PYRROLO[2,3d]PYRIMIDINE COMPOSITIONS AND THEIR USE

Description of the invention

The subject of the invention is a pyrrolo [2,3d] pyrimidine derivative of the formula I, methods for the preparation of this derivative, pharmaceutical preparations, and its use in diseases of the digestive system, respiratory system or for inhibiting the activity of an adenosine receptor in a cell.

Adenosine is a ubiquitin modulator of numerous physiological processes, especially those that take place in the cardiovascular and nervous systems. Adenosine acts through specific proteins of cell surface receptors. Adenosine modulates various physiological functions, including induction of drug-induced drowsiness, vasodilation, inhibition of heart rate and contractility, inhibition of platelet aggregation, stimulation of glucogenogenesis, and inhibition of lipolysis. It appears that adenosine, in addition to its effect on adenylate cyclase, opens potassium channels, reduces the flow through calcium channels, and inhibits or stimulates phosphoinositide turnover through receptor mediated mechanisms (see e.g. CE. Muller and B.Stein "Adenosine Receptor Antagonists). : Structures and Potential Therapeutic Applications, "Current Pharmaceutyical Design, 2: 501 (1996) and CE. Muller" A1-Adenosine Receptor Antagonists, "Exp. Opin. Ther. Patents 7 (5): 419 (1997)).

The adenosine receptors belong to the purine receptor superfamily, which are currently divided into P1 (adenosine) and P2 (ATP, ADP and other nucleotides) receptors. So far, four subtypes for adenosine nucleoside have been cloned, from different species including man. Two receptor subtypes (A1 and A2) exhibit affinity for adenosine in the nanomolar range, while the other two known subtypes, A2b and A3, are low affinity receptors with an affinity for adenosine in the low micromolar range. Activation of the adenosine A1 and A3 receptor can lead to the inhibition of adenylate cyclase activity, while activation of A2a and A2b results in the stimulation of adenylate cyclase.

Several A1 antagonists have been developed for the treatment of cognitive impairment, renal failure, and cardiac arrhythmias. It has been suggested that A2a antagonists may be of benefit to patients suffering from Morbus Parkinson (Parkinson's disease). In particular in terms of their topical administration, adenosine receptor antagonist compounds may be of value in the treatment of allergic inflammation and asthma. Information available (e.g. Nyce and Metzger, "DNA antisense Therapy for Asthma in an Animal Model" Nature (1997) 385: 721-5) indicate that in this pathological context, A1 antagonists may block constriction of smooth muscles beneath the respiratory epithelium, while A2b or A3 receptor antagonists can block mast cell degranulation, reducing the release of histamine and other inflammatory mediators. A2b receptors have been found in the gastrointestinal tract, especially in the colon and intestinal epithelium. A2b receptors have been suggested to mediate the corresponding cAMP (Stromeier et al., J.Bio.Chem. (1995) 270: 2387-94).

Adenosine receptors have also been found to exist on the retinas of various species of mammals, including cattle, pigs, monkeys, rats, guinea pigs, mice, rabbits and humans (see Blazynski et al., Discrete Distributions of Adenosine Receptors in Mammalian Retina, Journal of Neurochemistry , torn 54, pp. 648-655 (1990); Woods et al., Characterization of Adenosine A1-Receptor Binding Sites in Bovine Retinal Membranes, Experimental Eye Research, Vol. 53, pp. 325-331 (1991); and Braas et al., Endogenous adenosine and adenosine receptors localized to ganglion cells of the retina, Proceedings of the National Academy of Science, vol. 84, pages 3906-3910 (1987)). More recently, Williams reported the observation of adenosine transport sites in cultured human retinal cell lines (Williams et al., Nucleoside Transport Sites in a Cultured Human Retinal Cell Line Estabilished By SV-40 T Antigen Gene, Current Eye Research, Vol. 13, pp. 109-118 ( 1994)).

Compounds that regulate adenosine uptake have previously been suggested as potential therapeutic agents for the treatment of major retinal and optic nerve injuries. In US Patent No. 5,780,450 to Shade, Shade discusses the use of adenosine reuptake inhibitors in the treatment of eye disorders. Shade does not describe the use of specific inhibitors of the A3 receptor. The entire content of US Patent No. 5,780,450 is hereby incorporated by reference.

Other adenosine receptor antagonists are needed as pharmacological tools and of considerable interest as medicaments for the above-mentioned diseases and / or conditions.

The present invention relates to a pyrrolo [2,3d] pyrimidine derivative of formula I:

PL 204 628 B1

<img file="PL204628B1_D0001.tif" />

wherein R1 is H;

R2 is C1-4 alkyl which is unsubstituted or substituted with hydroxyl, carboxyl, amino, acetoxy, pyridyl or tert-butoxycarbonyl;

B-CONH-C1-4 alkylene wherein B is H, C1-3 alkyl, cyclopropyl, aminoethyl, carboxyethyl, amino, methylamino, ethylamino, acetyl, or tert-butyloxy;

NH2-CO-C1-3 alkylene wherein the hydrogen of the amino group may be replaced by methyl or cyclopropylmethyl;

CH2CH2NHSO2CH3; or cyclohexyl or cyclopentyl, the cyclohexyl or cyclopentyl being optionally substituted with hydroxyl, acetylamino, methylsulfonylamino, benzoyloxy, 2-aminoacetoxy, 2-aminomethylacetylamine, or 2-N-tert-butyloxyacylaminoacetoxyl; or

R1 and R2 together form a 3-acetylaminopiperidyl group,

R3 is phenyl, pyridyl, furanyl, or thienyl, wherein the phenyl is unsubstituted or substituted with halogen;

R4 is H;

R5 is H or methyl unsubstituted or substituted with phenoxy, 4-fluorophenoxy, 4-chlorophenoxy, 4-methoxyphenoxy, pyridin-2-one-1-yl, 2-pyridyloxy, phenylamino or N-methylphenylamino; and

R6 is H or methyl, or a pharmaceutically acceptable salt thereof.

Preferably the compound of formula:

<img file="PL204628B1_D0002.tif" />

PL 204 628 B1

Preferably the compound of formula:

Preferably the compound of formula:

<img file="PL204628B1_D0003.tif" />

<img file="PL204628B1_D0004.tif" />

Preferably the compound where

R2 is cyclohexyl or cyclopentyl, the cyclohexyl or cyclopentyl being optionally substituted with hydroxyl, acetylamino, methylsulfonylamino, benzoyloxy, 2-aminoacetoxy, 2-aminomethylacetylamine, or 2-N-tert-butyloxyacylaminoacetoxy.

More preferably, wherein R1 and R4 are hydrogen, R3 is unsubstituted or halogen-substituted phenyl, and R5 and R6 are each methyl.

More preferably wherein R2 is monohydroxycyclopentyl.

More preferably wherein R2 is monohydroxycyclohexyl.

PL 204 628 B1

More preferably of formula:

<img file="PL204628B1_D0005.tif" />

More preferably of formula:

<img file="PL204628B1_D0006.tif" />

More preferably of formula:

<img file="PL204628B1_D0007.tif" />

More preferably of formula:

<img file="PL204628B1_D0008.tif" />

PL 204 628 B1

More preferably of formula:

<img file="PL204628B1_D0009.tif" />

More preferably of formula:

<img file="PL204628B1_D0010.tif" />

Preferably, the compound wherein R2 is B-CONH-C1-4 alkylene and B is H, C1-3 alkyl, cyclopropyl, aminoethyl, carboxyethyl, amino, methyl amino, ethyl amino, acetyl, or tert-butyloxy.

Preferably the compound wherein R1 and R4 are hydrogen, R3 is unsubstituted or halogen-substituted phenyl, and R5 and R6 are each methyl.

PL 204 628 B1

Preferably the compound of formula:

<img file="PL204628B1_D0011.tif" />

Preferably the compound of formula:

<img file="PL204628B1_D0012.tif" />

Preferably the compound wherein R3 is unsubstituted or halogen-substituted phenyl.

More preferably, the compound where each of R5 and R6 is methyl.

More preferably the compound wherein R3 is unsubstituted phenyl.

More preferably the compound wherein R3 is halogen-substituted phenyl.

Preferably the compound wherein R3 is pyridyl, furanyl or thienyl.

More preferably, the compound where each of R5 and R6 is methyl.

Preferably the compound wherein R5 and R6 are each hydrogen.

Preferably the compound wherein each of R5 and R6 is methyl.

Preferably a compound selected from the group consisting of:

4- (cis-3-hydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine,

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

4- (3-acetamido) piperidinyl-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine,

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

PL 204 628 B1

4- (2-acetamidobutyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, 4- (2-N'-methylurea butyl) amino-5,6-dimethyl-2-phenyl -7H-pyrrolo [2,3d] pyrimidine, 4- (2-aminocyclopropylacetamidoethyl) amino-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, 4- (trans-4-hydroxycyclohexyl) amino-2- (3- chlorophenyl) -7H-pyrrolo [2,3d] pyrimidine, 4- (trans-4-hydroxycyclohexyl) amino-2- (3-fluorophenyl) -7H-pyrrolo [2,3d] pyrimidine, and 4- (trans-4- hydroxycyclohexyl) amino-2- (4-pyridyl) -7H-pyrrolo [2,3d] pyrimidine, 4- (trans-4-hydroxycyclohexyl) amino-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, and 4- [2- (3-fluoro-phenyl) -7H-pyrrolo [2,3d] pyrimidin-4 -ylamino] -cyclohexanol.

Preferably the compound of formula I:

<img file="PL204628B1_D0013.tif" />

R1 is H;

R2 is 2-acetylaminoethyl or 2-N'-methylureaethyl;

R3 is phenyl;

R4 is H;

R5 is H or methyl, methyl being unsubstituted or substituted with phenoxy, 4-fluorophenoxy, 4-chlorophenoxy, 4-methoxyphenoxy, pyridin-2-one-1-yl, 2-pyridyloxy, phenylamino or N-methylphenylamino; and R6 is H.

More preferably, a compound selected from the group consisting of:

4- (2-acetylaminoethyl) amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine;

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

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

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

4- (2-acetylaminoethyl) amino-6- (2-pyridyloxy) methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine;

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

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

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

The invention relates to the use of a compound as defined above for the manufacture of a medicament for the treatment of a disease or condition associated with elevated levels of adenosine, wherein the disease or condition is a central nervous system disorder, a kidney disorder, an inflammatory disorder, an allergic disorder, a gastrointestinal disorder, an ocular disorder or a medical condition. respiratory.

Preferably, the digestive disease is diarrhea and the respiratory disease is asthma, hay fever or chronic obstructive pulmonary disease.

The invention relates to a compound of the invention for use in a method of inhibiting the activity of an adenosine receptor in a cell.

Preferably a compound selected from the group consisting of:

4- (2-acetylaminoethyl) amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine;

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

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

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

4- (2-acetylaminoethyl) amino-6- (2-pyridyloxy) methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine;

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

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

PL 204 628 B1

4- (2-N'-methylureaethyl) amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. Preferably a compound selected from the group consisting of: 4- (cis-3-hydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, 4- (cis-3- (2-aminoacetoxy) cyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine trifluoroacetic acid salt,

4- (3-acetamido) piperidinyl-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine,

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

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

4- (2-N'-methylurea butyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, 4- (2-aminocyclopropylacetamidoethyl) amino-2-phenyl-7H-pyrrolo [2 , 3d] pyrimidine, 4- (trans-4-hydroxycyclohexyl) amino-2- (3-chlorophenyl) -7H-pyrrolo [2,3d] pyrimidine, 4- (trans-4-hydroxycyclohexyl) amino-2- (3- fluorophenyl) -7H-pyrrolo [2,3d] pyrimidine, and 4- (trans-4-hydroxycyclohexyl) amino-2- (4-pyridyl) -7H-pyrrolo [2,3d] pyrimidine, 4- (trans-4- hydroxycyclohexyl) amino-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, and

4- [2- (3-fluoro-phenyl) -7H-pyrrolo [2,3-d] pyrimidin-4-ylamino] -cyclohexanol.

The invention relates to a pharmaceutical preparation containing a therapeutically effective amount of a compound as defined above and a pharmaceutically acceptable carrier.

Preferably the compound is selected from the group consisting of:

4- (2-acetylaminoethyl) amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine;

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

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

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

4- (2-acetylaminoethyl) amino-6- (2-pyridyloxy) methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine;

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

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

4- (2-N'-methylureaethyl) amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. Preferably the compound is selected from the group consisting of: 4- (cis-3-hydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, 4- (cis-3- (2-aminoacetoxy) ) cyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, trifluoroacetic acid salt,

4- (3-acetamido) piperidinyl-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine,

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

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

4- (2-N'-methylurea butyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, 4- (2-aminocyclopropylacetamidoethyl) amino-2-phenyl-7H-pyrrolo [2 , 3d] pyrimidine, 4- (trans-4-hydroxycyclohexyl) amino-2- (3-chlorophenyl) -7H-pyrrolo [2,3d] pyrimidine, 4- (trans-4-hydroxycyclohexyl) amino-2- (3- fluorophenyl) -7H-pyrrolo [2,3d] pyrimidine, and 4- (trans-4-hydroxycyclohexyl) amino-2- (4-pyridyl) -7H-pyrrolo [2,3d] pyrimidine, 4- (trans-4- hydroxycyclohexyl) amino-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, and

4- [2- (3-fluoro-phenyl) -7H-pyrrolo [2,3-d] pyrimidin-4-ylamino] -cyclohexanol.

Preferably the formulation is an ophthalmic formulation.

Preferably the formulation is for periocular, retrobulbar or ocular injection purposes.

Preferably the formulation is a systemic formulation.

Preferably the formulation is a surgical irrigation solution.

The invention relates to a method for producing a compound

<img file="PL204628B1_D0014.tif" />

PL 204 628 B1 comprising the steps of:

a) reaction

<img file="PL204628B1_D0015.tif" />

wherein P is a removable protecting group;

b) treating the product of step a) under cyclization conditions to obtain

<img file="PL204628B1_D0016.tif" />

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

<img file="PL204628B1_D0017.tif" />

and

d) treating the chlorinated product from step c) with an amine to afford

<img file="PL204628B1_D0018.tif" />

R1 is H;

R2 is C1-4 alkyl which is unsubstituted or substituted with hydroxyl, carboxyl, amino, acetoxy, pyridyl or tert-butoxycarbonyl;

B-CONH-C1-4 alkylene wherein B is H, C1-3 alkyl, cyclopropyl, aminoethyl, carboxyethyl, amino, methylamino, ethylamino, acetyl, or tert-butyloxy;

PL 204 628 B1

NH2-CO-C1-3 alkylene wherein the hydrogen of the amino group may be replaced by methyl or cyclopropylmethyl;

CH2CH2NHSO2CH3; or cyclohexyl or cyclopentyl, the cyclohexyl or cyclopentyl being optionally substituted with hydroxyl, acetylamino, methylsulfonylamino, benzoyloxy, 2-aminoacetoxy, 2-aminomethylacetylamine, or 2-N-tertbutyloxyacylaminoacetoxy; or

R1 and R2 together form a 3-acetylaminopiperidyl ring,

R3 is phenyl, pyridyl, furanyl, or thienyl, wherein the phenyl is unsubstituted or substituted with halogen;

R6 is H or methyl.

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

A method of modulating the adenosine receptor in a mammal is to administer to the mammal a therapeutically effective amount of N-6 substituted 7-deazapurine such that modulation of adenosine receptor activity occurs. Suitable adenosine receptors include the A1, A2, or A3 families. In the illustrated embodiment, the N-6 substituted 7-deazapurine is an adenosine receptor antagonist.

The N-6-substituted 7-deazapurine also has use in the manufacture of a medicament for the treatment of a variety of conditions, e.g., asthma, bronchitis, allergic rhinitis, chronic obstructive airways disease, kidney disease, gastrointestinal disease, and eye disease, in a mammal. Wherein the mammal is administered a therapeutically effective amount of the N-6 substituted 7-deazapurine such that a condition for the mammal is treated. Suitable N-6 substituted 7 deazapurines include those illustrated by general formula I:

<img file="PL204628B1_D0019.tif" />

and pharmaceutically acceptable salts thereof. R1 and R2 may each be independently hydrogen or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety, or together may form a substituted or unsubstituted heterocyclic ring. R3 can be a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety. R4 can be a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety. R5 and R6 may be each independently halo, e.g., chlorine, fluoro, or bromo, hydrogen or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety, R5 and R6 may each independently be halo, e.g., chlorine, fluoro , or bromine, hydrogen, or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety, or R4 and R5 or R5 and R6 together may form a substituted or unsubstituted heterocyclic or carbocyclic ring.

In certain embodiments, R1 and R2 may each independently be substituted or unsubstituted cycloalkyl or heteroarylalkyl moieties. In other embodiments, R3 can be a hydrogen atom or a substituted or unsubstituted heteroaryl moiety. In still other embodiments, R4, R5, and R6 may each independently be heteroaryl moieties. W recommends12

In one embodiment, R1 is hydrogen, R2 is cyclohexanol, e.g., trans-cyclohexanol, R3 is phenyl, R4 is hydrogen, R5 is methyl and R6 is methyl. In yet another embodiment, R1 is hydrogen, R2 is

NHMe

R3 is phenyl, R4 is hydrogen and R5 and R6 are methyl groups.

The invention further relates to pharmaceutical formulations used for the treatment of a N-6 substituted 7-deazapurine responsive condition in a mammal, e.g., asthma, bronchitis, allergic rhinitis, chronic obstructive disease, kidney disease, gastrointestinal disorders, and eye disorders. . The pharmaceutical preparation comprises a therapeutically effective amount of the N-6 substituted 7-deazapurine and a pharmaceutically acceptable carrier.

Alternatively, the invention may relate to packaged pharmaceutical compositions / pharmaceutical preparations for the treatment of an N-6-substituted 7-deazapurine responsive condition in a mammal. The packaged pharmaceutical composition / pharmaceutical preparation comprises a container holding a therapeutically effective amount of at least one N-6 substituted 7-deazapurine and instructions for the use of the N-6 substituted 7-deazapurine to treat an N-6 substituted 7-deazapurine responsive condition in a mammal.

A compound of formula I may contain the following substituents among which:

R1 is hydrogen;

R2 is a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted alkyl, or R1 and R2 together form a substituted or unsubstituted heterocyclic ring;

R3 is unsubstituted or substituted aryl;

R4 is hydrogen; and

R5 and R6 are each independently hydrogen or alkyl, and pharmaceutically acceptable salts thereof. The deazapurines according to this embodiment may preferably be selective A3 antagonists. The compounds may be useful in numerous therapeutic applications, such as, for example, in the treatment of asthma, renal failure associated with heart failure, and glaucoma. In a particularly preferred embodiment, deazapurine is a water-soluble prime form that can be metabolized in vivo to active drug form by, e.g., esterase-catalyzed hydrolysis.

The method of inhibiting the activity of an adenosine (e.g., A3) receptor in a cell may be carried out by contacting the cell with an N-6 substituted 7-deazapurine (e.g., preferably, an adenosine receptor antagonist).

A method of treating an eye injury in an animal (e.g., human) can be carried out by administering to the animal an effective amount of an N-6 substituted 7-deazapurine of Formula I. Preferably, the N-6 substituted 7-deazapurine is an antagonist of A3 adenosine receptors in cells of the animal. The damage occurs to the retina or the optic nerve start, and can be acute or chronic. The damage may result from, for example, glaucoma, edema, ischemia, hypoxia, or trauma.

The invention also describes a pharmaceutical formulation comprising the N-6-substituted 7-deazapurines of Formula I. Preferably, the pharmaceutical formulation is an ophthalmic formulation (e.g., periocular, retrobulbar or intraocular injection formulation, systemic formulation, or surgical irrigation solution).

Deazapurine having formula II has also been described:

R

<img file="PL204628B1_D0020.tif" />

(H)

PL 204 628 B1 in which

X is N or CR6;

R1 and R2 are each independently hydrogen, or substituted or unsubstituted alkoxy, aminoalkyl, alkyl, aryl, or alkylaryl, or together form a substituted or unsubstituted heterocyclic ring provided that R1 and R2 are not both hydrogen;

R3 is substituted or unsubstituted alkyl, aralkyl, or aryl;

R4 is hydrogen or substituted or unsubstituted C1-C6 alkyl;

L is hydrogen, substituted or unsubstituted alkyl, or R4 and L together form a substituted or unsubstituted heterocyclic or carbocyclic ring;

R6 is hydrogen, substituted or unsubstituted alkyl, or halogen;

Q and CH2; O, S, or NR7, wherein R7 is hydrogen or substituted or unsubstituted C1-C6 alkyl; and

W is unsubstituted or substituted alkyl, cycloalkyl, aryl, arylalkyl, biaryl, heteroaryl, substituted carbonyl, substituted thiocarbonyl, or substituted sulfonyl; provided that when R3 is pyrrolidine then R4 is not methyl. The disclosure relates to the pharmaceutically acceptable salts and drug preforms of the compounds.

Preferably X may be CR6 and Q is CH2, O, S, or NH in formula II, wherein R6 is as defined above.

In another embodiment of Formula II, X is N.

The method of inhibiting the activity of an adenosine receptor (e.g., the adenosine A2b receptor) in a cell may be by contacting the cell with a compound. Preferably, the compound is a receptor antagonist.

A method of treating a gastrointestinal disease (e.g., diarrhea) or respiratory disease (e.g., allergic rhinitis, chronic obstructive disease) in an animal may be carried out by administering to the animal an effective amount of a compound of formula II (e.g., an A2b antagonist), preferably , the animal is man.

Features and other details of the invention will now be described in more detail and highlighted in the claims. It will be understood that particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. The essential features of the invention can be used in various embodiments without departing from the scope of the invention.

The present invention provides the use of a compound of the invention in the manufacture of a medicament for the treatment of an N-6 substituted 7-deazapurine responsive condition in a mammal. The methods of treatment comprise administering a therapeutically effective amount of an N-6 substituted 7-deazapurine, described below, to the mammal such that treatment of an N-6 substituted 7-deazapurine responsive condition in the mammal takes place.

The phrase "N-6 substituted 7-deazapurine responsive condition" is intended to include a disease state or condition characterized by a response to treatment with an N-6 substituted 7-deazapurine as described below, e.g., treatment comprises significantly reducing at least one symptom or the effect of the state achieved by the use of N-6-substituted 7-deazapurine. Typically such conditions are associated with an increase in the amount of adenosine in the host such that the host often experiences physiological symptoms which include, but are not limited to, toxin release, inflammation, coma, water retention, weight gain or loss, pancreatitis, emphysema, rheumatoid arthritis. arthritis, arthritis, multi-organ failure, respiratory distress syndrome in infants and adults, allergic rhinitis, chronic obstructive respiratory disease, eye disorders, digestive disorders, skin cancer stimulation, immunodeficiency and asthma. (See, e.g., CE. Muller and B. Stein "Adenosine Receptor Antagonists: Structures and Potential Therapeutic Applications," Current Pharmaceutical Design, 2: 501 (1996) and CE. Muller "A1-Adenosine Receptor Antagonists," Exp. Opin. Ther. Patents 7 (5): 419. (1997) and I. Feoktistove, R. Polosa, ST Holgate, and I. Biaggioni “Adenosine A2b receptors: a novel therapeutic target in asthma?” TIPS 19; 148 (1998)). Effects often associated with such symptoms include, but are not limited to, fever, shortness of breath, nausea, diarrhea, weakness, headache, and even death. In one embodiment, the N-6-substituted 7-deazapurine responsive state includes those disease states mediated by stimulation of adenosine receptors, e.g., A1, A1a, A2b, A3, etc., such that the concentration of calcium in the cells is modulated. and / or PLC (phospholipase C) activation. In a preferred embodiment, the N-6 substituted 7-deazapurine responsive state is bound to the adenosine receptor (s), e.g., the N-6 substituted 7-deazapurine acts as an antagonist. Examples

An appropriate response state that can be treated with the compounds, e.g., adenosine receptor subtypes that mediate biological effects include central nervous system (CNS) effects, cardiovascular effects, renal effects, respiratory effects , effects on immunity, effects on the digestive system and effects on metabolism. The relative amount of adenosine in the subject may be related to the effects listed below; that is, an increased level of adenosine can release the effect of, e.g., an unwanted physiological response, e.g., an asthma attack.

The effects on the CNS include decreased release of transmitters (A1), sedation (A1), decreased locomotor activity (A2a), anticonvulsant activity, chemoreceptor stimulation (A2) and excessive pain sensation. Therapeutic uses of the compounds include the treatment of dementia, Alzheimer's disease, and memory enhancement.

Cardiovascular effects include vasodilation (A2a), (A2b) and (A3), vasoconstriction (A1), bradycardia (A1), platelet inhibition (A2a), negative cardiac inotropy and dromotropy (A1), arrhythmia, tachycardia and utensils. Therapeutic uses of the compounds include, for example, prevention of ischemia-induced cardiac disorders and toning of the heart, preservation of cardiac tissue, and recovery of cardiac function.

The effects on the kidneys include reduction of GFR (A1), narrowing of mesangial cells (A1), reduction of urine volume (A1) and inhibition of renin release (A1). Suitable therapeutic uses for the compounds include the use of these compounds as diuretics, sodium excretioners, potassium sparing agents, protecting / preventing acute renal failure, antihypertensive, anti-edema and nephritis.

Effects on the respiratory system include bronchodilation (A2), bronchoconstriction (A1), chronic airway obstruction, allergic rhinitis, mucus secretion and respiratory depression (A2). Suitable therapeutic uses for the compounds include anti-asthma uses, treatment of post-transplant lung disease, and respiratory conditions.

Effects on the immune system include immunosuppression (A2), neutrophilic chemotaxis (A1), formation of neutrophilic peroxide (A2a) and mast cell ginning (A2b and A3). Therapeutic uses of antagonists include allerganic and non-allergic inflammation, e.g., release of histamine and other inflammatory mediators.

Effects on the digestive system include inhibition of acid secretion (A1). Therapeutic use may include gastric acid reflux and ulceration. The effects on the digestive system also include colon, bowel and diarrheal disease, e.g., diarrheal disease associated with enteritis (A2b).

Eye conditions include damage to the retina and the optic nerve origin, and trauma-related conditions (A3). In a preferred embodiment, the eye condition is glaucoma.

Other therapeutic uses for the compounds include treating obesity (lipolytic properties), hypertension, treating depression, drug-induced somnolence, as anxiolytics, as antieptic agents, and as laxatives, e.g., affecting gut motility without causing diarrhea.

The term "disease state" is intended to include those conditions which are caused or associated with undesirable levels of adenosine, adenosine cyclase activity, increased physiological activity associated with aberrant stimulation of adenosine receptors, and / or increased cAMP. In one embodiment, the disease condition is, e.g., asthma, chronic obstruction 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, infarction, cerebral ischemia, intermittent claudication, critical limb ischemia, venous hypertension, varicose veins, venous ulceration, and atherosclerosis. Disturbed reperfusion conditions include, for example, any postoperative trauma, such as reconstructive surgery, dissolution of a thrombus, or vasoplasty.

The phrase "treating an N-6 substituted 7-deazapurine responsive condition" or "treating an N-6 substituted 7-deazapurine responsive condition" is intended to include altering a disease state or condition as described above such that physiological symptoms in a mammal may be much smaller or minimized. The phrase also includes the control, prevention or inhibition of the physiological symptoms or the effects associated with the abnormal amount of adenosine. In one preferred embodiment, the control of the disease state or condition is such that it removes the disease or condition. In another preferred embodiment, the control is selective such that abnormal levels of adenosine receptor activity are controlled while other physiological systems and parameters remain unaffected.

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

<img file="PL204628B1_D0021.tif" />

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

In some embodiments, the N-6 substituted 7-deazapurine is not N-6 benzyl or N-6 phenylethyl substituted. In other embodiments, R4 is not benzyl or phenylethyl substituted. In preferred embodiments, Rl and R2 are not both hydrogen. In yet other preferred embodiments, R3 is not hydrogen.

The expression "therapeutically effective amount" of N-6 substituted 7-deazapurine, as described below, means that amount of a therapeutic compound as is necessary or sufficient to perform its intended function in a mammal, e.g., treating an N-6-substituted 7-responsive condition. -deazapurin, or a mammalian disease state. The effective amount of the therapeutic compound can be varied according to factors such as the amount of the causative agent already present in the mammal, the age, sex, and weight of the mammal, and the ability of the therapeutic compound to effect an N-6 substituted 7-deazapurine responsive state in the mammal. One skilled in the art would be able to investigate the above-mentioned factors and make the determination taking into account the effective amount of the therapeutic compound without undue experimentation. An in vitro or in vivo test can also be used to determine an "effective amount" of a therapeutic compound described below. The ordinarily skilled artisan would select the appropriate amount of the therapeutic compound to be used in the above-mentioned test or as a therapeutic treatment.

The therapeutically effective amount preferably reduces at least one symptom or effect associated with a condition corresponding to the N-6 substituted 7-deazapurine or the condition treated by at least about 20% (more preferably by at least about 40%, even more preferably by at least about 40%). 60%, and even more preferably by at least about 80%) of the untreated subjects. Those skilled in the art can schedule tests to measure the reduction of such symptoms and / or impacts. Any assay known in the art capable of measuring such parameters is intended to be included as part of this invention. For example, if the condition being treated is asthma, then the volume of air exiting the subject's lungs before and after treatment may be measured to measure the increase in volume, using a technique known in the art. Similarly, if the condition being treated is inflammation, then the area that is inflamed may be measured before and after treatment to measure reduction in the area affected by inflammation using techniques known in the art.

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

The term "animal" includes any organism possessing adenosine receptors or any organism susceptible to an N-6-substituted 7-deazapurine responsive state. Examples of animals include yeast, mammals, reptiles, and birds. They also include transgenic animals.

The term "mammal" is known in the art and is intended to include an animal, more preferably a warm blooded animal, most preferably dairy cattle, sheep, pigs, horses, dogs, cats, rats, mice and humans. Mammals susceptible to an N-6-substituted 7-deazapurine responsive state, e.g., inflammation, emphysema, asthma, conditions of the central nervous system, or acute respiratory distress syndrome, are included as part of this invention.

Also disclosed are methods of modulating the adenosine receptor (s) in a mammal by administering to the mammal a therapeutically effective amount of an N-6 substituted 7-deazapurine such that modulation of the mammalian adenosine receptor occurs. Suitable adenosine receptors include

From A1, A2, or A3 families. In a preferred embodiment, the N-6 substituted 7-deazapurine is an adenosine receptor antagonist.

The phrase "modulating the adenosine receptor" is intended to include those instances where the compound interacts with the adenosine receptor (s) to result in increased, diminished or abnormal adenosine receptor mediated physiological activity or a subsequent cascade of effects resulting from adenosine receptor modulation. The physiological activities associated with adenosine receptors include induction of sedation, vasodilation, suppression of heart rate and contractility, inhibition of platelet aggregation, stimulation of gluconeogenesis, inhibition of lipolysis, opening of potassium channels, reduction of calcium channel flow, etc.

The terms "modulate," "modulating", and "modulation" are intended to prevent, remove, or inhibit the resulting increase in undesirable physiological activity associated with aberrant stimulation of the adenosine receptor, eg, in the context of the disclosed therapeutic methods. The term modulate can include antagonistic effects, e.g., reduction in the activity or production of mediators of allergy and allergic inflammation that results from overstimulation of the adenosine receptor (s). For example, therapeutic deazapurines can interact with the adenosine receptor to inhibit, e.g., adenylate cyclase activity.

The phrase "condition characterized by aberrant adenosine receptor activity" is intended to include those diseases, conditions or conditions which are associated with inappropriate stimulation of the adenosine receptor, in that receptor stimulation causes a chain of biochemical and or physiological events that directly or indirectly bind to with a disease, illness or condition. This stimulation of the adenosine receptor need not be the sole causative agent of the disease, disease, or condition, but merely responsible for causing some of the symptoms usually associated with the disease, disease, or condition being treated. Abnormal receptor stimulation may be the only factor or at least one other factor that may be associated with the condition being treated. Examples of conditions include those disease states listed above, including inflammation, gastrointestinal disorders, and those symptoms manifested by the presence of increased adenosine receptor activity. Recommended examples include those symptoms that are associated with asthma, allergic rhinitis, chronic obstructive airways disease, emphysema, bronchitis, gastrointestinal disorders, and glaucoma.

The phrase "treating or treating a condition characterized by inappropriate adenosine receptor activity" is intended to include the abolition or reduction of at least one symptom usually associated with the condition. Treatment also includes eliminating or reducing more than one symptom. Preferably, the treatment cures, e.g., substantially eliminates the symptoms associated with the condition.

The application discloses N-6-substituted 7-deazapurin compounds that have the formula I:

<img file="PL204628B1_D0022.tif" />

where

R1 and R2 may optionally each be independently hydrogen, or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety, or together form a substituted or unsubstituted heterocyclic ring;

R3 can be a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety;

R4 can be a hydrogen atom or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety. R5 and R6 may each be independently halogen, e.g., chlorine, fluoro, or bromo, hydrogen or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety, or R4 and R5 or R5 and R6 together may form a substituted or unsubstituted heterocycle 204,628 B1 or carbocyclic. Pharmaceutically acceptable salts of the N-6 substituted 7-deazapurines are also included.

In some embodiments, R1 and R2 may each independently be a substituted or unsubstituted cycloalkyl or heteroarylalkyl moiety. In other embodiments, R3 may be a hydrogen atom or a substituted or unsubstituted heteroaryl moiety. In still other embodiments, R4, R5, and R6 may each be independently heteroaryl moiety.

In one embodiment, R1 is hydrogen, R2 is a substituted or unsubstituted cyclohexane, cyclopentyl, cyclobutyl or cyclopropane moiety, R3 is a substituted or unsubstituted phenyl moiety, R4 is hydrogen, and R5 and R6 are both methyl groups.

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

In yet another embodiment, R1 is hydrogen, R2 is cyclohexanol, R3 is a substituted or unsubstituted phenyl, pyridine, furan, cyclopentane, or thiophene moiety, R4 is hydrogen, a substituted alkyl, aryl or arylalkyl moiety, and R5 and R6 are each independently is hydrogen, or a substituted or unsubstituted alkyl, aryl, or alkylaryl moiety.

In yet another embodiment, R1 is hydrogen, R2 is a substituted or unsubstituted alkylamine, arylamine, or alkylarylamine, substituted or unsubstituted alkylamide, arylamide or alkylarylamide, substituted or unsubstituted alkylsulfonamide, arylsulfonamide or alkylarylsulfonamide, substituted or unsubstituted alkylurea, or alkylarylurea, substituted or unsubstituted alkylcarbamate, arylcarbamate, or alkylarylcarbamate, substituted or unsubstituted alkyl carboxylic acid, aryl carboxylic acid or alkylaryl carboxylic acid, R3 is a substituted or unsubstituted phenyl moiety, R4 is hydrogen and R5 and R6 are methyl groups.

In yet another embodiment, R2 is guanidine, modified guanidine, cyanoguanidine, thiourea, thioamide or amidines.

<img file="PL204628B1_D0023.tif" />

In one embodiment, R.₂ can be where R._2a-R_2c are each independently hydrogen or a saturated or unsaturated alkyl, aryl or alkylaryl moiety, and R2d is a hydrogen atom or a saturated or unsaturated alkyl, aryl, or alkylaryl moiety, NR2eR2f, or OR2g, where R2e-R2g are each independently hydrogen or saturated or unsaturated alkyl, aryl or alkylaryl moieties. Alternatively, R2a and R2b together may form a carbocyclic or heterocyclic ring with a ring size of between about 3 and 8 members, e.g., cyclopropyl, cyclopentyl, cyclohexyl.

Both R5 and R6 are not methyl. Preferably, one of R5 and R6 is an alkyl group, e.g., methyl and the other is hydrogen.

Furthermore, if R4 is 1-phenylethyl and R1 is hydrogen then R3 is not phenyl, -2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 3, 4-dichlorophenyl, 3-methoxyphenyl or 4-methoxyphenyl or if R4 and R1 are 1-phenylethyl then R3 is not hydrogen or if R4 is hydrogen and R3 is phenyl then R1 is not phenylethyl.

In another aspect of the invention, if R.₅ and r₆ together they form a carbocyclic ring, e.g.,

<img file="PL204628B1_D0024.tif" />

-methylphenyl) ethyl, phenylisopropyl, phenyl or 1-phenylethyl or if R3 is not hydrogen when R4 is 1-phenylethyl. The carbocyclic ring formed by R5 and R6 can be either aromatic or aliphatic and can have between 4 and 12 carbon atoms, e.g., naphthyl, phenylcyclohexyl, etc., preferably between 5 and 7 carbon atoms, e.g., cyclopentyl or cyclohexyl. Alternatively, R5 and R6 together may form a heterocyclic ring as described below. Typical heterocyclic rings

These include between 4 and 12 carbon atoms, preferably between 5 and 7 carbon atoms, and may be either aromatic or aliphatic. The heterocyclic ring may be further substituted, including the substitution of one or more carbon atoms of the ring structure with one or more heteroatoms. In yet another aspect of the invention, R1 and R2 form a heterocyclic ring. Representative examples include, but are not limited to, those heterocyclic rings which are mentioned below, such as morpholino, piperazine and others, e.g., 4-hydroxypiperidines, 4-aminopiperidines. If R₁

N and R₂ together they form a piperazine group,, R₇ it may be a hydrogen atom or a substituted or unsubstituted alkyl, aryl or alkylaryl moiety.

In yet another aspect of the invention, R4 and R5 together may form a heterocyclic ring, e.g.

<img file="PL204628B1_D0025.tif" />

The heterocyclic ring may be either aromatic or aliphatic and may form a ring having between 4 and 12 carbon atoms, e.g., naphthyl, phenylcyclohexyl, etc., and may be either aromatic or aliphatic, e.g., cyclohexyl, cyclopentyl. The heterocyclic ring may be further substituted, including the substitution of carbon atoms of the ring structure with one or more heteroatoms. Alternatively, R4 and R5 together may form a heterocyclic ring, such as those described below.

In some embodiments, the N-6 substituted 7-deazapurine is not N-6 benzyl or N-6 phenylethyl substituted. In other embodiments, R4 is not benzyl or phenylethyl substituted. In preferred embodiments, both R1 and R2 are not hydrogen. In yet other preferred embodiments, R3 is not hydrogen.

The compounds can include water-soluble drug preforms that are metabolized in vivo to the active drug form, e.g., by esterase-catalyzed hydrolysis. Examples of potential drug preforms include deazapurines, e.g., with R2 as cycloalkyl substituted with -OC (O) (Z) NH2, wherein Z is the side chain of a naturally or unnaturally occurring amino acid, or an analog thereof, the amino acids α, β, γ, or ω, or a 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.

In an additional embodiment, the invention describes deazapurines of formula (I) wherein:

R1 is hydrogen;

R2 is a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted alkyl, or R1 and R2 together form a substituted or unsubstituted heterocyclic ring;

R3 is unsubstituted or substituted aryl;

R4 is hydrogen; and

R5 and R6 are each independently hydrogen or alkyl, and pharmaceutically acceptable salts thereof. The deazapurines according to this embodiment may be potentially selective A3 receptor antagonists.

In one embodiment, R2 is substituted (e.g., substituted with hydroxy) or unsubstituted cycloalkyl. In a preferred minor embodiment, R1 and R4 are hydrogen, R3 is unsubstituted or substituted phenyl, and R5 and R6 are each alkyl. Preferably R2 is mono-hydroxycyclopentyl or mono-hydroxycyclohexyl. R2 may also be substituted with -NH-C (= O) E, wherein E is substituted or unsubstituted C1-C4 alkyl (e.g., alkylamine, e.g., ethylamine).

R1 and R2 may also form together a substituted or unsubstituted heterocyclic ring which may be substituted with an amine or an acetamide group.

PL 204 628 B1

In another aspect, R2 can be -A-NHC (= O) B, wherein 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 R1 and R4 are hydrogen, R3 is unsubstituted or substituted phenyl, and R5 and R6 are each alkyl. B can be a substituted or unsubstituted cycloalkyl, e.g., cyclopropyl or 1-aminocyclopropyl.

R3 may be substituted or unsubstituted phenyl, preferably R5 and R6 are each alkyl. Preferably, R3 may have one or more substituents (e.g., o-, m- or p-chlorophenyl, o-, m- and p-fluorophenyl).

Preferably, R3 may be substituted or unsubstituted heteroaryl, preferably R5 and R6 are each alkyl. Examples of the heteroaryl group include pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, pyrrolyl, triazolyl, thiazolyl, oxazolyl, oxadiazolyl, furanyl, methylenedioxyphenyl, and thiophenyl. Preferably,

R3 is 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, or 3-pyrimidyl.

Preferably, R5 and R6 are each hydrogen. In another, R5 and R6 are each methyl.

In a particularly preferred embodiment, the deazapurines are water-soluble drug preforms that can be metabolized in vivo to the active drug form, e.g. by esterase-catalyzed hydrolysis. Preferably the drug preform comprises a group R2 which is cycloalkyl substituted with -OC (O) (Z) NH2 where Z is a side chain of a naturally or unnaturally occurring amino acid, analogue thereof, amino acid α, β, γ, or ω, or a dipeptide . Examples of preferred side chains include the side chain of glycine, alanine, valine, leucine, isoleucine, lysine, α-methylalanine, aminocyclopropanecarboxylic acid, azetidine-2-carboxylic acid, β-alanine, γ-aminobutyric acid, alanine-alanine, or glycine-alanine .

In a particularly preferred embodiment, Z is the glycine side chain, R2 is cyclohexyl, R3 is phenyl, and R5 and R6 are methyl.

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

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

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

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

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

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

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

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

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

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

Also disclosed is a method of inhibiting the activity of an adenosine receptor (e.g., A1, A2a, A2b, or, preferably, A3) in a cell by contacting the cell with an N-6 substituted 7-deazapurine (e.g., preferably, an adenosine receptor antagonist).

In addition, there is also described a method of treating damage to an animal (e.g., human) eye by administering to the animal an effective amount of an N-6 substituted 7-deazapurine. Preferably, the N-6 substituted 7-deazapurine is an antagonist of the adenosine A3 receptors in cells of the animal. The damage occurs to the retina or the optic nerve start, and can be acute or chronic. The damage may result from, for example, glaucoma, edema, ischemia, hypoxia, or trauma.

Preferably deazapurine having formula II as above in which

X is N or CR6;

PL 204 628 B1

R1 and R2 are each independently hydrogen, or substituted or unsubstituted alkoxy, aminoalkyl, alkyl, aryl, or alkylaryl, or together form a substituted or unsubstituted heterocyclic ring provided that R1 and R2 are not both hydrogen;

R3 is substituted or unsubstituted alkyl, aralkyl, or aryl;

R4 is hydrogen or substituted or unsubstituted C1-C6 alkyl:

L is hydrogen, substituted or unsubstituted alkyl, or R4 and L together form a substituted or unsubstituted heterocyclic or carbocyclic ring;

R6 is hydrogen, substituted or unsubstituted alkyl, or halogen;

Q is CH2, O, S, or NR7 wherein R7 is hydrogen or substituted or unsubstituted C1-C6 alkyl; and

W is unsubstituted or substituted alkyl, cycloalkyl, alkynyl, aryl, arylalkyl, biaryl, heteroaryl, substituted carbonyl, substituted thiocarbonyl, or substituted sulfonyl, provided that if R3 is pyrrolidine, then R4 is not methyl.

In compounds of formula II, X may be CR6 and Q is CH2, O, S, or NH. In another embodiment, X is N.

Optionally, in an additional embodiment of compounds of formula II, W is a substituted or unsubstituted aryl, 5- or 6-membered heteroaryl, or biaryl. W may be substituted with one or more substituents. Examples of the substituents include: halogen, hydroxy, alkoxy, amino, aminoalkyl, aminocarboxamide, CN, CF3, CO2R8, CONHR8, CONR8R9, SOR8, SO2R8, and SO2NR8R9; wherein R8 and R9 are each independently hydrogen, or substituted or unsubstituted alkyl, cycloalkyl, aryl, or aralkyl. Preferably, W may be a substituted or unsubstituted phenyl, e.g., methylenedioxyphenyl. W can also be a substituted or unsubstituted 5-membered heteroaryl ring, e.g., pyrrole, pyrazole, oxazole, imidazole, triazole, tetrazole, furan, thiophene, thiazole, and oxadiazole. Preferably, W may be a 6-membered heteroaryl ring, e.g., pyridyl, pyrimidyl, pyridazinyl, pyrazinium, and thiophenyl. In a preferred embodiment, W is 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, or 5-pyrimidyl.

Preferably, in formula II, Q is NH and W is a 3-pyrazole ring which is unsubstituted or N-substituted with substituted or unsubstituted alkyl, cycloalkyl, aryl, or aralkyl.

In another embodiment of the compounds of Formula II, Q is oxygen, and W is a 2-thiazole ring that is unsubstituted or substituted with substituted or unsubstituted alkyl, cycloalkyl, aryl, or aralkyl.

In another embodiment of the compounds of Formula II, W is substituted or unsubstituted alkyl, cycloalkyl, e.g., cyclopentyl, or aralkyl. Examples of substituents include halogen, hydroxy, substituted or unsubstituted alkyl, cycloalkyl, aryl, aralkyl, or NHR10, where R10 is hydrogen, or substituted or unsubstituted alkyl, cycloalkyl, aryl, or aralkyl.

In yet another embodiment deazapurine of formula II, wherein optionally W is - (CH2) aC (= O) Y or - (CH2) aC (= S) Y, and a is an integer from 0 to 3, Y is aryl, alkyl , arylalkyl, cycloalkyl, heteroaryl, alkynyl, NHR11R12, or provided that Q is NH, OR13, wherein R11, R12a and R13 are each independently hydrogen, or unsubstituted or substituted alkyl, aryl, arylalkyl, or cycloalkyl. Preferably, Y is a 5- or 6-membered heteroaryl ring.

Furthermore, W can be - (CH2) bS (= O) jY where j is 1 or 2, b is 0, 1, 2, or 3, Y is aryl, alkyl, aralkyl, cycloalkyl, alkynyl, heteroaryl, NHR14R15 provided that when b is 1, Q is CH2, and wherein R14, R15, and R16 are each independently hydrogen, or unsubstituted or substituted alkyl, aryl, aralkyl, or cycloalkyl.

In another embodiment, R3 is selected from the group consisting of substituted and unsubstituted phenyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinal, pyrrolyl, triazolyl, thiazolyl, oxazolyl, oxadiazolyl, pyrazolyl, furanyenyl, methylenedioxyphenyl, and thioxy phenyl. When R3 is phenyl, it may be substituted, e.g., by hydroxy, alkoxy (e.g., methoxy), alkyl (e.g., tolyl), and halogen (e.g., o-, m-, or p-fluorophenyl or o- , m-, or p-chlorophenyl). Preferably, R3 may be 2-, 3-, or 4-pyridyl or 2- or 3-pyrimidyl.

R6 may be hydrogen or C1-C3 alkyl. Preferably, R6 is hydrogen.

R1 can be hydrogen, and R2 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 alkylarlamide, substituted or unsubstituted alkylsulfonamide, arylsulfonamide or alkylarylsulfonamide, substituted or unsubstituted alkylurea, arylurea or alkylaryl urea, substituted or unsubstituted alkylcarbamate, arylcarbamate or alkylarylcarbamate, or substituted or unsubstituted alkylcarboxylic acid, arylcarboxylic acid, or alkylarylcarboxylic acid. Preferably, R2 is a substituted or unsubstituted cycloalkyl, e.g., mono- or dihydroxy-substituted cyclohexyl or cyclopentyl (preferably, monohydroxy-substituted cyclohexyl or monohydroxy-substituted cyclopentyl).

Preferably, R.₂ may have the following formula:

<img file="PL204628B1_D0026.tif" />

wherein A is C1-C6 alkyl, C3-C7 cycloalkyl, a chain of one to seven atoms, or a ring of three to seven atoms, optionally substituted with C1-C6 alkyl, halogen, hydroxy, carboxyl, thiol, or amino;

B is methyl, N (Me) 2, N (Et) 2, NHMe, NHEt, (CH2) rNH3 +, NH (CH2) rCH3, (CH2) rNH2, (CH2) rCHCH3NH2, (CH2) rNHMe, (CH2) rOH , CH2CN, (CH2) mCO2H, CHR18R19, or CHMeOH where r is an integer from 0 to 2, m is 1 or 2, R18 is alkyl, R19 is NH3 + or CO2H or R₁₈ and r₂₉ together they mean:

—CH-NH— \ where p is 2 or 3; and

R17 is C1-C6 alkyl, C3-C7 cycloalkyl, a chain of one to seven atoms, or a ring of three to seven atoms, optionally substituted with C1-C6 alkyl, halogen, hydroxy, carboxyl, thiol, or amino.

Preferably, A is unsubstituted or substituted C1-C6 alkyl. B may be unsubstituted or unsubstituted C1-C6 alkyl.

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

The compounds can include water-soluble drug preforms that are metabolized in vivo to the active drug form, e.g., by esterase-catalyzed hydrolysis. Examples of potential drug preforms include deazapurines with, e.g., R2 in the cycloalkyl form substituted with -OC (O) (Z) NH2, wherein Z is the side chain of a naturally or unnaturally occurring amino acid, or an analogue thereof, amino acid α, β, γ, or ω or a 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.

In another embodiment, R.₁ and r₂ together they mean:

<img file="PL204628B1_D0027.tif" />

wherein n is 1 or 2, and wherein the ring may be optionally substituted with one or more hydroxy, amino, thiol, carboxyl, halogen, CH2OH, CH2NHC (= O) alkyl, or CH2NHC (=) NHalkyl groups. Preferably, n is 1 or 2 and said ring is substituted with -NHC (= O) alkyl.

In one preferred embodiment, R1 is hydrogen, R2 is substituted or unsubstituted C1-C6 alkyl, R3 is substituted or unsubstituted phenyl, R4 is hydrogen, L is hydrogen or substituted or unsubstituted C1-C6 alkyl, Q is O, S or NR7, wherein R7 is hydrogen or substituted or unsubstituted C1-C6 alkyl, and W is substituted or unsubstituted aryl. Preferably, R2 is -A-NHC (= O) B, wherein A and B are each independently unsubstituted or substituted C1-C4 alkyl. For example, A may be CH2CH2. B may be, for example, alkyl (e.g., methyl), or aminoalkyl (e.g., aminomethyl). Preferably, R3 is unsubstituted phenyl and L is hydrogen. R6 may be methyl or, preferably, hydrogen. Preferably, Q is O, S, or NR7 wherein R7 is hydrogen or substituted or unsubstituted C1-C6 alkyl, eg, methyl. W is unsubstituted or substituted phenyl (e.g., alkoxy, halogen-substituted). Preferably, W is p-fluorophenyl, p-chlorophenyl, or p-methoxyphenyl. W can also be heteroaryl, e.g., 2-pyridyl.

PL 204 628 B1

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

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

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

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

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

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

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

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

Also disclosed is a method of inhibiting the activity of an adenosine receptor (e.g., adenosine A2b receptor) in a cell by contacting the cell with a compound of the invention. As well as a method of treating a gastrointestinal disorder (e.g., diarrhea) in an animal by administering to the animal an effective amount of a compound of the invention (e.g., an A2b antagonist). Preferably, the animal is human.

A pharmaceutical composition comprising an N-6-substituted 7-deazapurine according to the invention and a pharmaceutically acceptable carrier has also been disclosed.

The method of treating an N-6 substituted 7-deazapurine responsive condition in an animal is carried out by administering to the mammal a therapeutically effective amount of deazapurine such that treatment of an N-6 substituted 7-deazapurine responsive condition in the animal occurs. Preferably, the disease state may be an adenosine mediated disease. Examples of the preferred disease states include: central nervous system disease, cardiovascular disease, kidney disease, inflammatory disease, allergic disease, gastrointestinal disease, eye disease, and respiratory disease.

The term "alkyl" refers to the radical of saturated aliphatic groups, including straight chain alkyl groups, branched chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. The term alkyl further includes alkyl groups which may additionally contain oxygen, nitrogen, sulfur or phosphorus atoms to replace one or more hydrocarbon backbone carbon atoms, e.g., oxygen, nitrogen, sulfur or phosphorus atoms. In preferred embodiments, a straight chain or branched alkyl has 30 or more carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched), and more preferably 20 or more. Likewise, preferred cycloalkyls have from 4-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbon atoms in their ring structure.

Furthermore, the term alkyl as used in the listing and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to an alkyl moiety having substituents replacing a hydrogen on one or more hydrocarbon backbone carbon atoms. Such substituents may include, for example, halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonoyl, phosphinyl, diamino, amino (dialkamino, aryl) , and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the hydrocarbon chain substituted moieties may themselves be substituted if desired. Cycloalkyls may be further substituted, e.g., with the substituents described above. An "alkylaryl" moiety is an alkyl substituted with an aryl (eg, phenylmethyl (benzyl)). The term "alkyl" also includes an unsaturated aliphatic group analogous in length and possible substitution to the alkyls described above, but which contain at least one double or triple bond, respectively.

PL 204 628 B1

The term "aryl" as used herein refers to the radical of an aryl group, including

Single-ring 5- and 6-membered aromatic groups that may contain from zero to four heteroatoms, e.g., benzene, pyrrole, furan, thiophene, imidazole, benzoxazole, benzothiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and others. Aryl groups also include polycyclic linked aromatic groups such as naphthyl, quinolyl, indolyl, and others. These aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles", "heteroaryls", or "heteroaromatic". The aromatic ring may be substituted at one or more ring positions with such substituents as described above, e.g., halogen, hydroxy, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, phosphinemocarbonyl, alkylcarbonyl, phosphinemarbonyl, , cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, or aromatic, heterocyclic, heterocyclic or heterocyclic. Aryl groups can also be linked or bridged to alicyclic or heterocyclic rings that are not aromatic so as to form a polycycle (e.g., tetralin).

The terms "alkenes" and "alkynyl" refer to an unsaturated aliphatic group analogous in length and possible substitution with the alkyls described above, but which contain at least one double or triple bond, respectively. For example, the invention contemplates cyano and propargyl groups.

Unless otherwise indicated in the number of carbon atoms, "lower alkyl" as used herein shall mean an alkyl group as defined above but having from one to ten carbon atoms, more preferably from one to six carbon atoms in its backbone structure, even more preferably one to three carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths.

The terms "alkoxyalkyl", "polyaminoalkyl" and "thioalkoxyalkyl" refer to alkyl groups as described above which additionally include oxygen, nitrogen or sulfur atoms replacing one or more hydrocarbon backbone carbon atoms, e.g., oxygen, nitrogen or sulfur atoms .

The terms "polycyclic" or "polycyclic" refer to a radical having two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and / or heterocycles) in which two or more carbon atoms are common to the two fused rings. for example, the rings are "linked rings". Rings that are joined by non-adjacent atoms are called "bridged" rings. Each of the polycyclic rings may be substituted with such substituents as described above, e.g., halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonylate, phosphinocarbonylate, phosphinocarbonylate, (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkyl aromatic or aromatic moiety or moiety.

The term "heteroatom" as used herein denotes an atom of any element other than carbon or hydrogen. The preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus. The term "amino acids" includes naturally occurring and unnaturally occurring amino acids found in proteins such as glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline , histidine, phenylalanine, tyrosine, and tryptophan. Amino acid analogs include amino acids with extended or truncated side chains or other side chains with appropriate functional groups. Amino acids also include D and L stereoisomers of an amino acid when the structure of the amino acid allows for stereoisomeric forms. The term "dipeptide" includes two or more amino acids linked together. Preferably, the dipeptidamia are two amino acids linked by a peptide bond.

Particularly preferred dipeptides include, e.g., alanine-alanine and glycine-alanine.

It should be noted that the structure of some compounds includes asymmetric carbon atoms. Accordingly, it is to be understood that isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are within the scope of this invention, unless otherwise stated. Such isomers

PL 204 628 B1 can be obtained in substantially pure form using classical separation techniques and by stereochemically controlled synthesis.

Pharmaceutical compositions for treating an N-6-substituted 7-deazapurine responsive condition in a mammal, include, e.g., respiratory conditions (e.g., asthma, bronchitis, chronic obstructive airways disease, and allergic rhinitis), renal disorders, gastrointestinal disorders, and medical conditions. eyes. The pharmaceutical composition comprises a therapeutically effective amount of the N-6 substituted 7-deazapurine described above and a pharmaceutically acceptable carrier. It should be understood that all deazapurines described above are used for the therapeutic treatment. It should additionally be understood that the deazapurines can be used alone or in combination with other deazapurines or in combination with other therapeutic compounds such as antibiotics, anti-inflammatory agents, or anti-cancer agents, e.g.

The term "antibiotic" is known in the art and is intended to include those substances which are produced by cultured microorganisms and their synthetic derivatives which eliminate or inhibit the growth of pathogens and are selectively toxic to pathogens with minimal or no harmful effects in the infected host . Suitable examples of antibiotics include, but are not limited to, the basic classes of aminoglycosides, cephalosporins, chloramphenicols, fuscidic acids, macrolides, penicillins, polymyxins, tetracyclines, and streptomycins.

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

The term "anti-neoplastic agent" is known in the art and is intended to include those agents that reduce, eliminate, or prevent the growth of neoplastic cells, preferably without deleteriously affecting other physiological functions. Representative examples include cisplatin and cyclophosphamide.

When the compounds are administered as pharmaceuticals to humans and mammals, they can be administered as such or as a pharmaceutical composition containing, e.g., 0.1 to 99.5% (more preferably, 0.5 to 90%) of the active ingredient in combination with a pharmaceutically active ingredient. an acceptable carrier. The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material associated with carrying or transporting the compound (s) within or into subject so that it can perform its intended function. Typically, such compounds are moved or transported from one organ, or part of an organism, to another organ, or part of an organism. Each carrier must be "acceptable" in the sense that it is compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatine; 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 glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's fluid; ethanol; phosphate buffered solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

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

PL 204 628 B1

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

The term "pharmaceutically acceptable esters" refers to the relatively non-toxic esterified products of the compounds. These esters can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound, in its free acid or hydroxyl form, with an appropriate esterifying agent. Carboxylic acids can be converted to esters by treatment with an alcohol in the presence of a catalyst. Hydroxy containing derivatives can be converted to esters by treatment with an esterifying agent such as alkanoyl halides. The term is intended to further include lower hydrocarbon groups capable of solvation under physiological conditions, e.g., alkyl esters, methyl, ethyl and propyl esters. (See, e.g., Berge et al., Supra).

Additionally, the use of drug preforms that are converted in vivo to therapeutic compounds has been considered (see, eg, RB Silverman, 1992, "The Organic Chemistry of Drug Design and Drug Action", Academic Press, Chapter 8). Such drug primers can be used to alter biodistribution (e.g., to allow compounds that would not normally enter the protease reactive site) or the pharmacokinetics of a therapeutic compound. For example, a carboxylic acid group may be esterified, e.g., with a methyl group or an ethyl group, or non-enzymatically, reducing or hydrolytically, to reveal an anionic group. The anionic group can be esterified with particles (e.g., acyloxymethyl esters) that are cleaved to reveal an intermediate which then decomposes to yield the active compound. In another embodiment, the drug preform is a reduced sulfate or sulfonate form, e.g. a thiol that is oxidized in vivo to a therapeutic compound. In addition, the anionic particle may be esterified to a group that is actively transported in vivo or that is selectively taken up by target organs. The ester may be selected to allow for the specific targeting of the therapeutic moieties to particular reactive sites, as described below for the carrier particles.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants, can 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; fat-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and others; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and others.

The formulations include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, rectal, vaginal and / or parenteral administration. The preparations may conveniently be presented in unit dosage form and may be prepared by any method known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, in addition to one hundred percent, the amount will be from about 1 percent to about ninety-nine percent of the active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

Methods of preparing these preparations or compositions include the step of bringing into association the compound with the carrier and, optionally, one or more accessory ingredients. In general, formulations are prepared by uniformly and intimately bringing the compound into association with liquid carriers, or by carefully dividing solid carriers or both, and then shaping the product if necessary.

PL 204 628 B1

Formulations suitable for oral administration may be capsules, cachets, pills, tablets, lozenges (using a flavored base, typically sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous fluid or non-aqueous, either in the form of an oil-in-water emulsion or a water-in-oil, or as an elixir or syrup, or in the form of lozenges (using an inert base such as gelatin and glycerin, or sucrose and acacia) and / or mouth rinses and others, each containing a predetermined amount of the compound as an active ingredient. The compound can also be administered as a bolus dose, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and others), the active ingredient is mixed with one or more pharmaceutically active carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: or bulking agents such as starches, lactose, sucrose, glucose, mannitol and / or alginates, gelatin, polyvinylpyrrolidone, sucrose and / or acacia; moisturizers such as glycerol; disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; dissolution retarding agents such as paraffin; absorption accelerators such as quaternary ammonium compounds; wetting agents, such as e.g. cetyl alcohol and glycerol monostearate; absorbents, such as kaolin or bentonite clay; lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical compositions can also include buffering agents. Solid compositions of a similar type can also be used as fillers in soft and hard gelatin capsules, using excipients such as lactose, milk sugars as well as high molecular weight polyethylene glycols and others.

A tablet may be made by compression or molding using one or more accessory ingredients. Compressed tablets may be made using a binder (e.g. gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surfactant or dispersant. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

Tablets and other solid dosage forms of pharmaceutical compositions such as dragees, pills, and granules can optionally be prepared or prepared with shells and shells such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They can also be formulated so as to provide slow or controlled release of the active ingredient, using e.g. hydroxypropyl methylcellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes, and / or microspheres. They may be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which may be dissolved in sterile water or some other sterile injectable medium just prior to use. These compositions may optionally contain opacifying agents and may be such that they release the active ingredient (s) only or primarily in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active ingredient may also be in microcapsule form with one or more of the above-described excipients, respectively.

Liquid dosage forms for oral administration of the compounds include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms can contain inert diluents commonly used in the art, such as, for example, water or other solvents, dissolving agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, propylene glycol, 1,3-butylene glycol, oils (in particular cotton, peanut, corn oils, from cereal germ, olive, castor, sesame), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, flavoring, and preserving.

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

PL 204 628 B1

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

Formulations suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or sprays containing such carriers as are known to be suitable in the art.

Dosage forms for topical or transdermal administration of a compound include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalers. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservative, buffer, or carrier gas.

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

Powders and sprays can contain, in addition to the compound, 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.

Transdermal patches have the added benefit of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flow can be controlled either by providing a flow-controlling membrane or by dispersing the active compound in a polymer matrix or gel.

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

The ophthalmic preparations can contain one or more deazapurines and a pharmaceutically acceptable carrier. Various types of media can be used. The carriers will generally be aqueous in nature. Aqueous solutions are generally recommended on a case-by-case basis as well as the patient's ability to easily administer such compositions by the instillation of one or two drops of the solutions into the affected eyes. However, deazapurines can also be readily incorporated into other types of compositions, such as suspensions, viscous or semi-viscous gels, or other types of solid or semi-solid compositions. The ophthalmic compositions can also include various other ingredients such as buffers, preservatives, cosolvents, and viscosity improvers.

An appropriate buffer system (e.g. sodium phosphate, sodium acetate or sodium borate) can be added to prevent pH changes under storage conditions.

Ophthalmic products are usually packaged in a multi-dose form. Preservatives are therefore required to prevent microbial contamination during use. Suitable preservatives include: benzalkonium chloride, thimerozal, 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 levels of from 0.001 to 1.0% w / v ("% w / v").

When deazapurines are administered during intraocular surgical procedures, such as by retrobulbar or periocular injection, and by ocular perfusion or injection, the use of balanced salt irrigation solutions as carriers is most preferred. Examples of physiologically balanced ocular irrigation solutions are BSS® Sterile Irrigating Solution and BSS Plus® Sterile Intraocular Irrigating Solution (Alcon Laboratories, Inc., Fort Worth, Tex. USA). A second type of solution is described in U.S. Patent No. 4,550,022 to Garabedian et al., The entire contents of which are hereby incorporated by reference. Retorbital and periocular injections are known to those skilled in the art and are described in numerous publications, including, e.g., Ophtalmic Surgery: Principles of Practice, Ed., GLSpaeth. WBSanders Co., Philadelphia, Pa., USA, pp. 85-87 (1990).

As noted above, the use of deazapurines to prevent or reduce damage to retinal or optic nerve conductive tissue at the cellular level is particularly

An important aspect of one embodiment. Ophthalmic conditions that can be treated include, but are not limited to, retinopathies, macular degeneration, ocular ischemia, glaucoma, and damage related to ophthalmic tissue injury such as ischemic reperfusion wounds, photochemical wounds, and wounds associated with eye surgery, particularly retinal injuries. or the beginning of the optic nerve by light exposure or surgical instruments. The compounds can also be used as an adjunct to ophthalmic surgery, such as by intravitreal injection or subconjunctival surgery after ophthalmic surgery. The compounds can be used for acute treatment of short-term conditions, or they can be administered chronically, especially in the case of a degenerative disease. The compounds can also be used prophylactically, especially prior to ocular surgery or non-invasive ophthalmic procedures or other types of surgery.

Pharmaceutical compositions suitable for parenteral administration include one or more compounds, in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders, which may be reconstituted into sterile, dispersible solutions or dispersions. injections just before use, which may contain antioxidants, buffers, bacteriostatic agents, solutes, which make the preparation isotonic with the blood of the intended recipient, or suspending or thickening agents.

Examples of suitable aqueous or non-aqueous vehicles that can be used in pharmaceutical compositions are water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and others), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. The proper fluidity can be maintained by using e.g. coating materials, such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. The prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, e.g., paraben, chlorobutanol, phenol sorbic acid, and others. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be provided by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

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

The injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers are poly (orthoesters) and poly (anhydrides). Injectable depot preparations are also made by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

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

The terms "parenteral administration" and "parenteral administration" as used herein mean modes of administration other than enteral or topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal injection or infusion. , intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intratracheal and intrasternal.

The terms "systemic administration", "systemically administered", "peripheral administration" and "peripherally administered" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system such that it enters the patient's system. hence, it undergoes metabolism and other similar processes, e.g. subcutaneous administration.

PL 204 628 B1

These compounds can be administered to humans and other animals for therapy by any suitable route of administration, including oral, nasal such as by spray, rectally, vaginally, parenterally, intra-reservoir and topically, such as by powders, ointments or drops, including buccal or sublingual use. .

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

The actual dosage levels of the active ingredients in the pharmaceutical compositions can be varied so as to obtain an amount of the active ingredient that is effective to produce the desired therapeutic response for the individual patient, composition, and mode of administration, so as to be non-toxic to the patient.

The dosage level selected will depend on various factors, including the activity of the particular compound or an ester, salt or amide thereof, route of administration, time of administration, removal rate of the particular compound used, duration of treatment, other drugs, compounds, and / or materials used in conjunction with the particular compound used. relationship used, age, gender, weight, condition, the general health and prior medical history of the treated patient; and other factors well known in the medical art.

A physician or veterinarian skilled in the art can readily determine and prescribe the effective amount of the required pharmaceutical composition. For example, a physician or veterinarian may initiate the doses used in the pharmaceutical composition at levels lower than required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above. Generally, intravenous and subcutaneous dosages of compounds for a patient, when used for the indicated pain relief effect, will be 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 as two, three, four, five, six or more sub-doses administered separately at appropriate times during the day, optionally in unit dosage forms.

Although it is possible to administer the compound singly, it is preferable to administer the compound as a pharmaceutical composition.

Packaged pharmaceutical compositions for treating an N-6 substituted 7-deazapurine responsive condition, e.g., undesirable increased activity of an adenosine receptor in a mammal include a container holding a therapeutically effective amount of at least one deazapurine as described above, and instructions for use of deazapurine for treating a deazapurine responsive condition in a mammal.

Deazapurines can be prepared using standard methods of organic synthesis. Deazapurines can be purified by reverse phase HPLC, chromatography, recrystallization etc. and their structures confirmed by mass spectrum analysis, elemental analysis, IR and / or NMR spectroscopy.

Typically, the synthesis of intermediates as well as deazapurines is carried out in solution. The addition and removal of one or more protecting groups is also customary in practice and is known to those skilled in the art. Typical synthetic schemes for the preparation of deazapurin intermediates are outlined below in Scheme I.

The invention is further illustrated by the following examples which should in no way be construed as additionally limiting. The content of all references, pending patent applications, and published patent applications cited in this application, including those referenced in the "Background" section, are hereby incorporated by reference. It should be understood that the models used in the examples are accepted models and that demonstration of efficacy in these models is supposed to be effective in humans.

Deazapurines can be prepared using standard methods of organic synthesis. Deazapurines can be purified by reverse phase HPLC, chromatography, recrystallization etc. and their structures confirmed by mass spectrum analysis, elemental analysis, IR and / or NMR spectroscopy.

Typically, synthesis of intermediates as well as deazapurines is carried out in solution. The addition and removal of one or more protecting groups is also standard practice, and it is

Known to those skilled in the art. Typical synthetic schemes for the preparation of deazapurine intermediates are outlined below in Scheme I.

<img file="PL204628B1_D0028.tif" />

wherein R3, R5 and R6 are as defined above.

Generally, protected 2-amino-3-cyanopyrrole can be treated with an acyl halide to form a carboxamido-3-cyanopyrrole which can be treated with acidic methanol affecting the ring closure of pyrrolo [2,3d] pyrimidin-4 (3H) -one (Muller, CE. Et al. J. Med. Chem. 40: 4396 (1997)). Removal of the pyrrole protecting groups followed by treatment with a chlorinating reagent, e.g., phosphorous oxychloride, gave substituted or unsubstituted 4-chloro-7H-pyrrolo [2,3d] pyrimidines. Treatment of chloropyrimidine with amines gave 7-deazapurines.

For example, as shown in Scheme I, N- (1-dl-phenylethyl) -2-amino-3-cyanopyrrole was treated with an acyl halide in pyridine and dichloromethane. The resulting N- (1-dl-phenylethyl) -2-phenylcarboxamido-3-cyanopyrrole was treated with a 10: 1 methanol / sulfuric acid mixture to effect ring closure, yielding dl-7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4 (3H) -one. Removal of the phenylethyl group by treatment of the pyrimidine with polyphosphoric acid (PPA) whereupon POCl3 gave the key intermediate, 4-chloro-7H-pyrrolo (2,3d] pyrimidine. Further treatment of 4-chloro-7H-pyrrolo [2,3d] pyrimidine with various amines listed in Table 1 gives compounds of formula (I) and (II).

PL 204 628 B1

M.⁺ + H.

M.⁺ + H.

<img file="PL204628B1_D0029.tif" />

343.2

<img file="PL204628B1_D0030.tif" />

351.27

<img file="PL204628B1_D0031.tif" />

343.18

<img file="PL204628B1_D0032.tif" />

430.35

<img file="PL204628B1_D0033.tif" />

337.21

<img file="PL204628B1_D0034.tif" />

359.44

<img file="PL204628B1_D0035.tif" />

364.19

<img file="PL204628B1_D0036.tif" />

404.32

<img file="PL204628B1_D0037.tif" />

330.18

<img file="PL204628B1_D0038.tif" />

330.45

<img file="PL204628B1_D0039.tif" />

347.22

<img file="PL204628B1_D0040.tif" />

339.47

<img file="PL204628B1_D0041.tif" />

350.28

<img file="PL204628B1_D0042.tif" />

353.41

<img file="PL204628B1_D0043.tif" />

344.19

<img file="PL204628B1_D0044.tif" />

324.45

<img file="PL204628B1_D0045.tif" />

349.16

<img file="PL204628B1_D0046.tif" />

359.38

<img file="PL204628B1_D0047.tif" />

371.12

<img file="PL204628B1_D0048.tif" />

379.40

PL 204 628 B1

<img file="PL204628B1_D0049.tif" />

cont. table 1

359.39

<img file="PL204628B1_D0050.tif" />

387.41

<img file="PL204628B1_D0051.tif" />

403.33

<img file="PL204628B1_D0052.tif" />

344.48

<img file="PL204628B1_D0053.tif" />

351.49

<img file="PL204628B1_D0054.tif" />

337.53

<img file="PL204628B1_D0055.tif" />

330.37

<img file="PL204628B1_D0056.tif" />

295.2

<img file="PL204628B1_D0057.tif" />

407.23

<img file="PL204628B1_D0058.tif" />

321.2

<img file="PL204628B1_D0059.tif" />

355.45

<img file="PL204628B1_D0060.tif" />

337.53

<img file="PL204628B1_D0061.tif" />

441.33

<img file="PL204628B1_D0062.tif" />

350.2

<img file="PL204628B1_D0063.tif" />

413.24

<img file="PL204628B1_D0064.tif" />

343.2

<img file="PL204628B1_D0065.tif" />

372.48

<img file="PL204628B1_D0066.tif" />

373.2

<img file="PL204628B1_D0067.tif" />

307.2

The general approach for making 6-substituted pyrroles is described in the following scheme (Scheme II).

PL 204 628 B1

Scheme II

<img file="PL204628B1_D0068.tif" />

wherein R1 to R5 are as defined above.

Transesterification and alkylation of ethyl cyanoacetate with α-haloketone gives the ketomethylester. Protection of the ketone followed by treatment with an amidine (e.g., alkyl, aryl or alkylaryl) hydrochloride resulted in the formation of a ketal protected pyrimidine. Removal of the protecting groups followed by cyclization and treatment with phosphorous oxychloride gave the chloride intermediate which can be further treated with an amine to yield a 6-substituted priol. Additionally, pyrrole-nitrogen alkylation can be achieved under conditions known in the art.

The general approach for making 5-substituted pyrroles is described in the following scheme (Scheme III).

PL 204 628 B1

Scheme III

<img file="PL204628B1_D0069.tif" />

wherein R1 to R6 are as defined above and R is removable protecting groups.

Condensation of malononitrile and excess ketone followed by bromination of the product gave a mixture of starting material, monobrominated and dibrominated products which were treated with alkylamine, arylamine or alkylarylamine. The resulting amine product was acylated with acid chloride and the monoacylated pyrrole was cyclized in the presence of acid to give the corresponding pyrimidine. The pyrrole protecting groups were removed with polyphosphoric acid and treated with phosphorous oxychloride to produce a chlorinated product. The chlorinated pyrrole can then be treated with an amine to form an amine substituted pyrrole at the 5-position. Alkylation of the pyrrole-nitrogen can be achieved under conditions known in the art.

Schemes IV and V describe the preparation of deazapurins 1 and 2.

<img file="PL204628B1_D0070.tif" />

where R5 and R6 are as described above, e.g., CH3.

Specific 6-methylpyrrolopyrimidine preparation:

The key reaction towards the 6-methylpyrrolopyrimidine (1) [R5 = CH3] was the cyclization of the cyanoacetate with benzamidine to the pyrimidine. It was believed that methyl cyanoacetate would cyclize more efficiently with benzamidine to pyrimidine than the corresponding ethyl ester. Thus, transesterification and alkylation of ethyl cyanoacetate in the presence of NaOMe and excess α-haloacetyl moiety, e.g., chloroacetone, gave the desired methyl ester (3) in 79% yield (Scheme IV). Ketoester (3)

PL 204 628 B1 was protected as acetal (4) in a yield of 81%. A new method of pyrimidine cyclization (5) was obtained using an amidine hydrochloride, e.g., benzamidine hydrochloride, with 2 equivalents of DBU giving an isolated 5 with a yield of 54%. This method improves the yield by 20% using published conditions that use NaOMe during cyclization with guanidine. Cyclization to pyrrolopyrimidine (6) was achieved by deprotecting the acetal in an aqueous HCl solution with a yield of 78%. Reaction of (6) with phosphorous oxychloride at reflux gave the corresponding 4-chloro derivative (7). Coupling with trans-4-aminocyclohexanol in dimethylsulfoxide at 135 ° C gave (1) an efficiency of 57% from (7). One skilled in the art will appreciate that the choice of reagents allows for great flexibility in selecting the desired R5 substituent.

Scheme IV

<img file="PL204628B1_D0071.tif" />

Specific 5-methylpyrrolopyrimidine preparation

Knoevengel condensation of malononitrile and excess ketone, e.g. acetone, at the reflux temperature of benzene gave 8 in 50% yield after distillation. Bromination of 8 with N-bromosuccinimide in the presence of benzoyl peroxide in chloroform gave a mixture of starting material, mono- (9), and di-brominated products (5/90/5) after distillation (70%). The mixture was reacted with α-methylalkylamine or with α-methylarylamine, e.g., α-methylbenzylamine, to provide the aminopyrrole (10). After passing through a short silica gel column, the partially purified amine (31% yield) was acylated with an acid chloride, e.g., benzoyl chloride to afford mono- (11), and diacylated (12) pyrroles which were separated by flash chromatography. The acid hydrolysis of the disubstituted pyrrole (12) produced a combined yield of 29% for the acylpyrrole (11). Cyclization in the presence of concentrated sulfuric acid and DMF gave (13) (23%) which was deprotected with polyphosphoric acid to give (14). Reaction of (14) with phosphorous oxychloride at reflux gave the corresponding 4-chloro derivative (15). Coupling with trans-4-aminocyclohexanol in dimethylsulfoxide at 135 ° C gave (2) [R6 = CH3] in 30% of (14) (see scheme V). One skilled in the art will appreciate that the choice of reagents allows for great flexibility in selecting the desired R6 substituent.

PL 204 628 B1

Scheme V

<img file="PL204628B1_D0072.tif" />

Alternative synthetic route for the preparation of R6-substituted pyrroles, e.g., 5-methylpyrrolopyrimidines:

This alternative route for the preparation of R6-substituted pyrroles, e.g., 5-methylpyrrolopyrimidine, involves the transesterification and alkylation of ethyl cyanoacetate to (16) (Scheme VI). Condensation of (16) with benzamidine hydrochloride using 2 equivalents of DBU gives the pyrimidine (17). Cyclization to the pyrrolopyrimidine (14) is achieved by deprotecting the acetal in an aqueous HCl solution. Reaction of (14) with phosphorous oxychloride at reflux gave the corresponding 4-chloro derivative (15). Coupling with trans-4-aminocyclohexanol in dimethylsulfoxide at 135 ° C gives 2. This procedure reduces the number of synthetic reactions towards the target compound (2) from 9 steps to 4. In addition, the yield is improved dramatically. Again, one skilled in the art will appreciate that the choice of reagents allows for great flexibility in selecting the desired R6 substituent.

PL 204 628 B1

Scheme VI

<img file="PL204628B1_D0073.tif" />

The general approach to make des-methylpyrrole is described in the following scheme (Scheme VII)

PL 204 628 B1

Scheme VII

<img file="PL204628B1_D0074.tif" />

Where R1 to R3 are defined as above.

Alkylation of the alkyl cyanoacetate with diethyl acetal in the presence of a base gave the cyanodiethyl acetal which was treated with an amidine salt to form the methylpyrrolopyrimidine precursor. The precursor was chlorinated and treated with an amine to form the target des-methylpyrrolopyrimidine as shown above.

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

PL 204 628 B1

Scheme VIII

<img file="PL204628B1_D0075.tif" />

Commercially available methyl cyanoacetate was alkylated with bromoacetoaldehyde diethyl acetal in the presence of potassium carbonate and Nal yielded c (19). Cyclization to pyrimidine (20) was achieved in two steps. Initially, a pyrimidino acetal was formed by reaction (19) using benzamidine hydrochloride with 2 equivalents of DBU. The obtained pyrimidine acetal was deprotected without purification using 1N HCl aqueous solution and the obtained aldehyde was cyclized to pyrrolopyrimidine (20) which was isolated by filtration. Reaction of (20) with phosphorous oxychloride at reflux gave the corresponding 4-chloro derivative (21). Coupling of the chlorine derivative with trans-4-aminocyclohexanol in DMSO at 135 ° C gave compound (18) from compound (21).

Schemes II-VIII show that it is possible to attach functionalities at the 5-position and 6-position of the pyrrolopyrimidine ring. By using different starting reagents and slight modification of the above reaction schemes, different functional groups can be introduced in the 5th and 6th positions of formula (I) and (II). Table 2 illustrates some examples.

PL 204 628 B1

Table 2. Selected list of 5- and 6-substituted pyrrolopyrimidines.

<td>Initial reagent</td><td>R5</td><td>R6</td>

<td>x-^ABOUT'y-^ABOUT\ cr</td><td>H.</td><td>H.</td>

<td>X) Cl— / \ = / \₃</td><td>H.</td><td>Substituted Ar</td>

<td>O</td><td>H.</td><td>CH2C (O) OCH3</td>

<td>0 0 Cl</td><td>C (O) OCH3</td><td>CH3</td>

<td>0 0 Α /, χ Cl ^H.</td><td>C (O) NHCH3</td><td>CH3</td>

The invention is further illustrated by the following examples which should in no way be construed as additionally limiting. The content of all references, pending patent applications, and published patent applications cited in this application, including those referenced in the "Background" section, are hereby incorporated by reference. It should be understood that the models used in the examples are acceptable models and that demonstration of efficacy in these models is supposed to be effective in humans.

Examples

Receiving 1:

₁

A modified Seel and Lupke alkylation method was used¹. NaOMe solution (25% w / v; 58.1 mmol) was slowly added to an ice-cooled (0 ° C) solution of O1 ethyl cyanoacetate (6.58 g, 58.1 mmol) and MeOH (20 mL). After 10 minutes, chloroacetone (5 ml; 62.8 mmol) was added slowly. After 4 hours, the solvent was removed. The brown oil was diluted with EtOAc (100 mL) and washed with H 2 O (100 mL). The organic fraction was dried, filtered, and concentrated to 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.

NMR ¹H (200 MHz, CDCl3) δ 4.24 (q, J = 7.2 Hz, OCH2), 3.91 (dd, 1H, J = 7.2, 7.0 Hz, CH), 3.62 ( s, 3H, OCH3), 3.42 (dd, 1H, J = 15.0, 7.1Hz, 1x CH2); 3.02 (dd, 1H, J = 15.0, 7.0Hz, 1x CH2); 2.44 (s, 3H, CH3), 1.26 (t, J = 7.1Hz, CH3 ester).

¹Seela, F .; Lupke, U. Chem. Ber. 1977, 110, 1462-1469.

Receiving 2:

₁

The Seel and Lupke procedure was used.¹ So, protection of ketone (3) (Scheme IV; 5.0 g, 32.2 mmol) with ethylene glycol (4 mL, 64.4 mmol) in the presence of TsOH (100 mg) gave (4) as an oil (Scheme IV ; 5.2 g, 81.0) after flash chromatography (SiO2; 3/7 EtOAc / Hex, Rf 0.35). It still contains ~ 5% ethyl ester.

NMR ¹H (200 MHz, CDCl3) δ 4.24 (q, J = 7.2 Hz, OCH2), 3.98 (s, 4H, 2 x acetal-CH2), 3.79 (s, 3H, OCH3), 3.62 (dd, 1H, J = 7.2, 7.0Hz, CH), 2.48 (dd, 1H, J = 15.0, 7.1Hz, 1 x CH2), 2.32 ( dd, 1H, J = 15.0, 7.0Hz, 1x CH2); 1.35 (s, 3H, CH3), 1.26 (t, J = 7.1Hz, ester-CH3);

MS (ES): 200.1 (M⁺+1).

¹Seela, F .; Lupke, U. Chem. Ber. 1977, 110, 1462-1469.

Receiving 3:

A solution of acetal (4) (Scheme IV, 1 g, 5.02 mmol), benzamidine (786 mg, 5.02 mmol), and DBU (1.5 ml, 10.04 mmol) in dry DMF (15 ml) was heated to 85 ° C for 15 hours. The mixture was diluted with CHCl3 (30 ml) and washed with 0.5 N NaOH (10 ml) and H2O (20 ml). The organic fraction was dried, filtered and concentrated to a brown oil. Flash chromatography (SiO2; 1/9 EtOAc / CH2Cl2, Rf 0.35) was attempted but material crystallized on the column. The silica gel was washed with MeOH. Fractions containing product (5) (Scheme IV) were concentrated and used without further purification (783 mg, 54.3%):

NMR ¹H (200 MHz, CDCl3) δ 8.24 (m, 2H, Ar-H), 7.45 (m, 3H, Ar-H), 5.24 (br s, 2H, NH2), 3.98 ( s, 4H, 2xacetal-CH2), 3.60-3.15 (m, 2H, CH2), 1.38 (s, 3H, CH3);

MS (ES): 288.1 (M⁺+1).

Preparation of compound (20) (Scheme VIII): Acetal solution (19) (4.43 g, 20.6 mmol)¹, benzamine hydrochloride (3.22 g, 20.6 mmol), and DBU (6.15 mL, 41.2 mmol) in dry DMF (20 mL) was heated to 85 ° C for fifteen hours. The mixture was diluted with 100 mL of CHCl3, and washed with HBO (2 x 50 mL). The organic fraction was dried, filtered, and concentrated to a dark brown oil. The dark brown oil was stirred in 1N HCl (100 mL) for 2 hours at room temperature. The resulting suspension was filtered to afford the HCl salt (20) as a tan solid (3.60 g, 70.6%);

NMR ¹H (200 MHz, DMSO-d6) 11.92 (s, 1H), 8.05 (m, 2H, Ar-H), 7.45 (m, 3H, Ar-H), 7.05 (s, 1H , pyrrole-H);

MS (ES): 212.1 (M⁺+1).

Receiving 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 provide the 2-phenyl-6-methyl-7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one HCl salt as a tan solid (498 mg, 78.0%):

NMR ¹H (200 MHz, DMSO-d6) 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, CH3);

MS (ES): 226.1 (M⁺+1).

Receiving 5:

A modified cyclization method was used by Chen et al.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 stirred for 15 hours. The mixture was diluted with EtOAc (200 mL) and washed with 0.5 N NaOH (50 mL). The organic fraction was dried, filtered, and concentrated to black tar (19.2 g; 94%). The residue was partially purified by flash chromatography (SiO2; 4/96 MeOH / CH2Cl2, Rf 0.35) to give a black solid (6.38 g, 31%) which is dl-1- (1-phenylethyl) -2-amino- 3-cyano-4-methylpyrrole:

MS (ES): 226.1 (M⁺+1).

¹Chen, YL; Mansbach, RS; Winter, SM; Brooks, E .; Collins, J .; Corman, ML; Dunaiskis, AR; Faraci, WS; Gallaschun, RJ; Schmidt, A .; Schulz, DWJ Med. Chem. 1997, 40, 1749-1754.

Receiving 6:

For dl-1- (1-phenylethyl) -2-amino-3-cyano-4,5-dimethylpyrrole solution¹ (14.9 g, 62.5 mmol) and pyridine (10.0 mL) in dichloromethane (50.0 mL) were added benzoyl chloride (9.37 g, 66.7 mmol) at 0 ° C. After stirring at 0 ° C for 1 hour, hexane (10.0 mL) was added to aid in the precipitation of the product. The solvent was removed under reduced pressure and the solid was recrystallized from EtOH / H2O to give 13.9 g (65%) of dl-1- (1-phenylethyl) -2-phenylcarbonylamino-3-cyano-4,5-dimethylpyrrole.

Melting point 218-221 ° C;

NMR ¹H (200 MHz, CDCl3) δ 1.72 (s, 3H), 1.76 (d, J = 7.3 Hz, 3H), 1.98 (s, 3H), 5.52 (q, J = 7.3 Hz, 1H), 7.14-7.54 (m, 9H), 7.68-7.72 (dd, J = 1.4 Hz, 6.9 Hz, 2H), 10.73 ( s, 1H);

MS (ES): 344.4 (M⁺+1).

¹Liebigs Ann. Chem. 1986, 1485-1505.

The following compounds were obtained in a similar manner to those of the preparation of 6: dl-1- (1-phenylethyl) -2- (3-pyridyl) carbonylamino-3-cyano-4,5-dimethylpyrrole.

NMR ¹H (200 MHz, CDCl3) δ 1.83 (d, J = 6.8 Hz, 3H), 2.02 (s, 3H), 2.12 (s, 3H), 5.50 (q, J = 6.8

Hz, 1H), 7.14-7.42 (m, 5H), 8.08 (m, 2H), 8.75 (m, 3H);

MS (ES): 345.2 (M⁺+1).

dl-1- (1-phenylethyl) -2- (2-furyl) carbonylamino-3-cyano-4,5-dimethylpyrrole.

NMR ¹H (200 MHz, CDCl3) δ 1.84 (d, J = 7.4 Hz, 3H), 1.92 (s, 3H), 2.09 (s, 3H), 5.49 (q, J = 7.4 Hz, 1H), 6.54 (dd, J = 1.8 Hz, 3.6 Hz, 1H), 7.12-7.47 (m, 7H);

MS (ES): 334.2 (M⁺+1), 230,1.

dl-1- (1-phenylethyl) -2- (3-furyl) carbonylamino-3-cyano-4,5-dimethylpyrrole.

PL 204 628 B1

NMR ¹H (200 MHz, CDCl3) δ 1.80 (d, J = 7 Hz 3H), 1.89 (s, 3H), 2.05 (s, 3H), 5.48 (q, J = 7 Hz, 1H), 6.59 (s, 1H), 7.12-7.40 (m, 6H), 7.93 (s, 1H);

MS (ES): 334.1 (M⁺+1), 230,0.

dl-1- (1-phenylethyl) -2-cyclopentylcarbonylamino-3-cyano-4,5-dimethylpyrrole.

NMR ¹H (200 MHz, CDCl3) δ 1.82 (d, J = 7.4 Hz, 3H), 1.88 (s, 3H), 2.05 (s, 3H), 1.63-1.85 ( m, 8H), 2.63 (m, 1H), 5.43 (q, J = 7.4Hz, 1H), 6.52 (s, 1H), 7.05-7.20 (m, 5H );

MS (ES): 336.3 (M⁺+1).

dl-1- (1-phenylethyl) -2- (2-thienyl) carbonylamino-3-cyano-4,5-dimethylpyrrole.

NMR ¹H (200 MHz, CDCl3) δ 1.82 (d, J = 6.8 Hz, 3H), 1.96 (s, 3H), 2.09 (s, 3H), 5.49 (q, J = 6.8 Hz, 1H), 7.05-7.55 (m, 8H);

MS (ES): 350.1 (M⁺+1), 246,0.

dl-1- (1-phenylethyl) -2- (3-thienyl) carbonylamino-3-cyano-4,5-dimethylpyrrole.

NMR ¹H (200 MHz, CDCl3) δ 1.83 (d, J = 7.0 Hz, 3H), 1.99 (s, 3H), 2.12 (s, 3H), 5.49 (q, J = 7.0 Hz, 1H), 6.90 (m, 1H), 7.18-7.36 (m, 6H), 7.79 (m, 1H);

MS (ES): 350.2 (M⁺+1), 246,1.

dl-1- (1-phenylethyl) -2- (4-fluorophenyl) carbonylamino-3-cyano-4,5-dimethylpyrrole.

NMR ¹H (200 MHz, CDCl3) δ 1.83 (d, J = 7.4 Hz, 3H), 1.96 (s, 3H), 2.08 (s, 3H), 5.51 (q, J = 7.4 Hz, 1H), 7.16-7.55 (m, 9H);

MS (ES): 362.2 (M⁺+1), 258,1.

dl-1- (1-phenylethyl) -2- (3-fluorophenyl) carbonylamino-3-cyano-4,5-dimethylpyrrole.

NMR ¹H (200 MHz, CDCl3) δ 1.83 (d, J = 7.4 Hz 3H), 1.97 (s, 3H), 2.10 (s, 3H), 5.50 (q, J = 7 , 4 Hz, 1H), 7.05-7.38 (m, 7H), 7.67-7.74 (m, 2H);

MS (ES): 362.2 (M⁺+1), 258,1.

dl-1- (1-phenylethyl) -2- (2-fluorophenyl) carbonylamino-3-cyano-4,5-dimethylpyrrole.

NMR ¹H (200 MHz, CDCl3) δ 1.85 (d, J = 7.2 Hz, 3H), 1.94 (s, 3H), 2.11 (s, 3H), 5.50 (q, J = 7.2 Hz, 1H), 7.12-7.35 (m, 6H), 7.53 (m, 1H), 7.77 (m, 1H), 8.13 (m, 1H);

MS (ES): 362.2 (M⁺+1), 258.0, dl-1- (1-phenylethyl) -2-isopropylcarbonylamino-3-cyano-4,5-dimethylpyrrole.

NMR ¹H (200 MHz, CDCl3) δ 1.19 (d, J = 7.0 Hz, 6H), 1.82 (d, J = 7.2 Hz, 3H), 1.88 (s, 3H), 2 . 06 (s, 3H), 2.46 (m, 1H), 5.39 (m, J = 7.2Hz, 1H), 6.64 (s, 1H), 7.11-7.36 ( m, 5H);

MS (ES): 310.2 (M⁺+1), 206,1.

In the case of acylation of dl-1- (1-phenylethyl) -2-amino-3-cyano-4-methylpyrrole, the monoacylated dl-1- (1-phenylethyl) -2-benzoylamino-3-cyano-4-dimethylpyrrole was obtained and diacylated pyrrole dl-1- (1-phenylethyl) -2-dibenzoylamino-3-cyano-4-methylpyrrole.

Monoacylated pyrrole: NMR ¹H (200 MHz, CDCl3) δ 7.69 (d, 2H, J = 7.8 Hz, Ar-H), 7.58-7.12 (m, 8H, Ar-H), 6.18 (s , 1H, pyrrol-H), 5.52 (q, 1H, J = 7.2Hz, CH-CH3), 2.05 (s, 3H, pyrrole-CH3), 1.85 (d, 3H, J = 7.2 Hz, CH-CH3);

MS (ES): 330.2 (M⁺+1);

Diacylated pyrrole: NMR ¹H (200 MHz, CDCl3) δ 7.85 (d, 2H, J = 7.7 Hz, Ar-H), 7.74 (d, 2H, J = 7.8 Hz, Ar-H), 7, 52-7.20 (m, 9H, Ar-H), 7.04 (m, 2H, Ar-H), 6.21 (s, 1H, pyrrol-H), 5.52 (q, 1H, J = 7.2 Hz, CH-CH3), 1.77 (d, 3H, J = 7.2 Hz, CH-CH3), 1.74 (s, 3H, pyrrole-CH3);

MS (ES): 434.1 (M⁺+1).

Receiving 7:

To a solution of dl-1- (1-phenylethyl) -2-phenylcarboxamido-3-cyano-4,5-dimethylpyrrole (1.0 g, 2.92 mmol) in methanol (10.0 ml) was added concentrated sulfuric acid (1 0.0 ml) at 0 ° C. The resulting mixture was heated to reflux for 15 hours and cooled to room temperature. The precipitate was filtered to give 0.48 g (48%) of dl-5,6-dimethyl-2-phenyl-7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, CDCl3) δ 2.02 (d, J = 7.4 Hz, 3H), 2.04 (s, 3H), 2.41 (s, 3H), 6.25 (q, J = 7.4 Hz, 1H), 7.22-7.50 (m, 9H), 8.07-8.12 (dd, J = 3.4 Hz, 6.8 Hz, 2H), 10.51 ( s, 1H);

MS (ES): 344.2 (M⁺+1).

The following compounds were obtained in a similar manner to those of the preparation of 7: dl-5,6-dimethyl-2- (3-pyridyl) -7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4 (3H) - he.

NMR ¹H (200 MHz, CDCl3) δ 2.03 (d, J = 7.2 Hz, 3H), 2.08 (s, 3H), 2.42 (s, 3H), 6.24 (q, J = 7.2

Hz, 1H), 7.09-7.42 (m, 5H), 8.48 (m, 2H), 8.70 (m, 3H);

MS (ES): 345.1 (M⁺+1).

Dl-5,6-dimethyl-2- (2-furyl) -7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, CDCl3) δ 1.98 (d, J = 7.8 Hz, 3H), 1.99 (s, 3H), 2.37 (s, 3H), 6.12 (q, J = 7.8 Hz, 1H), 6.48 (dd, 3 = 1.8 Hz, 3.6 Hz, 1H), 7.17-7.55 (m, 7H), 9.6 (s, 1H) ;

MS (ES): 334.2 (M⁺+1).

dl-5,6-dimethyl-2- (3-furyl) -7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, CDCl3) δ 1.99 (d, J = 7 Hz, 3H), 2.02 (s, 3H), 2.42 (s, 3H), 6.24 (q, J = 7 Hz , 1H), 7.09 (s, 1H), 7.18-7.32 (m, 5H), 7.48 (s, 1H), 8.51 (s, 1H);

MS (ES): 334.2 (M⁺+1).

dl-5,6-dimethyl-2-cyclopentyl-7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, CDCl3) δ 1.95 (d, J = 7.4 Hz, 3H), 2.00 (s, 3H), 2.33 (s, 3H), 1.68-1.88 ( m, 8H), 2.97 (m, 1H), 6.10 (q, J = 7.4 Hz, 1H); 7.16-7.30 (m, 5H); 9.29 (s, 1H);

MS (ES): 336.3 (M⁺+1).

dl-5,6-dimethyl-2- (2-thienyl) -7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, CDCl3) δ 2.02 (d, J = 7.2 Hz, 3H), 2.06 (s, 3H), 2.41 (s, 3H), 6.13 (q, J = 7.2 Hz, 1H), 7.12 (dd, J = 4.8, 2.8 Hz, 1H), 7.26-7.32 (m, 5H), 7.44 (d, J = 4 , 8 Hz, 1H), 8.01 (d, J = 2.8 Hz, 1H) 11.25 (s, 1H);

MS (ES): 350.2 (M⁺+1).

dl-5,6-dimethyl-2- (3-thienyl) -7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, CDCl3) δ 2.00 (d, J = 7.4 Hz, 3H), 2.05 (s, 3H), 2.43 (s, 3H), 6.24 (q, J = 7.4 Hz, 1H), 7.24-7.33 (m, 5H), 7.33-7.39 (m, 1H), 7.85 (m, 1H), 8.47 (m, 1H ), 12.01 (s. 1H);

MS (ES): 350.2 (M⁺+1).

dl-5,6-dimethyl-2- (4-fluorophenyl) -7N-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, CDCl3) δ 2.01 (d, J = 6.8 Hz, 3H), 2.05 (s, 3H), 2.42 (s, 3H), 6.26 (q, J = 6.8 Hz, 1H), 7.12-7.36 (m, 7H), 8.23-8.30 (m, 2H), 1.82 (s, 1H);

MS (ES): 362.3 (M⁺+1).

dl-5,6-dimethyl-2- (3-fluorophenyl) -7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, CDCl3) δ 2.02 (d, J = 7.4 Hz, 3H), 2.06 (s, 3H), 2.44 (s, 3H), 6.29 (q, J = 7.4 Hz, 1H), 7.13-7.51 (m, 7H), 8.00-8.04 (m, 2H), 11.72 (s, 1H);

MS (ES): 362.2 (M⁺+1).

dl-6,6-dimethyl-2- (2-fluorophenyl) -7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, CDCl3) δ 2.00 (d, J = 7.2 Hz, 3H), 2.05 (s, 3H), 2.38 (s, 3H), 6.24 (q, J = 7.2 Hz, 1H), 7.18-7.45 (m, 8H), 8.21 (m, 1H), 9.54 (s, 1H);

MS (ES): 362.2 (M⁺+1).

dl-5,6-dimethyl-2-isopropyl-7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, CDCl3) δ 1.30 (d, J = 6.8 Hz, 3H), 1.32 (d, J = 7.0 Hz, 3H), 2.01 (s, 3H), 2 . 34 (s, 3H), 2.90 (m, 1H), 6.13 (m, 1H), 7.17-7.34 (m, 5H), 10.16 (s, 1H);

MS (ES): 310.2 (M⁺+1).

Receiving 8:

A solution of dl-1- (1-phenylethyl) -2-benzoylamino-3-cyano-4-dimethylpyrrole (785 mg, 2.38 mmol) with concentrated H2SO4 (1 ml) in DMF (13 ml) was stirred at 130 ° C for 48 hours. The black solution was diluted with CHCl3 (100 mL) and washed with 1N NaOH (30 mL), and brine (30 mL). The organic fraction was dried, filtered, concentrated, and purified by flash chromatography (SiO2; 8/2 EtOAc / Hex, Rf 0.35) to a brown solid (184 mg, 24%) i.e. dl-5-methyl-2-phenyl- 7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, CDCl3) δ 8.18 (m, 2H, Ar-H), 7.62-7.44 (m, 3H, Ar-H), 7.40-7.18 (m, 5H, ArH), 6.48 (s, 1H, pyrrole-H), 6.28 (q, 1H, J = 7.2Hz, CH-CH3), 2.18 (s, 3H, pyrrole-CH3), 2 .07 (d, 3H, J = 7.2Hz, CH-CH3);

MS (ES): 330.2 (M⁺+1).

Receiving 9:

A mixture of dl-1- (1-phenylethyl) -2-amino-3-cyano-4,5-dimethylpyrrole (9.60 g, 40.0 mmol) and formic acid (50.0 ml, 98%) was heated to reflux for 5 hours. After cooling to room temperature and scraping the sides of the flask, a copious precipitate was formed and filtered. The material was washed with water until the washes were neutral pH, yielding dl-5,6-dimethyl-7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, CDCl3) δ 1.96 (d, J = 7.4 Hz, 3H), 2.00 (s, 3H), 2.38 (s, 3H), 6.21 (q, J = 7.4 Hz, 1H), 7.11-7.35 (m, 5H), 7.81 (s, 1H), 11.71 (s, 1H);

PL 204 628 B1

MS (ES): 268.2 (M⁺+1).

Receiving 10:

dl-5,6-dimethyl-2-phenyl-7H-7- (1-phenylethyl) pyrrolo [2,3d] pyrimidin-4 (3H) -one (1.0 g, 2.91 mmol) was suspended in polyphosphoric acid (30.0 ml). The mixture was heated at 100 ° C for 4 hours. The hot slurry was poured onto ice water, stirred vigorously to disperse the slurry, and basified to pH 6 with solid KOH. The resulting solid was filtered and collected to give 0.49 g (69%) of 5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, DMSO-d6) δ 2.17 (s, 3H), 2.22 (s, 3H), 7.45 (br, 3H), 8.07 (br, 2H), 11.49 ( s, 1H), 11.82 (s, 1H);

MS (ES): 344.2 (M⁺+1).

The following compounds were obtained in a similar manner to those of Preparation 10:

5-methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one.

MS (ES): 226.0 (M⁺+1).

5.6-dimethyl-2- (3-pyridyl) -7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one.

MS (ES): 241.1 (M⁺+1).

5.6-dimethyl-2- (2-furyl) -7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, DMSO-d6) δ 2.13 (s, 3H), 2.18 (s, 3H), 6.39 (dd, J = 1.8, 3.6 Hz, 1H), 6, 65 (dd, J = 1.8 Hz, 3.6 Hz, 1H), 7.85 (dd, J = 1.8, 3.6 Hz, 1H,), 11.45 (s, 1H), 1 . 60 (s. 1H);

MS (ES): 230.1 (M⁺+1).

5.6-dimethyl-2- (3-furyl) -7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, DMSO-d6) δ 2.14 (s, 3H), 2.19 (s, 3H), 6.66 (s, 1H), 7.78 (s, 1H), 8.35 ( s, 1H), 11.3 (s, 1H), 11.4 (s, 1H);

MS (ES): 230.1 (M⁺+1).

5,6-dimethyl-2-cyclopentyl-7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, DMSO-d6) δ 1.57-1.91 (m, 8H), 2.12 (s, 3H), 2.16 (s, 3H), 2.99 (m, 1H), 11.24 (s, 1H), 11.38 (s, 1H);

MS (ES): 232.2 (M⁺+1).

5.6-dimethyl-2- (2-thienyl) -7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, DMSO-d6) δ 2.14 (s, 3H), 2.19 (s, 3H), 7.14 (dd, J = 3.0, 5.2 Hz, 1H), 7, 70 (d, J = 5.2 Hz, 1H), 8.10 (d, J = 3.0 Hz, 1H), 11.50 (s, 1H);

MS (ES): 246.1 (M⁺+1).

5.6-dimethyl-2- (3-thienyl) -7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, DMSO-d6) δ 2.17 (s, 3H), 2.21 (s, 3H), 7.66 (m, 1H), 7.75 (m, 1H), 8.43 ( m, 1H), 11.47 (s, 1H), 11.69 (s, 1H);

MS (ES): 246.1 (M⁺+1).

5.6-dimethyl-2- (4-fluorophenyl) -7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, DMSO-d6) δ 2.17 (s, 3H), 2.21 (s, 3H), 7.31 (m, 2H), 8.12 (m, 2H), 11.47 ( s, 1H);

MS (ES): 258.2 (M⁺+1).

5.6-dimethyl-2- (3-fluorophenyl) -7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, DMSO-d6) δ 2.18 (s, 3H), 2.21 (s, 3H), 7.33 (m, 1H), 7.52 (m, 1H), 7.85- 7.95 (m, 2H), 11.56 (s, 1H), 11.80 (s, 1H);

MS (ES): 258.1 (M⁺+1).

5.6-dimethyl-2- (2-fluorophenyl) -7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, DMSO-d6) δ 2.18 (s, 3H), 2.22 (s, 3H), 7.27-7, 37 (m, 2H), 7.53 (m, 1H), 7.68 (m, 1H), 11.54 (s, 1H), 11.78 (s, 1H);

MS (ES): 258.1 (M⁺+1).

5.6-dimethyl-2-isopropyl-7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, DMSO-d6) δ 1.17 (d, J = 6.6 Hz, 6H), 2.11 (s, 3H), 2.15 (s, 3H), 2.81 (m, 1H),

11.20 (s, 1H), 11.39 (s, 1H);

MS (ES): 206.1 (M⁺+1).

5.6-dimethyl-7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one.

NMR ¹H (200 MHz, DMSO-d6) δ 2.13 (s, 3H), 2.17 (s, 3H), 7.65 (s, 1H);

MS (ES): 164.0 (M⁺+1).

PL 204 628 B1

Receiving 11:

A solution of 5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidin-4 (3H) -one (1.0 g, 4.2 mmol) in phosphorous oxychloride (25.0 mL) was heated to reflux for 6 hours then concentrated to dryness under reduced pressure. Water was added to the residue to induce crystallization, and the resulting solid was filtered and collected to give 0.90 g (83%) of 4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, DMSO-d6) δ 2.33 (s, 3H), 2.33 (s, 3H), 7.46-7.49 (m, 3H), 8.30-8.35 (m , 2H),

12.20 (s. 1H);

MS (ES): 258.1 (M⁺+1).

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

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

MS (ES): 244.0 (M⁺+1).

4-chloro-6-methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.

MS (ES): 244.0 (M⁺+1).

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

NMR ¹H (200 MHz, DMSO-d6) 8.35 (2, 2H), 7.63 (br s, 1H), 7.45 (m, 3H), 6.47 (br s, 1H);

MS (ES): 230.0 (M⁺+1).

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

MS (ES): 259.0 (M⁺+1).

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

NMR ¹H (200 MHz, DMSO-d6) δ 2.35 (s, 3H), 2.35 (s, 3H), 6.68 (dd, J = 1.8, 3.6 Hz, 1H), 7, 34 (dd, J = 1.8 Hz, 3.6 Hz, 1H), 7.89 (dd, J = 1.8, 3.6 Hz, 1H);

MS (ES): 248.0 (M⁺+1).

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

NMR ¹H (200 MHz, DMSO-d6) δ 2.31 (s, 3H), 2.31 (s, 3H), 6.62 (s, 1H), 7.78 (s, 1H), 8.18 ( s, 1H), 12.02 (s, 1H);

MS (ES): 248.1 (M⁺+1)

4-chloro-5,6-dimethyl-2-cyclopentyl-7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, DMSO-d6) δ 1.61-1.96 (m, 8H), 2.27 (s, 3H), 2.27 (s, 3H), 3.22 (m, 1H), 11.97 (s. 1H);

MS (ES): 250.1 (M⁺+1).

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

NMR ¹H (200 MHz, DMSO-d6) δ 2.29 (s, 3H), 2.31 (s, 3H), 7.14 (dd, J = 3.1Hz, 4.0Hz, 1H), 7, 33 (d, J = 4.9 Hz, 1H), 7.82 (d, J = 3.1 Hz, 1H), 12.19 (s, 1H);

MS (ES): 264.1 (M⁺+1).

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

NMR ¹H (200 MHz, DMSO-d6) δ 2.32 (s, 3H), 2.32 (s, 3H), 7.62 (dd, J = 3.0, 5.2 Hz, 1H), 7, 75 (d, J = 5.2 Hz, 1H), 8.20 (d, J = 3.0 Hz, 1H);

MS (ES): 264.0 (M⁺+1).

4-chloro-5,6-dimethyl-2- (4-fluorophenyl] -7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, DMSO-d6) δ 2.33 (s, 3H), 2.33 (s, 3H), 7.30 (m, 2H), 8.34 (m, 2H), 12.11 ( s, 1H);

MS (ES): 276.1 (M⁺+1).

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

NMR 1R (200 MHz, DMSO-d6) δ 2.31 (s, 3H), 2.33 (s, 3H), 7.29 (m, 1H), 7.52 (m, 1H), 7.96 (m, 1H), 8.14 (m, 1H), 11.57 (s, 1H);

MS (ES): 276.1 (M⁺+1).

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

NMR ¹H (200 MHz, DMSO-d6) δ 2.34 (s, 3H), 2.34 (s, 3H), 7.33 (m, 2H), 7.44 (m, 1H), 7.99 ( m, 1H). I 2.23 (s, 1H);

MS (ES): 276.1 (M⁺+1).

4-chloro-5,6-dimethyl-2-isopropyl-7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, DMSO-d6) δ 1.24 (d, J = 6.6 Hz, 6H), 2.28 (s, 3H), 2.28 (s, 3H), 3.08 (q, J = 6.6 Hz, 1H), 11.95 (s, 1H);

MS (ES): 224.0 (M⁺+1).

4-chloro-5,6-dimethyl-7H-pyrrolo [2,3d] pyrimidine.

PL 204 628 B1

NMR ¹H (200 MHz, DMSO-d6) δ 2.31 (s, 3H), 2.32 (s, 3H), 8.40 (s, 1H);

MS (ES): 182.0 (M⁺+1).

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

Receiving 12:

To a solution of dl-1,2-diaminopropane (1.48 g, 20.0 mmol) and sodium carbonate (2.73 g, 22.0 mmol), dioxane (100.0 ml) and water (100.0 ml) was added di-tert-dicarbonate (4.80 g, 22.0 mmol) at room temperature. The resulting mixture was stirred for 14 hours. Dioxane was removed under reduced pressure. The precipitate was filtered off and the filtrate was concentrated under reduced pressure to dryness. The residue was triturated with EtOAc then filtered. The filtrate was concentrated under reduced pressure to dryness to give a mixture of dl-1-amino-2- (1,1-dimethylethoxy) carbonylaminopropane and dl-2-amino-1- (1,1-dimethylethoxy) carbonylaminopropane, which could not be separated by the normal method. chromatographic. The mixture was used for the reaction in Example 8.

Receiving 13:

To a solution of Fmoc-e-Ala-OH (1.0 g, 3.212 mmol) and oxalyl chloride (0.428 g, 0.29 ml, 3.373 mmol) in dichloromethane (20.0 ml) was added some N, N-dimethylformamide at a temperature of 0 ° C. The mixture was stirred at room temperature for 1 hour then cyclopropylmethylamine (0.229 g, 0.28 mL, 3.212 mmol) and triethylamine (0.65 g, 0.90 mL, 6.424 mmol) were added. After 10 minutes, the mixture was treated with 1 M hydrochloride (10.0 ml) and the aqueous mixture was extracted with dichloromethane (3 x 30.0 ml). The organic solution was concentrated under reduced pressure to dryness. The residue was treated with a solution of 20% piperidine in N, N-dimethylformamide (20.0 ml) for 0.5 h. After the solvent was removed under reduced pressure, the residue was treated with 1 M hydrochloride (20.0 mL) and ethyl acetate (20.0 mL). The mixture was separated and the aqueous layer was basified with solid sodium hydroxide to pH = 8. The precipitate was removed by filtration and the aqueous solution was subjected to an ion exchange column eluted with 20% pyridine to yield 0.262 g (57%) of N-cyclopropylmethyl-e-alanine amide.

NMR ¹H (200 MHz, CD3OD) δ 0.22 (m, 2H), 0.49 (m, 2H), 0.96 (m, 2H), 2.40 (t, 2H), 2.92 (t, 2H), 3.05 (d, 2H);

MS (ES): 143.1 (M⁺+1).

Receiving 14:

N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine. trans-1,4-cyclohexyldiamine (6.08 g, 53.2 mmol) was dissolved in dichloromethane (1.00 mL). A solution of di-tert-butyl dicarbonate (2.32 g, 10.65 mmol in 40 mL of dichloromethane) was added via cannula. After 20 hours, the reaction was partitioned between CHCl3 and water. The layers were separated and the aqueous layer was extracted with CHCl3 (3x). The combined organic layers were dried over MgSO4, filtered, and concentrated to provide 1.20 g of a white solid (53%).

NMR ¹H (200 MHz, CDCl3): δ 1.0-1.3 (m, 4H), 1.44 (s, 9H), 1.8-2.1 (m, 4H), 2.62 (brm, 1H), 3.40 (br s, 1H), 4.37 (br s, 1H);

MS (ES): 215.2 (M⁺+1).

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

N-tert-butoxycarbonyl-trans-1,4-cyclohexyl diamine (530 mg, 2.47 mmol) was dissolved in dichloromethane (20 mL). Acetic anhydride (250 mg, 2.60 mmol) was added dropwise. After 16 hours, the reaction was diluted with water and CHCl3. The layers were separated and the aqueous layer was extracted with CHCl3 (3 ×). The combined organic layers were dried over MgSO4, filtered, and concentrated. Recrystallization (EtOH / H20) gave 190 mg of white crystals (30%).

NMR ¹H (200 MHz, CDCl3): δ 0.9 - 1.30 (m, 4H), 1.43 (s, 9H), 1.96-2.10 (m, 7H), 3.40 (brs, 1H), 3.70 (br s, 1H), 4.40 (br s, 1H), 4.40 (br s, 1H);

MS (ES): 257.2 (M⁺+1), 242.1 (M⁺ - 15), 201.1 (M⁺ - 56).

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

4- (N-acetyl) -N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine (190 mg, 0.74 mmol), dissolved in dichloromethane (5 ml) and diluted with TFA (6 ml). After 16 hours, the reaction was concentrated. Crude solid, DMSO (2 mL), NaHCO3 (200 mg, 2.2 mmol) and 4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine (35 mg, 0.14 mmol) ) was combined in a flask and heated to a temperature of 130 ° C. After 4.5 hours, the reaction was cooled to room temperature and diluted with EtOAc and water. The layers were separated and the aqueous layer was extracted with EtOAc (3 ×). The combined organic layers were dried over MgSO4, filtered and concentrated. Chromatography (preparative silica plate; 20: 1 CHCl3: EtOH) gave 0.3 mg of a tan solid (1% yield).

MS (ES): 378.2 (M⁺+1).

4- (N-methanesulfonyl) -N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine. trans-1,4-cyclohexyldiamine (530 mg, 2.47 mmol) was dissolved in dichloromethane (20 mL) and diluted with pyridine (233 mg, 3.0 mmol). Methanesulfonyl chloride (300 mg, 2.60 mmol) was added dropwise. After 16 hours, the reaction was diluted with water and CHCl3. The layers were separated and the aqueous layer was extracted with CHCl3 (3x). The combined organic layers were dried over MgSO4, filtered, and concentrated. Recrystallization (EtOH / H2O) gave 206 mg of white crystals (29%).

NMR ¹H (200 MHz, CDCl3): δ 1.10-1.40 (m, 4H), 1.45 (s, 9H), 2.00-2.20 (m, 4H), 2.98 (s, 3H), 3.20-3.50 (br s, 2H), 4.37 (br s, 1H);

MS (ES) 293.1 (M⁺+1), 278.1 (M⁺-15), 237.1 (M⁺-56).

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

4- (N-sulfonyl) -N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine (206 mg, 0.71 mmol), dissolved in dichloromethane (5 ml) and diluted with TFA (6 ml). After 16 hours, the reaction was concentrated. The crude reaction mixture, DMSO (2 mL), NaHCO3 (100 mg, 1.1 mmol), and 1-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine were combined in a flask and heated to a temperature 130 ° C. After 15 hours, the reaction was cooled to room temperature, and diluted with EtOAc (3 ×). The combined organic layers were dried over MgSO4, filtered, and concentrated. Chromatography (preparative silica plate, 20: 1 CHCl3 / EtOH) gave 2.6 mg of a tan solid (5%).

MS (ES): 414.2 (M⁺+1).

Example 1:

A solution of 4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine (0.50 g, 1.94 mmol) and 4-transhydroxycyclohexylamine (2.23 g, 19.4 mmol) in methylsulfoxide (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 EtOAc (3 x 10.0 mL). The combined EtOAc solution was dried (MgSO4) and filtered, the filtrate was concentrated in vacuo to dryness, the residue was chromatographed on silica gel to give 0.49 g (75%) of 4- (4-transhydroxycyclohexyl) amino-5,6-dimethyl-2- phenyl-7H-pyrrolo [2,3d] pyrimidine. Melting point 197-199 ° C;

NMR ¹H (200 MHz, CDCl3) δ 1.25-1.59 (m, 8H), 2.08 (s, 3H), 2.29 (s, 3H), 3.68-3.79 (m, 1H ), 4.32-4.38 (m, 1H), 4.88 (d, J = 8Hz, 1H), 7.26-7.49 (m, 3H), 8.40-8.44 ( dd, J = 2.2.8 Hz, 2H), 10.60 (s, 1H);

MS (ES): 337.2 (M⁺+1).

The following compounds were prepared in a similar manner to Example 1:

4- (4-transhydroxycyclohexyl) amino-6-methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, CDCl3) δ 11.37 (s, 1H, pyrrole-NH), 8.45 (m, 2H, Ar-H), 7.55 (m, 3H, Ar-H), 6.17 (s, 1H, pyrrol-H), 4.90 (br d, 1H, NH), 4.18 (m, 1H, CH-O), 3.69 (m, 1H, CH-N), 2, 40-2.20 (m, 2H), 2.19-1.98 (m, 2H), 2.25 (s, 3H, CH3) 1.68-1.20 (m, 4H);

MS (ES): 323.2 (M⁺+1).

4- (4-transhydroxycyclohexyl) amino-5-methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, CDCl3) δ 11.37 (s, 1H, pyrrol-NH), 8.40 (m, 2H, Ar-H), 7.45 (m, 3H, Ar-H), 5.96 (s, 1H, pyrrol-H), 4.90 (br d, 1H, NH), 4.18 (m, 1H, CH-O), 3.69 (m, 1H, CH-N), 2, 38-2.20 (m, 2H), 2.18-1.98 (m, 2H), 2.00 (s, 3H, CH3) 1.68-1.20 (m, 4H);

MS (ES): 323.2 (M⁺+1).

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

Melting point 245.5-246.5 ° C;

NMR ¹H (200 MHz, CD3OD) δ 8.33 (m, 2H, Ar-H), 7.42 (m, 3H, Ar-H), 7.02 (d, 1H, J = 3.6 Hz, pyrrole -H), 6.53 (d, 1H, J = 3.6Hz, pyrrole-H), 4.26 (m, 1H, CH-O), 3.62 (m, 1H, CH-N), 2.30-2.12 (m, 2H), 2.12-1.96 (m, 2H), 1.64-1.34 (m, 4H);

MS, M⁺1=309,3;

Anal (C18H20N4O) C, H, N.

4- (4-transhydroxycyclohexyl) amino-5,6-dimethyl-2- (3-pyridyl) -7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, CDCl3) δ 1.21-1.54 (m, 8H); 2.28 (s. 3H); 2.33 (s. 3H); 3.70 (m, 1H), 4.31 (m, 1H), 4.89 (d, 1H), 7.40 (m, 1H), 8.61 (m, 2H), 9.64 (m , 1H);

MS (ES): 338.2 (M⁺+1).

PL 204 628 B1

4- (4-transhydroxycyclohexyl) amino-5,6-dimethyl-2- (2-furyl) -7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, CDCl3) δ 1.26-1.64 (m, 8H), 2.22 (s, 3H) 2.30 (s, 3H), 3.72 (m, 1H), 4.23 (m, 1H), 4.85 (d, 1H) 6.52 (m, 1H), 7.12 (m, 1H), 7.53 (m, 1H), 9.28 (s, 1H);

MS (ES): 327.2 (M⁺+1).

4- (4-transhydroxycyclohexyl) amino-5,6-dimethyl-2- (3-furyl) -7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, CDCl3) δ 1.25-1.63 (m, 8H), 2.11 (s, 3H) 2.27 (s, 3H), 3.71 (m, 1H), 4.20 (m, 1H), 4.84 (d, 1H) 7.03 (m, 1H), 7.45 (m, 1H), 8.13 (m, 1H), 10.38 (m, 1H);

MS (ES): 327.2 (M⁺+1).

4- (4-transhydroxycyclohexyl) amino-5,6-dimethyl-2-cyclopentyl-7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, CDCl3) δ 1.26-2.04 (m, 16H), 2.26 (s, 3H), 2.27 (s, 3H), 3.15 (m, 1H), 3, 70 (m, 1H), 4.12 (m, 1H), 4.75 (d, 1H);

MS (ES): 329.2 (M⁺+1).

4- (4-transhydroxycyclohexyl) amino-5,6-dimethyl-2- (2-thienyl) -7H-pyrrolo [2,3d] pyrimidin-4-amine.

NMR ¹H (200 MHz, CDCl3) δ 1.28-1.59 (m, 8H), 2.19 (s, 3H) 2.29 (s, 3H), 3.74 (m, 1H), 4.19 (m, 1H), 4.84 (d, 1H) 7.09 (m, 1H), 7.34 (m, 1H), 7.85 (m, 1H), 9.02 (s, 1H);

MS (ES): 343.2 (M⁺+1).

4- (4-transhydroxycyclohexyl) amino-5,6-dimethyl-2- (3-thienyl) -7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, CDCl3) δ 1.21-1.60 (m, 8H), 1.98 (s, 3H) 2.23 (s, 3H), 3.66 (m, 1H), 4.22 (m, 1H), 7.27 (m, 1H) 7.86 (m, 1H), 8.09 (m, 1H), 11.23 (s, 1H);

MS (ES): 343.2 (M⁺+1).

4- (4-transhydroxycyclohexyl) amino-5,6-dimethyl-2- (4-fluorophenyl) -7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, CDCl3) δ 1.26-1.66 (m, 8H), 1.94 (s, 3H), 2.28 (s, 3H), 3.73 (m, 1H), 4, 33 (m, 1H), 4.92 (d, 1H), 7.13 (m, 2H), 8.41 (m, 2H), 11.14 (s, 1H);

MS (ES): 355.2 (M⁺+1).

4- (4-transhydroxycyclohexyl) amino-5,6-dimethyl-2- (3-fluorophenyl) -7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, CDCl3) δ 1.26-1.71 (m, 8H), 2.06 (s, 3H), 2.30 (s, 3H), 3.72 (m, 1H), 4, 30 (m, 1H), 4.90 (d, 1H), 7.09 (m, 1H), 7.39 (m, 1H), 8.05 (m, 1H), 8.20 (m, 1H ), 10.04 (s. 1H);

MS (ES): 355.2 (M⁺+1).

4- (4-transhydroxycyclohexyl) amino-5,6-dimethyl-2- (2-fluorophenyl) -7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, CDCl3) δ 1.30-1.64 (m, 8H), 2.17 (s, 3H), 2.31 (s, 3H), 3.73 (m, 1H), 4, 24 (m, 1H), 4.82 (d, 1H), 7.28 (m, 2H), 8.18 (m, 1H), 9.02 (m, 1H), 12.20 (s, 1H );

MS (ES): 355.3 (M⁺+1).

4- (4-transhydroxycyclohexyl) amino-5,6-dimethyl-2-isopropyl-7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, CDCl3) δ 1.31 (d, J = 7.0 Hz, 6H), 1.30-1.65 (m, 8H), 2.27 (s, 3H), 2.28 ( s, 3H), 3.01 (m, J = 7.0 Hz, 1H), 3.71 (m, 1H), 4.14 (m, 1H), 4.78 (d, 1H);

MS (ES): 303.2.

dl-4- (2-transhydroxycyclohexyl) amino-5,6-dimethyl-2-isopropyl-7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, CDCl3) δ 1.31-1.42 (br, 4H), 1.75-1.82 (br, 4H), 2.02 (s, 3H), 2.29 (s, 3H ), 3.53 (m, 1H), 4.02 (m, 1H), 5.08 (d, 1H), 7.41-7.46 (m, 3H), 8.30 (m, 2H) . 10.08 (s. 1H);

MS (ES): 337.2 (M.⁺+1).

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

MS (ES): 353.2 (M⁺+1).

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

MS (ES): 353.2 (M⁺+1).

4- (2-acetylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. Melting point 196-199 ° C;

NMR ¹H (200 MHz, CDCl3) δ 1.72 (s, 3H), 1.97 (s, 3H), 2.31 (s, 3H), 3.59 (m, 2H), 3.96 (m, 2H), 5.63 (br, 1H), 7.44-7.47 (m, 3H), 8.36-8.43 (dd, J = 1Hz, 7Hz, 2H), 10.76 (s , 1H);

MS (ES): 324.5 (M⁺+1).

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

NMR ¹H (200 MHz, CDCl3) δ 1.62 (m, 2H), 1.79 (br, 4H), 1.92 (s, 3H), 2.29 (s, 3H), 4.11 (m, 1H), 4.23 (m, 1H), 5.28 (d, 1H), 7.41-7.49 (m, 3H), 8.22 (m, 2H), 10.51 (s, 1H );

MS (ES): 323.2 (M⁺+1).

For the preparation of 2-transhydroxycyclopentylamine, see PCT 9417090.

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

PL 204 628 B1

NMR ¹H (200 MHz, CDCl3) δ 1.58-1.90 (br, 6H), 2.05 (s, 3H), 2.29 (s, 3H), 4.48-4.57 (m, 1H ),

4.91-5.01 (m, 2H), 7.35-7.46 (m, 3H), 8.42-8.47 (m, 2H), 10.11 (s, 1H);

MS (ES): 323.2 (M⁺+1).

¹For the production of 3-transhydroxycyclopentylamine, see EP-A-322242.

₁ dl-4- (3-cis-hydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.¹NMR ¹H (200 MHz, CDCl3) δ 1.82-2.28 (br, 6H), 2.02 (s, 3H), p 2.30 (s, 3H), 4.53-4.60 (m, 1H),

4.95-5.08 (m, 1H), 5.85-5.93 (d, 1H), 7.35-7.47 (m, 3H), 8.42-8.46 (m, 2H ), 10.05 (s. 1H);

MS (ES): 323.2 (M⁺+1).

¹For the preparation of 3-cis-hydroxycyclopentylamine, see EP-A-322242.

₁

4- (3,4-trans-dihydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.¹NMR ¹H (200 MHz, CDCl3) δ 1.92-1.99 (br, 2H), 2.14 (s, 3H), 2.20 (br, 2H), 2.30 (s, 3H), 2.412, 52 (br, 2H), 4.35 (m, 2H), 4.98 (m, 2H), 7.38-7.47 (m, 3H), 8.38-8.42 (m, 2H) . 9.53 (s. 1H);

MS (ES): 339.2 (M⁺+1).

¹For the preparation of 3,4-trans-dihydroxycyclopentylamine, see PCT 9417090. 4- (3-amino-3-oxopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, CDCl3) δ 2.02 (s, 3H), 2.29 (s, 3H), 2.71 (t, 2H), 4.18 (m, 2H), 5.75-5, 95 (m, 3H), 7.38-7.48 (m, 3H), 8.37-8.41 (m, 2H), 10.42 (s, 1H);

MS (ES): 310.1 (M⁺+1).

4- (3-N-cyclopropylmethylamino-3-oxopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] -pyrimidine.

NMR ¹H (200 MHz, CD3OD) δ 0.51 (q, 2H), 0.40 (q, 2H), 1.79-1.95 (br, 1H), 2.36 (s, 3H), 2, 40 (s, 3H), 2.72 (t, 2H), 2.99 (d, 2H), 4.04 (t, 2H), 7.58-7.62 (m, 3H), 8.22 -8.29 (m, 2H);

MS (ES): 364.2 (M⁺+1).

4- (2-amino-2-oxoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d) pyrimidine

NMR ¹H (200 MHz, CD3OD) δ 2.31 (s, 3H), 2.38 (s, 3H), 4.26 (s, 2H), 7.36 (m, 3H), 8.33 (m, 2H);

MS (ES): 396.1 (M⁺+1).

4- (2-N-methylamino-2-oxoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, CDCl3) δ 1.99 (s, 3H), 2.17 (s, 3H), 2.82 (d, 3H), 4.39 (d, 2H), 5.76 (t, 1H),

6.71 (br, 1H), 7.41-7.48 (m, 3H), 8.40 (m, 2H), 10.66 (s, 1H);

MS (ES): 310.1 (M⁺+1).

4- (3-tert-butyloxyl-3-oxopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, CDCl3) δ 1.45 (s, 9H), 1.96 (s, 3H), 2.29 (s, 3H), 2.71 (t, 2H), 4.01 (q, 2H),

5.78 (t, 1H), 7.41-7.48 (m, 3H), 8.22-8.29 (m, 2H);

MS (ES): 367.2 (M⁺+1).

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

NMR ¹H (200 MHz, CDCl3) δ 1.92 (s, 3H), 2.29 (s, 3H), 3.81-3.98 (br, 4H), 5.59 (t, 1H), 7.397, 48 (m, 3H), 8.37 (m, 2H), 10.72 (s, 1H);

MS (ES): 283.1 (M⁺+1).

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

NMR ¹H (200 MHz, CDCl3) δ 1.84 (m, 2H), 1.99 (s, 3H), 2.32 (s, 3H), 3.62 (t, 2H), 3.96 (m, 2H),

3.35 (t, 1H), 7.39-7.48 (m, 3H), 8.36 (m, 2H), 10.27 (s, 1H);

MS (ES): 297.2 (M⁺+1).

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

NMR ¹H (200 MHz, CDCl3) δ 1.71-1.82 (m, 4H), 1.99 (s, 3H), 2.31 (s, 3H), 3.68-3.80 (m, 4H ),

5.20 (t, 1H), 7.41-7.49 (m, 3H), 8.41 (m, 2H), 10.37 (s, 1H);

MS (ES): 311.2 (M⁺+1).

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

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

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

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

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

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

PL 204 628 B1

Example 2:

To a stirred suspension of triphenylphosphine (0.047 g, 0.179 mmol) and benzoic acid (0.022 g, 0.179 mmol) in THF (1.0 mL) cooled to 0 ° C was added 4- (4-transhydroxycyclohexyl) amino-5,6-dimethyl -2-phenyl-7H-pyrrolo [2,3d] 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. The reaction mixture was then allowed to warm to room temperature. After completion of the reaction by TLC, the reaction mixture was neutralized with an aqueous solution of sodium bicarbonate (3.0 mL). The aqueous phase was separated and extracted with ether (2 x 5.0 ml). The organic extracts were combined, dried, and concentrated under reduced pressure to dryness. Ether (2.0 mL) and hexane (5.0 mL) were added to the residue, and most of the triphenylphosphine oxide was filtered off. Concentration of the filtrate gave a sticky oil which was purified by column chromatography (hexane: ethyl acetate = 4: 1) to give 5.0 mg (7.6%) of 4- (4-cis-benzoyloxycyclohexyl) amino-5,6-dimethyl-2 -phenyl-7H-pyrrolo [2,3d] pyrimidine.

MS (ES): 441.3 (M⁺+1).

The reaction also produced 50.0 mg (84%) 4- (3-cyclohexenyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.

MS (ES): 319.2 (M⁺+1).

Example 3:

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

MS (ES): 337.2 (M⁺+1).

The following compounds were prepared in a similar manner to those of Example 3:

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

NMR ¹H (200 MHz, CDCl3) δ 2.01 (s, 3H), 2.31 (s, 3H), 2.73 (t, 2H), 2.97 (s, 6H), 4.08 (m, 2H), 6.09 (t, 1H), 7.41-7.48 (m, 3H), 8.43 (m, 2H), 10.46 (s, 1H);

MS (ES): 338.2 (M⁺+1).

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

NMR ¹H (200 MHz, CDCl3) δ 2.26 (s, 3H), 2.37 (s, 3H), 3.59-3.78 (m, 2H), 3.88-4.01 (m, 2H ),

5.48-5.60 (m, 1H), 7.38-7.57 (m, 3H), 8.09 (s, 1H), 8.30-8.45 (m, 2H), 8. 82 (s. 1H);

MS (ES): 310.1 (M⁺+1).

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

MS (ES): 338.2 (M⁺+1).

Example 4:

4- (3-tert-butyloxy-3-oxopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine (70.0 mg, 0.191 mmol) was dissolved in trifuloroacetic acid: dichloromethane (1: 1, 5.0 ml). The resulting solution was stirred at room temperature for 1 hour and then heated to reflux for 2 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure to dryness. The residue was subjected to preparative thin layer chromatography (EtOAc: hexane: AcOH = 7: 2.5: 0.5) to give 40.0 mg (68%) of 4- (3-hydroxy-3-oxopropyl) amino-5,6-dimethyl- 2-phenyl-7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, CD3OD) δ 2.32 (s, 3H), 2.38 (s, 3H), 2.81 (t, 2H), 4.01 (t, 2H), 7.55 (m, 3H). 8.24 (m, 2H);

MS (ES): 311.1 (M⁺+1).

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

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

MS (ES): 296.1 (M⁺+1), 278.1 (M⁺-NH3).

Example 5:

4- (3-hydroxy-3-oxopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo (2,3d] pyrimidine (50.0 mg, 0.161 mmol) was dissolved in a mixture of N, N-dimethylformamide ( 0.50 ml), dioxane (0.50 ml) and water (0.25 ml) To this solution was added methylamine (0.02 ml, 40% by weight in water, 0.242 mmol), triethylamine (0.085 ml) and tetrafluoroborate. N, N, N'N'-tetramethyl uronium (61.2 mg, 0.203 mmol). After stirring at room temperature for 10 minutes, the solution was concentrated and the residue was subjected to preparative thin layer chromatography (EtOAc) to give 35.0 mg (67%) of 4- (3-N-methyl-3-oxopropyl) amino-5. 6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, CDCl3) δ 1.92 (s, 3H), 2.30 (s, 3H), 2.65 (t, 2H), 4.08 (t, 2H), 5.90 (t, 1H), 6.12 (m, 1H), 7.45 (m, 3H), 8.41 (m, 2H), 10.68 (s, 1H);

MS (ES): 311.1 (M⁺+1).

The following compounds were prepared in a similar manner to those of Example 5:

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

MS (ES): 350.2 (M⁺+1).

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

MS (ES): 352.2 (M⁺+1).

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

NMR ¹H (200 MHz, CDCl3) δ 1.00-1.08 (t, 3H), 1.71-2.03 (m, 4H), 2.08 (s, 3H), 2.37 (s, 3H ), 3.26-3.40 (m, 2H), 3.79-3.96 (m, 2H), 5.53-5.62 (m, 1H), 6.17-6.33 (m , 1H), 7.33-7.57 (m, 3H), 8.318.39 (m, 2H), 9.69 (s, 1H);

MS (ES): 352.2 (M⁺+1).

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

NMR ¹H (200 MHz, CDCl3) δ 2.18 (s, 3H), 2.27 (s, 3H), 2.92 (s, 3H), 3.39-3.53 (m, 2H), 3.713, 88 (m, 2H), 5.31-5.39 (m, 1H), 6.17-6.33 (m, 1H), 7.36-7.43 (m, 3H), 8.20- 8.25 (m, 2H); 9.52 (s, 1H);

MS (ES): 360.2 (M⁺+1).

Example 6:

A mixture of 4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine (0.70 g, 2.72 mmol) and 1,2-diaminoethane (10.0 mL, 150 mmol) heated to reflux under an inert atmosphere for 6 hours. Excess amine was removed under reduced pressure, the residue was washed sequentially with ether and hexane to give 0.75 g (98%) of 4- (2-aminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine .

MS (ES); 282.2 (M⁺+1), 265.1 (M⁺-NH3).

Example 7:

To a solution of 4- (2-aminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine (70.0 mg, 0.249 mmol) and triethylamine (50.4 mg, 0.498 mmol) in dichloromethane (2.0 mL), propionyl chloride (25.6 mg, 0.024 mL, 0.274 mmol) was added at 0 ° C. After 1 hour, the mixture was concentrated under reduced pressure and the residue was subjected to preparative thin layer chromatography (EtOAc) to provide 22.0 mg (26%) of 4- (2-propionylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [ 2,3d] pyrimidines.

MS (ES): 338.2 (M⁺+1).

The following compounds were prepared in a similar manner to those of Example 7:

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

NMR ¹H (200 MHz, CDCl3) δ 2.13 (s, 3H), 2.32 (s, 3H), 3.53 (d, 3H), 3.55 (m, 2H), 3.88 (m, 2H), 4.29 (m, 1H), 5.68 (t, 1H), 5.84 (m, 1H), 7.42 (m, 3H), 8.36 (dd, 2H), 9. 52 (s. 1H);

MS (ES): 339.3 (M⁺+1).

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

MS (ES): 353.2 (M⁺+1).

Example 8:

To a solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (41.1 mg, 0.215 mmol), dimethylaminopyridine (2.4 mg, 0.020 mmol) and 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-7H-pyrrolo [2,3d] pyrimidine (55.0 mg, 0.196 mmol). The mixture was stirred at room temperature for 4 hours. Ordinary work-up and column chromatography (EtOAc) then afforded 10.0 mg (15%) of 4- (2'-pyruvylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.

MS (ES): 352.2 (M⁺+1).

Example 9:

N-isocyanate was added to a solution of 4- (2-aminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine (60.0 mg, 0.213 mmol) in dichloromethane (2.0 mL). -terimethylsilyl (43.3 mg, 0.051 mL, 0.320 mmol). The mixture was stirred at room temperature for 3 hours and then an aqueous sodium bicarbonate solution was added. After filtration through a small amount of silica gel, the filtrate was concentrated in vacuo to dryness to give 9.8 mg (14%) of 4- (2-ureaethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2, 3d] pyrimidines.

PL 204 628 B1

MS (ES): 325.2 (M⁺+1).

The following compounds were prepared in a similar manner to those of Example 9: dl-4- (2-acetylaminopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d) pyrimidine.

NMR ¹H (200 MHz, CDCl3) δ 1.28-1.32 (d, J = 8 Hz, 3H), 1.66 (s, 3H), 1.96 (s, 3H), 2.30 (s , 3H) 3.76-3.83 (m, 2H), 4.10-4.30 (m, 1H), 5.60-5.66 (t, J = 6Hz, 1H), 7.40 -7.51 (m, 3H), 8.36-8.43 (m, 2H), 10.83 (s, 1H);

MS (ES): 338.2 (M⁺+1).

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

NMR ¹H (200 MHz, CDCl3) δ 1.31 (d, 3H), 1.66 (s, 3H) 1.99 (s, 3H), 2.31 (s, 3H), 3.78-3.83 (m, 2H), 4.17-4.22 (m, 1H), 5.67 (t, 1H), 7.38-7.5 (m, 3H), 8.39 (m, 2H), 10.81 (s. 1H);

MS (ES): 338.2 (M⁺+1).

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

NMR ¹H (200 MHz, CDCl3) δ 1.41 (d, 3H), 1.68 (s, 3H), 2.21 (s, 3H), 2.34 (s, 3H), 3.46-3, 52 (br, m, 2H), 4.73 (m, 1H), 5.22 (d, 1H), 7.41-7.46 (m, 3H), 8.36-8.40 (m, 2H), 8.93 (s, 1H);

MS (ES): 338.2 (M⁺+1).

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

NMR ¹H (200 MHz, CDCl3) δ 1.31 (d, 3H), 1.66 (s, 3H) 2.26 (s, 3H), 2.35 (s, 3H), 3.78-3.83 (m, 2H), 4.17-4.22 (m, 1H), 5.67 (t, 1H), 7.38-7.5 (m, 3H), 8.39 (m, 2H), 8.67 (s. 1H);

MS (ES): 338.2 (M⁺+1).

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

NMR ¹H (200 MHz, CDCl3) δ 1.41 (d, 3H), 1.68 (s, 3H), 2.05 (s, 3H), 2.32 (s, 3H), 3.46-3, 52 (m, 2H), 4.73 (m, 1H), 5.22 (d, 1H), 7.41-7.46 (m, 3H), 8.36-8.40 (m, 2H) . 10.13 (s. 1H);

MS (ES): 338.2 (M⁺+1).

Example 10:

Reaction of 4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine with a mixture of dl-1-amino-2- (1,1-dimethylethoxy) carbonylaminopropane and dl-2-amino-1 - (1,1-dimethylethoxy) carbonylaminopropane proceeded in a similar manner to that of Example 1. The reaction mixture gave a mixture of dl-4- (1-methyl-2- (1,1-dimethylethoxy) carbonylamino) ethylamino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine and dl-4- (2-methyl-2- (1,1-dimethylethoxy) carbonylamino) ethylamino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine which was separated by column chromatography (EtOAc: hexanes = 1: 3). The first fraction was dl-4- (1-methyl-2- (1,1-dimethylethoxy) carbonylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine:

NMR ¹H (200 MHz, CDCl3) δ 1.29 - 1.38 (m, 12H), 1.95 (s, 3H), 2.31 (s, 3H) 3.34-3.43 (m, 2H) , 4.62-4.70 (m, 1H), 5.36-5.40 (d, J = 8Hz, 1H), 5.53 (br, 1H), 7.37-7.49 (m , 3H), 8.37-8.44 (m, 2H), 10.75 (s, 1H).

MS 396.3 (M⁺+1);

The second fraction was dl-4- (2- (1,1-dimethylethoxy) carbonylaminopropyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine:

NMR ¹H (200 MHz, CDCl3) δ 1.26-1.40 (m, 12H), 2.00 (s, 3H), 2.31 (s, 3H) 3.60-3.90 (m, 2H ), 3.95-4.10 (m, 1H), 5.41-5.44 (d, J = 6.0 Hz, 1H), 5.65 (br, 1H), 7.40-7, 46 (m, 3H), 8.37-8.44 (m, 2H), 10.89 (s, 1H);

MS (ES): 396.2 (M⁺+1).

The following compounds were prepared in a similar manner to those of Example 10:

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

NMR ¹H (200 MHz, CDCl3) δ 1.43 (m, 4H), 1.60 (s, 3H), 1.83 (m, 2H), 2.18 (s, 3H), 2.30 (m, 2H), 2.32 (s, 3H), 3.73 (br, 1H), 4.25 (br, 1H), 5.29 (d, 1H), 7.43-7.48 (m, 3H ), 8.35-8.40 (m, 2H), 9.05 (s, 1H).

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

NMR ¹H (200 MHz, CDCl3) δ 1.51 (s, 6H), 1.56 (s, 3H), 2.07 (s; 3H), 2.36 (s, 3H), 3.76 (d, 2H), 5.78 (t, 1H), 7.41-7.48 (m, 3H), 7.93 (s, 1H), 8.39 (m, 2H), 10.07 (s, 1H );

MS (ES): 352.3 (M⁺+1).

Example 11:

dl-4- (1-methyl-2- (1,1-dimethylethoxy) carbonylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine (60.6 mg, 0.153 mmol) treated with trifluoroacetic acid (0.5 mL) in dichloromethane (2.0 mL) for 14 hours. The organic solvent was removed under reduced pressure to dryness. The residue was dissolved in N, N-dimethylformamide (2.0 ml) and triethylamine (2.0 ml). Down

Acetic anhydride (17.2 mg, 0.016, 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 under reduced pressure to dryness. The residue was subjected to preparative thin layer chromatography (EtOAc) to provide 27.0 mg (52%) of dl-4- (1-methyl-2-acetylaminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d ] pyrimidines.

NMR ¹H (200 MHz, CDCl3) δ 1.38-1.42 (d, J = 8 Hz, 3H), 1.69 (s, 3H), 2.01 (s, 3H), 2.32 (s, 3H) 3.38-3.60 (m, 2H), 4.65-4.80 (m, 1H), 5.23-5.26 (d, J = 6 Hz, 1H), 7.40- 7.51 (m, 3H), 8.37-8.43 (m, 2H), 10.44 (s, 1H);

MS (ES): 338.2 (M⁺+1).

Example 12:

(R, R) -4- (2-aminocyclohexyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, prepared in a similar manner to those of Example 1 from 4-chloro-5 , 6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine (0.15 g, 0.583 mmol) and (1R, 2R) - (-) - 1,2-diaminocyclohexane (0.63 g, 5.517 mmol), treated with triethylamine (0.726 g, 7.175 mmol) and acetic anhydride (0.325 g, 3.18 mmol) in N, N-dimethylformamide (10.0 mL) at room temperature for 2 hours. After the solvent was removed under reduced pressure, ethyl acetate (10.0 mL) was added to the residue and water (10.0 mL) was added to the residue. The mixture was separated and the aqueous layer was extracted with ethyl acetate (2 x 10.0 ml). The combined ethyl acetate solution was dried (MgSO4) and filtered. The filtrate was concentrated under reduced pressure to dryness, and the residue was subjected to column chromatography (EtOAc: hexane = 1: 1) to give 57.0 mg (26%) of (R, R) -4- (2-acetylaminocyclohexyl) amino-5,6-dimethyl -2-phenyl-7H-pyrrolo [2,3d] pyrimidine.

NMR ¹H (200 MHz, CDCl3) δ 1.43 (m, 4H), 1.60 (s, 3H), 1.84 (m, 2H), 2.22 (s, 3H), 2.30 (m, 2H), 2.33 (s, 3H), 3.72 (br, 1H), 4.24 (br, 1H), 5.29 (d, 1H), 7.43-7.48 (m, 3H ), 8.35-8.39 (m, 2H), 8.83 (s, 1H);

MS (ES): 378.3 (M⁺+1).

Example 13:

To a solution of 4- (2-hydroxyethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine (40.0 mg, 0.141 mmol) in pyridine (1.0 mL) was added acetic anhydride (0.108 g, 1.06 mmol) at 0 ° C. The mixture was stirred at room temperature for 4 hours and the solvent was removed under reduced pressure. The residue was subjected to preparative thin layer chromatography (EtOAc: hexane = 1: 1) to provide 32.3 mg (71%) of 4- (2-acetyloxyethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidines.

NMR ¹H (200 MHz, CDCl3) δ 1.90 (s, 3H), 2.08 (s, 3H), 2.31 (s, 3H), 4.05 (m, 2H), 4.45 (t, 2H), 5.42 (m, 1H), 7.41-7.49 (m, 3H), 8.42 (m, 2H), 11.23 (s, 1H).

Example 14:

A solution of Fmoc-e-Ala-OH (97.4 mg, 0.313 mmol) and oxalyl chloride (39.7 mg, 27.3 µL, 0.313 mmol) in dichloromethane (4.0 ml) with 1 drop of N, N-dimethylformamide stirred at 0 ° C for 1 hour, then 4- (2-aminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine (80.0mg, 0.285mmol) was added. and triethylamine (57.6 mg, 79.4 µL, 0.570 mmol) at 0 ° C. After 3 hours, the mixture was concentrated under reduced pressure and the residue was treated with a solution of 20% piperidine in N, N-dimethylformamide (2.0 mL) for 0.5 hours. After removing the solvent under reduced pressure, the residue was washed with diethyl ether: hexane (1: 5) to give 3.0 mg (3%) of 4- (6-amino-3-aza-4-oxohexyl) amino-5,6-dimethyl -2-phenyl-7H-pyrrolo [2,3d] pyrimidine.

MS (ES): 353.2 (M⁺+1).

Example 15:

A solution of 4- (2-aminoethyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine (70.0 mg, 0.249 mmol) and succinic anhydride (27.0 mg, 0.274 mmol) in dichloromethane (4.0 ml) with 1 drop of N, N-dimethylformamide was stirred at room temperature for 4 hours. The reaction mixture was extracted with 20% sodium hydroxide (3 x 5.0 ml). The aqueous solution was acidified with 3M hydrochloride to pH = 7.0. The entire mixture was extracted with ethyl acetate (3 x 10 ml). The combined organic solution was dried (MgSO4) and filtered. The filtrate was concentrated under reduced pressure to dryness to give 15.0 mg (16%) of 4- (7-hydroxy-3-aza-4,7-dioxoheptyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2 , 3d] pyrimidines.

MS (ES): 382.2 (M⁺+1).

PL 204 628 B1

Example 16:

700 mg of 4-cis-3-hydroxycyclopentyl) amino-2-phenyl-5,6-dimethyl-7H-pyrrolo (2,3d] pyrimidine were added to 10 ml of dimethylformamide (DMF) at room temperature, followed by 455 mg of N-Boc. -glycine, 20 mg of N, N-dimethylaminopyridine (DMAP), 293 mg of hydroxybenzotriazole (HOBT) and 622 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCl) The reaction mixture was allowed to stir overnight. The 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, elution with ethyl acetate / hexane gave 410 mg of the desired product: 4- (cis-3- (Nt-butoxycarbonyl-2-aminoacetoxy) cyclopentyl) amino-2-phenyl-5.6, -dimethyl-7H- pyrrolo [2,3d] pyrimidines.

MS (ES) (M⁺+1) = 480,2.

The ester was then treated with 5 mL of 20% trifluoroacetic acid in dichloromethane at room temperature, allowed to stand overnight and then concentrated. Trituration with ethyl acetate gave 300 mg of an off-white solid;

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

MS (ES) (M⁺+1)=380,1.

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

4- (cis-3-hydroxycyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine

MS (ES) (M⁺+1)= 323,1.

4- (cis-3- (2-arainoacetoxy) cyclopentyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine, trifluoroacetic acid salt

MS (ES) (M⁺+1)= 380,1.

4- (3-acetamido) piperidinyl-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine

MS (ES) (M⁺+1)= 364,2.

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

MS (ES) (M⁺+1)=353,4.

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

MS (ES) (M⁺+1)= 352,4.

4- (2-N'-methylurea butyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine

MS (ES) (M⁺+1)= 367,5

4- (2-aminocyclopropylacetamidoethyl) amino-2-phenyl-7H-pyrrolo [2,3d] pyrimidine

MS (ES) (M⁺+1)= 309,1.

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

MS (ES) (M⁺+1) =342,8.

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

MS (ES) (M⁺+1)=327,2.

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

MS (ES) (M⁺+1)=310,2.

PL 204 628 B1

Example 17

Scheme IX

<img file="PL204628B1_D0076.tif" />

The pyrrole nitrogen (7) (Scheme IX) was protected with di-tert-butyldicarbonate under basic conditions to give the corresponding carbamate (22). Radical bromination (22) proceeded regioselectively, obtaining bromide (23). Overall, compound (23) has served as a key electrophilic intermediate for various nucleophilic coupling partners. Displacement of the alkyl bromide with sodium phenate trihydrate gave compound (24). Subsequent replacement of the aryl chloride and removal of the tert-butyl carbamate protecting group occurred in one step to afford the desired compound (25).

Detailed synthesis of compounds (22) - (25) according to scheme IX

<img file="PL204628B1_D0077.tif" />

Di-tert-butyl dicarbonate (5.37 g, 24.6 mmol) and dimethylaminopyridine (1.13 g, 9.2 mmol) were added to the solution containing (7) (1.50 g, 6.15 mmol) and pyridine (30 ml). After 20 hours, the reaction was concentrated and the residue was partitioned between CH2Cl2 and water. The CH2Cl2 layer was separated, dried over MgSO4, filtered and concentrated to give a black solid. Flash chromatography (SiO2; 1/9 EtOAc / Hexanes, Rf 0.40) gave 1.70 g (80%) of a white solid (22).

NMR ¹H (200 MHz, CDCl3) δ 8.50 (m, 2H, Ar-H), 7.45 (m, 3H, Ar-H), 6.39 (s, 1H, pyrrol-H), 2.66 (s, 3H, pyrrole-CH3),

<img file="PL204628B1_D0078.tif" />

1.76 (s, 9H, carbamate-CH3);

MS, M + 1 = 344.1;

Melting point = 175-177 ° C.

PL 204 628 B1

N-Bromosuccinimide (508 mg, 2.86 mmol) and AIBN (112 mg, 0.68 mmol) were added to a solution containing (22) (935 mg, 2.71 mmol) and CCl4 (50 ml). The solution was heated to reflux. After 2 hours, the reaction was cooled to room temperature and concentrated in vacuo to give a white solid. Flash chromatography (SiO2; 1/1 CH2Cl2 / Hexanes, Rf 0.30) gave 960 mg (84%) of a white solid (23).

NMR ¹H (200 MHz, CDCl3) δ 8.52 (m, 2H, Ar-H), 7.48 (m, 3H, Ar-H), 6.76 (s, 1H, pyrrol-H), 4.93 (s, 2H, pyrrole-CH2Br), 1.79 (s, 9H, carbamate-CH3);

MS, M + 1 = 423.9;

Melting point = 155-157 ° C.

<img file="PL204628B1_D0079.tif" />

Sodium phenoxide trihydrate (173 mg, 1.02 mmol) was added in one part to a solution of bromide (23) (410 mg, 0.97 mmol) dissolved in CH2Cl2 (5 mL) and DMF (10 mL). After 2 hours, the reaction solution was partitioned between CH2Cl2 and water. The aqueous layer was extracted with CH2Cl2. The combined CH2Cl2 layers were washed with water, dried over MgSO4, filtered and concentrated to give a yellow solid. Flash chromatography (SiO2; 1/6 EtOAc / Hexanes, Rf 0.30) gave 210 mg (50%) of a white solid (24).

NMR ¹H (200 MHz, CDCl3) δ 8.53 (m, 2H, Ar-H), 7.48 (m, 3H, Ar-H), 7.34 (m, 2H, Ar-H), 7.03 (m, 3H, Ar-H), 6.83 (s, 1H, pyrrol-H), 5.45 (s, 2H, ArCH2O), 1.76 (s, 9H, carbamate-CH3);

MS, M + = 436.2.

<img file="PL204628B1_D0080.tif" />

A solution containing (24) (85 mg, 0.20 mmol), N-acetylethylenediamine (201 mg, 1.95 mmol) and DMSO (3 mL) was heated to 100 ° C. After 1 hour, the temperature was raised to 130 ° C. After 3 hours, the reaction was cooled to room temperature and partitioned between EtOAc and water.

The aqueous layer was extracted with EtOAc (2x). The combined EtOAc layers were washed with water, dried over MgSO4, filtered, and concentrated. Flash chromatography (SiO2; 1/10 EtOH / CHCl3, Rf 0.25) gave 73 mg (93%) of a white foamy solid (25).

NMR ¹H (200 MHz, DMSO-d5) δ 11.81 (br s, 1H, NH), 8.39 (m, 2H, Ar-H), 8.03 (br t, 1H, NH), 7.57 (br t, 1H, NH), 7.20-7.50 (m, 5H, Ar-H), 6.89-7.09 (m, 3H, Ar-H), 6.59 (s, 1H , pyrrole-H), 5.12 (s, 2H, ArCH2O), 3.61 (m, 2H, NCH2), 3.36 (m, 2H, NCH2), 1.79 (s, 3H, COCH3);

MS, M⁺1 = 402,6

The following compounds were prepared in a similar manner to Example 17:

4- (2-acetylaminoethyl) amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.

Melting point 196-197 ° C;

PL 204 628 B1

MS (ES): 401.6 (M⁺+1).

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

MS (ES): 420.1 (M⁺+1).

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

MS (ES): 436.1 (M⁺+1).

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

MS (ES): 432.1 (M⁺+1).

4- (2-acetylaminoethyl) amino-6- (N-pyridin-2-one) methyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine.

MS (ES): 403.1 (M⁺+1).

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

MS (ES): 400.9 (M⁺+1).

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

MS (ES): 414.8 (M⁺+1).

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

MS (ES): 416.9 (M⁺+1).

yeast β-galactosidase reporter gene assays for human A1 and A2a adenosine receptor

Yeast strains (S. cerevisiae) were transformed with human adenosine A1 (A1R; strain CADUS CY12660) with human A2a (A2a; strain CADUS CY8362) and the lacZ reporter gene (β-galactosidase) was added to be used as a functional reading. A full description of the transformation is listed below (see yeast strains). NECA (5'-N-ethylcarboxamide adenosine), a potent adenosine receptor agonist with similar affinity for A1 and A2a receptors, was used as a ligand for all assays. Test compounds were tested at 8 concentrations (0.1 - 10,000 nM) for the ability to inhibit NECA-induced beta-galactosidase activity by CY12660 or CY8362.

Obtaining seed yeast cultures. The respective yeast strains, CY 12660 and CY8362, were each streaked on an LT agar plate and incubated at 30 ° C until colonies were observed. The yeast from these colonies was added to LT fluid (pH 6.8) and grown overnight at 30 ° C. Each yeast strain was then diluted to OD600 = 1.0-2.0 (approximately 1-2 x 10⁷ cells / ml) as determined spectrophotometrically (Molecular Devices VMAX). For each 6 ml of liquid yeast culture, 4 ml of 40% glycerol (1: 1.5 v / v) ("yeast / glycerol starting solution") was added. Ten volumes of 1 ml were made from this yeast / glycerol starting solution and stored at -80 ° C until needed for the test.

Yeast test A1R and A2aR. One vial of each of the yeast / glycerol CY8362 and CY12660 starting solutions was melted and used to inoculate the Supplemented LT liquid, pH 6.8 (92 ml LT fluid to which was added: 5 ml 40% glucose, 0.45 ml 1M KOH and 2.5 ml Pipes, pH 6.8). The liquid cultures were grown for 16-18 hours (overnight) at 30 ° C. Equal amounts from overnight cultures where then diluted in LT media containing 4 U / ml adenosine deaminase (type VI or VII from calf mucosa, Sigma) to obtain OD600 = 0.15 (1.5 X 10⁶ cells / ml) for CY8362 (A2aR) and OD600 = 0.50 (5X10⁶ cells / ml) for CY12660 (AIR).

Assays were performed with a final volume of 100 µl in 96-well microtiter plates such that a final concentration of 2% DMSO was achieved in all wells. For the first screening, 1-2 concentrations of test compounds (10 μΜ, 1 μΜ) were used. For compound profiling, 8 concentrations (10,000, 1,000, 500, 100, 50, 10, 1 and 0.1 nM) were tested. To each microtiter plate, 10 µl of 20% DMSO was added to the "control" and "total" wells while 10 µl of test compound (in 20% DMSO) was added to the "unknown" wells. Then, 10 µl of NECA (5 µΜ for A1R, 1 µΜ for A2aR) was added to the "complete" and "unknown" wells; 10 µl of PBS was added to the "control" wells. During the final addition, 80 µl of yeast strain, CY8362 or CY12660, was added to all wells. All plates were then briefly shaken (LabLine orbital shaker 2-3 minutes) and allowed to incubate for 4 hours at 30 ° C in a dry oven.

Β-galactosidase activity can be assessed using either colorimetric (e.g., ONPG, CPRG), luminescent (e.g., Galacton-Star) or fluorometric (e.g., FDG, Resorufin) substrates. Currently, fluorescence detection is recommended based on the principal signal: noise ratio, freedom from overlap and low cost. Fluorescein digalactopyranoside (FDG, Molecular Probes, or Marker Gene Technologies), a beta-galactosidase fluorescent substrate, was added to all wells at 20 µl / well (final concentration = 80 µM). The plates were shaken for 5-6 seconds (LabLine orbital shaker) then incubated at 37 ° C for 90 minutes (95% O2 / 5% CO2 incubator). At the end of the 90 minute incubation period, beta-galactosidase activity is present

The coating was stopped with 20 µ / well of 1M Na₂WHAT₃ and all plates were shaken for 5-6 seconds. The plates were then mixed for 6 seconds and the relative fluorescence intensity was determined using a fluorometer (Tecan Spectrafluor; excitation = 485 nm, emission = 535 nm).

Calculations. Relative fluorescence values ​​for the "control" wells were interpreted as background and separated from the "total" and "unknown" values. Compound profiles were analyzed by log transformation (X-axis: compound concentration) followed by a single site competition curve fitting the calculated ICSO (GraphPad Prism) values.

Yeast strains. The strains of Saccharomyces cerevisiae CY12660 (far1 * 1442 tbt1-1 fus1-HIS3 canl ste14: trp1 :: LYS2 ste3 * 1156 gpa1 (41) -Gai3 lys2 ura3 leu2 trp1: his3; LEU2 PGKpMfalLmuer-PH5P3MR-hA1R-PHO5P3R-hA1R 2 and CY8362 [gpa1p-rGasE10K far1 * 1442 tbt1-1 fus1-HIS3 can1 ste14: trp1: LYS2 ste3 * 1156 lys2 ura3 leu2 trp1 his3; LEU2 PGKp-hA2aR 2mu-ori REP3 Ampr].

LT media. LT media (supplemented with Leu-Trp) consists of 100 g yeast nitrogen base DIFCO supplemented with the following ingredients: 1.0 g valine, 1.0 g aspartic acid, 0.758 phenylalanine, 0.9 g lysine, 0.45 g tyrosine, 0.45 g 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 of cysteine, 0.3 g of arginine, 0.9 g of histidine and 1.0 g of threonine.

Construction of yeast strains expressing the human A1 adenosine receptor

In this example, the construction of yeast strains expressing the human A1 adenosine receptor operably integrated into the yeast pathway of the pheromone system is described.

I. Construction of the expression vector

To construct a yeast expression vector for the human A1 adenosine receptor, the A1 adenosine receptor cDNA was obtained by PCR with human hippocampal mRNA reverse transcriptase using primers designed from the published human A1 adenosine receptor sequence and standard techniques. The PCR product was subcloned into the NcoI and XbaI sites of the yeast pMP15 expression plasmid. Plasmid pMP15 was generated from pLPXt as follows: Xbal site from YEP51 (Broach, JR et al. (1983) "Vectors for high-level, inducible expression of cloned genes in yeast" pages 83-117 in M. Inouye (ed.), Experimental Manipulation of Gene Expression. Academic Press, New York) was removed by digestion, end-fill and ligation to form Yep51NcoDXba. Another Xbal site was created at the BamHI site by digesting with BamHI, padding the ends, adding a linker (New England Biolabs, # 1081), digesting with Xbal and re-ligating to form YEP51NcoXt. This plasmid was digested with Esp31 and NcoI and ligated with the PCR generated Leu2 and PGKp fragments. A 2 kb PCR product of Leu2 was generated by amplification from YEP51Nco using primers containing the Esp31 and BglII sites. A 660 bp PCR product of PGKp was generated by amplification from pPGKas (Kung, Y.-S. et al. (1990) Mol. Cell. Biol. 10: 25822590) using PCR primers containing BglII and NcoI sites. The resulting plasmid is called pLPXt. pLPXt was modified by inserting the factor coding region a leader sequence for the prepro protein into the NcoI site. The leader sequence for the prepro protein was introduced so that the NcoI cloning site at the 3 'end of the leader was preserved but not regenerated at the 5' end. In this way, the receptors can be cloned by digesting the plasmid with NcoI and Xbal. The resulting plasmid is called pMP15.

The plasmid pMP15 into which the human adenosine A1 receptor cDNA was introduced was designated p5095. In this vector, the receptor cDNA binds to the 3 'end of the yeast α-factor prepro leader sequence. During protein maturation, the prepro peptide sequences are cleaved to form a full-length mature receptor. This occurs during the receptor being transformed in the yeast secretory pathway. This plasmid is maintained by Leu selection (i.e. growth in leucin-free medium). The sequence of the cloned coding region was determined and found to be equivalent to that published in the literature (GenBank accession numbers S45235 and S56143).

II. Construction of yeast strain

To create a yeast strain expressing the human A1 adenosine receptor, yeast strain CY7967 was used as the starting parent strain. The genotype of CY7967 is as follows:

MATa gpaD1163 gpa1 (41) Gai3 far1D1442 tbt-1 FUS1-HIS3 can1 ste14 :: trp1 :: LYS2 ste3D1156 lys2 ura3 leu2 trp1 his3

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The genetic markers are described below:

MATa ...................... matching type a.

gpa1D1163 ............. Endogenous GPA1 G protein has been removed.

gpa1 (41) Gai3 ......... gpa1 (41) -Gai3 was integrated into the yeast genome. This Ga chimeric protein consists of the first 41 amino acids of the endogenous yeast GPA1 Ga subunit fused to the Gai3 G protein of a mammal in which the related N-terminal amino acids have been removed.

Far1D1442 .............. FAR1 (responsible for cell cycle arrest) was removed (thereby preventing cell cycle arrest during activation of the pheromone response pathway).

tbt-1 .......................... strain with high transformation efficiency by electroporation.

FUSI-HISS ................ fusion between the FUSI promoter and the HIS3 coding region (thereby creating the pheromone-induced HISS gene). can 1 ........................ arginine / canavin permease.

ste14 :: trp1 :: LYS2 ..... disruption of the STE14 gene, C-farnesyl methyltransferase (thereby lowering the basic signaling via the pheromone pathway).

ste3D1156 ............... discontinued endogenous yeast STR, factor a pheromone receptor (STE3).

Iys2 ......................... defect in 2-aminoapidate reductase, yeast needs lysine to grow.

urA3 ........................ defect in orotidine-5'-phosphate decarboxylase, yeast needs uracil to grow.

Ieu2 ......................... defect in b-isopropyl malate dehydrogenase, yeast need leucin for growth, trp1 .......... ................. defect in phosphoribosylanthranilate, yeast need tryptophan to grow.

his3 ......................... defect in imidazolycerolphosphate dehydrogenase, yeast needs they stimulate histidine to grow.

Two plasmids were transformed into strain CY7967 by electroporation: plasmid p5095 (encoding human A1 adenosine receptor; described above) and pl584, which is a plasmid of the FUS1-β-galactosidase reporter gene. Plasmid p1584 was obtained from plasmid pRS426 (Christianson, TW et al. (1992) Gene 110: 119-1122). The plasmid pRS426 contains a polylinker site at nucleotides 2004-2016. A fusion was introduced between the FUS1 promoter and the β-galactosidase gene at the EagI and XhoI restriction sites to create plasmid p1584. Plasmid p1584 is maintained by Trp selection (i.e. growth in leucin-free medium).

The resulting p5095 and p1584-bearing strain, referred to as CY12660, expresses the human A1 adenosine receptor. For the cultivation of this strain in liquid or on agar plates, minimal media without leucine and tryptophan was used. To perform the plate growth assay (designated FUSI-HIS3), the plates were kept at pH 6.8 and contained 0.5-2.5 mM 3-amino-1,2,4-triazole and contained no leucine, tryptophan and histidine. As a check for specificity, all experiments included a comparison with one or more of the seven yeast-based transmembrane receptor screens.

Construction of yeast strains expressing the human A1a adenosine receptor

In this example, the construction of yeast strains expressing the human A1a adenosine receptor operably integrated into the yeast pathway of the pheromone system is described.

I. Construction of the expression vector

To construct a yeast expression vector for the human A1a adenosine receptor, human A2a receptor cDNA was obtained from Dr. Phil Murphy (NIH). Upon receipt of this clone, the A2a receptor insert was sequenced and found to be identical to the published sequence (GenBank Accession 546950). Receptor CDNA was cleaved from the plasmid by PCR using VENT polymerase and cloned into plasmid pLPBX, which directs expression of the receptor by the constitutive Phosphoglycerate Kinase (PGK) promoter in yeast. The sequence of the entire insert was re-sequenced and found to be identical to the published sequence. However, due to the cloning strategy employed, there were three amino acids attached to the carboxyl terminus of the receptor, GlySerVal.

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II. Construction of a yeast strain

To create a yeast strain expressing the human adenosine A2a receptor, yeast strain CY8342 was used as the starting parental strain. The genotype of CY8342 is as follows: MATa far1D1442 tbt1-1 lys2 ura3 leu2 trp1 his3 fus1-HIS3 can1 ste3D1156 gpaD1163 ste14 :: trp1 :: LYS2 gpalp-rGasE10K (or gpa1p-rG_aSD229S or gpa1p-rG_aSE10K + D229S)

The genetic markers are as described in Example 1, except for the G protein variant. To express the human A2a receptor, a yeast strain in which the endogenous yeast GPA1 G protein was removed and replaced with the mammalian Gas protein was used. Three mutant rats were used

Gas. These variants contain one or two point mutations that transform them into proteins that couple efficiently with yeast βγ. Identified as G "_sE10K (with the ten glutamic acid replaced by lysine), G_asD229S (in which the aspartic acid at position 229 has been replaced by serine) and G._asE10K + D229S (which includes both point mutations).

Strain CY8342 (carrying one of the three mutant rat G proteins_as) transformed with either the parental vector pLPBX (Receptor-) or pLPBX-A2a (Receptor +). A plasmid with the FUS1 promoter was added and linked to the β-galactosidase coding sequences (described above) to estimate the amount of activation of the pheromone response pathway.

Functional test using yeast strains expressing the human A1 adenosine receptor

In this example, the use of functional screening assays in yeast for modulators of the human A1 adenosine receptor is described.

I. Ligands used in the test

Adenosine, a natural agonist of this receptor, was used to perform this test, as well as two other synthetic agonists. Adenosine, reported to have an EC50 value of about 75 nM, and (-) - N6- (2-phenylisopropyl) adenosine (PIA), reported to have a reported affinity of about 50 nM, were used in a subset of the experiments. 5'-N-ethylcarboxamidoadenosine (NECA) was used in all growth tests. To prevent signaling caused by the presence of adenosine in the growth media, adenosine deaminase (4 U / ml) was added to all tests.

II. Biological response in yeast

The ability of the adenosine A1 receptor to functionally conjugate in a heterologous yeast system was assessed by introducing the A1 receptor expression vector (p5095, described above) into a series of yeast strains that expressed different G protein subunits. Most of these transformants expressed G subunits._and of the subtype G_ai or G_a0. Additional G proteins were also tested_and in terms of possible identification of random G protein receptor coupling_and. In different strains, an integrated STE18 or a chimeric STE18-Gy2 construct with the yeast genome. The yeast strain contained the defective HIS3 gene and an integrated copy of FUS1-HIS3, thus allowing selection in selective media containing 3-amino-1,2,4-triazole (tested at 0.2, 0.5 and 1.0 mM) and no histidine. Transformants were isolated and monolayers were prepared on media containing 3-amino-1,2,4-triazole, 4 U / ml adenosine deaminase and no histidine. Five microliters of different ligand concentrations were used (e.g., NECA at 0, 0.1, 1.0 and 10 mM). Growth was monitored for 2 days. Ligand-dependent growth responses were thus tested in various yeast strains. The results are summarized in Table 1 below. The symbol (-) indicates that ligand-dependent receptor activation was not detected while (+) indicates a ligand-dependent response. The term "LIRMA" indicates ligand-independent receptor mediated activation.

Table 3

<td>Strain yeast</td><td>Subunit Ga</td><td>Subunit Gy</td><td>Variations strains</td><td>Results</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td>

<td>CY1316</td><td>GPA1</td><td>STE18</td><td></td><td> -</td>

<td></td><td>GPA41-Gai1</td><td></td><td></td><td> +</td>

<td></td><td>GPA41-Gai2</td><td></td><td></td><td> +</td>

PL 204 628 B1 cont. table 3

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td>

<td></td><td>GPA41-Gai3</td><td></td><td></td><td> +</td>

<td></td><td>GPA41 -Gai2-GaOB</td><td></td><td></td><td>LIRMA</td>

<td></td><td>GPA41-G_andSE10K</td><td></td><td></td><td> -</td>

<td></td><td>GPA41-Ga3D229s</td><td></td><td></td><td> -</td>

<td>CY7967</td><td>GPA41-G_andi3-integrated</td><td>STE18</td><td></td><td> +++</td>

<td>CY2120</td><td>GPA1</td><td>STE18</td><td>sst2Δ</td><td> +</td>

<td></td><td>GPA41-Gai1</td><td></td><td></td><td> +</td>

<td></td><td>GPA41-Gai2</td><td></td><td></td><td> +</td>

<td></td><td>GPA41-Gai3</td><td></td><td></td><td> +</td>

<td></td><td>GPA41 -G_andi2-G_andOB</td><td></td><td></td><td>LIRMA</td>

<td></td><td>GPA41-G_andSE10K</td><td></td><td></td><td> -</td>

<td></td><td>GPA41-GaSD229S</td><td></td><td></td><td> -</td>

<td>CY9438</td><td>GPA1</td><td>STE18-Gy2</td><td></td><td> -</td>

<td></td><td>GPA41-Gai1</td><td></td><td></td><td> +</td>

<td></td><td>GPA41-Gai2</td><td></td><td></td><td> +</td>

<td></td><td>GPA41-Gai3</td><td></td><td></td><td> +</td>

<td></td><td>GPA41 -G_andi2-G_andOB</td><td></td><td></td><td>LIRMA</td>

<td></td><td>GPA41-G_andSE10K</td><td></td><td></td><td> -</td>

<td></td><td>GPA41-GaSD229s</td><td></td><td></td><td> -</td>

<td>CY10560</td><td>GPA1-integrated</td><td></td><td>sst2Δ</td><td> + +</td>

As noted in Table 3, it was found that the strongest signal was found in the yeast strain expressing the GPA 1 (41) -Ga chimera._ai3.

III. Fus1-LacZ test

To more fully characterize the activation of the pheromone response pathway, the synthesis of β-galactosidase by fus1 LacZ was measured in response to agonist stimulation. To perform the β-galactosidase assay, increasing ligand concentrations were added to mid-arrhythmic cultures of the human A1 adenosine receptor expressed in yeast strains co-expressing the Ste18-GY2 and GPA41-G chimeras_ai3. Transformants were isolated and grown overnight in the presence of histidine and 4 U / ml of adenosine deaminase. After five hours of incubation with 4 U / ml adenosine deaminase and ligand, the induction of β-galactosidase was measured using CPRG as a substrate for β-galactoside. 5 x 10 were used for the test⁵ cells.

The results obtained for the NECA stimulation indicated that at a NECA concentration of 10^-8 M, about 2-fold stimulation of β-galactosidase activity was obtained. In addition, an approximately 10-fold stimulation index was observed at a NECA concentration of 10^-5 M.

The use of this test was extended to assess the value of antagonist activity on this strain. Two known adenosine antagonists, XAC and DPCPX, were tested for their ability to compete with NECA (at 5 mM) for activity in the β-galactosidase assay. In these assays, the induction of β-galactosidase was measured using FDG as a substrate and 1.6 x 10⁵ cells per test. The results indicated that both XAC and DPCPX served as potent antagonists of adenosine A1 receptor expression in yeast, with IC50 values ​​of 44 nM and 49 nM, respectively.

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To determine whether this inhibitory effect was specific to the A1 subtype, a series of experiments complementary to the yeast-based A2a receptor assay (described in Example 4) were performed. The results obtained with the yeast A2a assay indicated that XAC was a relatively effective A2a receptor antagonist according to published reports. In contrast, DPCPX was relatively inert at this receptor as expected from published reports.

IV. Radioligand binding

The adenosine A1 receptor assay was further characterized by measuring the receptor parameters in terms of radioligand binding. The replacement bond [³H] CPX by several compounds relevant for the adenosine receptor, XAC, DPCPX, and CGS, using yeast-derived membranes expressing the human A1 adenosine receptor. The results with yeast membranes expressing the human A1 adenosine receptor were compared to the results with yeast membranes expressing the human A2a adenosine receptor or the human A3 receptor to test the binding specificity. To perform the assay, fifty mg of membranes were incubated with 0.4 nM [³H] CPX and increasing concentrations of adenosine receptor ligands. Incubation took place in 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10 mM MgCl2, 0.25% BSA and 2 U / ml adenosine deaminase in the presence of protease inhibitors for 60 minutes at room temperature. Binding was terminated by adding ice cold 50 mM Tris-HCl, pH 7.4 plus 10 mM MgCl2, followed by rapid filtration through GFB filters pre-soaked with 0.5% polyethyleneimine, using a Packard 96-well harvester. Data was analyzed by a nonlinear least squares curve fitting procedure using Prism 2.01 software. The IC50 values ​​obtained in this experiment are listed in Table 4 below:

Table 4

<td></td><td></td><td>IC50 [nM]</td><td></td>

<td>Relationship</td><td>hA1R</td><td>hA2aR</td><td>hA3R</td>

<td>XAC</td><td> 6,6</td><td> 11,7</td><td> 53,1</td>

<td>DPCPX</td><td> 8,5</td><td> 326,4</td><td> 1307,0</td>

<td>CGS-15943</td><td> 13,1</td><td> 15,8</td><td> 55,5</td>

<td>NECA</td><td> 215,5</td><td> 294,9</td><td> 34,9</td>

<td>R-PlA</td><td> 67,6</td><td> 678,1</td><td> 23,6</td>

<td>IB-MECA</td><td> 727,7</td><td> 859,4</td><td> 3,1</td>

<td>Aloxysin</td><td> 1072,0</td><td> 1934,0</td><td> 8216,0</td>

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

Functional test using yeast strains expressing the human adenosine A2a receptor

In this example, the use of functional screening assays in yeast for modulators of the human A1 adenosine receptor is described.

I. Ligands used in the test

The natural ligand adenosine was used to study the human A2a receptor functionally expressed in yeast, as well as other well-described and commercially available ligands. Three ligands were used to establish this assay. They include:

<td>Ligand</td><td>Featured Ki</td><td>Function</td>

<td>Adenosine</td><td>500 nM</td><td>agonist</td>

<td>5'-N-ethylcarboxamide adenosine (NECA)</td><td>10-15 nM</td><td>agonist</td>

<td>(-) - N6- (2-phenylisopropyl) adenosine (PIA)</td><td>100-125 nM</td><td>agonist</td>

To prevent a signal due to the presence of adenosine in the growth media, adenosine deaminase (4 U / ml) was added to all tests.

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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 G_asE10K, G_asD229S or G_asE10K + D229S. The ability of the ligand to stimulate the pheromone response pathway in a receptor-dependent manner was marked by a change in the yeast phenotype. Receptor activation modified the phenotype from histidine auxotrophy to histidine prototrophy (fus1-HIS3 activation). Three independent transformants were isolated and grown overnight in the presence of histidine. Cells were washed to remove histidine and diluted to 2 x 10⁶ cells / ml. 5 microls of each transformant were spotted onto non-selective media (containing histidine) or selective media (1 mM AT) in the absence or presence of 4 U / ml adenosine deaminase. Plates were grown at 30 ° C for 24 hours. In the presence of histidine, both Receptor strains⁺ (R⁺) and Receptor^- (R^-) were able to grow. However, in the absence of histidine, only R cells grew⁺. Since no ligand was added to these plates, there were two possible explanations for these results. One possible interpretation was that receptor binding yeast grew due to ligand independent receptor mediated activation (LIRMA). Alternatively, the yeast may have synthesized the adenosine ligand. To distinguish between the two, an enzyme that breaks down the ligand, adenosine deaminase (ADA), was added to the cultured yeast and platelets. In the presence of adenosine deaminase, R.⁺ they did not grow in the absence of histidine, indicating that the yeast did indeed synthesize the ligand.

This interpretation was confirmed by the A2a liquid growth test. In this experiment, the R + yeast (strain G_asE10K expressing A2a receptor) was seeded at three densities (1 x 10⁶ cells / ml; 3 x 10⁵ cells / ml; or 1 x 10⁵ cells / ml) in the presence or absence of adenosine deaminase (4 U / ml). The accuracy of the test increased with increasing concentrations (0, 0.1, 0.2 or 0.4 mM) of 3-amino-1,2,4-triazole (AT), a competitive antagonist of imidazolglycerol-P dehydratase, a protein product of the HIS3 gene. In the presence of adenosine deaminase and 3-amino-1,2-triazole, yeast grew less. However, in the absence of 3-amino-1,2,4-triazole, adenosine deaminase had little effect. Thus, adenosine deaminase alone has no direct effect on the pheromone response pathway.

An alternative approach to measure growth and one that can be miniaturized for a large number of screened items is the A2a receptor ligand spot test. The strain G._asE10K expressing the A2a receptor (A2aR +) or without the receptor (R-) was grown overnight in the presence of histidine and 4 U / ml adenosine deaminase. Cells were washed to remove histidine and diluted to 5 x 10⁶ cells / ml. 1 x 10⁶ cells were spread on selective plates containing 4 U / ml adenosine deaminase and 0.5 or 1.0 mM 3-amino-1,2,4-triazole (AT) and allowed to dry for 1 hour. A single layer was loaded with 5 microls of the following reagents: 10 mM adenosine, 38.7 mM histidine, dimethyl sulfoxide (DMSO), 10 mM PIA and 10 mM NECA. Cells were grown for 24 hours at 30 ° C. The results showed that receptor-deficient cells could only grow when histidine was added to the medium. In contrast, R + cells only grew in the areas where the A2a receptor ligands PIA and NECA were spotted. As the plates contained adenosine deaminase, the lack of growth where adenosine was spotted confirmed that adenosine deaminase was active.

III. LacZ fus1 test

To assess activation of the yeast association pathway, the synthesis of β-galactosidase by fus1 LacZ was measured. Yeast strains expressing G._asE10K, G_asD229S or G_asE10K + D229S was transformed with a plasmid encoding the human A2a receptor (R +) or a plasmid without the receptor (R-). Transformants were isolated and grown overnight in the presence of histidine and 4 U / ml of adenosine deaminase. 1 x 10⁷ cells were diluted to 1 x 10⁶ cells / ml and exposed to increasing concentrations of NECA for 4 hours, after which the β-galactosidase activity in the cells was determined. The results showed that substantially no β-galactosidase activity was detected in the R- strains, while an increasing β-galactosidase activity was detected in the R + strains expressing either G_asE10K, G_asD229S or G_asE10K + D229S with increasing NECA concentration, indicating an increase in dose-dependent β-galactosidase units detected in response to exposure to increasing ligand concentration. This dose-dependency was only observed in cells expressing the A2a receptor. Moreover, the strongest design of the G._as for the A1a receptor was G_asE10K. Construction G_asThe D229S was the second-strongest G construction_as for the A2a receptor, while the G_asThe E10K + D229S was the weakest of the three G designs tested_as, even though even the G_asE10K + D229S stimulated the easily detectable β-galactosidase activity.

PL 204 628 B1

For an additional description of identified tests, see US Application No. 09/088985, titled "Functional Expression of Adenosine Receptors in Yeast," filed June 2, 1998 (agent number CPI-093), the entire contents of which are hereby incorporated by reference.

Pharmacological characterization of the human adenosine receptor subtypes

Materials and methods

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

Yeast strains. Strains of Saccharomyces cerevisiae CY12660 [fur1 * 1442 tbt1-1 fus1-HIS3 can1 ste14: trp1 :: LYS2 ste3 * 1156 gpa1 (41) -Gai3 lys2 ura3 leu2 trp1: his3; LEU2 PGKp-Mfa1LeaderhA1R-PHO5term 2mu-orig REP3 Ampr] and CY8362 [gpa1p-rGasE10K far1 * 1442 tbt1-1 fus1-HIS3 can1 ste14: trp1: LYS2 ste3 * 1156 lys2 ura3 his3 leu2 trp1; LEU2 PGKp-hA2aR 2mu-ori REP3 Ampr] was used as described above.

Yeast culture. Transformed yeast was grown in Leu-Trp [LT] media (pH 5.4) supplemented with 2% glucose. For membrane preparation, 250 ml of LT medium were inoculated with a starting titer of 1-2 x 10⁶ cells / ml from 30 ml overnight culture and incubated at 30 ° C with constant aeration by rotation. After 16 hours of growth, cells were harvested by centrifugation and membranes were prepared as described below.

Mammalian cell culture. HEK-293 cells stably expressing the human adenosine 2a receptor subtype (Cadus # 5 clone) were grown in Dulbeco Minimal Essential Medium (DMEM) supplemented with 10% fetal bovine serum and penicillin IX / streptomycin IX under selective pressure using 500 mg / ml of the antibiotic G418. at 37 ° C in a humid atmosphere containing 5% CO2.

Yeast cell membrane preparations. 250 ml cultures were harvested after an overnight incubation, by centrifugation at 2,000 xg in a Sorvall RT6000 centrifuge. The cells were washed with ice water, centrifuged at 4 ° C and the pellet was resuspended in 10 ml ice-cold lysis buffer [5 mM TrisHCl, pH 7.5; 5mM EDTA; and 5 mM EGTA] supplemented with protease inhibitor mixture tablets (1 tablet per 25 ml buffer). Glass beads (17 g; Mesh 400-600; Sigma) were added to the suspension and the cells disrupted by vigorous centrifugation at 4 ° C for 5 minutes. The homogenate was diluted with an additional 30 ml of lysis buffer plus protease inhibitors and centrifuged at 3.000 xg for 5 minutes. The membranes were then granulated at 36,000 xg (Sorvall RC5B, rotor type SS34) for 45 minutes. The obtained membrane pellet was resuspended in 5 ml of membrane buffer [50 mM Tris-HCl, pH 7.5; 0.6mM EDTA; and 5 mM MgCl2] supplemented with protease inhibitor mixture tablets (1 tablet per 50 ml buffer) and stored at -80 ° C for additional experiments.

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

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

Adenosine receptor subtype 1 saturation and competitive radioligand binding.

PL 204 628 B1

Saturation and competitive binding were performed on membranes from yeast cells transformed with the human receptor A1 subtype using an antagonist [³H] DPCPX as radioactive ligand. Membranes were diluted in binding buffer [50 mM Tris-HCl, pH 7.4; containing 10 mM MgCl2; 1.0mM EDTA; 0.25% BSA; 2 U / ml of adenosine deaminase and 1 tablet of a mixture of protease inhibitor / 50 ml] with a concentration of 1.0 mg / ml. In saturating binding, membranes (50 pg / well) were incubated with increasing concentrations [³H] DPCPX (0.05-25 nM) in a final volume of 100 microliters of binding buffer at 25 ° C for 1 hour in the absence and presence of 10 pM unlabeled XAC in 96-well microtiter plates:

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

Semi-competitive radioligand binding of the 2a adenosine receptor subtype

Competition binding was performed on membranes from HEK293 cells stably expressing the human A2a receptor subtype using an agonist [³H] CGS-21680 as radioactive ligand. Membranes were diluted in binding buffer [50 mM Tris-HCl, pH 7.4; containing 10 mM MgCl2; 1.0mM EDTA; 0.25% BSA; 2 U / ml adenosine deaminase and 1 tablet protease inhibitor mixture / 50 ml] at concentrations of 0.2 mg / ml. Membranes (10 μg / well) were incubated with [³H] CGS-21680 (100 nM) in a final volume of 100 ml binding buffer at 25 ° C for 1 hour in the absence and presence of 50 μM unlabeled NECA or increasing concentrations of competing compounds, in a 96-well microtiter plate .

Semi-competitive binding of adenosine receptor radioligand³

Competition binding was performed on membranes from HEK293 cells stably expressing the human A3 receptor subtype using an agonist [¹²⁶I] AB-MECA as radioactive ligand. Membranes were diluted in binding buffer [50 mM Tris-HCl, pH 7.4; containing 10 mM MgCl2; 1.0mM EDTA; 0.25% BSA; 2 U / ml adenosine deaminase and 1 tablet of protease inhibitor / 50 ml mixture] at a concentration of 0.2 mg / ml. Membranes (10 μg / well) were incubated with [¹²⁵I] AB-MECA (0.75 nM) in a final volume of 100 μl of binding buffer at 25 ° C for 1 hour in the absence and presence of 10 μM unlabeled IB-MECA or increasing concentrations of competing compounds in a 96-well plate for microtiteration.

At the end of incubation, radioligand binding assays for receptor subtypes A1, A2a, and A3 were terminated by adding ice-cold 50 mM Tris-HCl (pH 7.4) buffer supplemented with 10 mM MgCl2, followed by rapid filtration through glass fiber filters (96-well GFB UniFilters; Packard) previously pre-impregnated with 0.5% polyethyleneamine in a Filtermate 196 cell harvester (Packard). The filter plates were dried, coated with 50 µl / well of scytylation fluid (MicroScint-20, Packard) and counted in a TopCount (Packard). The tests were performed in triplicate. Non-specific binding was 5.6 ± 0.5%, 10.8 ± 1.4%, and 15.1 ± 2.6% of total binding, respectively, in the A1R, A2aR, and A3R binding assay.

Semi-competitive radioligand binding of the adenosine 2b receptor

Competition binding was performed on membranes from HEK293 cells stably expressing the human A2b receptor subtype using an A1 receptor antagonist [³H] DPCPX as radioactive ligand. The membranes were diluted in binding buffer (10 mM Hepes-KOH, pH 7.4; containing 1.0 mM EDTA; 0.1 mM benzamidine and 2 U / ml adenosine deaminase] at a concentration of 0.3 mg / ml. Membranes. (15 μg / well) was incubated with [³H] DPCPX (15 nM) in a final volume of 100 µl binding buffer at 25 ° C for 1 hour in the absence and presence of 10 µM unlabeled XAC or increasing concentrations of competing compounds in a 96-well microtiter plate. At the end of the incubation, the assay was terminated by the addition of ice-cold 10 mM Hepes-KOH (pH 7.4) buffer followed by rapid filtration through glass fiber filters (96-well GF / C UniFilters, Packard) pre-saturated with 0.5% polyethyleneimine in Filtermate 196 cell harvester (Packard). The dried filter plates were coated with 50 µl / well with scytillation fluid (MicroScint-20, Packard) and counted in a TopCount (Packard). The tests were performed in triplicate. Non-specific binding was 14.3 2.3% of total binding. Specific binding [³H] DPCPX; [³H] CGS-21680 and (¹²⁵I] AEI-MECA was defined as the difference between total binding and non-specific binding. The percent inhibition of the compounds against complete binding was calculated. Competition data was analyzed by iterative fit 66

The Ki values ​​were calculated from the IC50 values ​​(Cheng and Prusof, Biochem. Pharmacol. 22, 3099-3109, 1973) using GraphPad Prizm 2.01 software.

Results

The primary function of some cell surface receptors is to recognize the appropriate ligands. Accordingly, we determined ligand binding affinities to establish the functional integrity of the yeast-expressed adenosine subtype 1 receptor. Crude membranes prepared from Saccharomyces cerevisiae transformed with the human adenosine subtype 1 receptor construct showed specific saturating binding [³H] DPCPX with a K D of 4.0 0.19 nM. KD and Bmax values ​​were calculated from the saturation isotherm and the Scatchard transformation of the data, indicating a single class of binding sites. The adenosine binding site density in the yeast membrane preparations was estimated to be 716.8 43.4 fmol / mg membrane protein.

The pharmacological subtype characterization of recombinant yeast cells transformed with human A1 receptor subtype selective adenosine ligands (XAC, DPCPX; CGS-15943; CDS-046142; CDS-046123; NECA, (R) -PIA; IB-MECA and Alloxazine) was investigated. They competed with [³H] DPCPX in expected order of sequence. The exchange curves recorded for these compounds show a typical slope for all ligands, and the data for each ligand can be modeled by a single site fit. The apparent dissociation constants estimated for the individual compound from the curves (Table 5) are consistent with the published values ​​for the receptor obtained from other sources.

Table 5

Ki values ​​for membranes from yeast cells transformed with the human A1 receptor subtype

Ligands

XAC

DPCPX

CGS-1594

NECA (R) -PIA

IB-MECA

Alloxazine

CDS-046142

CDS-046123

Ki (nM)

5.5 7,1 10,8 179,6 56,3

606.5 894,1 13,9 9,8

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

PL 204 628 B1

Table 6

Activity of the CDS-046142 series: influence of the substituent in the N position,

<img file="PL204628B1_D0081.tif" />

<td></td><td></td><td colspan="2">A1</td>

<td>Code</td><td>R</td><td>Ki binding (nM)</td><td>Yeast IC50 (nM)</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>CDS-046142</td><td>..... OH</td><td> 13.9</td><td> 97.2</td>

<td>CDS-062365</td><td></td><td> 1423</td><td> &gt; 10,000</td>

<td>CDS-069533</td><td>, OH κΖ&gt; ···^οΗ</td><td> 483.5</td><td> &gt; 10,000</td>

<td>CDS-069534</td><td>OH hC&gt; -^oh</td><td> 196.6</td><td> 4442.0</td>

<td>CDS-056176</td><td>, _. H 0 1-0-4-</td><td> &gt; 10,000</td><td> &gt;10000</td>

<td>CDS-056175</td><td>ηΟ-Ϊ-L</td><td> &gt;10000</td><td> &gt;10000</td>

<td>CDS-062352</td><td>, -K θ 0 — Ph</td><td> 297.9</td><td> &gt;10000</td>

<td>CDS-062351</td><td></td><td> 309.7</td><td></td>

<td>CDS-090909</td><td>ξ Z \ ..... OH KJ ©</td><td> 29.1</td><td></td>

<td>CDS-090910</td><td></td><td> 193.9</td><td></td>

PL 204 628 B1 cont. table 6

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>CDS-090913</td><td>- 1 (+) OH</td><td> 411.5</td><td></td>

<td>CDS-062352</td><td></td><td> 786.6</td><td> &gt;10000</td>

<td>CDS-092474</td><td>X i SHh Trans (S, S)</td><td> 64.8</td><td></td>

<td>CDS-092475</td><td>JiHAc Trans (R, R)</td><td> 6726.0</td><td></td>

<td>CDS-091175</td><td>^H0...... (dl)</td><td> 32.1</td><td></td>

<td>CDS-062351</td><td>(dl)</td><td> 816.9</td><td> 2577.0</td>

<td>CDS-090914</td><td>sO ..... 'oh</td><td> 34.3</td><td></td>

PL 204 628 B1

Table 7

Activity of the CDS-046142 series: effect of the substituent at C2 position.

<img file="PL204628B1_D0082.tif" />

<td></td><td></td><td colspan="2">A1</td>

<td>Code</td><td>R</td><td>Ki binding (nM)</td><td>Yeast IC50 (nM)</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>CDS-069532</td><td>Ύ</td><td> 604.5</td><td> &gt;10000</td>

<td>CDS-090895</td><td>Y</td><td> 157.7</td><td> 763.1</td>

<td>CDS-065564</td><td></td><td> 198.5</td><td> 2782.5</td>

<td>CDS-090896</td><td></td><td> 443.6</td><td> &gt;10000</td>

<td>CDS-090903</td><td></td><td> 61.1</td><td> 297.0</td>

<td>CDS-090890</td><td></td><td> 30.1</td><td> 194.7</td>

<td>CDS-090915</td><td>Y F.</td><td> 19.9</td><td></td>

PL 204 628 B1 cont. table 7

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>CDS-090912</td><td>. C '</td><td> 62.8</td><td></td>

<td>CDS-090936</td><td> &gt;+</td><td> 2145</td><td></td>

<td>CDS-090177</td><td>F. 0 '</td><td> 48.7</td><td></td>

Table 8

Activity of the CDS-046142 series: effect of a pyrrole ring substituent

<img file="PL204628B1_D0083.tif" />

R '

<td></td><td></td><td></td><td></td><td></td><td></td><td>A1</td>

<td>Code</td><td>R</td><td>R '</td><td>R "</td><td>R '"</td><td>Tie Ki (nM)</td><td>Yeast IC50 (nM)</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td>

<td>CDS-078187</td><td> 0*</td><td>Me</td><td>Me</td><td>Me</td><td> 3311</td><td> &gt;10000</td>

<td>CDS-090905</td><td> ©</td><td>H.</td><td>Me</td><td>H.</td><td> 22.3</td><td> 148.3</td>

<td>CDS-090921</td><td>about</td><td>H.</td><td>H.</td><td>Me</td><td> 8.9</td><td></td>

<td>CDS-090902</td><td></td><td>«I&gt; -0</td><td>Me</td><td>Me</td><td> 2210</td><td> &gt;10000</td>

<td>CDS-056090</td><td> 0</td><td>x&gt;</td><td>Me</td><td>Me</td><td> 863.1</td><td></td>

<td>CDS-056091</td><td>σ</td><td></td><td>Me</td><td>Me</td><td> 4512</td><td></td>

PL 204 628 B1 cont. table 8

<td> 1</td><td> 2</td><td colspan="2"> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td>

<td>CDS-056089</td><td>X σ</td><td></td><td>-ο</td><td>Me</td><td>Me</td><td> 8451</td><td></td>

<td>CDS-056092</td><td>Cf</td><td></td><td>about</td><td>Me</td><td>Me</td><td> 35.3</td><td></td>

Table 9

<img file="PL204628B1_D0084.tif" />

<td></td><td></td><td colspan="2">A1</td>

<td>Code</td><td>R</td><td>Tie Ki (nM)</td><td>Yeast IC50 (nM)</td>

<td>CDS-056090</td><td></td><td> 863.1</td><td></td>

<td>CDS-056091</td><td>X)</td><td> 4512</td><td></td>

<td>CDS-056089</td><td>Λ ^ -Μ ^ ΝΗΛς</td><td> 8451</td><td></td>

<td>CDS-056092</td><td></td><td> 35.3</td><td></td>

Table 10 CDS-046123 series activity: effect of substituent at position N6

<img file="PL204628B1_D0085.tif" />

<td></td><td></td><td colspan="2">A1</td>

<td>Code</td><td>R</td><td>Ki binding (nM)</td><td>Yeast IC50 (nM)</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>CDS-062354</td><td></td><td> 1789</td><td> &gt;10000</td>

PL 204 628 B1 cont. table 10

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>CDS-067146</td><td>H. X-V^H. 0</td><td> 54.4</td><td> 1865</td>

<td>CDS-046123</td><td>H. 1 0</td><td> 9.8</td><td> 82.8</td>

<td>CDS-062357</td><td> 0</td><td> 26.7</td><td> 195.7</td>

<td>CDS-062355</td><td>c- / 0</td><td> 32.8</td><td> 545.8</td>

<td>CDS-062356</td><td> 0</td><td> 147.5</td><td> 3972</td>

<td>CDS-067325</td><td> 0 0</td><td> 151.7</td><td> 2918</td>

<td>CDS-062392</td><td> 0 11 * 0</td><td> 692.5</td><td> &gt;10000</td>

<td>CDS-062393</td><td>Vv^NHy ^^C00H 0</td><td> 93.1</td><td> 3217</td>

<td>CDS-062394</td><td>about</td><td> 475.3</td><td> &gt;10000</td>

<td>CDS-067227</td><td>V ^ - ^ NHAc</td><td> 674.9</td><td> 9376.0</td>

<td>CDS-065568</td><td></td><td> 121.9</td><td> 2067.5</td>

<td>CDS-066956</td><td> 0</td><td> 233.9</td><td> 3462</td>

<td>CDS-067038</td><td> 0</td><td> 270.1</td><td> 3009.5</td>

<td>CDS-062358</td><td>χ - \ /^0Η</td><td> 384.9</td><td> 2005</td>

<td>CDS-062359</td><td></td><td> 179.3</td><td> 3712</td>

<td>CDS-062360</td><td>Χχ / '- χ / θπ</td><td> 176.1</td><td> 5054</td>

PL 204 628 B1

Table 11

Activity of CDS-046123 series: effect of substituent at position N6

<img file="PL204628B1_D0086.tif" />

<td></td><td></td><td colspan="2">A1</td>

<td></td><td></td><td>Ki binding (nM)</td><td>Yeast IC50 (nM)</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>CDS-046123</td><td>ν—. ΝΗ ΧΗ- 0</td><td> 9,8</td><td> 115.4</td>

<td>CDS-069535</td><td>νη .νη, r 0</td><td> 53,9</td><td> 551.0</td>

<td>CDS-090894</td><td>ΝΗ .NHMe γ- 0</td><td> 10.3</td><td> 101.3</td>

<td>CDS-062301</td><td>ν— \ ΝΗ ΝΗΕί '' γ0</td><td> 71.1</td><td> 3217</td>

<td>CDS-090904</td><td>Η 1 ηΎ^Η' Me 0 (±)</td><td> 6.5</td><td> 58.7</td>

<td>CDS-090906</td><td>Η 1 / γνγ⁰^ MC 0 (ί)</td><td> 105.4</td><td> 472.1</td>

<td>CDS-090908</td><td>Mc Η⁰ (and)</td><td> 27.8</td><td> 162.4</td>

<td>CDS-090907</td><td>Me Η ν-Χ, Ν ./0-/ V— Υ Υ ~ ° (i)</td><td> 126.5</td><td> 1297.0</td>

<td>CDS-092473</td><td>γ ^, ΝΗΑο</td><td> 2.3</td><td></td>

PL 204 628 B1 cont. table 11

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>CDS-095450</td><td>I ^ L ^ NHAc S.</td><td> 9.0</td><td></td>

<td>CDS-095451</td><td>^ nhac</td><td> 17.3</td><td></td>

<td>CDS-091183</td><td>and ^R</td><td> 2.5</td><td></td>

<td>CDS-091184</td><td>IA ^ / Nhac * R</td><td> 213</td><td></td>

Table 12 "Retro-amide" analogs of CDS-046123

<img file="PL204628B1_D0087.tif" />

<td></td><td></td><td colspan="2">A1</td>

<td>Code</td><td>R</td><td>Ki binding (nM)</td><td>Yeast IC50 (nM)</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>CDS-065567</td><td> 0</td><td> 16.5</td><td> 189.4</td>

<td>CDS-090891</td><td>ABOUT</td><td> 7.4</td><td> 45.7</td>

<td>CDS-062373.</td><td>0 Η</td><td> 95.8</td><td> 3345.0</td>

<td>CDS-090893</td><td>ABOUT</td><td> 529.1</td><td> 4040.0</td>

<td>CDS-062371</td><td> 0</td><td> 1060.0</td><td> &gt;10000</td>

PL 204 628 B1 cont. table 12

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>CDS-062372</td><td> 0</td><td> 1272</td><td> &gt;10000</td>

<td>CDS-065566</td><td>νγ^ΝΗ2 0</td><td> 50.8</td><td> 4028</td>

<td>CDS-065565</td><td>^ χ-χ ^ -NHMe * · T 0</td><td> 48.5</td><td> 701.5</td>

Table 13

Profile of selective adenosine antagonists

<td colspan="2">R WITH HN and ^{Me N}'iT ^ X 1 L ^ Me H.</td><td colspan="4">Ki binding (nM)</td>

<td></td><td>R</td><td>A1</td><td>A2a</td><td>A2b</td><td>A3</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td>

<td>CDS-046123</td><td>γγ ^^ΝΗΑε</td><td> 9.8-25.1</td><td> 18.0-48.6</td><td> 80.3</td><td> 513.0</td>

<td>CDS-090908</td><td>Me jA ^ NHAc</td><td> 27.8</td><td> 50.7</td><td> 84.6</td><td> 429.8</td>

<td>CDS-090894</td><td>H. 1 V \ Yk .NHMe^: Y 0</td><td> 20.2</td><td> 75.6</td><td> 20.1</td><td> 4.3</td>

<td>CDS-090891</td><td>0 Y ^ - ^^ NHNle</td><td> 17.4</td><td> 111.3</td><td> 120.6</td><td> 44.6</td>

<td>CDS-046142</td><td>..... OH</td><td> 13.9-30.9</td><td> 933.7</td><td> 138.0</td><td> 21.5</td>

<td>CDS-090890¹</td><td></td><td> 46.6</td><td> 730.9</td><td> 30%</td><td> 9.9</td>

PL 204 628 B1 cont. table 13

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td>

<td>CDS-090905²</td><td>..... OH</td><td> 16.4</td><td> 766.3</td><td> 168.3</td><td> 71.7</td>

<td>CDS-090909</td><td> .....^0H(dl)</td><td> 29.1</td><td> 190.6</td><td> 1143.0</td><td> 3.1</td>

<td>CDS-90910</td><td>κΧ</td><td> 180</td><td> 230</td><td> 670</td><td> 1.0</td>

<td>CDS-116676</td><td>H. 1 OY '</td><td> 40</td><td> 109</td><td> 109</td><td> 0.3</td>

<td>CDS-121180</td><td>1 H.</td><td> 255</td><td> 76%</td><td> 275</td><td> &lt;2.6</td>

<td>CDS-121178</td><td>about^V‘^CH^ -Y_M.e 1 H.</td><td> 531</td><td> 981</td><td> 736</td><td> 5.3</td>

<td>CDS-121179</td><td>^y(CHi ^ Jl_NH,_e1 H.</td><td> 443</td><td> 2965</td><td> 375</td><td> &lt;6.2</td>

<td>CDS-123264³</td><td>0 YcH ^_NX [7_NtV1 H.</td><td> 30%</td><td> 65%</td><td> 515</td><td> 24</td>

<td>CDS-062391</td><td>Υκ ^ Υ_ΝΗΞι1 H.</td><td> 87</td><td> 204</td><td> 30</td><td> 0.02</td>

<td>CDS-121181</td><td>AND H.</td><td> 75,000</td><td> 720,000</td><td> 3,400</td><td> 507</td>

PL 204 628 B1 cont. table 13

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td>

<td>CDS-121268</td><td>0 AND Η</td><td> 333</td><td> 710,000</td><td> 710,000</td><td> 97</td>

<td>CDS-121272</td><td>Η XrV^N</td><td> 710,000</td><td> 710,000</td><td> 720,000</td><td> 369</td>

<td>CDS-096370⁴</td><td>..... OH FF</td><td> 3.7±0.5</td><td> 63 0± 56.4</td><td> 2307± 926</td><td> 630±76</td>

<td>CDS-113760^4,5</td><td>..... OH xU</td><td> 1.8</td><td> 206</td><td> 802</td><td> 270</td>

<td>CDS-116665^4,5</td><td>..... OH</td><td> 8.0</td><td> 531</td><td> 530</td><td> 419</td>

<td>CDS-131921^4,7</td><td>...... OH</td><td> 8.0</td><td> 131</td><td> 1031</td><td> 54%⁸</td>

¹2-thienyl-2-yl; ²water-soluble; ³R5 and R6 are hydrogen; ⁵R3 is fluorophenyl; ⁶R3 is fluorophenyl; ⁷R3 is pyridyl;

⁸% activity @ 10 µM

PL 204 628 B1

Table 14: Profile of the selective A2b antagonists

<img file="PL204628B1_D0088.tif" />

<td rowspan="2">Code</td><td rowspan="2">XR1</td><td rowspan="2">R2</td><td colspan="4">Ki binding data (nM)</td>

<td>A1</td><td>A2a</td><td>A2B</td><td>Aa</td>

<td>CDS-129851</td><td>-O-Ph</td><td>Me</td><td> 41,7</td><td> 21</td><td> 0,3</td><td> 14,6</td>

<td>CDS-143995</td><td>-O-Ph (p) F</td><td>Me</td><td> 33</td><td> 58</td><td> 0,01</td><td> 18</td>

<td>CDS-143994</td><td>-O-Ph (p) Cl</td><td>Me</td><td> 825</td><td> 591</td><td> 0,3</td><td> 60</td>

<td>CD5-143988</td><td>-N-pyridin-2-one</td><td>Me</td><td> 63</td><td> 41</td><td> 47</td><td> 48</td>

<td>CDS-143996</td><td>-NH-Ph</td><td>Me</td><td> 49</td><td> 31</td><td> 109</td><td> 57</td>

Incorporation as reference

All patents, published patent applications, and other references described herein are hereby formally incorporated herein by reference.

Equivalents

Those skilled in the art will recognize, or be able to determine, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to fall within the scope of the following claims.