change in the phenotype of yeast. Receptor activation modified the phenotype from histidine auxotrophy to histidine prototrophy (activation 1i81-Sh83). Three independent transformants were isolated and grown overnight in the presence of histidine. Cells were washed to remove histidine and diluted at a rate of 2x10 ⁶ cells / ml. 5 μl of each transformant was spotted on a non-selective medium (including histidine) or selective medium (1 mM AT) in the absence or presence of 4 IU / ml adenosine deaminase. The tablets were grown at 30 ° C for 24 hours. In the presence of histidine, both the Receptor (B) and the Receptor (B ^- ) strains were able to grow. However, in the absence of histidine, only B ⁺ cells grew. Since no ligand was added to these plates, there are two possible explanations for this result. One possible explanation is that receptor-containing yeast had a growth advantage due to ligand-independent receptor-mediated activation (HBMA).

Alternatively, yeast may be able to synthesize adenosine. In order to distinguish between these two possibilities, an enzyme that destroys the ligand, adenosine deaminase (AEA), was added to the growing yeast and tablets. In the presence of adenosine deaminase B, the cells no longer grew in the absence of histidine, indicating that the yeast did synthesize the ligand.

This interpretation was confirmed by analysis of A2a growth in the liquid. In this experiment, yeast (strain O _a8 E10K expressing the A2a receptor) was inoculated at three densities (1x10 ⁶ cells / ml; 3x10 ⁵ cells / ml or 1x10 ⁵ cells / ml) in the presence or absence of adenosine deaminase (4

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IU / ml). The assurance of the analysis was increased by increasing the concentrations (0, 0.1, 0.2, or 0.4 mmol) of 3-amino-1,2,4-triazole (AT), a competitive antagonist of the imidazol glycerol-P dehydratase, the protein product of the Ш83 gene. In the presence of adenosine deaminase and 3-amino-1,2,4-triazole, the yeast grew less intensively. However, in the absence of 3-amino-1,2,4-triazole, adenosine deaminase had a negligible effect. Thus, adenosine deaminase itself does not directly affect the path of the pheromone response.

An alternative approach to measuring growth, such as can be made miniature for high throughput screening, is a spot analysis of the A2a receptor ligand. C _aa E10K strain expressing the A2a receptor (A2aK ⁺ ) or lacking the receptor (K ^- ) was grown overnight in the presence of histidine and 4MU / ml adenosine deaminase. Cells were washed to remove histidine and diluted to 5x10 ⁶ cells / ml. On selection plates containing 4 IU / ml adenosine deaminase and from 0.5 to 1.0 mM 3-amino-1,2,4-triazole (AT), 1 × 10 ⁶ cells were applied and allowed to dry for 1 h. The following was applied to the monolayer reagents: 10 mM adenosine, 38.7 mM histidine, dimethyl sulfoxide (DMSO), 10 mM PTA or 10 mM HECA. Cells were grown for 24 hours at 30 ° C. The results demonstrated that cells without a receptor could only grow when histidine was added to the medium. In contrast, K ⁺ cells can grow only in areas where the PTA or HESA receptor ligands were applied as an A2a spot. Since the tablets contained adenosine deaminase, a lack of growth, where adenosine was applied as a spot, confirmed that adenosine deaminase was active.

III. §§1 Ε; κΖ analysis.

To quantitatively activate the yeast crossing path, β-galactosidase synthesis was measured through Γι.ΐ51Ε; κΖ. Yeast strains expressing C _aa E10K, C _aa E2298 or C _aa E10K + P2298 were transformed with a plasmid encoding the human A _2a receptor (K ⁺ ) or a plasmid without a receptor (K ^- ). Transformants were isolated and grown overnight in the presence of histidine and 4MU / ml adenosine deaminase. 1x10 ⁷ cells were diluted to 1x10 ⁷ cells / ml and exposed to an increasing concentration of NECA for 4 hours, followed by the formation of β-galactosidase activity in the cells. The results showed that, in essence, β-galactosidase activity was not detected in K ^- strains, while increased amounts of β-galactosidase activity were found in K ⁺ strains expressing either C _aa E10K, C _aa E2298, or C _aa E10K + P2298, as an increase in the concentration of No. ECA, indicating a dose-dependent increase in units of β-galactosidase, detected as a response to the effect of an increased concentration of the ligand. Such dose dependence was observed only in cells expressing the A2a receptor. In addition, C _aa E10K was found to be the most effective C _a8 construct for the A2a receptor. The C _aa E2298 construct was the second most effective C _a8 construct for the A2A receptor, while the C _aa E10K + P2298 construct was the least effective of the three C _a8 constructs tested, although even the C _aa E10K + P2298 construct stimulated easily detectable amounts of β-galactosidase activity.

For a further description of these analyzes, see U.S. Patent Application Publication No. I8-2002-0015967-A1, published February 7, 2002, entitled Ει.ιικΙίοηη1 Β.χρίΌδδίοη οΓ Aye ^^ te Keserfga ίη Wyei, which has now been liquidated, the entire contents of which incorporated herein by reference.

Pharmacological characteristics of subtypes of human adenosine receptor Materials and methods

Materials ^[3 H] ^ RSRX [cyclopentyl-1,3-dipropilksantin, 8- [dipropyl-2,3- ³ H (N)] (120.0

Ki / mmol); [ ³ H] -CC8 21680, [carboxyethyl ^ H ^)] (30 Ci / mmol); and [ ¹²⁵ Σ] -AB-MECA ([ ¹²⁵ Σ] -4aminobenzyl-5'-Y-methylcarboxamidoadenosine) (2200 Ci / mmol) were obtained from No. \ ν EpDaiy Kis1eag (Βοκΐοη, МА). XAS (belonging to the class of xanthine amine); JESA (5-S-ethyl-carboxamide-adenoidemic) and ΣΒMESA were obtained from Kekeagsy Vusjietkak [Schep · ^^ ·· ^ (EVE No. 1 ^, MA). Mixed pills of adenosine deaminase and a complete protease inhibitor were purchased from Mapie & Tg. (Ση ^^ ρο ^, ГЫ). Membranes of HEK-293 cells stably expressing A2 adenosine [KB-HA2a], 2b 1 adenosine [KBNA2b] or adenosine 3 [KB-HA3] subtypes of human receptors, respectively, were obtained from Keserig Βίο1ο§γ (Beetet, Mo). Cell culture reagents were obtained from IgE TesNηο1οβ ^ e5 (Sgai Shai, CT) with the exception of serum obtained from Hus ^ e (^ Odaη, IT).

Yeast strains: used strains of Zassyagotusek segeuye SU 12660 [Gag1 * 1442 1Ы1-1 Γι.ΐ51Ш83 san1 51e14 :: 1gr1 :: EU82 §1e3 * 1156 dra1 (41) -Οαί3 1y§2 yga3 1ey2 1gr1: §§3; LIE2 RbKr-MEaSheayegLA1K-RN051egt 2ti-ο ^^ d KEP3 Atrg] and SU8362 [dray1r-gSaE10K Gag1 * 1442 1Y-1 Gi51-H! 83 san1 51e14 :: 1gr1 :: EU82 §1e3 * 1156 1§1 §§3; LIE2 RSKr-NA ₂ aK 2ιηι.ι-οπ KEP3 Atrg], as described above.

Yeast culture: Transformed yeast was grown in Le-Tgr (Lt) medium (pH 5.4) supplemented with 2% glucose. To obtain membranes, 250 ml of L medium was inoculated with an initial titer of 12 x 10 ⁶ cells / ml 30 ml of culture obtained overnight and incubated at 30 ° C with constant supply of oxygen by rotation. After 16 hours of growth, cells were harvested by centrifugation and membranes were obtained as described below.

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Human tissue culture: HEK-293 cells stably expressing the subtype 2a of the human adenosine receptor (clone Sabi8 # 5) were grown in Dulbecco's minimal essential medium (EMEM) supplemented with 10% fetal calf serum and IX penicillin / streptomycin at the selected pressure using 500 mg / ml C418 antibiotic at 37 ° C in a humidified atmosphere containing 5% CO ₂ .

Obtaining yeast cell membranes: 250 ml of culture were collected after incubation overnight by centrifugation at 2000d in a 8ogua11 VT6000 centrifuge. Cells were washed with ice-cold water, centrifuged at 4 ° C, and the pellet from the centrifugation was resuspended in 10 ml of ice-cold lysis buffer [5 mM Tg-HC1, pH 7.5; 5 mM ΕΌΤΆ and 5 mM ECTA] supplemented with mixed tablets of a protease inhibitor (1 tablet per 25 ml of buffer). Glass beads (17 g; 400-600 mesh; 8 mm) were added to the suspension and the cells were destroyed by vortex mixing at 4 ° C for 5 min. The homogenate was diluted with an additional 30 ml of lysis buffer plus protease inhibitors and centrifuged at 3000 d for 5 minutes. Subsequently, the membranes were besieged at 36000 d (8ogua1 BC5V type 8834 rotor) for 45 min. The resulting membrane pellet was resuspended in 5 ml of membrane buffer [50 mm Tg18-HC1, pH 7.5; 0.6 mm ΕΌΤΑ and 5 mm MDS1 ₂ ], supplemented with mixed tablets of a protease inhibitor (1 tablet in 50 ml of buffer) and stored at -80 ° C for subsequent experiments.

Obtaining mammalian cell membranes: HEK-293 cell membranes were prepared as previously described (Όιιζχ E. E1 a1., GVuESet., 267, 9844-9851, 1992). Briefly, cells were washed with phosphate buffered saline (PB8) and harvested using a rubber collector. Cells were besieged at 4 ° C and 200 d in a centrifuge 8ogua11 VT6000. The centrifugation pellet was resuspended in 5 ml per lysis buffer plate at 4 ° C [5 mM Tg18-HCl, pH 7.5; 5 mM EOTA; 5 mM ESTA; 0.1 mM phenylmethylsulfonyl fluoride, 10 mg / ml pepstatin A and 10 mg / ml aprotinin], and homogenized in an Ehonns homogenizer. The cell lysate was then centrifuged at 36000 d (8ogua1 BC5B type 8834 rotor) for 45 minutes and the pellet obtained by centrifugation was resuspended in 5 ml of membrane buffer [50 mM Tg-HC1, pH 7.5; 0.6 mM EOTA; 5 mM MDS1 ₂ ; 0.1 mM phenylmethylsulfonyl fluoride, 10 mg / ml pepstatin A and 10 mg / ml aprotinin] and stored at -80 ° C for subsequent experiments.

Vyu-VaD protein analysis kits based on the Bradford dye binding technique (VGADEogD M., AiaEBusies. 72: 248 (1976)) were used to determine the total protein concentration in the membranes of yeast and mammals.

Saturation and competitive radioligand binding of adenosine receptor subtype 1: Saturation and competitive binding on the membranes of yeast cells transformed with the A1 human receptor subtype was performed using the [ ³ H] ORPCX antagonist as a radioactive ligand. Membranes were diluted in binding buffer [50 mM Tg18-HCl, pH 7.4; containing 10 mm MDS1 ₂ ; 1.0 mM EOTA; 0.25% BSA; 2 IU / ml adenosine deaminase and 1 mixed tablet of a protease inhibitor / 50 ml] at concentrations of 1.0 mg / ml.

With saturated binding, membranes (50 μg / well) were incubated with increasing concentrations of [ ³ H] ORPCX (0.05–25 nM) in a final volume of 100 μl of binding buffer at 25 ° C. for 1 h in the absence or presence of 10 μM unlabeled XAS in a 96-well microtiter plate.

In competitive binding, membranes (50 μg / well) were incubated with [ ³ H] ORPCX (1.0 nM) in a final volume of 100 μl of binding buffer at 25 ° C. for 1 h in the absence or presence of 10 μM unlabeled HAC or increasing concentrations of competing compounds in a 96-well microtiter plate.

Competitive radioligand binding of the adenosine receptor subtype 2a: Competitive binding on HEK293 membranes of cells stably expressing the human A2a subtype receptor was performed using the [ ³ H] CC8-21680 agonist as a radioactive ligand. Membranes were diluted in binding buffer [50 mM Tg18-HCl, pH 7.4; containing 10 mm MDS1 ₂ ; 1.0 mM EOTA; 0.25% BSA; 2 IU / ml adenosine deaminase and 1 mixed tablet of a protease inhibitor / 50 ml] at concentrations of 0.2 mg / ml. Membranes (10 μg / well) were incubated with [ ³ H] CC8-21680 (100 nM) in a final volume of 100 μl of binding buffer at 25 ° C. for 1 h in the absence or presence of 50 μM unlabeled MECA or increasing concentrations of competing compounds in a 96 well microtiter plate.

Competitive radioligand binding of adenosine receptor subtype 3: Competitive binding on HEK293 membranes of cells stably expressing the human subtype A3 subtype was performed using the [ ¹²⁵ 1] AB-MECA agonist as a radioactive ligand. Membranes were diluted in binding buffer [50 mM Tg18-HCl, pH 7.4; containing 10 mm MDS1 ₂ ; 1.0 mM EOTA; 0.25% BSA; 2 IU / ml adenosine deaminase and 1 mixed tablet of a protease inhibitor / 50 ml] at concentrations of 0.2 mg / ml. Membranes (10 μg / well) were incubated with [ ¹²⁵ 1] AB-MECA (0.75 nM) in a final volume of 100 μl of binding buffer at 25 ° C. for 1 h in the absence or presence of 50 μM

- 60 007254 unlabeled W-MESA or increasing concentrations of competing compounds in a 96-well microtiter plate.

At the end of the incubation, analyzes of the radioligand binding of A _1b A _2a and A ₃ receptor subtypes were completed by adding ice-cold 50 mM Tg18-HCl (pH 7.4) supplemented with '0 mM MdC1 ₂ followed by rapid filtration through glass fiber filters (96 well, CE / B IsHezhegs, Raskagb), previously washed with 0.5% polyethyleneimine in a harvester of E111egta! e '96 cells (Raskagb). The filter plates were dried, coated at a rate of 50 μl / well with scintillation liquid (M1gozs1p1-20, Raskagb) and counted in TorCoip! (Raskagb). Analyzes were performed in three sequences. Nonspecific binding was 5.6 ± 0.5%, '0.8 ±', 4%, and '5,' ± 2.6% of the total binding in the A'K, A2aK, and A3K binding assays, respectively.

Competitive radioligand binding of the adenosine receptor subtype 2b: Competitive binding on HEK293 membranes of cells stably expressing the human A2b subtype receptor was performed using the A ′ receptor antagonist [ ³ H] ORPCX as a radioactive ligand. Membranes were diluted in binding buffer [0 mM Nerez-KOH, pH 7.4; containing ', 0 mM EOTA; 0.1 mM benzamidine and 2 IU / ml adenosine deaminase] at concentrations of 0.3 mg / ml. Membranes (15 μg / well) were incubated with [ ³ H] ORPCX ('5 nM) in a final volume of '00 μl binding buffer at 25 ° C for' h in the absence or presence of '0 μM unlabeled HAC or increasing concentrations of competing compounds in a 96-well microtiter plate. At the end of the incubation, the analysis was completed by adding ice-cold '0 mM Nerez-KOH (pH 7.4) buffer, followed by rapid filtration through glass fiber filters (96-well, CE / V Ischegz, Raskagb), pre-washed with 0.5% polyethyleneimine in the harvester of E111egta cells! e '96 (Raskagb). The filter plates were dried, coated at a rate of 50 μl / well with scintillation liquid (M1gozs1p1-20, Raskagb) and counted in TorCoip! (Raskagb). Analyzes were performed in three sequences. Nonspecific binding accounted for 14.3 + 2.3% of total binding.

Specific binding of ^[3 H] ORSRH, ^[3 H] SS8-21680 and [ ^'25 Σ] AB-MECA was defined as the difference between total binding and nonspecific binding. The percentage inhibition of compounds was calculated relative to total binding. Competitive data were analyzed using an iterative curve corresponding to a single-site model, and the values of K were calculated from the values of Kec (CEepd apb PrzizoG, VusEet.Reagtaso 1.22, 3099-3'09, '973) using the software Рп / т 2.0'.

results

The primary function of some cell surface receptors is the recognition of suitable ligands. Accordingly, the authors of the present invention determined the affinity of ligand binding to establish the functional integrity of subtype 1 of the adenosine receptor expressed by yeast. The untreated membranes obtained from 8assEagotuses of segeu181ae transformed with the construct of the subtype 1 human adenosine receptor showed specific saturated binding of [ ³ H] ORPCX with K _about 4.00.19 nM. The values of K _o and B _{max were} calculated from the saturation isotherm and data conversion according to Scatchard, indicating one class of binding sites. The density of adenosine binding sites in yeast membrane preparations was estimated as 7'6.8 ± 43.4 fmol / mg membrane protein.

Pharmacological characteristics of the subtype of recombinant yeast cells transformed with subtype A! receptor, was investigated using subtype-selective adenosine ligands (XAS, OPCX, CC8-15943; Compound 600; Compound '002; No. CA, (K) -P! A, W-MECA and alloxazine), which competed with [ ³ H ] ORSX in the expected rank order. Substitution curves recorded for these compounds showed typical steepness for all ligands, and data for each ligand could be modeled using a single-site approach. The apparent dissociation constants estimated from the curves for the individual compound (Table 5) correspond to the published value for the receptor obtained from other sources.

Table 5. Values of K! for yeast cell membranes transformed with subtype A! human receptor

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In the table. 6-12 show the profiles of the effectiveness and structural activity of the desazapurines of the invention. Tables 13-14 show the selectivity that can be achieved for human adenosine receptor sites by modulating functional groups near the desazapurine structure. Table 14 also shows the unexpected discovery that the compounds described herein have subnanomolar activity and higher selectivity for the A ₂ receptor compared to the compounds in table 13.

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608 ____...BUT • bO " 29, 1 609 LEN 193, 9 610 Ό ί ω §Η 411.5 611 ο 785, 6 > 10,000 612 X BNAS Trans (3.3) 64.8 613 £) ΝΗΑο Trans (K, K) 6726.0 614 ⁿ h (W) 32.1 615 xO ⁴ (<I) 816, 9 2577.0 616 Ζ ^0Η *> he 34.3

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Table 7. The effect of C ₂ substituent

n

A1 Compound TO Binding Κι (nM) Yeast 1C50 (nM) 700 604.5 > 10,000 701 0 157.7 763, 1 702 BUT 198.5 2782.5 703 443,6 > 10,000 704 ABOUT 5 61.1 297.0 705 <> | 30.1 194.7 706 at G 19, 9 707 62.8 708 > + 2145 709 from 48.7

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Table 8. The effect of the substituent of the pyrrole ring

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Table 9

A1 Compound TO Linking To ₄ (nm) Yeast 1C50 (nM) 900 BUT 863.1 901 4512 902 8451 903 γ 35.3

Table 10. The effect of ^ -substituent

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1008 93.1 3217 1009 . ό 475.3 > 10,000 1010 674.9 9376.0 1011 121, 9 2067.5 1012 233, 9 3462 1013 ^At Η 270.1 3009.5 1014 ν \ ^ ^0Η 384.9 2005 1015 179.3 3712 1016 176, 1 5054

Table 11. The effect of ^ -substituent

Α1 Compound Κ Binding (nM) Κί Yeast 1C50 (nM) 1100 9.8 115.4 1101 0 53, 9 551,0

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1102 10.3 101.3 1103 71.1 3217 1104 Μ 1 Me 0 _(<) 6, 5 58.7 1105 Η ΑΑΑ Μ · 0 <* " 105,4 472.1 1106 me Η ΑΑ'ά ⁰ (*) 27.8 162.4 1107 Μ »Η ο (*) 126, 5 1297.0 1108 ^ X * 2,3 1109 L ^ IAe ⁷ 5 9.0 1110 νγ; ^ΝΗΑ0 17.3 1111 5 K- 2,5 1112 213

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Table 12. "Retroamide" analogues

A1 Compound TO Binding Ku (nM) Yeast 1C50 (nM) 1200 ABOUT 16.5 189, 4 1201 Ha A G1NMe 7.4 45.7 1202 BUT/ and 95.8 3345.0 1203 529.1 4040.0 1204 ABOUT 1060.0 > 10,000 1205 ABOUT 1272 > 10,000 1206 ABOUT 50.8 4028 1207 ABOUT 48.5 701.5

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Table 13

Profile of Selective Adenosine Antagonists

ΝΗΗ .. | Me about ⁿ Binding Κι (nM) Compound TO. A1 A2a A2b AZ 1300 Λ ^ \ / - ^ΝΗΑο 9.8- 25.1 18.0- 48 b 80.3 513.0 1301 Me 27.8 50.7 84.6 429.8 1302 Η ^ · ^Ν \ / ^ΝΗΜβ γ τ 0 20,2 75 b 20.1 4.3 1303 ha A LHNMe 17.4 111.3 120, 6 44.6 1304 At 13, 9- 30, 9 933.7 138.0 21.5 1305 ¹ > ^he 46.6 730, 9 thirty% 9.9

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1306 ² 16,4 766, 3 168.3 71.7 1307 (άΐ) 29.1 190.6 1143.0 3,1 1308 (±) 180 230 670 1,0 1309 n / 'ν / ’γ * one lhh one 40 109 109 0.3 1310 1 Χ. _Ν · ^ ^ ΝΗΜβ Η 255 76% 275 <2.6 1311 α. Λ ι 531 981 736 5, 3 1312 χ 1 1 Α. \ _Ν - ^ ^ ΝΗΜβ [ 443 2965 375 <6.2 1313 ³ ι thirty% 65% 515 24 1314 χ 1 (CH _g ), Α. \ _Ν - <^ ΝΗΕ (1 87 204 thirty 0.02 1315 0 one Η 75,000 720,000 3,400 507

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1316 I 333 710,000 710,000 97 1317 710,000 710,000 720,000 369 1318 ⁴ Λ ,> ^he 3.7 ± 0.5 63O ± 56.4 2307 ± 926 630 ± 76 3319 ⁴⁷³ "from > ^he 1.8 206 802 270 1320 ⁴ ' ⁶ BUT 8, 0 531 530 419 1321 ⁴ ' ⁷ <0 l ™ 8.0 131 1031 54% ^{8 x} 2-thienyl-2 -il; ² C ₅ -H; ^Z water soluble ; ⁴ B <5 and Kb represent hydrogen; '' K ₃ represents 3- fluorophenyl; ⁶ K ₆ is 3- chlorophenyl; ⁷ H ₃ represents 4-pyridyl; ⁸ % activity @ 10 μM

Table 14. Selectivity profile of A _2b antagonists

Compound ΧΚι to ₂ Data Αι binding Κι (nM) Az A ₂ a A ₂ in 1400 -O-RI Me 41.7 21 10.3 14.6 1401 -O-RI (P) g Me 33 58 8.8 eighteen 1402 -O-Pb (p) C1 Me 825 591 22 60 1403 -Y-pyridin-2-one Me 60 41 eighteen 48 1404 -ΝΗ-ΡΓ Me 49 31 4.6

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Table 15. Compounds selective for A _g adenosine receptor * are at least 10 times more selective than the other three subtypes

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1501 ^ • nn * 1502 NM - \ n-CH 1503 'but * 1504 Τι ol ★

Pages 176-201 relate to compounds specific for the A _2a receptor

Summary of the invention

The present invention also relates to compounds that selectively bind to the A _2a adenosine receptor, thereby treating a disease associated with the A _2a adenosine receptor in a subject by administering to the subject a therapeutically effective amount of such compounds. The disease being treated is associated, for example, with a central nervous system disease, cardiovascular disease, kidney disease, inflammatory disease, gastrointestinal disease, eye disease, allergic disease or respiratory tract disease.

This invention also relates to a compound having the structure:

BUT,\

VI where ΝΚ ₁ Κ ₂ represents a substituted or unsubstituted 4-8 membered ring;

where K3 represents a substituted or unsubstituted four-six-membered ring;

where K5 is H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, where said substituted alkyl is C (K ₇ ) (Κ ₈ ) ΧΚ ₉ , where X is O, 8 or ΝΚιο, where each of K ₇ and K ₈ independently represents H or alkyl, where each of K ₉ and K ₁₀ independently represents alkyl or cycloalkyl, or K ₉ , K ₁₀ and nitrogen together form a substituted or unsubstituted ring containing from 4 up to 7 members;

where K6 represents H, alkyl, substituted alkyl or cycloalkyl;

- 74 007254 provided that ΝΚ ₁ Κ _{2 is} not 3-acetamidopiperidino, 3-hydroxypyrrolidino, 3-methoxycarbonylmethylpyrrolidino,

3-aminocarbonylmethyl or pyrrolidino; provided that ΝΚ ₁ Κ ₂ is 3-hydroxymethylpiperidino only if K ₃ is 4-pyridyl.

The present invention also relates to a method for inhibiting the activity of an A _2a adenosine receptor in a cell, which comprises contacting said cell with the above compounds.

This invention also relates to a compound having the structure:

where ΝΚ ₁ Κ ₂ represents a substituted or unsubstituted 4-8 membered ring;

where K ₃ represents a substituted or unsubstituted four-six membered ring;

where K ₅ represents H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, wherein said substituted alkyl is —C (K ₇ ) (K ₈ ) XK ₉ , where X is O, 8 or ΝΚ ₁₀ where each of K ₇ and K ₈ independently is H or alkyl, where each of K ₉ and K ₁₀ independently is alkyl or cycloalkyl, or K ₉ , K ₁₀ and nitrogen together form a substituted or unsubstituted ring, containing from 4 to 7 members;

where KB is H, alkyl, substituted alkyl or cycloalkyl;

provided that ΝΚ ₁ ΝΚ _{2 is} not 3-acetamidopiperidino, 3-hydroxypyrrolidino, 3-methoxycarbonylmethylpyrrolidino, 3-aminocarbonylmethyl or pyrrolidino; provided that ΝΚ ₁ Κ ₂ is 3-hydroxymethylpiperidino only if K ₃ is 4-pyridyl.

In one embodiment, Compound K ₃ is a substituted or unsubstituted four-membered six ring, phenyl, pyrrole, thiophene, furan, thiazole, imidazole, pyrazole, 1,2,4triazole, pyridine, 2 (1H) -pyridone, 4 (1H) -pyridone, pyrazine, pyrimidine, pyridazine, isothiazole, isoxazole, oxazole, tetrazole, naphthalene, tetraline, naphthyridine, benzofuran, benzothiophene, indole, 2,3-dihydroindole, 1H-indole, indoline, benzopyrazole, 1,3-benzodiazole , purine, coumarin, chromon, quinoline, tetrahydroquinoline, isoquinoline, benzimidazole, quinazoline, pyrido [2,3-b] pyrazine, pi rido [3,4-b] pyrazine, pyrido [3,2-b] pyridazine, pyrido [3,4-b] pyridine, 1H-pyrazole [3,4-b] pyrimidine, pteridine, 2 (1H) -quinolone , 1 (2H) -isoquinolone, 1,4-benzisoxazine, benzothiazole, quinoxaline, quinoline-no-oxide, isoquinoline-^ oxide, quinoxaline-no-oxide, quinazoline-^ oxide, benzoxazine, phthalazine, cinnoline, or has the structure:

where Υ represents carbon or nitrogen;

where K ₃ 'represents H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, halogen, methoxy, methylamino, methylthio.

In another embodiment of the compound, the compound has the structure: Κ ₈ \ (/) πί \ Υα

where m is 1 or 2; where K _A and _K are each independently H, -OH, -CH ₂ OH, -CH ₂ CH ₂ OH, -C (= N ^ 0 ₂ heteroatom, or -C (= 0) ΝΚ ₁₁ Κ ₁₁ '; where K ₁₁ represents aryl, substituted aryl, or heteroaryl; where K ₁₁ ' represents alkyl or XK ₁₁ , where X represents 0 or Ν, and Kp represents substituted alkyl or aryl.

In another embodiment, the compound Κ ₁ Κ ₂ Ν is (E) -2-aminocarbonylpyrrolidino, (E) -2-hydroxymethylpyrrolidino, (E) -2-hydroxymethyl-trans-4-hydroxypyrrolidino, piperazino or 3-hydroxymethylpiperidino.

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

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where m is 0, 1, 2 or 3; where Υ represents O, 8 or ΝΚ, where K represents K _A or K _in ; where K _A and K _in , each independently, represents H, —OH, —CH ₂ OH, —CH ₂ CH ₂ OH, —C (= O ^ H ₂ , heteroatom or —C (= O) KK ₁₁ K ₁₁ '; where K ₁₁ represents aryl, substituted aryl, or heteroaryl; where Kp' represents alkyl or XK ₁₁ , where X represents 0 or Ν, and K ₁₁ represents substituted alkyl or aryl.

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

(Compound 1600)

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

(Compound 1601)

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

(Compound 1602)

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

ABOUT.....

(Compound 1603)

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

N

(Compound 1604)

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

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(Compound 1605)

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

ABOUT-/

(Compound 1606)

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

(Compound 1607)

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

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

This invention further relates to a compound having structure (V):

where K ₅ represents H or methyl.

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

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(Compound 1608)

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

The invention also relates to a method for treating a disease associated with the A _2a adenosine receptor in a subject, comprising administering to the subject a therapeutically effective amount of any of compounds IV or V.

In one embodiment of the method, the compound cures these diseases by stimulating adenylate cyclase.

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

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

In another embodiment of the method, said A _2a adenosine receptor is associated with Parkinson's disease and diseases associated with motor activity, vasodilation, platelet inhibition, neutrophilic superoxide generation, impaired cognition, or senile dementia.

Diseases associated with A ₁ , A _2a , A _2b and A ₃ adenosine receptors are described in No. O-99/06053 and No. 0-09822465, No. 0-09705138, No. 0-09511681, No. 0-09733879, No. 09291089 , PCT / I8 98/16053, and US Pat. No. 5,516,894, the entire contents of which are incorporated herein by reference in their entirety.

The present invention also relates to a water-soluble prodrug of compounds IV or V, wherein said water-soluble prodrug metabolizes ΐη νΐνο to an active drug that selectively inhibits the A2a adenosine receptor.

In one embodiment of the prodrug, said prodrug metabolizes ΐη νΐνο by esterase-catalyzed hydrolysis.

This invention also relates to a pharmaceutical composition comprising a prodrug and a pharmaceutically acceptable carrier.

The present invention also relates to a method for inhibiting the activity of A _2a adenosine receptor in a cell, which comprises contacting the cell with compound IV or V.

In one embodiment of the method, the compound is an antagonist of said A _2a adenosine receptor.

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

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

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

The present invention also relates to a method for treating a gastrointestinal disease in a subject, comprising administering an effective amount of compounds IV or V.

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

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

In another embodiment of the method, the compound is an antagonist of A _2a adenosine receptors.

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The invention further relates to a method for treating a respiratory tract disease in a subject, comprising administering to the subject an effective amount of a compound IV or V.

In one embodiment of the method, said disease is asthma, chronic obstructive pulmonary disease, allergic rhinitis, or upper respiratory tract disease.

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

In another embodiment of the method, the compound is an antagonist of A _2a adenosine receptors.

The present invention also relates to a method for treating eye damage in a subject, which comprises administering to said subject an effective amount of a compound IV or V.

In one embodiment of the method, said damage includes damage to the retina or optic head nerve.

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

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

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

In another embodiment of the method, the compound is an antagonist of A _2a adenosine receptors.

The invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of compounds IV or V and a pharmaceutically acceptable carrier.

In one embodiment of the pharmaceutical composition, said therapeutically effective amount is effective for treating Parkinson's disease and diseases associated with motor activity, vasodilation, platelet inhibition, neutrophilic generation of superoxide, impaired cognition, or senile dementia.

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

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

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

In another embodiment of the pharmaceutical composition, said pharmaceutical composition is a surgical irrigation solution.

This invention also relates to combination therapy for Parkinson's disease, comprising compound IV or V and any of the dopamine enhancing agents.

This invention also relates to combination therapy for cancer, including compound IV or V and any of the cytotoxic agents.

This invention also relates to combination therapy for glaucoma, comprising a compound IV or V and a prostaglandin agonist, a muscarinic agonist or a β-2 antagonist.

This invention also relates to a packaged pharmaceutical composition for treating a subject with an A _2a adenosine receptor disease, comprising: (a) a container containing a therapeutically effective amount of compounds IV or V; and (b) instructions for using said compound for treating said disease in a subject.

The present invention also relates to a process for preparing compound IV, comprising the steps of a) reacting

cyclization to obtain

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

where ΝΚ'Κ ₂ represents a substituted or unsubstituted 4-8 membered ring;

where K ₃ represents a substituted or unsubstituted four-six membered ring;

where Κ5 is H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, wherein said substituted alkyl is —C (Κ ₇ ) (K ₈ ) XK ₉ , where X is O, 8 or ΝΚ ₁₀ , where each of K ₇ and Κ ₈ , independently, is H or alkyl, where each of K ₉ and Κ ₁₀ , independently, is alkyl or cycloalkyl, or Κ ₉ , Κ ₁₀ and the nitrogen atom together form a ring system of 4- 7 members;

where represents H, alkyl, substituted alkyl or cycloalkyl;

provided that ΝΚ'Κ _{2 is} not 3-acetamidopiperidino, 3-hydroxypyrrolidino, 3-methoxycarbonylmethylpyrrolidino, 3-aminocarbonylmethyl or pyrrolidino; provided that ΝΚ'Κ ₂ is 3-hydroxymethylpiperidino only if K ₃ is 4-pyridyl.

This invention also relates to a method for producing compound V, comprising the steps of

a) interactions

obtaining where P represents a removable protecting group; B) processing the product of stage a) under cyclization to obtain

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

6) treating the chlorinated product of step c) initially with dimethylamine and formaldehyde, then with Ν-methylbenzylamine and finally with N11 ₂ 1 <| with receipt

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where In ₁ represents acetamidoethyl; where B ₃ represents 4-pyridyl, where B ₅ represents H or methyl; where Kb is Ν-methyl-K-benzylaminomethyl.

As used herein, the expression “compound is A _2a selective” means that the compound has a binding constant for the A _2a adenosine receptor at least ten times the constant for A _b A _2b or A ₃ adenosine receptors.

The invention is further illustrated by the following examples, which should in no way be construed as being further limiting. The contents of all references pending patent applications and published patent applications cited in this application, including those cited in the "Background of the invention", are incorporated into this description by reference. It should be understood that the models used in the examples are generally accepted models, and demonstrating performance on these models predicts performance in humans.

The invention will be better understood from the following experimental details. However, one skilled in the art will readily understand that the specific methods and results discussed are merely illustrative of the invention, as described in more detail in the following claims.

Example 22. Synthesis of A _2a adenosine antagonists, compounds 1601, 1602 and 1603.

connection 26 connection 27 connection 28 connection 1601

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 successively added and the reaction mixture was heated to 85 ° C. After 22 hours, the reaction mixture was cooled to room temperature and DMF was removed in vacuo. The dark oil was diluted with 2M HC1 (80 ml). The reaction mixture was left to undergo a reaction. After 2 hours, the solution was cooled to 10 ° C and filtered. The solid was washed with cold water and dried, yielding 7.40 g of a yellow solid, compound 27 (69%). Ή-NMR (200 MHz, b ₆ DMSO) b 6.58 (s, 1H), 7.27 (s, 1H), 8.53 (d, 2H, 1 = 5.6 Hz), 9.00 ( d, 2H, 1 = 5.2 Hz), 12.35 (br s, 1H). Mass spectrum (E8): 212.8 (M ⁺ +1).

Compound 27 (7.4 mmol, 29.8 mmol) was diluted with POC13 and heated to 105 ° C. After 18 hours, the reaction mixture was cooled to room temperature and POC1 _{3 was} removed in vacuo. The thick dark oil was diluted with MeOH (75 ml) followed by ether (120 ml). The amorphous red solid was filtered off and washed with ether to give 3.82 g of a red solid. The crude solid, compound 28, is approximately 80% pure and is used without further purification in the next reaction. Ή-NMR (200 MHz, b _6- DMSO) b 6.58 (s, 1H), 7.27 (s, 1H), 8.53 (d, 2H, 1 = 5.6 Hz), 9.00 (d, 2H, 1 = 5.2 Hz), 12.35 (br s, 1H). Mass spectrum (e8): 212.8 (M ⁺ +1).

Compound 1601: DMSO (5 ml) and Ό-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 HUAc and H ₂ O. The layers were separated and the aqueous layer was extracted with HUAc (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 golden brown solid. The solid was recrystallized from ESAc to give 82 mg of a golden yellow solid. Я-NMR (200 MHz, 66-DMSO) b 2.05 (m, 4H), 3.43 (m, 1H), 3.70-4.00 (m, 3H), 4.50 (br.s , 1H), 4.92 (br s, 1H), 6.62 (m, 1H), 7.22 (m, 1H), 8.22 (d, 2H, 1 = 6.0 Hz), 6 64 (d, 2H, 1 = 6.2 Hz); mass spectrum (E8) 296.0 (M ⁺ +1), mp = 210-220 ° C (decomposition).

Compound 1602: Chromatography (silica, 9: 1, CHCl ₃ / MeOH) afforded 10 mg of a golden brown solid (2%). ¹ H-NMR (200 MHz, b6-DMSO) b 2.00-2.50 (m, 4H), 4.05 (m, 1H), 4.21 (m, 1H), 6.71 (d, 1H, 1 = 3.2 Hz), 7.18 (d, 1H, 1 = 3.2 Hz), 8.37 (d, 2H, 1 = 4.8 Hz), 8.56 (d, 2H, 1 = 5.0 Hz). Mass spectrum (E8): 309.1 (M ⁺ +1).

Compound 1603: Chromatography (silica, 20: 1, hexanes / EuAc) afforded 135 mg of a golden brown solid (53%). ¹ H-NMR (200 MHz, b6-DMSO) b 2.00-2.50 (m, 4H), 4.05 (m, 1H),

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4.21 (m, 1H), 6.71 (d, 1H, 1 = 3.2 Hz), 7.18 (d, 1H, 1 = 3.2 Hz), 8.37 (d, 2H, 1 = 4.8 Hz), 8.56 (d, 2H, 1 = 5.0 Hz). Mass spectrum (E8): 309, 1 (M ⁺ +1).

Compound 1605: 60 mg of 2- (4'-pyridyl) 4-chloropyrimidinopyrrole salt in 2 ml of anhydrous DMSO was dissolved in a 50 ml round bottom flask. To the solution was added the salt of trifluoroacetic acid 3- (K) -hydroxy- (O) -prolinol (380 mg) and 500 mg of sodium bicarbonate. Then the mixture was purged with a stream of nitrogen gas for 5 min and heated to 130 ° C. After 2 hours, the reaction mixture was cooled to room temperature and DMSO was removed in vacuo. The residue was partitioned between E! OAc (15 ml) and saturated aqueous sodium bicarbonate (15 ml). The organic layer was separated and washed with brine (15 ml) and dried over Pa ₂ 8O ₄ . After removing the solvent, the crude product was purified by preparative TLC (CH ₂ Cl ₂ / MeOH = 95/5) to obtain 35 mg (50%). ¹ H-NMR (200 MHz, ATP1 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, 2H); 8.7 (d, 2H); 11.0 (1H). Mass spectrum (E8): 312 (M ⁺ +1).

Example 23. Synthesis of A _2a adenosine antagonist, compound 1606.

Compound 28 (200 mg) was treated with DMF (30 ml), 2,2-dimethylglycine methyl ester (73 mg HC1 salt in 2 ml of water) and 500 mg sodium bicarbonate. After 18 h, DMF was removed in vacuo. The residue was partitioned between E1OAc (30 ml) and saturated aqueous sodium bicarbonate (15 ml). The organic layer was washed with brine (15 ml), dried over sodium sulfate, filtered and concentrated. Chromatography (silica, 10: 4, hexanes / E! OAc) afforded 150 mg of pure product, compound 29 (69%). ¹ H-NMR (200 MHz, SPS1 ₃ ) 1.4 (s, 6H); 3.8 (s, 3n); 3.9 (s, 2H); 6.4 (s, 1H); 7.4-7.5 (m, 3H); 8.4 (m, 2H); 9.8 (s, 1H).

Compound 1606: The production method is the same as for compound 1605 (72%). ¹ H-NMR (200 MHz, ATP1 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, 1 = 8.5 Hz, 2H), 10 (s, 1H). Mass spectrum (E8): 424.0 (m ⁺ +1).

The following compounds can be synthesized in a similar manner.

Compound 1600: (51%), mass spectrum (E8) 326.0 (M ⁺ +1).

Compound 1607: ¹ H-NMR (200 MHz, SPS1 ₃ ) 1.40-1.80 (m, 5H), 2.80-3.50 (m, 3H), 4.60-4.80 (m, 3H), 6.66 (d, 1H, 1 = 6.2 Hz), 7.26 (m, 1H), 8.21 (d, 2H, 1 = 6.3 Hz), 8, 65 (d, 2H, 1 = 5.8 Hz), 11.90 (s, 1H). M8 (E8): 310.1 (M ⁺ +1).

Compound 1608: (64%) ¹ H-NMR (200 MHz, d _6- 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 (br s, 1H), 8.23 (d, 2H, 1 = 6.0 Hz), 8.63 ( d, 2H, 1 = 5.4 Hz), 8.82 (broad s, 1H), 11.56 (broad s, 1H). Mass spectrum (E8): 444.0 (M ⁺ +1).

Compound 1609: ¹ H-NMR (200 MHz, SP ₃ OD) 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). M8 (E8): 357.0 (M ⁺ +1).

Table 16. Compounds selective for A _2a- adenosine receptor

* at least 5 times more selective than the other three subtypes Connection Structure Corresponding Κϊ-Α! K ₄ -A _2a Corresponding to _{4th 2b} Corresponding Κχ-Az 1600 ζ “ ^! γγ a / kAA / ^{Ν N} *

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1601 Oh Ν ·· .. ,,, ζ ^0Η * Γ5 ίΤ ^ Ί 'Ν Η \ Ζ 1602 ο ΝΗ _} * \ χ Ύ I 11C 5 II Ί Η II 1 1603 Ο / ^0Η ν / Ζ * ^Μ ^ 1ί ο '' ‘'• Ν Η

- 83 007254

- 84 007254

Pages 202-256 relate to compounds specific for the A ₃ receptor.

Summary of the invention

The present invention is also based on compounds that selectively bind to the A ₃ adenosine receptor, thereby treating a disease associated with the A ₃ adenosine receptor in a subject by administering to the subject a therapeutically effective amount of such compounds. The disease to be treated, include, for example, asthma hypersensitivity, rhinitis, hay fever, serum sickness, allergic vasculitis, atopic dermatitis, dermatitis, psoriasis, eczema, idiopathic pulmonary fibrosis, eosinophilic cholecystitis, chronic airways disease, hypereosinophilic syndromes, eosinophilic gastroenteritis edema, urticaria, eosinophilic myocardial disease, periodic angioedema with eosinophilia, inflammatory bowel disease, ulcers colitis, allergic granulomatosis, carcinomatosis, eosinophilic granuloma, hereditary histiocytosis, hypertension, mast cell degranulation, tumor, cardiac hypoxia, cerebral ischemia, diuresis, renal failure, neurological disease, mental impairment, impaired bronchitis and cognitive impairment , autoimmune disease, Crohn’s disease, Grave’s disease, diabetes, multiple sclerosis, anemia, psoriasis, impaired reproductive ability, systemic lupus erythematosus, reperfusion injury, diameter of brain arterioles, release of allergic mediators, scleroderma, stroke, general ischemia, central nervous system disorder, cardiovascular disease, kidney disease, inflammatory disease, gastrointestinal disease, eye disease, allergic disease, ways or immunological disease.

This invention also relates to a compound having the structure:

where K1 is H and K2 is cyclopropylmethylaminocarbonylethyl, cis3-hydroxycyclopentyl, acetamidobutyl, methylaminocarbonylaminobutyl, ethylaminocarbonylaminopropyl, methylaminocarbonylaminopropyl, 2-acetylamino-3-methylbutylaminoethylaminoethylaminoethylaminoethylaminoethylamine pyrrolylcarbonylaminoethyl, 2-imidazolidinoneethyl, 1-aminocarbonyl-2-methylpropyl, 1-aminocarbonyl-2phenylethyl, 3-hydroxyazetidino, 2-imidazolylethyl, acetamidoethyl, 1- (K) -phenyl-2-hydroxyethyl l, Ν methylaminocarbonylpyridyl-2-methyl, or Cd, K ₂ and nitrogen together represent 3-acetamidopiperidino, 3-hydroxypyrrolidino, 3-methyloxycarbonylmethylpyrrolidino, 3-aminocarbonylmethylpyrrolidino or 3-hydroxymethylpiperidino four-membered, where ₃ ring, pyrrole, thiophene, furan, thiazole, imidazole, pyrazole, 1,2,4triazole, pyridine, 2 (1 N) -pyridone, 4 (1 N) -pyridone, pyrazine, pyrimidine, pyridazine, isothiazole, isoxazole, oxazole, tetrazole, naphthalene, tetralin, naphthyridine, benzofuran, benzothiophene, indole, 2, Dihydroindole, 1H-indole, indoline, benzopyrazole, 1,3-benzodioxole, benzoxazole, purine, coumarin, chromone, quinoline, tetrahydroquinoline, isoquinoline, benzimidazole, quinazoline, pyrido [2,3-b] pyrazine, pyrido [3,4 B] pyrazine, pyrido [3,2-b] pyridazine, pyrido [3,4-b] pyridine, 1H-pyrazole [3,4-b] pyrimidine, pteridine, 2 (1H) -quinolone, 1 (2H) - isoquinolone, 1,4-benzisoxazine, benzothiazole, phthalazine or cinnoline, where K5 is H, alkyl, substituted alkyl, aryl or substituted aryl, where K6 is H, alkyl, substituted alkyl or cycloalkyl.

The present invention also relates to a method for inhibiting the activity of adenosine A3 receptor in a cell, which comprises contacting the cell with the above compounds.

Typical synthesis schemes for preparing the intermediate compounds of desazapurin according to the invention are outlined below in scheme I.

This invention also relates to a method for producing compound IV, comprising the steps of

a) interaction with receipt

- 85 007254 where P is a removable protecting group; B) processing the product of stage a) under cyclization to obtain

c) treating the product from step b) under suitable conditions to obtain

c) treating the chlorinated product of step c) NHΚ ₁ Κ ₂ to obtain

where Κι is H and K ₂ is cyclopropylmethylaminocarbonylethyl, cis3-hydroxycyclopentyl, acetamidobutyl, methylaminocarbonylaminobutyl, ethylaminocarbonylaminopropyl, methylaminocarbonylaminopropyl, 2-acetylamino-3-methylbutyl, di-3-ethylaminoethylamino -pyrrolylcarbonylaminoethyl, 2-imidazolidinoneethyl, 1-aminocarbonyl-2-methylpropyl, 1-aminocarbonyl-2phenylethyl, 3-hydroxyazetidino, 2-imidazolylethyl, acetamidoethyl, 1- (K) -phenyl-2-hydroxyethyl yl, Νmetilaminokarbonilpiridil-2-methyl, or Κι, K ₂ and the nitrogen together are 3-acetamidopiperidine, 3-hydroxypyrrolidine, 3-metiloksikarbonilmetilpirrolidino, 3-aminokarbonilmetilpirrolidino or 3-hydroxymethylpiperidine, where R ₃ represents a substituted or unsubstituted four to six membered ring ;

where K ₅ represents H, alkyl, substituted alkyl or cycloalkyl;

where Κ6 represents H, alkyl, substituted alkyl, aryl or substituted aryl.

This invention also relates to a method for producing compound V, comprising the steps of

a) interactions

with receipt

where P represents a removable protecting group;

B) processing the product of stage a) under cyclization to obtain OH

c) treating the product from step b) under suitable conditions to obtain

c) processing the chlorinated product of step c)

- 86 007254

where K ₁ represents cyclopropylmethyl, methyl, methylamino or aminomethyl;

where K ₃ represents aryl, substituted aryl, heteroaryl;

where K ₅ represents H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, where said substituted alkyl is —C (K ₇ ) (K ₈ ) K ₉ K ₁₀ , where each of K ₇ and K ₈ represents H or alkyl, where each of K ₉ and K ₁₀ represents alkyl or cycloalkyl, or K ₉ , K ₁₀ and the nitrogen atom together form a ring system of 4-7 members, where K ₆ represents H, alkyl, substituted alkyl or cycloalkyl.

This invention additionally relates to a method for producing compound VI, comprising a) interaction

P to obtain

P where P is a removable protecting group;

B) processing the product of stage a) under cyclization to obtain

c) treating the product from step b) under suitable conditions to obtain

c) processing the chlorinated product of step c)

with receipt

where K ₃ represents unsubstituted aryl,

- 87 007254 where Κ ₆ represents H, alkyl, substituted alkyl or cycloalkyl;

where K ₅ represents H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, where said substituted alkyl is —C (K ₇ ) (Κ ₈ ) ΝΚ ₉ Κι ₀ , where each of K ₇ and K ₈ represents H or alkyl, where each of K ₉ and Κ ₁₀ represents alkyl or cycloalkyl, or Κ ₉ , Κ ₁₀ and the nitrogen atom together form a ring system of 4-7 members.

This invention also relates to a compound having the structure:

IV where Κι is H and K ₂ is cyclopropylmethylaminocarbonylethyl, cis3-hydroxycyclopentyl, acetamidobutyl, methylaminocarbonylaminobutyl, ethylaminocarbonylaminopropyl, methylaminocarbonylaminopropyl, ethyl-3-ethyl-3-di-ethyl-3-di-ethyl-3-di-ethyl 2-pyrrolylcarbonylaminoethyl, 2-imidazolidinoneethyl, 1-aminocarbonyl-2-methylpropyl, 1-aminocarbonyl-2-phenylethyl, 3-hydroxyazetidino, 2-imidazolylethyl, acetamidoethyl, 1- (K) -phenyl-2-hydroxy Teal, №metilaminokarbonilpiridil-2-methyl, or

Κι, K ₂ and the nitrogen atom together represent 3-acetamidopiperidino, 3-hydroxypyrrolidino, 3methyloxycarbonylmethylpyrrolidino, 3-aminocarbonylmethylpyrrolidino or 3-hydroxymethylpiperidino, where P3 is substituted or unsubstituted benzene, pyrrole, thiazole, thiazole, thiazole, thiazole 1,2,4-triazole, pyridine, 2 (1H) -pyridone, 4 (1H) -pyridone, pyrazine, pyrimidine, pyridazine, isothiazole, isoxazole, oxazole, tetrazole, naphthalene, tetraline, naphthyridine, benzofuran, benzothiophene, indole, 2,3-dihydroindole, 1H-indole, indoline, benzopyrazole, 1,3-benzodiox l, benzoxazole, purine, coumarin, chromon, quinoline, tetrahydroquinoline, isoquinoline, benzimidazole, quinazoline, pyrido [2,3b] pyrazine, pyrido [3,4-b] pyrazine, pyrido [3,2-c] pyridazine, pyrido [ 3,4-b] pyridine, 1H-pyrazole [3,4-b] pyrimidine, pteridine, 2 (1H) -quinolone, 1 (2H) -isoquinolone, 1,4-benzisoxazine, benzothiazole, quinoxaline, hipolipA-oxide, isoquinoline - ^ oxide, quinoxaline - ^ oxide, quinazoline - ^ oxide, benzoxazine, phthalazine or cinnoline, where P5 is H, alkyl, substituted alkyl, aryl or substituted aryl;

where P6 represents H, alkyl, substituted alkyl or cycloalkyl.

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

In another embodiment, compound K ₃ is phenyl.

In another embodiment, compound P6 is hydrogen or methyl.

In another embodiment, Compound K ₅ is hydrogen, methyl, phenyl, 3chlorophenyloxymethyl or trans-2-phenylaminomethylpyrrolidinomethyl.

The present invention further relates to a compound having the structure:

- 88 007254 where t is 0, 1 or 2;

where K ₁ represents cyclopropylmethyl, methyl, methylamino or aminomethyl;

where K ₃ represents aryl, substituted aryl or heteroaryl;

where K ₅ represents H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, where said substituted alkyl is —C (K ₇ ) (K ₈ ) ΝΚ ₉ Κ ₁₀ , where each of K ₇ and K ₈ represents H or alkyl, where each of K ₉ and K ₁₀ represents alkyl or cycloalkyl, or K ₉ , K ₁₀ and nitrogen together form a ring system containing from 4 to 7 members;

where K ₆ represents H, alkyl, substituted alkyl or cycloalkyl.

In one embodiment of compound V, t is 0, and K ₃ represents phenyl.

In another embodiment of compound V, t is 1, and K ₃ is phenyl.

In another embodiment of compound V, t is 2, and K ₃ is phenyl.

In another embodiment, compounds V, K ₅ and K ₆ are methyl.

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

(Compound 1316)

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

(Compound 1311)

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

(Compound 1202)

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

’ΝΗ (Compound 1310)

- 89 007254

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

The invention further relates to a compound having the structure: n ^.

Eun

(Compound 609)

The invention also relates to a compound having the structure:

where K ₃ represents unsubstituted aryl;

where K ₆ represents H, alkyl, substituted alkyl or cycloalkyl;

where K ₅ represents H, alkyl, substituted alkyl, aryl, arylalkyl, amino, substituted aryl, where said substituted alkyl is ^ (Κ ₇ ) (Κ ₈ ) ΝΚ ₉ Κ ₁₀ , where each of K ₇ and K ₈ represents H or alkyl, where each of K ₉ and K ₁₀ represents alkyl or cycloalkyl, or K ₉ , K ₁₀ and nitrogen together form a ring system containing from 4 to 7 members.

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

(Compound 1309)

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

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

- 90 007254

This invention also relates to a compound having the structure:

where K ₁ represents

3-hydroxycyclopentylethylaminocarbonylaminopropyl, Ν, Ν-diethylaminocarbonylaminoethyl, thioacetamidoethyl, 3-aminoacetyloxycyclopentyl, 3-hydroxycyclopentyl, 2-pyrrolylcarbonylaminoethyl-2-aminomethyl-2-amyl-2-ethyl-2-amyl-2-ethyl-2-amyl-ethyl-2-amyl-ethyl-2-ethyl-2-adeno-2-ethyl-2-ethyl-2-adeno-2-amino-2-amino-ethylbutylaminoethyl-2-amino-2-amino-ethylbenzyl. acetamidoethyl, 1- (K) -phenyl-2-hydroxyethyl or Ν-methylaminocarbonylpyridyl-2-methyl;

where K ₅ and K ₆ independently represent H, substituted or unsubstituted alkyl or aryl.

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

(Compound 1700)

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

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

(Compound 1702)

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

ΝΗ

(Compound ’704)

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

(Compound 1705)

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

(Compound 1706)

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

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

- 92 007254

In (Compound 1707) another embodiment of the compound, the compound has the structure:

(Compound 1708) in another embodiment of the compound, the compound has the structure:

(Compound 1709) in another embodiment of the compound, the compound has the structure:

- 93 007254

(Compound 1710)

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

(Compound 1712)

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

(Compound 1713)

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

(Compound 1714)

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

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

- 94 007254

(Compound 1715)

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

This invention also relates to a compound having the structure:

To ’\ /

where B _s B ₂ and the nitrogen atom together represent a 3-hydroxypyrrolidine, 3-metiloksikarbonilmetilpirrolidino, 3-aminokarbonilmetilpirrolidino or 3-hydroxymethyl;

where B ₅ and Bb independently represent H, substituted or unsubstituted alkyl or aryl.

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

(Compound 1711)

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

- 95 007254

(Compound 1703)

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

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

(Compound 1716)

In another embodiment of the compound, the compound has the structure: H ₃ C

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

- 96 007254

(Compound 1717)

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

(Compound 1718)

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

- 97 007254

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

The present invention also relates to a method for treating a disease associated with an A3 adenosine receptor in a subject, comprising administering to the subject a therapeutically effective amount of any of compounds IV, V, VII or VIII.

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

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

In another embodiment of the method, said A3 adenosine receptor is associated with a central nervous system disease, cardiovascular disease, asthma, hypersensitivity, rhinitis, hay fever, serum sickness, allergic vasculitis, atopic dermatitis, dermatitis, psoriasis, eczema, idiopathic pulmonary fibrosis, eososis cholecystitis, chronic respiratory disease, hypereosinophilic syndrome, eosinophilic gastroenteritis, edema, urticaria, eosinophilic disease myocardial infarction, recurrent angioedema with eosinophilia, inflammatory bowel disease, ulcerative colitis, allergic granulomatosis, carcinomatosis, eosinophilic granuloma, hereditary histiocytosis, hypertension, mast cell degranulation, tumor, cardiac neurocystic disease, cerebral neurocystic disease, violation, cognitive impairment, myocardial ischemia, bronchostenosis, arthritis, autoimmune disease, disease Crohn's disease, Grave’s disease, diabetes, multiple sclerosis, anemia, psoriasis, impaired reproductive ability, systemic lupus erythematosus, reperfusion injury, diameter of the arteriole of the brain, release of allergic mediators, scleroderma, stroke, general ischemia, impaired central nervous system, cardiovascular disease kidney disease, inflammatory disease, gastrointestinal disease, eye disease, allergic disease, respiratory disease or immunological disease.

Diseases associated with L1, A2a, A2b, and A3 by adenosine receptors are described in No. 0 99/06053 and No. 0-09822465, No. 0-09705138, No. 0-09511681, No. 0-09733879, No. 09291089, PCT / i8 98 / 16053 and SCL patent No. 5516894, the full contents of which in their entirety are incorporated into this description by reference.

This invention also relates to a water-soluble prodrug of any of compounds IV,

V, VI, VII or VIII; where said water-soluble prodrug metabolizes ίη νίνο to an active drug that selectively inhibits the A3 adenosine receptor.

In one embodiment of the prodrug, said prodrug metabolizes ίη νίνο by esterase-catalyzed hydrolysis.

This invention also relates to a pharmaceutical composition comprising a prodrug and a pharmaceutically acceptable carrier.

This invention also relates to a method for inhibiting the activity of adenosine receptor A3 in a cell, which comprises contacting said cell with any of compounds IV, V,

VI, VII or VIII.

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

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

- 98 007254

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

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

The invention also relates to a method for treating a gastrointestinal disease in a subject, comprising administering an effective amount of any of compounds IV, V, VI, VII or VIII.

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

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

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

The invention further relates to a method for treating a respiratory tract disease in a subject, comprising administering to the subject an effective amount of any of compounds IV, V, VI, VII or VIII.

In one embodiment of the method, said disease is asthma, chronic obstructive pulmonary disease, allergic rhinitis, or upper respiratory tract disease.

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

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

The present invention also relates to a method for treating eye damage in a subject, which comprises administering to said subject an effective amount of any of compounds IV, V, VI, VII or VIII.

In one embodiment of the method, said damage includes damage to the retina or optic head nerve.

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

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

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

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

This invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of any of compounds IV, V, VI, VII or VIII and a pharmaceutically acceptable carrier.

In one embodiment of the pharmaceutical composition, said therapeutically effective amount is effective for treating a respiratory or gastrointestinal disease.

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

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

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

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

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

In another embodiment of the pharmaceutical composition, said pharmaceutical composition is a surgical irrigation solution.

This invention also relates to a packaged pharmaceutical composition for treating a disease associated with an A3 adenosine receptor in a subject, comprising: (a) a container containing a therapeutically effective amount of any of compounds IV, V, VI, VII or VIII; and (b) instructions for using said compound for treating said disease in a subject.

Compounds represented by formulas IV, V, VI, VII or VIII can be synthesized in accordance with schemes EX.

As used herein, the expression “compound is A ₃ selective” means that the compound has a binding constant for the A3 adenosine receptor that is at least ten times the constant for A ', A _2a or A _2b adenosine receptors.

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The invention is further illustrated by the following examples, which should in no way be construed as being further limiting. The contents of all references pending patent applications and published patent applications cited in this application, including those cited in the "Background of the invention", are incorporated into this description by reference. It should be understood that the models used in the examples are generally accepted models, and a demonstration of effectiveness on these models predicts human performance.

One skilled in the art will know that the metabolism of the compounds described herein in a subject produces certain biologically active metabolites that can serve as medicaments.

The invention will be better understood from the following experimental details. However, one skilled in the art will readily understand that the specific methods and results discussed are merely illustrative of the invention, as described in more detail in the following claims.

Example 24. Experimental L3 adenosine antagonists.

Compound 1700 (table 17, below): mass spectrum (E8) 3bb, 1 (M ⁺ +1).

Compound 1710 (table 17, below): mass spectrum (E8) 381.1 (M ⁺ +1).

Compound 131b (table 17, below): mass spectrum (E8) 353.2 (M ⁺ +1).

Compound 1703 (table 17, below): mass spectrum (E8) 357.1 (M ⁺ +1).

Compound 1719 (table 17, below): ¹ H-NMR (200 MHz, non-DMSO) 1.75 (m, 2H), 3.11 (m, 2H), 3.35 (s, 3H), 3, 59 (m, 2H), 5.72 (m, 1H), 5.9b (m, 1H), b, 55 (s, 1H), 7.15 (s, 1H), 7.49 (m, 2H) ), 8.32 (m, 2H).

Compound 1704 (table 17, below): mass spectrum (E8) 3b7.0 (M ⁺ +1).

Compound 170b (table 17, below): ¹ H-NMR (200 MHz, CISE) b 1.22 (m, 2H), 1, b0-2.40 (m, 4H), 4.53 (m, 1H) 4.94 (m, 1H), 5.70 (d, 1H, 1 = 8.2 Hz), b, 35 (d, 1H, 1 = 2.8 Hz), b, 97 (d, 1H, 1 = 2.0 Hz), 7.50 (m, 3H), 8.40 (m, 2H), 10.83 (br s, 1H).

Compound 1707 (table 17, below): mass spectrum (E8) 347.0 (M ⁺ +1).

Compound 1708 (table 17, below): mass spectrum (E8) 399.0 (M ⁺ +1).

Compound 1709 (table 17, below): mass spectrum (E8) 385.9 (M ⁺ +1).

Compound 1710 (table 17, below): mass spectrum (E8) 434.0 (M ⁺ +1).

Compound 1711 (table 17, below): ¹ H-NMR (200 MHz, C1JOO) b 3.95 (d, 2H, 1 = 5.8 Hz), 4.23-4.31 (m, 2H), 4 53 (t, 2H, 1 = 8.8 Hz), b, 30 (d, 1H, 1 = 3.0 Hz), b, 98 (d, 1H, 1 = 3.0 Hz), 7.45 -7.48 (m, 3H), 7.83-8.42 (m, 2H), 9.70 (br s, 1H). Mass spectrum (E8) 281.1 (M ⁺ +1).

Compound 1712 (Table 17, below): ¹ H-NMR (200 MHz, C1) _; O1)) b 3.02 (m, 2H), 3.92 (m, 2H), 5.09 (2.2H), b, 53 (s, 1H), b, 90-7.04 (br. s, 1H), b, 92 (m, 2H), 7.02 (m, 1H), 7.21 (dd, 1H, 1 = 8.2 Hz), 7.40 (m, 3H), 7, 50-7.80 (br s, 1H); 8.33 (m, 2H). Mass spectrum (E8) 445.1 (M ⁺ +1).

Compound 1713 (table 17, below): ¹ H-NMR (200 MHz, b 1, b5-1.80 (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, 1b-3.38 ( m, 1H), 3.45 (d, 1H, 1 = 14 Hz), 3.533, b0 (m, 2H), 3.84-3.92 (m, 2H), 3.97 (d, 1H, 1 = 14 Hz), 5.55 (t, 1H, 1 = 5.8 Hz), b, 17 (s, 1H), b, 55-b, 59 (m, 2H), b, b4-b, 71 (m , 1H), 7.11-7.19 (m, 2H), 7.43-7.4b (m, 3H), 8.38-8.42 (m, 2H). Mass spectrum (E8) 484.0 (M ⁺ +1).

Compound 1714 (table 17, below): Mass spectrum (E8) 471.0 (M ⁺ +1).

Compound 1715 (table 17, below): Mass spectrum (E8) 505.0 (M ⁺ +1).

Compound 171b (table 17, below): ¹ H-NMR (200 MHz, C1JOO) b 1, b5 (m, 1H), 2.18 (m, 1H), 2.49 (broad d, 2H, 1 = b, 2 Hz), 2, b4 (m, 1H), 3.38 (m, 1H), 3, b9 (s, 3H), 3.72 (m, 1H), 3.93 (m, 1H) 4.10 (m, 1H), 5.0b (2.2H), b, 58 (s, 1H), b, 92 (m, 2H), 7.02 (m, 1H), 7.23 ( dd, 1H, 1 = 8.1 Hz), 7.39 (m, 3H), 8.32 (m, 2H).

Compound 1717 (Table 17, below): ¹ H-NMR (200 MHz, C1) _; O1)) b 1, b9 (m, 1H), 2.2b (m, 1H), 2.42 (d, 2H, 1 = 7.4Hz), 2.72 (m, 1H), 3.53 ( m, 1H), 3.83 (m, 1H), 4.02 (m, 1H), 4.14 (dd, 1H, 1 = 10, b, 7.0Hz), 5.14 (2.2H) b, b9 (s, 1H), b, 9b (m, 2H), 7.0b (m, 1H), 7.25 (dd, 1H, 1 = 8.0t), 7.39 (m, 3H ), 8.35 (m, 2H). Mass spectrum (E8) 4b2.2 (M ⁺ +1).

Compound 1718 (Table 17, below): ¹ H-NMR (200 MHz, C1) _; O1)) b 1.40-2.00 (m, 5H), 3.52 (d, 2H, 7, b Hz), 3.80-4.00 (m, 1H), 4.00-4, 20 (m, 3H), 4.50 (m, 2H), b, 3b-b. 50 (m, 2H), b, 54 (s, 1H), b, 84-b, 92 (m, 1H) 7.05 (t, 1H, 1 = 8.2 Hz), 7.30-7.45 (m, 3H), 8.24 (d, 2H, 1 = 9.8 Hz). Mass spectrum (E8) 449.0 (M ⁺ +1).

- 100 007254

Table 17. Compounds selective for the A ₃ adenosine receptor * are at least 10 times more selective than the other three subtypes

Compound Structure Κι-Αχ K1-A _2a Kx-A _2b Κχ-az 1202 X one * BUT Cm, 'y - see SU- N 1700 V g 2 sn SU- N 1309 ABOUT ★ about l Ag one CH " ί 1Ϊ L- ^ n and

- 101 007254

1701 n> s * / ^{n, s} \ / ⁿ \ ^ o ΝΗ 1 Sn> Λ BUT ΧΥ Η 1311 0 = 0 ΗΝ * ^ ΝΗ Ο Α Η 1312 / ^sn> * ΗΝ ΗΝ _ θΛ Sn W-sn. Η

- 102 007254

1310 n, s ΝΗ 0 ^<:! ί ^!! ί ^ · ΝΗ Α sA CH Γ> - «· Η * 1316 Η _? About Ν ^{'!! :::} * ^## ^ ^Xv HH I * V Γ CH οΑ Η 1702 ^Η ' ^ς η ^ ⁵ ΗΝ * ★ α _νμ Λ ’ ^ α0 2 CH _a 0 ^x Η

- 103 007254

1703 b KA'u-- ' x / l * σ -L / L AND 1704 g * Mon 0 <Ά V Sun ₅ one ·> Ag CH * ^ Ν M 1705 ΝΜ, * at. = 0 0 from to NN I Sn ό At - Sleep ABOUT' n 1706 but l ’ΙΜ > * ΓΊ · / N

- 104 007254

1707 BUT ΗΝ .. oh " * 1708 .0 X ^ ^m \ ^ ΝΗ * 1709 ^CM> y - * \ θΛ, Α-yk- / * 1710 οχ θΧ / Ο ~ ΧΙ ^ *

- 105 007254

1711 n ° ό Ν * 1712 0— to 1713 sn »^ 4 θ '^ ιΛ— ^Η \ γ to 1714 od to

- 106 007254

- 107 007254

This invention relates to a compound having the structure:

1505

This invention also relates to a compound having the structure:

1506

This invention also relates to a compound having the structure:

1507

This invention also relates to a compound having the structure:

1508

This invention also relates to a compound having the structure:

1509

This invention also relates to a compound having the structure:

- 108 007254

1510

This invention also relates to a compound having the structure:

1511

This invention also relates to a compound having the structure:

1512

This invention also relates to a compound having the structure:

1513

This invention also relates to a compound having the structure:

1514

This invention also relates to a compound having the structure: OH

ΗΝ

1515

- 109 007254

This invention also relates to a compound having the structure: OH

1516

This invention also relates to a compound having the structure:

IT

ΗΝ

1517

This invention also relates to a compound having the structure:

1518

This invention also relates to a compound having the structure:

This invention also relates to a compound having the structure: OH

ΗΝ

1520

In a further embodiment, the invention relates to a method for treating a disease associated with an A ₁ adenosine receptor in a subject, comprising administering to the subject a therapeutically effective amount of compounds 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1516, 1517 , 1518, 1519 or 1520.

- 110 007254

In a further embodiment, the invention relates to the aforementioned method, wherein the subject is a mammal.

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

In a further embodiment, the invention relates to the aforementioned method, wherein said A1 adenosine receptor is associated with impaired cognitive ability, renal failure, cardiac arrhythmia, airway epithelium, transmitter release, sedation, constriction of blood vessels, bradycardia, negative cardiac inotropy, and dromotropia, bronchostenosis, neutrophilic chemotaxis, reflux or peptic ulcer.

In a further embodiment, the invention relates to a water-soluble prodrug of compound 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1516, 1517, 1518, 1519 or 1520, where the water-soluble prodrug metabolizes ίη νίνο to give an active drug an agent that selectively inhibits the A ₁ adenosine receptor.

In a further embodiment of the invention, said prodrug metabolizes ίη νίνο by esterase-catalyzed hydrolysis.

In a further embodiment, the invention relates to a pharmaceutical composition comprising the aforementioned prodrug and a pharmaceutically acceptable carrier.

In a further embodiment, the invention relates to a method for inhibiting A1 activity of an adenosine receptor in a cell, which comprises contacting the cell with compounds 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1516, 1517, 1518, 1519 or 1520.

In a further embodiment, the invention relates to the above method of inhibiting A1 activity of an adenosine receptor in a cell, wherein the compound is an A1 antagonist of the adenosine receptor.

In a further embodiment, the invention relates to the above method of inhibiting the activity of the A ₁ adenosine receptor in a cell, wherein the cell is a human cell.

In a further embodiment, the invention relates to the above method of inhibiting activity A! an adenosine receptor in a human cell, where the compound is an A1 antagonist of the adenosine receptor.

In a further embodiment, the invention relates to a method for treating a subject with an A1 adenosine receptor disease, wherein said disease is asthma, chronic obstructive pulmonary disease, allergic rhinitis or upper respiratory tract disease.

In a further embodiment, the invention relates to a method for treating a subject with an A1 adenosine receptor disease, wherein said disease is asthma, chronic obstructive pulmonary disease, allergic rhinitis or upper respiratory tract disease, and where the subject is a human.

In a further embodiment, the invention relates to a method for treating the above disease, wherein said compound is an A ₁ antagonist _of adenosine receptors.

In a further embodiment, the invention relates to combination asthma therapy comprising a compound of 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1516, 1517, 1518, 1519 or 1520 and a steroid, β2 agonist, glucocorticoid, lucotriene antagonist or anticholinergic agonist.

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

In a further embodiment, the invention relates to a method for treating a respiratory tract disease using compound 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1516, 1517, 1518, 1519 or 1520, wherein said disease is asthma, allergic rhinitis, or chronic obstructive pulmonary disease.

In a further embodiment, the invention relates to the aforementioned pharmaceutical composition (s), wherein said pharmaceutical composition is a preparation for periocular, retrobulbar or intraocular injection.

In a further embodiment, the invention relates to the aforementioned pharmaceutical composition (s), wherein said pharmaceutical composition is a systemic preparation.

In a further embodiment, the invention relates to the aforementioned pharmaceutical composition (s), wherein said pharmaceutical composition is a surgical irrigation solution.

In a further embodiment, the invention relates to a packaged pharmaceutical composition for treating a disease associated with the A1 adenosine receptor in a subject, 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

- 111 007254 (b) instructions for using said compound for treating said disease in a subject.

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

In yet another further embodiment, the invention relates to a method for treating a disease associated with an A ₁ adenosine receptor in a subject, wherein the A ₁ adenosine receptor is associated with congestive heart failure.

Illustrative example

Example 21. Synthesis of amide 1- [6- (4-hydroxy-4-phenylpiperidin-1-ylmethyl) -2-phenyl-7H-pyrrolo [2,3-y] pyrimidin-4-yl] pyrrolidin-2-carboxylic acid (1505 )

Compound 1505 was synthesized in a manner analogous to the method of example 17, using the synthesis scheme of IX and L-prolinamide and 4-phenylpiperidin-4-ol, obtaining:

'H-NMR (th _6- DMSO) th 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, 1 = 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 (br s , 1H); mass spectrum (E8): 305.1 (M ⁺ +1); mp = 234-235 ° C.

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

Compound 1506 was synthesized using synthesis scheme VII and P-prolinamide, obtaining:

1506 'H-NMR (th _6- DMSO) th 2.05 (m, 4H), 3.85 (m, 1H), 4.05 (m, 1H), 4.70 (d, 1H, 1 = 8 , 0 Hz), 6.58 (broad s, 1H), 6.95 (broad s, 1H), 7.15 (d, 1H, 1 = 3.4 Hz), 7.40 (m, 3H ), 7.50 (br s, 1H), 8.40 (m, 2H), 11.6 (br s, 1H); mass spectrum (E8): 308.3 (m ⁺ +1). Mp = 236-238 ° C.

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

Compound 1507 was prepared using compound 23 as synthesis IX, yielding:

predecessor scheme

23 Bromide (4.23 g, 10 mmol) was dissolved in anhydrous methanol (60 ml) and DCM (120 ml) and treated with Ag0 ₂ CCP ₃ in an N2 atmosphere at room temperature for 1 h. The solid was removed by filtration and washed DXM (2x20 ml). The filtrate was concentrated in vacuo. The residue was redissolved in DCM (80 ml). The resulting solution was then washed with saturated NaHCO ₃ solution and brine, dried over MD80 ₄ , filtered and concentrated to give 3.71 g (4, 99%) of an off-white solid. 'H-NMR (SES 1 _h ) 1.75 (s, 9H), 3.51 (s, 3H), 4.83 (s, 2H), 6.70 (s, 1H), 7.47 (m 3H); 8.52 (m, 2H).

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1507

Aryl chloride 4 (2.488 g, 6.55 mmol), DMSO (15 ml), L-prolinamide (4.0 g, 35.0 mmol) and No. HCO ₃ (2.9 g) were combined and heated to 120 ° C. nitrogen atmosphere. After 4 hours, the reaction mixture was cooled to room temperature and diluted with water (60 ml). The resulting suspension was extracted with DCM (10x). The combined organic layers were washed with saturated HCO ₃ solution and brine, dried over MgSO ₄ , filtered and concentrated to give 2.4-8 g of a brown solid. The pure product (1.86 g, 81%) was obtained after flash chromatography as a white solid. White crystals obtained from a mixture of THF / hexane. Mp = 213-215 ° C. ¹ H-NMR (SES1 ₃ ) 4 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, 1 = 8.2 Hz), 5.49 (broad s, 1H), 6.48 (s, 1H), 7 08 (br s, 1H), 7.42 (m, 3H), 8.38 (m, 2H), 9.78 (br s, 1H); mass spectrum (E8): 352.2 (M ⁺ +1).

Example 24. Synthesis of 4-hydroxy-1- (2-phenyl-7H-pyrrolo [2,3-4] pyrimidin-4-yl) pyrrolidin-2 carboxylic acid amide (1508).

Compound 1508 received hydroxyproline amide, receiving:

using cis

¹ H-NMR (4 _6- DMSO) 4 1.90 (m, 1H), 3.85 (d, 1H, 1 = 9.2 Hz), 4.08 (m, 1H), 4.37 (s , 1H), 4.67 (dd, 1H, 1 = 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 (br s, 1H); mass spectrum (E8): 324.2 (M ⁺ +1); mp = 268-271 ° C.

Example 25. Synthesis of 3- [4 - ((8) -2-carbamoyl-pyrrolidin-1-yl) -2-phenyl-7H-pyrrolo [2,3-4] pyrimidine-

6-yl] propionic acid (1509).

Compound 1509 was prepared using compound 23 as a precursor in synthesis scheme IX, obtaining:

Tert-Butoxycarbonyl protected, aryl bromide 23 (4.0 g, 9.5 mmol), dry DMSO (25 ml), No. H ₂ PO ₄ (454 mg, 3.79 mmol) and No. H ₂ PO ₄ (1.62 g , 11.4 mmol) were combined and heated at 50 ° C under argon for approximately 3.5 hours. The mixture was then poured into water (200 ml) and extracted with three portions of 100 ml of E! OAc. The combined organic layers were washed thoroughly with water, brine, dried over MgSO ₄ , filtered and concentrated to give a yellow solid, which was purified by trituration with ethanol to obtain 1.55 g of a pale yellow solid (7). The mother liquor was purified by flash chromatography (10% E! OAc in hexane) to give an additional 454 mg (60%). ¹ H-NMR (COS1 ₃ ) 4 1.77 (s, 9H), 7.25 (s, 1H), 7.48 (m, 3H), 8.52 (m, 2H) 10.39 (s, 1H); mp = 156 ° C (decomp.).

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Aldehyde 7 (600 mg, 1.7 mmol) was dissolved in anhydrous THF (20 ml) and cooled to 0 ° C in argon atmosphere. A solution of tert (butoxycarbonylmethylene) triphenylphosphorane (694 mg, 1.8 mmol) in 10 ml of dry THF was added dropwise through a cannula through a cannula. After 3 hours, the mixture was concentrated and purified by trituration with ethanol to give 565 mg (73%) of a white solid (8). ¹ H-NMR (SPS1 ₃ ) b 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 of THF was diluted to 100 ml of EuAc. After adding 600 mg of the catalyst (5 wt.% Pb, 50% H ₂ O) and purging with argon, the mixture was hydrogenated at atmospheric pressure. After 8 hours, the mixture was filtered, concentrated and purified by flash chromatography (10% EJAc in hexane), isolating 200 mg (35%) 9 as a clear oil, which crystallized upon standing. ¹ H-NMR (SBSl) b 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 E-prolinamide (440 mg, 4.4 mmol) were combined and heated to 85 ° C in 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 was washed with EyAc (3x). The combined organic layers were washed thoroughly with water, 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%). Mass spectrum (E8): 435.8 (M ⁺ +1).

1509

Ester 10 (30 mg, mmol) in 5 ml of dioxane was hydrolyzed by adding 0.5 ml of concentrated HCl. After 3 hours, the mixture was concentrated in vacuo and recrystallized from a mixture of EUN / EYAc, getting 1509 as a white solid (20 mg, 61%). Mass spectrum (E8): 380 (M ⁺ +1).

Example 26. Synthesis of [X- (2-phenyl-6-aminocarbonylmethoxymethyl-7H-pyrrolo [2,3-b] pyrimidin-4yl) - (E)] - prolinamide (1510).

Compound 1510 was prepared using compound 23 as a precursor to the scheme.

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23 Bromide (', 27 g, 3 mmol) and molecular sieves (5 g) were stirred in anhydrous methyl glycolate (5.8 g, 60 mmol) and DCM (40 ml). The solution was treated with AaOTH in an atmosphere of Ν ₂ and allowed to stir for 3 hours. The solid was removed by filtration and washed with DCM (2 ml). The filtrate was concentrated in vacuo. The residue was redissolved in DCM (80 ml). The resulting solution was then washed with water, saturated NaHCO3 solution and brine, dried over MgSO ₄ , filtered and concentrated to obtain ', 35 g (99%) of an off-white solid (' 2). 'H-NMR (SBS _{1 3} ) 6', 75 (s, 9H), 3.80 (s, 3H), 5.0 (s, 2H), 6.78 (s, 'H), 7.47 ( m, 3H); 8.52 (m, 2H).

Aryl chloride '2 ('77 mg, 0.4' mmol), DMSO ('0 ml), L-prolinamide (466 mg, 4 mmol) and \ a11CO ₃ (500 mg) were combined and heated to '20 ° C in the atmosphere argon. After 4 hours, the reaction mixture was cooled to room temperature and diluted with water (60 ml). The resulting suspension was extracted with DCM (5x30 ml). The combined organic layers were washed with saturated sodium NaNSOz and saturated brine, dried over Md8O _4, filtered and concentrated to give a brown solid. The pure product ('54 mg, 92%) was obtained after flash chromatography as a white solid ('3). 'H-NMR (ΟϋΟ1 ₃ ) 6 2,' 5 (m, 3H), 2.52 (m, 'H), 3.55 (s, 3H), 4.58 (s, 2H), 5.08 (s, 'H,), 5.85 (broad s,' H), 6.48 (s, 'H), 7.08 (broad s,' H), 7.42 (m, 3H) 8.40 (m, 2H); 0.58 (br s, 'H); mass spectrum (E8): 4'0, '(M ⁺ +').

Methyl ester 13 (124 mg, 0.3 mmol) was dissolved in HOSN ₃ (15 ml). Ammonia was bubbled through the solution for 0.5 hours. The reaction mixture was then stirred for another 3 hours at room temperature. After removal of the solvent, 111 mg of a white solid (1510, 93%) was obtained. 'H-NMR (ΟϋΟ1 ₃ ) 6', 82 (m, 3H), 2.20 (m, 'H), 2.80 (m,' H), 3, 0 (m, 'H), 3 , 63 (dd, 2H, 1 '=' 3.8Hz,

1 ₂ = '9.4Hz), 3.87 (m,' H), 4.07 (m, 'n), 4.97 (m,' H), 5.96 (m, 2n), 6, 35 (s, 'H), 6.86 (broad s,' H), 7, '' (broad s, 'H), 7.37 (m, 3H), 8.28 (m, 2H) , '', 46 (broad s, 'H); mass spectrum (E8): 394.8 (M ⁺ + ').

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

Compound 1511 was prepared using compound 15 as a precursor to synthetic scheme VII, obtaining:

Pyrrolopyrimidine 15 (2.00 g, 7) was added to a suspension of sodium hydride (780 mg of a 60% suspension in oil, 9.5 mmol) in anhydrous DMF (20 ml), cooled in an ice / water bath, in a stream of nitrogen. , 52 mmol) in DMF (10 ml) for 5 min. After 15 minutes, benzenesulfonyl chloride (1.2 ml, 9.40 mmol) was added, then the cooling bath was removed. After 4 h, the reaction mixture was poured into a mixture of ice and saturated solution \ a11SO _3, the precipitated solid was filtered and triturated with acetone (3) and methanol (2) to give 2.37 g of beige solid. This solid contains approximately '0 mol.% DMF (on this basis, the yield was 83%) and can be used in the next stage; a pure sample can be obtained by chromatographic purification on silica gel using acetone as eluent. 'H-NMR (COS1 ₃ ) 6 6.70 (d, 1 = 4.2Hz,' H), 7.47-7.68 (m, 6H),

- ’’ 5 007254

7.76 (d, 1 = 4.2 Hz, 1H), 8.24-8.32 (m, 2H), 8.48-8.56 (m, 2H); IR (solid): n = 3146 cm ^-1 , 1585, 1539, 1506, 1450, 1417, 1386, 1370, 1186, 1176, 1154, 1111, 1015, 919, 726, 683, 616, 607; Mass spectrum (EB): 372/370 (MH ⁺ ); mp = 226-227 ° C.

To a solution of Ν-sulfonyl substituted compound 16 (337 mg, 0.911 mmol) in dry THF (34 ml) cooled with a dry ice / acetone mixture was added BEA · THF (1.0 ml, 1.5 M solution in cyclohexane, 1.5 mmol). After 45 minutes, carbon dioxide was bubbled into the solution for 5 minutes, then the cooling bath was removed. When the solution reached ambient temperature, the solvents were evaporated, yielding 398 mg of salt 17 containing approximately 0.5 equiv. (1Pr) ^ CO ₂ B1, in the form of a yellow solid. The salt was used without purification in the next step. ¹ H-NMR (b _6- DMSO) b 6.44 (s, 1H), 7.50-7.75 (m, 6H), 8.33-8.40 (m, 2H), 8.53 ( dd, 6 = 8.0, 1.6Hz, 2H).

A solution of lithium salt 17 (50 mg) and B-prolinamide (122 mg, 1.07 mmol) in DMSO (1.5 ml) was heated under a stream of nitrogen at 80 ° C for 15.5 hours. 4% was added to the cooled solution aqueous acetic acid (10 ml) and the mixture was extracted with EyAc (5x10 ml). The combined organic layers were washed with 4% aqueous acetic acid (10 ml), water (10 ml) and brine (10 ml) and dried over MDBO ₄ . Filtration and concentration gave 40 mg of 18 as a yellowish solid, which was used without purification in the next step. ¹ H-NMR (SP ₃ OE) b 1.95-2.36 (m, 4H), 3.853.95 (m, 1H), 3.95-4.17 (m, 1H), 4.72 (broad .s, 1H), 7.14 (s, 1H), 7.35-7.45 (m, 3H), 7.45-7.70 (m, 3H), 8.338.50 (m, 4H).

A solution of sodium hydroxide in methanol (1.5 ml, 5M, 7.5 mmol) was added to a solution of pyrrolopyrimidine 18 (40 mg, 0.081 mmol) in methanol (2 ml). After 2 h, the pH was adjusted to 5, most of the methanol was evaporated, the mixture was extracted with ЕУАс (5x10 ml), the combined organic layers were washed with brine and dried over MDBO ₄ . Filtration and concentration gave 24 mg of a pale yellow solid, which was triturated with toluene / E! OAc / MeOH to give 15.6 mg (55%) of acid 1511 as a slightly yellow solid. ¹ H-NMR (CO ₃ OD) b 2.05-2.20 (m, 4H), 3.954.10 (m, 1H), 4, 15-4.25 (m, 1H), 4.85 (broad s, 1H), 7.14 (s, 1H), 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; mass spectrum (EB): 352 (M ⁺ +1); mp = 220 ° C (decomp.).

Example 28. Synthesis of amide (1- (6-methyl-2-phenyl-7H-pyrrolo [2,3-b] pyrimidin-4-yl)) - (B) pyrrolidine-2-carboxylic acid (1512).

Compound 1512 was synthesized using the following steps:

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Aryl chloride 20 (3 g, 10.7 mmol), DMSO (50 ml) and (8) -prolinamide were combined and heated at 85 ° C in argon atmosphere. After stirring overnight (14 hours), the mixture was cooled to room temperature and poured into 800 ml of water. This mixture was extracted three times with 200 ml of ESA each.

The combined organic layers were washed thoroughly with water (3x300 ml), brine, dried over MD80 ₄ , filtered and concentrated to give a dark brown solid. The solid was recrystallized twice from ELS, yielding 1.95 g (57%) of a golden red solid (1512). ¹ H-NMR / _6- DMSO) ά 1.8-2.2 (m, 4H), 2.3 (s, 3H), 3.8 (m, 1H), 4.0 (m, 1H), 4.6 (d, 1H) 6.2 (s, 1H), 6.9 (s, 1H), 7.2 (m, 3H), 7.3 (s, 1H), 8.4 (m, 2H), 11.5 (s, 1H); mass spectrum (E8): 322 (M ⁺ +1).

Example 29. Synthesis of 1- [6- (2-hydroxyethoxymethyl) -2-phenyl-7H-pyrrolo [2,3-0] pyrimidin-4yl] pyrrolidine-2-carboxylic acid amide (1513).

Compound 1513 was synthesized in a manner analogous to the method of example 17, in accordance with synthetic scheme IX using L-prolinamide and ethane-1,2-diol, receiving:

1513

Mass spectrum (E8) 382 (M ⁺ +1).

Example 30. Synthesis of 4- (6-imidazol-1-ylmethyl-2-phenyl-7H-pyrrolo [2,3-0] pyrimidin-4-ylamino) cyclohexanol (1514).

Compound 1514 was synthesized in a manner analogous to the method of example 17, in accordance with synthetic scheme IX using Ν-6-aminocyclohexanol and imidazole, obtaining:

Mass spectrum (E8) 389 (M ⁺ +1).

Example 31: Synthesis of 4- (4-hydroxycyclohexylamino) -2-phenyl-7H-pyrrolo [2,3-0] pyrimidine-6-carboxylic acid (1515).

Compound 1515 was synthesized in a manner analogous to the method of example 27, in accordance with synthetic scheme IX using Ν-6-aminocyclohexanol, obtaining:

IT

ΗΝ

Mass spectrum (E8) 353 (M ⁺ +1)

Example 32. Synthesis of 4- [6- (2-hydroxyethoxymethyl) -2-phenyl-7H-pyrrolo [2,3-0] pyrimidin-4-ylamino] cyclohexanol (1516).

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Compound 1516 was synthesized by a method similar to the method for producing compound 1513, in accordance with the synthetic scheme X using Ν-6-aminocyclohexanol, obtaining:

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

A solution of lithium salt 17 (0.13 mmol) in anhydrous DMF (4 ml) was stirred with methyl iodide (0.1 ml, 1.6 mmol) at 20 ° C. under argon for 3 hours. The DMF was evaporated and an aqueous solution was added. ammonium chloride (15 ml). The mixture was extracted with E! OAc (3x15 ml), the combined organic layers were washed with water (2x10 ml) and brine (10 ml) and dried over MgO ₄ . Filtration and concentration give 21 mg (38%) of methyl ester 22.

A solution of methyl ester 22 (24.5 mg, 0.057 mmol) and 4-trans-aminocyclohexanol (66 mg, 0.57 mmol) in DMSO (1.5 ml) was heated under nitrogen at 80 ° C for 5 hours, then the heating was stopped and stirring at 20 ° C was continued for 13.5 hours. To the cooled solution was added 4% aqueous acetic acid (10 ml) and the mixture was extracted with E! OAc (3x10 ml). The combined organic layers were washed with 4% aqueous acetic acid (10 ml), water (10 ml), 2N. HaON (10 ml), water (10 ml), and saturated brine (10 ml) and dried over MgO ₄ . To a solution of the crude substance obtained after filtration and concentration ( ¹ H NMR indicates approximately 50% removal of the benzenesulfonyl group) in THF (2 ml), a solution of HaOH in MeOH (0.5 ml of a 5M solution, 2.5 mmol) is added at ambient temperature. After 20 minutes, water and saturated HaNCO ₃ solution (5 ml each) are added and the mixture is extracted with E! OAc (4x15 ml). The combined organic layers were washed with 2N HaON (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 / E! OAc 1: 1 1: 2 gives 8.6 mg (41%)

1517 as a white solid, mp. 225-227 ° C. ¹ H-NMR (CO ₃ OD) d 1.38-1.62 (m, 4H), 1.95-2.10 (m, 2H), 2.10-2.25 (m, 2H), 3 55-3.70 (m, 1H), 3.91 (s, 3H), 4.20-4.35 (m, 1H), 7.32 (s, 1H), 7.35-7.47 (m, 3H); 8.358.42 (m, 2H); IR (solid): η = 3352 cm ^-1 , 3064, 2935, 2860, 1701, 1605, 1588, 1574, 1534, 1447, 1386, 1333, 1263, 1206, 1164, 1074, 938, 756, 705; mass spectrum (E8): 367 (MH ⁺ ).

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

Compound 1518 was synthesized in a manner analogous to the method of example 26, using compound 12 as a precursor, obtaining:

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Mass spectrum (E8) 410 (M ⁺ +1).

Example 35. Synthesis of [4- (2-carbamoylpyrrolidin-1-yl) -2-phenyl-7H-pyrrolo [2,3-th] pyrimidin-6ylmethoxy] acetic acid (1519).

Compound 1519 was synthesized in a manner analogous to the method for producing compound 1518, where the ester methyl group was hydrolyzed with a base to give:

Mass spectrum (E8) 396 (M ⁺ +1).

Example 36. Synthesis of amide 4- (4-hydroxycyclohexylamino) -2-phenyl-7H-pyrrolo [2,3-th] pyrimidine-

6-carboxylic acid (1520)

Ammonia gas is condensed into a solution of pyrrolopyrimidine 23 (7.8 mg, 0.021 mmol) in methanol (6 ml), cooled with a dry ice / acetone mixture, until a total volume of 12 ml is reached, after stirring for 10 days at 20 ° The solvents were evaporated and the residue was purified by preparative TLC on silica gel, eluting with a mixture of 5% MeOH in CH ₂ Cl ₂ . The material thus obtained was triturated with ether to give 6.5 mg (88%) of amide 1520 as a white solid, mp. 225-227 ° C (decomp.). ¹ H-NMR (CB30B) ά 1.40-1.60 (m, 4H), 2.00-2.15 (m, 2H), 2.15-2.25 (m, 2H), 3.55 -

3.70 (m, 1H), 4.20-4.35 (m, 1H), 7.16 (s, 1H), 7.35-7.47 (m, 3H), 8.34-8, 40 (m, 2H); IR (solid): η = 3358 cm ¹ , 3064, 3025, 2964, 2924, 2853, 1652, 1593, 1539, 1493, 1452, 1374, 1326, 1251, 1197, 1113, 1074, 1028, 751, 699; mass spectrum (E8): 352 (MH ⁺ ).

Connection Activity

Saturation and competitive radioligand binding of the adenosine (A!) Receptor subtype 1 was performed for compounds 1505, 1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1516, 1517, 1518, 1519 and 1520, as described here and along with another on p. 152-153 of this description. All of the above compounds are equal to or superior to the binding affinity of the A1 receptor of reference compounds 1318 or 1319, as described here and among others in table 13, on page 171 of this description.

It is assumed that the solubility in water of the above compounds listed in Table 18 will be better than the reference compounds 1318 or 1319 due to their cEodP values, which were calculated using the C8 computer program CESHIGAA, CESHIGAA E'EGA, developed 6.0 ®1999, developed SAShpide8th Sogrogayop, 100 Saspyde ragk Ightue, Saspyde, MA 021140.

The compounds specific for the A1 receptor listed in Table 18 had lower syodR values between about 1.5 and about 3.4, compared to reference compounds 1318 or 1319 having syodR values of about 3.8. It was impossible to predict that more polar A! the receptor compounds listed in table 18 and having lower cEodP values than reference compounds 1318 or 1319 will still retain the efficiency and selectivity of A1 receptor binding compared to these reference compounds.

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Pages 288-293 relate to additional compounds specific for the A _2a receptor. This invention relates to a compound having the structure:

1609

This invention also relates to a compound having the structure:

<

1610

In a further embodiment, the invention relates to a method for treating a disease in a subject associated with an A _2a adenosine receptor, comprising administering to the subject a therapeutically effective amount of compounds 1609 or 1610.

In a further embodiment, the invention relates to the aforementioned method, wherein the subject is a mammal.

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

In a further embodiment, the invention relates to a method for treating a disease associated with an A _2a adenosine receptor in a subject, wherein the A _2a adenosine receptor is associated with motor activity, vasodilation, platelet inhibition, neutrophilic superoxide generation, cognitive impairment, senile dementia or Parkinson's disease .

The invention relates to the above method, where the compound treats the disease by stimulating adenylate cyclase.

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The invention also relates to a water-soluble prodrug of compound 1609 or 1610, wherein the water-soluble prodrug metabolizes ΐη νίνο to give an active drug that selectively inhibits the A _2a adenosine receptor.

The invention also relates to a water-soluble prodrug of compound 1609 or 1610, wherein the prodrug metabolizes ΐη νίνο by esterase-catalyzed hydrolysis.

The invention also relates to a pharmaceutical composition comprising a water-soluble prodrug of compound 1609 or 1610 and a pharmaceutically acceptable carrier.

The invention also relates to a method for inhibiting the activity of A _2a adenosine receptor in a cell, which comprises contacting the cell with compounds 1609 or 1610.

The invention also relates to a method for inhibiting A2a adenosine receptor activity in a cell, which comprises contacting the cell with compounds 1609 or 1610, wherein the compound is an antagonist of said A2a adenosine receptor.

The invention also relates to the above method, wherein the cell is a human cell.

The invention also relates to the above method, wherein the cell is a human cell and the compound is an antagonist of A _2a adenosine receptors.

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

The invention also relates to the aforementioned pharmaceutical composition, wherein a therapeutically effective amount is effective for the treatment of Parkinson's disease and a disease associated with motor activity, vasodilation, platelet inhibition, neutrophilic superoxide generation, cognitive impairment, or senile dementia.

The invention also relates to the aforementioned pharmaceutical composition, wherein the pharmaceutical composition is an ophthalmic preparation.

The invention also relates to the aforementioned pharmaceutical composition, wherein the pharmaceutical composition is a preparation for periocular, retrobulbar or intraocular injection.

The invention also relates to the aforementioned pharmaceutical composition, wherein the pharmaceutical composition is a systemic preparation.

The invention also relates to the above pharmaceutical composition, wherein the pharmaceutical composition is a surgical irrigation solution.

The invention also relates to combination therapy for Parkinson's disease, comprising compounds 1609 or 1610 and any of the dopamine enhancing agents.

The invention also relates to combination therapy for cancer, including compounds 1609 or 1610 and any of the cytotoxic agents.

The invention also relates to combination therapy for glaucoma, comprising compounds 1609 or 1610 and a prostaglandin agonist, muscarinic agonist or β-2 antagonist.

The invention also relates to a packaged pharmaceutical composition for treating a subject with a disease associated with an A ₂ adenosine receptor, including:

(a) a container containing a therapeutically effective amount of compounds 1609 or 1610, and (b) instructions for using said compound for treating said disease in a subject.

Illustrative example

Example 41. Synthesis of 1- (6-phenyl-2-pyridin-4-yl-7H-pyrrolo [2,3-b] pyrimidin-4-yl] pyrrolidine-2-carboxylic acid amide (1609).

Compound 1609 was synthesized by reacting b-prolinamide with a suitable chloride intermediate described in Scheme II on page 82 to give:

1609 ¹ H-NMR (ά6-DMSO) ά 1.95-2.15 (m, 4H), 4.00 (broad s, 1H), 4.15 (broad s, 1H), 4.72 ( broad s, 1H), 6.90 (broad s, 1H), 7.19 (broad s, 1H), 7.30 (t, 1H, 1 = 7.0 Hz), 7.44 (t , 2H, 1 = 7.0 Hz), 7.59 (s, 1H), 7.92 (br s, 2n), 8.26 (d, 2H, 1 = 6.2 Hz), 8.65 (d, 2H, 1 = 6.2 Hz); mass spectrum (E8): 384.9 (M + 1); mp = 280-316 ° C (decomp.).

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

- 121 007254

Compound 1610 was synthesized by reacting L-prolinamide with a suitable chloride intermediate described in Scheme II on page 82 to give:

1610

Я-NMR (66-DMSO) b 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, 1 = 8.2 Hz), 7.53 (s, 2H), 7.60 (s, 1H), 8.28 (d, 2H, 1 = 5.8 Hz), 8.67 (d, 2H, 1 = 5.8 Hz), 12.37 (s, 1H); mass spectrum (E8): 415.0 (M ⁺ +1).

Connection Activity

Competitive radioligand binding of the adenosine (A _2a ) receptor subtype 2a was performed for compounds 1609 and 1610, as described herein and, along with another, on page 153 of this description. Compounds 1609 and 1610 were found to have affinity and selectivity for binding to the A ₂ receptor.

Pages 294-300 relate to additional compounds specific for the A ₃ receptor. This invention also relates to a compound having the structure:

1720

In a further embodiment, the invention relates to a method for inhibiting the activity of an A ₃ adenosine receptor in a cell, which comprises contacting the cell with compound 1720.

In a further embodiment, the invention relates to a method for inhibiting the activity of an A ₃ adenosine receptor in a cell, wherein the compound is an antagonist of the A ₃ adenosine receptor.

In a further embodiment, the invention relates to the above method of inhibiting the activity of an A ₃ adenosine receptor in a cell, wherein the cell is a human cell.

In a further embodiment, the invention relates to a method for inhibiting the activity of an A ₃ adenosine receptor in a cell, wherein the cell is a human cell and where the compound is an antagonist of the A ₃ adenosine receptor.

In a further embodiment, the invention relates to a method for treating eye damage in a subject, comprising administering to the subject a composition comprising a therapeutically effective amount of compound 1720.

In a further embodiment, the invention relates to the above method, wherein the damage includes damage to the retina or optic head nerve.

In a further embodiment, the invention relates to the treatment of glaucoma, comprising administering to a subject a therapeutically effective amount of compound 1720.

In a further embodiment, the invention relates to the treatment of glaucoma comprising one or more adenosine receptor antagonists, preferably comprising an A ₃ adenosine receptor antagonist (preferably N-6 substituted 7-deazapurin, most preferably [2- (3H-imidazol-4-yl) ethyl ] - (2-phenyl-7H-pyrrolo [2,3-b] pyrimidin-4-yl) amine).

In an alternative embodiment, the invention relates to combination therapy for glaucoma comprising an A ₃ antagonist _{of an} adenosine receptor (preferably N-6 substituted with 7 dezazapurin, most preferably [2- (3H-imidazol-4-yl) ethyl] - (2-phenyl-7H-pyrrolo [2,3-b] pyrimidin-4-yl) amine) and one or more other compounds selected from the group consisting of beta-adrenergic antagonists (ie beta-adrenergic antagonists or beta-blockers) (eg, timolol maleate, betaxolol, karterolol, levobunolol, metipranolol, k-653328 (acetate k- 652698), beta-1-adrenoceptor antagonists), alpha-2-adrenoceptor agonists (e.g. aplaclonidine, brimony

- 122 007254 dynes, Λ6Ν-195795, Λ6Ν-190837 (analogue of Wow-a-6781)), carbonic anhydrase inhibitors (brinzolamide, dorzolamide, MK-927 (inhibitor of the human carboxylic anhydrase II isoenzyme), inhibitors of the carbonic anhydrase isoenzyme IV) agonists (e.g. muscarinic cholinergic agonists, carbachol, pilocarpine HC1, pilocarpine nitrate, pilocarpine prodrugs (e.g., PP-22A), prostaglandins and prostaglandin receptor agonists (e.g. latanoprost, unoproston isopropylanone, agonist RS1 propanolide, agonist a distinct RK receptor, RS agonists such as antihypertensive prostamides), angiotensin converting enzyme (ACE) inhibitors (e.g. spirapril, spiraprilate), AMPA receptor antagonists, 5-HT agonists (e.g., a selective 5-HT 1A receptor agonist, such as MKS-242 (5- [3 - ((28) -1,4-benzodioxan-2ylmethyl) amino] propoxy-1,3-benzodioxol НС1), angiogenesis inhibitors (for example, anecortave steroid), KhMEA antagonists (for example, NI- 211, memantine, agonist of the cannabinoid MMEA receptor dexanabinol, prodrugs and analogues of dexanabinol, ΝΚ2 B-selective antagonists (e.g., eliprodil (8B-82.0715)), renin inhibitors (e.g., CCP-38560, 8K-43845), cannabinoid receptor agonists (e.g. tetrahydrocannabinol (THS) and THC analogs, selective CB2 cannabinoid receptor agonists (e.g., b- 768242, b-759787), compounds such as anandamide that bind both brain-specific CB1 receptors and peripheral CB2 receptors), angiotensin receptor antagonists (e.g. angiotensin II receptor antagonists (e.g., C8-088), selective angiotensin antagonists II AT4 receptor such as potassium losartan, hydrochlorothiazide (HCT2), somatostatin agonists (e.g. non-peptide somatostatin agonist ΝΝΕ-269100), glucocorticoid antagonists, mast cell degranulation inhibitors (e.g. nedocromil), alpha-adrenergic receptor blockers (e.g., 2-adrenergic dipole adrenergic receptors) antagonists, alpha-1-adrenoceptor (e.g. bunazosin)), alpha-2adrenotseptora antagonists, thromboxane _A2 mimetics, protein kinase inhibitors (e.g. H7), prostaglandin F derivatives (e.g., 8-1033) antagonists rostaglandin-2 alpha (for example, RyXA-34), dopamine agonists Ό1 and 5-HT2 (phenoldopam), nitric oxide releasing agents (for example, NXX-904 or YXX-905, releasing nitric oxide derivatives of timolol), 5-HT2 antagonists (e.g., sarpogrelate), IMEA antagonists (e.g., prodrugs and dexanabinol analogues), alpha-1-adrenoceptor antagonists (e.g., bunazosin), cyclooxygenase inhibitors (e.g., diclofenac or the non-steroidal compound nefafenac), inosine receptors, 2-inosamines and 2-agonists adrenoceptor (e.g., thalipexole), antagonist of the dopamine Ό1 receptor and agonists of the Ό2 receptor (e.g., 8О2-SBS-756), antagonists of a vasopressin receptor (e.g., antagonists of vasopressin V2 receptor (e.g., 8К-121463)), endothelial antagonists (e.g., TBS-2576), 1 (3 -hydroxy-2-phosphonylmethoxypropyl) cytosine (HPMRS) and related analogs and prodrugs, thyroid hormone receptor ligands (e.g. KV-130015), muscarinic M1 agonists, HMOA receptor antagonists (e.g., X-receptor antagonist, X-cannabinoid receptor, cannabinoid receptor) agonists such as hypotensive lipids, prostamides, sodium channel blockers, XMEA antagonists, mixed-ion channel blockers, combinations of beta-adrenoceptor antagonists and PCP2 alpha agonists (e.g. latanoprost and thymodol), guanylate cyclase activators (e.g. atrial natriuretic peptide mimetic peptide or AKP) ), inhibitors of A№ neutral endopeptidase, nitrovasodilators (e.g. nitroglycerin, hydralazine, sodium nitroprusside), endothelin receptor modulators (e.g. ET-1 or non-peptide mimetics and, sarafotoxin-86c), ethacrylic acid, other analogues of phenoxyacetic acid (e.g. indacrinone, ticrinafen), actin disruptors (e.g. latrunculin), calcium channel blockers (e.g. verapamil, nifedipine, brovincamine, nivalidipine) and neutron protective agents.

Combination glaucoma therapy comprising compound 1720 and one or more compounds selected from the group consisting of beta-adrenoceptor antagonists, alpha-2-adrenoceptor agonists, carbonic anhydrase inhibitors, cholinergic agonists and prostaglandin receptor agonists.

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

In a further embodiment, the invention relates to a packaged pharmaceutical composition for treating a disease associated with an A3 adenosine receptor in a subject, comprising:

(a) a container containing a therapeutically effective amount of compound 1720; and (b) instructions for using said compound for treating said disease in a subject.

In a further embodiment, the invention relates to a method for preparing a composition that comprises a compound 1720, the method comprising mixing a compound 1720 with a suitable carrier.

- 123 007254

In a further embodiment, the invention relates to a pharmaceutically acceptable salt of compound 1720, wherein the pharmaceutically acceptable salt comprises an anion selected from the group consisting of maleate, fumarate, tartrate, acetate, phosphate and mesylate.

Illustrative example.

Example 43. Synthesis of [2- (3H-imidazol-4-yl) ethyl] - (2-phenyl-7H-pyrrolo [2,3-6] pyrimidin-4-yl) amine (1720).

Compound 1720 was synthesized using compound 1 as a precursor according to synthetic scheme VII, obtaining:

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 in a stream of nitrogen. After 6.5 hours, the reaction mixture was cooled to room temperature and partitioned between ESAc and water. The layers were separated and the aqueous layer was extracted with EuAc (3x). The combined organic layers were washed with brine (2x), dried over MgSO ₄ , filtered and concentrated to give 494 mg of a brown solid. The solid was washed with cold MeOH and recrystallized from MeOH to give 197 mg (43%) of an off-white solid (1720). 'H-NMR (SP3OP) 6 3.05 (t, 2H, 1 = 7.0 Hz), 3.94 (t, 2H, 1 = 7.0 Hz), 6.50 (d, 1H, 1 = 3.5 Hz), 6.88 (br s, 1H), 7.04 (d, 1H, 1 = 3.5 Hz), 7.42 (m, 3H), 7.57 (s, 1H) 8.34 (m, 2H); mass spectrum (E8): 305.1 (M ⁺ +1); mp = 234-235 ° C.

Connection Activity

Competitive radioligand binding of the adenosine-3 (A ₃ ) receptor was performed for compound 1720, as described here and, among others, on pages 153-154 of this description. Compound 1720 was found to have an A3 receptor binding affinity 10 times greater than that of reference compound 1308, as described herein and, inter alia, in table 13 on page 169 of the description.

Inclusion as a reference

All patents, published patent applications, and other references cited herein are expressly incorporated herein by reference.

Equivalents

Those skilled in the art will recognize or be able to verify, using nothing more than routine experiments, that there are many equivalents to the specific embodiments of the invention specifically described herein.

It is implied that such equivalents are covered by the scope of the following claims. This invention also relates to compounds having the formula:

where Κ'ΝΚ ₂ together form a ring having the structure:

- 124 007254

or Cd represents H, and K ₂ represents

K ₅ represents H or substituted or unsubstituted alkyl or alkylaryl.

Claims (74)

1. A compound having a structure or Cd represents H and K ₂ represents - (СН2) 2С (0) ОН, -С М) С1 ΙΓ (0) ΝΊ К -С (О) ОН, -СН3, -СН20 (СН2) 2ОН, -СН2ОСН2С (0) ОСН3 or -СН2ОСН2С (О) IT; or if Cd is H and K ₂ is he / then K5 is —C1 Ν'ΝΉΛΙ 1 _; ), -C (O) OH, -CH20 (CH2O0H, -C (0) OCH3 or -C (0 #, or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, having the structure - 125 007254 3. The compound according to claim 1, having the structure 4. The compound according to claim 1, having the structure 5. The compound according to claim 1, having the structure 6. The compound according to claim 1, having the structure 7. The compound according to claim 1, having the structure 8. The compound according to claim 1, having the structure - 126 007254 9. The compound according to claim 1, having the structure 10. The compound according to claim 1, having the structure 11. The compound according to claim 1, having the structure 12. The compound according to claim 1, having the structure IT ΗΝ 13. The compound according to claim 1, having the structure 14. The compound according to claim 1, having the structure - 127 007254 15. Connection on 16. Connection by 17. Connection on 18. The use of a compound according to claim 1 for the manufacture of a medicament for the treatment of a disease associated with A ₁ adenosine receptor in a subject. 19. The application of claim 18, wherein said A ₁ adenosine receptor is associated with cognitive impairment, renal failure, cardiac arrhythmia, respiratory epithelium, release of transmitters, sedation, constriction of blood vessels, bradycardia, negative cardiac inotropy and dromotropia, bronchostenosis, neutronrophy chemotaxis, reflux, or peptic ulcer. 20. The water-soluble prodrug of the compounds according to claim 1, where the water-soluble prodrug metabolizes ίη νίνο, giving an active drug that selectively inhibits the A ₁ adenosine receptor. 21. The use of a compound according to claim 1 for the manufacture of a medicament for inhibiting the activity of A ₁ adenosine receptor in a cell. 22. The application of claim 18, wherein said disease is asthma, chronic obstructive pulmonary disease, allergic rhinitis, or upper respiratory tract disease. 23. The pharmaceutical composition comprising the compound according to claim 1, a pharmaceutically acceptable carrier and at least one steroid, a B2 agonist, glucocorticoid, lucotriene antagonist or anticholinergic agonist. 24. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 1 and a pharmaceutically acceptable carrier. 25. The application of claim 18, wherein said disease is asthma, allergic rhinitis, or chronic obstructive pulmonary disease. - 128 007254 26. A packaged pharmaceutical composition for treating a disease associated with an A’adenosine receptor in a subject, comprising: (a) a container containing a therapeutically effective amount of a compound according to claim 1; and (b) instructions for using said compound for treating said disease in a subject. 27. A pharmaceutically acceptable salt of a compound according to claims 6, 8, 12, 15 or 16, wherein said salt contains a cation selected from the group consisting of sodium, calcium and ammonium. 28. The application of claim 18, wherein A ’adenosine receptor is associated with congestive heart failure. 29. The compound having the structure 30. The compound having the structure 31. The use of the compounds of clause 29 or 30 for the manufacture of a medicament for the treatment of a disease associated with A _2a adenosine receptor in a subject. 32. The application of claim 18 or 31, wherein the subject is a human. 33. The application of claim 32, wherein said A _2a adenosine receptor is associated with motor activity, vasodilation, platelet inhibition, neutrophilic superoxide generation, cognitive impairment, senile dementia or Parkinson's disease. 34. The application of clause 31, where the compound treats the specified disease by stimulating adenylate cyclase. 35. The water-soluble prodrug of the compounds of claim 29 or 30, wherein said water-soluble prodrug metabolizes ίη νίνο to give an active drug that selectively inhibits the A2a adenosine receptor. 36. The prodrug of claim 35, wherein said prodrug metabolizes ίη νίνο via esterase-catalyzed hydrolysis. 37. A pharmaceutical composition comprising the prodrug of claim 35 and a pharmaceutically acceptable carrier. 38. The use of a compound according to claim 29 or 30 for the manufacture of a medicament for inhibiting A2a adenosine receptor activity in a cell. 39. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 29 or 30 and a pharmaceutically acceptable carrier. 40. The pharmaceutical composition according to § 39, where the specified therapeutically effective amount is effective for the treatment of Parkinson's disease and diseases associated with motor activity, vasodilation, inhibition of platelets, neutrophilic generation of superoxide, cognitive impairment or senile dementia. 41. The pharmaceutical composition according to § 39, where the specified pharmaceutical composition is an ophthalmic drug. 42. The pharmaceutical composition according to paragraph 24 or 39, where the specified pharmaceutical composition is a preparation for periocular, retrobulbar or intraocular injection. 43. The pharmaceutical composition according to paragraph 24 or 39, where the specified pharmaceutical composition is a systemic preparation. 44. The pharmaceutical composition according to paragraph 24 or 39, where the specified pharmaceutical composition is a surgical irrigation solution. 45. A pharmaceutical composition comprising the compounds of Claim 29 or 30, a pharmaceutically acceptable carrier, and any of the dopamine enhancing agents. - 129 007254 46. A pharmaceutical composition comprising the compound of Claim 29 or 30, a pharmaceutically acceptable carrier, and any of the cytotoxic agents. 47. A pharmaceutical composition comprising the compound of Claim 29 or 30, a pharmaceutically acceptable carrier, and at least one prostaglandin agonist, muscarinic agonist, or β2 antagonist. 48. A packaged pharmaceutical composition for treating a disease associated with A _2a adenosine receptor in a subject, comprising: (a) a container containing a therapeutically effective amount of a compound according to claim 29 or 30; and (b) instructions for using said compound for treating said disease in a subject. 49. The pharmaceutically acceptable salt of a compound according to claim 29 or 30. 50. The compound having the structure N ΝΗ or a pharmaceutically acceptable salt thereof. 51. The use of a compound according to claim 50 for the manufacture of a medicament for inhibiting the activity of A ₃ adenosine receptor in a cell. 52. The use of claims 21, 38 or 51, wherein the cell is a human cell. 53. The use of a compound according to claim 50 for the manufacture of a medicament for treating eye damage in a subject. 54. The application of claim 53, wherein the damage includes damage to the retina or optic head nerve. 55. The use of a compound according to claim 50 for the manufacture of a medicament for the treatment of glaucoma in a subject. 56. The application of claim 55, wherein the drug further comprises one or more compounds selected from the group consisting of beta-adrenoseceptor antagonists, alpha-2 adrenoceptor agonists, carboxylic anhydrase inhibitors, cholinergic agonists, prostaglandins and prostaglandin receptor agonists, angiotensin- converting enzyme (ACE), AMPA receptor antagonists, 5-HT agonists, angiogenesis inhibitors, ΝΜΌΑ antagonists, renin inhibitors, cannabinoid receptor agonists, antagonists hyotensin receptor, hydrochlorothiazide (HCT2), somatostatin agonists, glucocorticoid antagonists, mast cell degranulation inhibitors, alpha-adrenergic receptor blockers, alpha-2 adrenoceptor antagonists, thromboxane A ₂ mimetics, prostate kinase agonists ₂ -dopamine kinase agonists, doprotein kinase agonists Όι and 5-HT2, nitric oxide releasing agents, 5-HT2 antagonists, cyclooxygenase inhibitors, inosine, dopamine agonists Ό ₂ receptor and alpha 2 adrenoceptor antagon the dopamine Όι receptor and agonists Ό ₂ receptors, vasopressin receptor antagonists, endothelial antagonists, 1-3-hydroxy-2phosphonylmethoxypropylcytosine (HPMRS) and related analogs and prodrugs, thyroid hormone receptor ligands, muscarinic M1 channel blockers of agonist channel ion agonists actions, beta adrenoceptor antagonists and combinations of POP2 alpha agonists, guanylate cyclase activators, nitrovasodilators, endothelin receptor modulators, ethacrine acid, etc. carbon analogues of phenoxyacetic acid, actin disruptors, calcium channel blockers and neutron protective agents. 57. The application of claim 55, wherein the drug further comprises one or more compounds selected from the group consisting of beta-adrenoseceptor antagonists, alpha-2 adrenoceptor agonists, carbonic anhydrase inhibitors, cholinergic agonists and prostaglandin receptor agonists. 58. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 50 and a pharmaceutically acceptable carrier. 59. A packaged pharmaceutical composition for treating a disease associated with an A3 adenosine receptor in a subject, comprising: (a) a container containing a therapeutically effective amount of a compound according to claim 50; and (b) instructions for using said compound for treating said disease in a subject. - 130 007254 60. A method for producing a composition comprising a compound according to claim 50, said method comprising mixing the compound with a suitable carrier. 61. The pharmaceutically acceptable salt of a compound according to claims 29, 30 or 50, wherein the pharmaceutically acceptable salt comprises an anion selected from the group consisting of maleate, fumarate, tartrate, acetate, phosphate and mesylate. 62. The pharmaceutical composition of claim 58, further comprising one or more compounds selected from the group consisting of beta-adrenoseceptor antagonists, alpha-2 adrenoceptor agonists, carboxylic anhydrase inhibitors, cholinergic agonists, prostaglandins and prostaglandin receptor agonists, angiotensin converting enzyme inhibitors (ACE), AMPA receptor antagonists, 5-HT agonists, angiogenesis inhibitors, Ν \ -1Ι) Λ antagonists, renin inhibitors, cannabinoid receptor agonists, angioten antagonists zine receptor, hydrochlorothiazide (HCT /), somatostatin agonists, glucocorticoid antagonists, mast cell degranulation inhibitors, alpha-adrenergic receptor blockers, alpha-2 adrenoceptor antagonists, thromboxane A ₂ mimetics, protein kinase inhibitors, prostaglandin agonists Όι dopamine and 5-HT2, agents that release nitric oxide, 5-HT2 antagonists, cyclooxygenase inhibitors, inosine, dopamine agonists Ό ₂ receptor and alpha 2 adrenoceptor antagonist d paminovogo Όι receptor agonists and Ό ₂ receptor, vasopressin receptor antagonists, endothelin antagonist, 1-3-hydroxy-2fosfonilmetoksipropiltsitozina (NRMRS) and related analogs and prodrugs, thyroid hormone receptor ligands, muscarinic _M1 agonists, sodium channel blockers, ion channel blockers, mixed actions, beta-adrenoceptor antagonists and combinations of HOG2 alpha agonists, guanylate cyclase activators, nitrovasodilators, endothelin receptor modulators, ethacrine acid, other taxes of phenoxyacetic acid, actin disruptors, calcium channel blockers and neutron protective agents. 63. The pharmaceutical composition according to § 58, further comprising one or more compounds selected from the group consisting of beta-adrenoseceptor antagonists, alpha-2 adrenoceptor agonists, carbonic anhydrase inhibitors, cholinergic agonists and prostaglandin receptor agonists. 64. The use of the compound of claim 50 for the manufacture of a medicament for the treatment of a disease associated with an A ₃ adenosine receptor in a subject where said disease associated with the A ₃ adenosine receptor is asthma, bronchitis, chronic obstructive pulmonary disease or bronchostenosis. 65. A method for producing a compound according to claim 50, comprising the steps of: (a) reacting the compound with histamine in the presence of dimethyl sulfoxide; (b) heating the reaction mixture in an inert atmosphere; (c) cooling the reaction mixture and separating the organic layer from the aqueous layer; (b) washing the organic layer with saturated brine, drying and filtering to obtain the target compound. 66. The method of claim 65, wherein in step (b), the reaction mixture is heated under a nitrogen atmosphere. 67. The method according to p, in which at stage (b) the organic layer is dried MDBO ₄ . 68. The use of a compound according to claim 1 for the manufacture of a medicament for the treatment of a disease associated with A! an adenosine receptor, in a subject where the disease is water retention, bradycardia, bronchitis, bronchospasm, Alzheimer's disease, cardiac arrhythmias, cardiac hypoxia, hypertension, inflammation, negative cardiac inotropy and dromotropia, renal failure, sedative disease or release respiratory epithelium, smooth muscle contraction under the respiratory epithelium, narrowing of blood vessels, or mast cell degranulation. 69. The use of a compound according to claim 1 for improving memory in a subject. 70. The use of a compound according to claim 29 or 30 for the manufacture of a medicament for the treatment of a disease associated with A _2a adenosine receptor in a subject where the disease associated with A _2a adenosine receptor is Parkinson's disease or glaucoma. 71. The use of a compound of claim 50 for the manufacture of a medicament for the treatment of a disease associated with an A3 adenosine receptor in a subject where the disease associated with an A3 adenosine receptor is myocardial ischemia, bronchitis or bronchospasm. - 131 007254 72. The use of a compound according to claim 50 for the manufacture of a medicament for the treatment of ocular inflammation associated with the A ₃ adenosine receptor in a subject. 73. The use of a compound of claim 50 for the manufacture of a medicament for the treatment of a disease associated with an A ₃ adenosine receptor in a subject where the disease associated with an A ₃ adenosine receptor is associated with mast cell degranulation. 74. The use of the compound of claim 50 for the manufacture of a medicament for the treatment of a disease associated with an A3 adenosine receptor in a subject where the disease associated with the A3 adenosine receptor is asthma, glaucoma, retinopathy, eye damage due to ischemia or yellow degeneration spots.