RU2258071C2 - Derivatives of 2-alkynyladenosine used for control inflammatory response - Google Patents

Abstract

FIELD: organic chemistry, medicine.

SUBSTANCE: invention relates to compound of the formula (I)

wherein each among R represents independently hydrogen atom, (C₁-C₆)-alkyl, (C₃-C₇)-cycloalkyl, phenyl or phenyl-(C₁-C₃)-alkyl; X and X' represent -CH₂OH, -CO₂R², -OC(O)R², -CH₂OC(O)R² or C(O)NR³R⁴ wherein R², R³ and R⁴ represent independently hydrogen atom (H), (C₁-C₆)-alkyl substituted optionally with one-three (C₁-C₆)-alkoxy-groups, (C₁-C₆)-alkylthio-groups, halogen atoms, hydroxy-, amino-, mono-(C₁-C₆)-alkyl)-amino-, di-(C₁-C₆)-alkyl)-amino-group; Z and Z' represent independently (C₁-C₆)-alkyl broken optionally with one-three sulfur atoms (S) or non-peroxide oxygen atom (O), or they absent; n = 1-3; or to its pharmaceutically acceptable salt. Compounds are agonists of adenosine A_2A-receptors and can be used for inhibition of inflammatory response or inflammation treatment.

EFFECT: valuable medicinal properties of compounds.

56 cl, 1 tbl, 21 dwg, 37 ex

Description

Technical field

The present invention relates to methods and compositions for preventing tissue damage as a result of an inflammatory effect.

BACKGROUND OF THE INVENTION

The present invention was created with funding from the US government (NIH Grant ROL HL37942). The US government has certain rights to this invention.

An inflammatory reaction serves to eliminate harmful agents from the body. There is a wide range of pathogenic lesions that can trigger an inflammatory response, including infection, allergens, autoimmune stimuli, an immune response to transplanted tissue, harmful chemicals and toxins, ischemia / reperfusion, hypoxia, and mechanical and thermal injury. Inflammation is usually a very localized action, which serves to remove, mitigate dilution and remove the damaging agent and damaged tissue. The reaction of the body becomes a factor in the disease in cases where it leads to undesirable damage to the tissues of the host in the process of eliminating the target agent or response to traumatic injury.

By way of example, inflammation is a component of pathogenesis in some vascular diseases or lesions. Similar examples include ischemia / reperfusion injury (NGFrangogiannis et al., In Myocardial Ischemia: Mechanisms, Reperfusion, Protection, M. Karmazyn, ed., Birkhuser Verlag (1996) at 236-284; HS Sharma et al., Med. Of Inflamm., 6, 175 (1987), atherosclerosis (R. Ross, Nature, 362, 801 (1993)), inflammatory aortic aneurysms (N. Girardi et al., Ann. Thor. Surg., 64, 251 (1997) ; DI Walker et al., Brit. J. Surg., 59, 609 (1972); RL Pennel et al., J. Vasc. Surg., 2, 859 (1985)), as well as restenosis after balloon angioplasty (see R. Ross, cited above.) Cells involved in the inflammatory process include white blood cells (i.e. cells of the immune system - neutrophils, eosinophils, lymphocytes, monocytes , Basophils, macrophages, dendritic cells and mast), vascular endothelium, vascular smooth muscle cells, fibroblasts, and myocytes.

The release of inflammatory cytokines, such as tumor necrosis factor alpha (TNFα), by white blood cells is a means by which the immune system resists pathogenic invasions, including infections. TNFα stimulates the expression and activation of adhesion factors to leukocytes and endothelial cells, primates neutrophils for an enhanced anti-inflammatory response to secondary stimuli, and also enhances the adhesive oxidative activity of neutrophils. See Sharma et al., Cited above. In addition, macrophages / dendritic cells act as auxiliary cells, processing the antigen for presentation to lymphocytes. Lymphocytes, in turn, receive a stimulus to act as pro-inflammatory cytotoxic cells.

et al., Rev. Infect.Dis., 9 (suppl. 5), S 602-S 606 (1987)). Например, TNFα может индуцировать адгезию нейтрофилов к стенкам кровеносных сосудов, а затем их миграцию по сосуду к месту повреждения и высвобождение их окислительных и неокислительных продуктов воспаления.In general, cytokines stimulate neutrophils, enhancing oxidative (e.g., superoxide and secondary products) and non-oxidative (e.g., myeloperoxidase and other enzymes) inflammatory activity. Unwanted and excessive release of cytokines can cause a counterproductive enhanced pathogenic effect as a result of the release of tissue-damaging oxidative and non-oxidative inflammatory products (K. G. Tracey et al., J. Exp. Med., 167, 1211 (1988); and.

et al., Rev. Infect. Dis., 9 (suppl. 5), S 602-S 606 (1987)). For example, TNFα can induce adhesion of neutrophils to the walls of blood vessels, and then their migration along the vessel to the site of damage and the release of their oxidative and non-oxidative products of inflammation.

Despite the fact that monocytes slowly collect in the foci of inflammation, under favorable conditions they develop into long-term, permanent auxiliary cells and macrophages. When triggered by an inflammation trigger, monocytes / macrophages also produce and secrete a number of cytokines (including TNFα), complement, lipids, reactive oxygen species, proteases, and growth factors that rearrange tissue and regulate tissue-surrounding functions.

et al., Rev. Infect. Dis., 9 (suppl. 5), S602-S606 (1987)); астме (N.M. Cembrzynska et al., Am. Rev. Respir. Dis., 147, 291 (1993)); отторжении при трансплантации органов (D.K. Imagawa et al., Transplantation, 51, 57 (1991); рассеянном склерозе (Н.Р. Hartung, Ann. Neurol., 33, 591 (1993)); спиде (Т. Matsuyama et al., AIDS, 5, 1405 (1991) и ожоге глаз щелочью (F. Miyamoto et al., Opthalmic Res., 30, 168 (1997)). Кроме того, образование супероксида в лейкоцитах способствует репликации вируса иммунодефицита человека (ВИЧ) (S. Legrand-Poels et al., AIDS Res. Hum. Retroviruses, 6, 1389 (1990)).For example, the pathogenicity of inflammatory cytokines in arthritis has been established (C. A. Dinarello, Semin. Immunol., 4, 133 (1992)); ischemia (A. Seekamp et al., Agents-Actions-Supp., 41, 137 (1993)); septic shock (

et al., Rev. Infect. Dis., 9 (suppl. 5), S602-S606 (1987)); asthma (NM Cembrzynska et al., Am. Rev. Respir. Dis., 147, 291 (1993)); organ transplant rejection (DK Imagawa et al., Transplantation, 51, 57 (1991); multiple sclerosis (N.P. Hartung, Ann. Neurol., 33, 591 (1993)); AIDS (T. Matsuyama et al. , AIDS, 5, 1405 (1991) and an alkali eye burn (F. Miyamoto et al., Opthalmic Res. 30, 168 (1997)). In addition, the formation of superoxide in leukocytes promotes the replication of human immunodeficiency virus (HIV) (S Legrand-Poels et al., AIDS Res. Hum. Retroviruses, 6, 1389 (1990)).

It is well known that adenosine and some of its analogues, non-selectively activating subtypes of adenosine receptors, reduce the production of neutrophils of inflammatory oxidative products (BNCronstein et al., Ann. NYAcacd Sci., 451, 291 (1985); PARoberts et al., Biochem. J. , 227, 669 (1985); DJ Schrier et al., J. Immunol., 137, 3284 (1986); B.N. Cronstein el al., Clinical Immunol and Immunopath., 42, 76 (1987); MAIannone et al ., in Topics and Perspective in Adenosine Research, E. Gerlach et al., cds., Springer-Verlag, Berlin, p. 286 (1987); STMcGarrity et al., J. Leukocyte Biol., 44, 411421 (1988) ; J. De La Harpe et al., J. Immunol, 143, 596 (1989); S. T. McGarrity el al., J. Immunol., 142, 1986 (1989); and C. P. Nielson et al ., Br.J. Pharmacol, 97, 882 (1989)). For example, adenosine has been found to inhibit the release of superoxide from neutrophils stimulated by chemoattractants, such as synthetic mimicking bacterial peptides, f-met-leu-phe (fMLP), and the complement component C ₅ a (BNCronstein et al., J. Immunol ., 135, 1366 (1985). Adenosine can reduce the greatly increased oxidative emission (focus) of PMN (neutrophil), first primed with THF-α and then stimulated with a second stimulus such as f-met-leu-phe (GW Sullivan et al ., Clin. Res., 41, 172A (1993)). In addition, it was found that adenosine is able to reduce the level of replication PI in the T cell line (S. Sipka et al., Acta. Biochim. Biopys. Hung., 23, 75 (1998)). However, the fact that in vivo adenosine has anti-inflammatory activity (GS Firestein et al. , Clin. Res., 41, 170A (1993); and BNCronstein et al., Clin. Res., 244A (1993)).

It has been suggested that neutrophils have more than one subtype of an adenosine receptor, which may have the opposite effect on superoxide release (BNCronstein et al., J. Clin. Invest. 85, 1150 (1990)). The existence of the A _2A receptor on neutrophils was originally established by Van Calker et al. (D. Van Calker et al., Eur. J. Pharmacology, 206, 285 (1991).

Gradually, more and more effective and / or selective compounds were developed as agonists of A _2A adenosine receptors (ARs) based on assays for radioligand binding and physiological reactions. Initially, compounds were developed that lack selectivity or have low selectivity for A _2A receptors, such as adenosine or 5'-carboxamides of adenosine, such as 5'-N-ethylcarboxamidoadenosine (NECA) (BNCronstein et al., J. Immunol., 135, 1366 (1985)). Later it was found that the addition of 2-alkylamino substituents increases the efficiency and selectivity, for example, CV1808 and CGS21680 (MF Jarvis et al., J. Pharmacol. Exp. Ther., 251, 888 (1989)). Derivatives of 2-alkoxy-substituted adenosine, such as WRC-0090, are even more effective and selective as agonists of the A _2A receptor in the coronary artery (M. Ueeda et al., J. Med. Chem., 34, 1334 (1991)). Derivatives of 2-alkylhydrazinoadenosine, for example, SHA 211 (also called WRC-0474), have also been found to be agonists of the A _2A receptor in the coronary artery (K. Niiya et al., J. Med. Chem., 35, 4557 ( 1992)).

There is one description of the combination of relatively non-specific analogues of adenosine: R-phenylisopropyladenosine (R-PIA) and 2-chloradenosine (CI-Ado) with a phosphodiesterase inhibitor (PDE), leading to a decrease in the oxidative activity of neutrophil (M.A. Iannone et al., Topics and Perspectives in Adenosine Research, E. Garlach et al., eds., Springer-Verlag, Berlin, pp. 286-298 (1987)). However, the analogues of R-PIA and CI-Ado are indeed more effective activators of A ₁ receptors for adenosine than A _2A receptors for adenosine, and, therefore, are likely to cause side effects due to the activation of A ₁ receptors on the heart muscle and other tissues, causing a heart block.

RAOlsson et al. (US patent No. 5278150) describe selective agonists of the A ₂ receptor of adenosine of the formula:

where Rib represents ribosyl, R ₁ may represent H, and R ₂ may represent cycloalkyl. It is indicated that these compounds can be used to treat hypertension, atherosclerosis, and also as a vasodilator.

Olsson et al. (US patent No. 5140015) describe specific agonists of the A ₂ receptor of adenosine of the formula:

where C (X) BR ₂ may represent CH ₂ OH, and R ₁ may represent alkyl or alkoxyalkyl. It is indicated that these compounds can be used as vasodilators or antihypertensives.

Linden et al. (US Pat. No. 5,877,180) are based on the discovery that certain inflammatory diseases, such as arthritis and asthma, can be effectively treated by administering compounds that are selective agonists of A _2A adenosine receptors, preferably in combination with a phosphodiesterase inhibitor, type IU . Embodiment Linden et al. provides a method for treating inflammatory diseases by administering an effective amount of an A _2A adenosine receptor of the following formula:

where R and X have the meanings indicated in the patent.

A preferred embodiment of the invention (Linden et al.) Involves the administration of a phosphodiesterase inhibitor (PDE), type IU, in combination with an adenosine A _2A receptor agonist. Phosphodiesterase (PDE) inhibitor, type IU, includes racemic and optically active 4- (polyalkoxyphenyl) -2-pyrrolidones of the following formula:

where R ^' , R ¹⁸ , R ¹⁹ and X have the meanings specified in US patent No. 4193926. Rolipram is an example of a suitable PDE type 1U inhibitor represented by the above formula.

G. Cristalli (US Pat. No. 5,593,975) describes 2-arylethynyl, 2-cycloalkylethynyl or 2-hydroxyalkylethynyl derivatives in which the riboside residue is substituted by carboxyamino or substituted carboxyamino (R ₃ HNC (O) -).

Derivatives of 2-alkynylpurine are described by Miyasaka et al. (US patent No. 4956345), while the 2-alkynyl group is substituted by (C ₃ -C ₁₆ ) alkyl. It is indicated that the compounds in accordance with US patent No. 5593975 dilate blood vessels and inhibit platelet aggregation, thus, they can be used as anti-ischemic, anti-atherosclerotic and antihypertensive agents.

However, there remains a need for selective agonists of A ₂ receptors of adenosine for medical use, which have less side effects.

SUMMARY OF THE INVENTION

The present invention includes compounds and methods of their use for treating inflammation in mammalian tissue. Tissue inflammation can be caused by pathological agents, either by physical, chemical or thermal trauma, or by trauma from medical procedures, such as organ, tissue or cell transplantation, angioplasty (PCTA), inflammation after ischemia / reperfusion or implantation. These compounds include a new class of 2-alkynyladenosine derivatives substituted at the ethine position with substituted cycloalkyl moieties. The riboside residue is preferably substituted at position 5 '("X") by an N-alkyl- (or cycloalkyl) carboxyamino ("aminocarbonyl") moiety. Thus, the present invention provides a method for suppressing an inflammatory response in mammals such as humans, as well as protecting inflamed tissue by administering an effective amount of one or more compounds in accordance with this invention.

The compounds of this invention have the following general formula (I):

where (a) each of R independently represents hydrogen, C ₁ -C ₆ alkyl, C ₃ -C ₇ cycloalkyl, phenyl or phenyl (C ₁ -C ₃ ) alkyl;

(b) X is —CH ₂ OH, —CO ₂ R ² , —OC (O) R ² , —CH ₂ OC (O) R ^2, or C (O) NR ³ R ⁴ ;

(c) each of R ² , R ³ and R ⁴ separately represents H, C _1-6 alkyl; C _1-6 alkyl substituted with one to three C _1-6 alkoxy, C _3-7 cycloalkyls, C _1-6 alkylthio, halogens, hydroxy, amino, mono (C _1-6 alkyl) amino, di ( C _1-6 alkyl) amino or C _6-10 aryls, where aryl may be substituted with one to three halogens, C _1-6 alkyl, hydroxy, amino, mono (C _1-6 alkyl) amino or di ( C _1-6 alkyl) amino; C _6-10 aryl; or C _6-10 aryl substituted with one to three halogens, hydroxy, amino, mono (C _1-6 alkyl) amino, di (C _1-6 alkyl) amino or C _1-6 alkyl;

(d) R ¹ represents (X- (Z) -) _n [(C ₃ -C ₁₀ ) cycloalkyl] - (Z ') -, where Z and Z' independently represent (C ₁ -C ₆ ) alkyl, optionally interrupted one to three S or non-peroxide O, or absent, and n is 1-3,

or a pharmaceutically acceptable salt thereof.

       This invention provides a compound of formula I, which can be used for medical treatment, preferably for the treatment or protection of tissues from inflammation, such as an inflammatory reaction, as well as the use of a compound of formula I for the manufacture of medicines for the treatment of an inflammatory reaction caused by a pathological condition or symptom in mammals, such as humans, associated with inflammation.

Despite the description of some agonists of A _2A adenosine receptors as vasodilators that can be directly used to treat hypertension, thrombus, atherosclerosis, etc., the tissue protective effect of the compounds of formula (I) is not mentioned in known sources.

The invention also provides for the use of these compounds with phosphodiesterase inhibitors, type IU, to synergistically reduce the inflammatory response of immune cells.

The invention also provides a pharmaceutical composition comprising an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable diluent or carrier, and optionally in combination with a phosphodiesterase inhibitor (PDE), type IU. The composition is preferably presented in unit dosage form.

In addition, the present invention provides a therapeutic method for the prevention or treatment of a pathological condition or symptom in mammals, such as humans, when activity of adenosine A _2A receptors is suspected and an agonism of said activity is desired, comprising administering to a mammal in need of such treatment an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. It is believed that activation of the A _2A adenosine receptors inhibits inflammation by acting on neutrophils, mast cells, monocytes / macrophages, T cells and / or eosinophils. Inhibition of such inflammatory cells leads to the protection of tissues after their defeat.

Inflammatory reactions that can be treated (including prophylactic) with a compound of formula I, optionally with a PDE inhibitor, type IU, include inflammation caused by:

(a) autoimmune stimulation (autoimmune diseases) such as lupus erythematosus, multiple sclerosis, endometriosis infertility, type I diabetes mellitus, including destruction of pancreatic islets leading to diabetes, and the inflammatory effects of diabetes, including leg ulcers, Crohn’s disease, ulcerative colitis, inflammatory bowel disease, osteoporosis and rheumatoid arthritis;

(b) allergic diseases such as asthma, hay fever, rhinitis, vernal conjunctivitis and other eosinophil-mediated conditions;

(c) skin diseases such as psoriasis, contact dermatitis, eczema, infectious skin ulcers, open wounds, cellulite;

(d) infectious diseases, including sepsis, septic shock, encephalitis, infectious arthritis, endotoxic bacterial toxic shock, gram-negative shock, Jarisch-Herxheimer reaction, herpes zoster, toxic shock, cerebral malaria, bacterial meningitis, adult respiratory distress syndrome ( ARDS), Lyme disease, HIV infection (enhanced TNFa-HIV replication, inhibition of TNFα activity of a reverse transcriptase inhibitor);

(e) debilitating diseases: cachexia after cancer and HIV;

(f) transplantation of an organ, tissue or cell (eg, bone marrow, cornea, kidney, lung, liver, heart, skin, pancreatic islets), including transplant rejection and graft versus host disease;

(g) the side effect of drug therapy, including the side effect of treatment with amphotericin B, the immunosuppressive therapy, for example, treatment with interleukin-2, the side effect of treatment with OKTZ, GM-CSF, cyclosporine, and the side effect of treatment with aminoglycosides, stomatitis and mucositis, caused by suppression of immunity;

(h) cardiovascular conditions, including circulatory diseases induced or caused by an inflammatory reaction, such as ischemia, atherosclerosis, peripheral vascular disease, post-angioplasty restenosis, inflammatory aortic aneurysm, vasculitis, stroke, spinal cord injury, congestive heart failure, hemorrhagic shock, and / reperfusion injury, vasospasm after subarachnoid hemorrhage, vasospasm after stroke, pleurisy, pericarditis, as well as cardiovascular complications of diabetes eta;

(i) dialysis, including pericarditis caused by peritoneal dialysis;

(j) gout and

(k) chemical or thermal injury caused by burns, acid, alkali, etc.

Especially interesting and effective is the use of these compounds for the treatment of inflammatory reactions caused by transplantation of an organ, tissue or cell, i.e. transplantation of allogeneic or xenogenic tissue to a mammalian recipient, autoimmune diseases and inflammatory conditions caused by circulatory pathologies and their treatment, including angioplasty, stent insertion, shunt or implantation. It has been unexpectedly discovered that the introduction of one or more compounds of formula (I) is effective after the occurrence of an inflammatory reaction, for example, after an object is damaged by a pathology or trauma that initiates an inflammatory reaction.

The present invention also includes a method for measuring the response, or binding a compound of formula I or sites designated A _2A adenosine receptors comprising said receptors, in vivo or in vitro, with an amount of a compound of formula I, effective to bind said receptors. Tissues or cells, including ligand bound receptor sites, can be used to measure the selectivity of test compounds for specific receptor subtypes, the amount of bioactive compound in the blood or other physiological fluids, or can be used as a tool to identify potential therapeutic agents for treating diseases or conditions associated with activation of the receptor site by contacting these agents with the indicated ligand-receptor complexes and measuring the degree neither ligand substitution and / or binding of the agent, or a cellular reaction to the agent (e.g., cAMP accumulation).

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise indicated, the definitions have the following meanings. Halogen means fluorine, chlorine, bromine or iodine. Alkyl, alkoxy, aralkyl, alkylaryl, etc. mean both straight and branched alkyl groups, however, reference to a single radical, such as "propyl", refers only to a straight chain radical, with a branched chain isomer such as "isopropyl" is specifically indicated. Aryl includes a phenyl radical or an ortho-fused bicyclic, carbocyclic radical having from about nine to ten ring atoms in which at least one ring is aromatic. Heteroaryl includes a radical attached through a carbon ring of a monocyclic aromatic ring containing from five to six ring atoms consisting of carbon and from one to four heteroatoms, each of which is selected from the group consisting of non-peroxide oxygen, sulfur and N (X), where X absent or represents H, O, (C ₁ -C ₄ ) alkyl, phenyl or benzyl, as well as the radical of an ortho-condensed bicyclic heterocycle containing from about eight to ten ring atoms derived from it, especially a benzene derivative or a derivative resulting from its condensation with propylene, trimethylene or tetramethylene diradicals.

One skilled in the art will appreciate that the compounds of formula (I) have several chiral centers and can be isolated in optically active and racemic forms. A riboside fragment of formula (I) is preferably obtained from D-ribose, i.e. 3 ', 4'-hydroxyl groups are alpha groups with respect to the sugar ring, and 2'- and 5'-groups are beta groups (3R, 4S, 2R, 5S). If the two groups on the cyclohexyl group are in position 4, then they are preferably trans groups. Some compounds may exhibit polymorphism. It is intended that the present invention include any racemic, optically active, polymorphic, or stereoisomeric forms or mixtures thereof of the compounds of the invention having the useful properties described herein, and it is well known in the art how to obtain optically active forms (e.g., by resolving the racemic forms by recrystallization or enzymes, synthesis from optically active starting materials, chiral synthesis or chromatographic separation using chiral stationary phase) and how to determine the activity of an adenosine agonist using the tests described here or other similar studies well known in the art.

The specific and preferred meanings given below for radicals, substituents and ranges are for illustrative purposes only; they do not exclude other specific values or other values falling within the established ranges for radicals and substituents.

Specifically, (C ₁ -C ₆ ) alkyl may be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, pentyl, 3-pentyl or hexyl. As used herein, the term “cycloalkyl” means bicycloalkyl (norbornyl, 2.2.2-bicyclooctyl, etc.) and tricycloalkyl (adamantyl, etc.), optionally including one or two N, O or S. Cycloalkyl also means (cycloalkyl ) alkyl. Thus, (C ₃ -C ₆ ) cycloalkyl may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; (C ₃ -C ₆ ) cycloalkyl (C ₁ -C ₆ ) alkyl may be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl; 2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl or 2-cyclohexylethyl.

(C ₁ -C ₆ ) alkoxy may be methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, pentoxy, 3-pentoxy or hexyloxy; (C ₂ -C ₆ ) alkenyl may be vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl or 5-hexenyl; (C ₂ -C ₆ ) alkynyl may be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1- hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl or 5-hexynyl; (C ₁ -C ₆ ) alkanoyl may be acetyl, propanoyl or butanoyl; halogen (C ₁ -C ₆ ) alkyl may be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl or pentafluoroethyl; hydroxy (C ₁ -C ₆ ) alkyl may be hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl, 1 -hydroxyhexyl or 6-hydroxyhexyl; (C ₁ -C ₆ ) alkoxycarbonyl (CO ₂ R ² ) may be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl or hexyloxycarbonyl; (C ₁ -C ₆ ) alkylthio may be methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, pentylthio or hexylthio; (C ₂ -C ₆ ) alkanoyloxy may be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy or hexanoyloxy; aryl may be phenyl, indenyl or naphthyl, and heteroaryl may be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyraxolyl, pyrrolyl, pyrazinyl, tetrazolyl, puridyl (or its N-oxide, tyridyl, tyridyl, tyridyl, tyri) or its N-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (or its N-oxide).

A particular value of R is amino, monomethylamino or cyclopropylamino.

A particular value of R ¹ is carboxy- or (C ₁ -C ₄ ) alkoxycarbonylcyclohexyl (C ₁ -C ₄ ) alkyl.

A particular value of R ² is H or (C ₁ -C ₄ ) alkyl, i.e. methyl or ethyl.

The specific value of R ³ is H, methyl or phenyl.

The specific value of R ⁴ is H, methyl or phenyl.

The specific value of Z is —CH ₂ - or —CH ₂ —CH ₂ -.

A particular value of X is CO ₂ R ² , (C ₂ -C ₅ ) alkanoylmethyl or amido.

The specific value of n is 1.

Preferred compounds of formula (I) are those in which each R represents H, X represents ethylaminocarbonyl, and R ¹ represents 4-carboxycyclohexylmethyl (DWH-146a), R ¹ represents 4-methoxycarbonylcyclohexylmethyl (DWH-146e) or R ¹ represents 4-acetoxymethylcyclohexylmethyl (JMR-193). They are presented below (DWH-146 (acid) and methylether (e)), as well as JMR-193.

DWH-146 (acid, X = H; ether, X = Me)

Synthesis of methyl 4- [3- (6-amino-9- (5 - [(ethylamino) carbonyl] -3,4-dihydroxytetrahydro-Z-furanyl-9H-2-purinyl) -2-propynyl] -1-cyclohexanecarboxylate ( DWH-146e) is carried out by cross-linking the iodadenosine derivative (N-ethyl-1'-deoxy-1 '- (amino-2-iodo-9H-purin-9-yl) -β-D-ribofuranuramide) with methyl 4- (2- propynyl) -1-cyclohexanecarboxylate, using Pd ¹¹ catalyst. The synthesis of iodadenozina performed from guanosine. guanosine is first treated with acetic anhydride, which atsetaliruet sugar hydroxyls, followed by the 6-position is chlorinated tetramethyl chloride mmonium and phosphorus oxychloride. Iodination of position 2 is carried out as a result of a modified Sandmeyer reaction followed by the replacement of 6-Cl and sugar acetates with ammonia. Hydroxyls 2 'and 3' are protected as acetonide, and hydroxyl 5 'is iodinated to acid with potassium permanganate. Deprotection of acetonides 2 'and 3', Fischer's esterification of 5 'acid with ethanol and conversion of the resulting ethyl ester to ethylamide with ethylamine gives N-ethyl-1'-deoxy-1' - (amino-2-iodo-9H-purin-9-yl) - β-D-ribofuranuoramide.

Acetylene (methyl 4- (2-propynyl) -1-cyclohexanecarboxylate) is synthesized based on trans-1,4-cyclohexanedimethanol. First, trans-diol is monotosylated, followed by the substitution of tosylate with an acetylene anion. The hydroxyl of the resulting hydroxylacetylene species is oxidized to acid with Jones reagent followed by methylation of (trimethylsilyl) diazomethane to give 4- (3-propynyl) -1-cyclohexanecarboxylate.

The crosslinking reaction is carried out under the following conditions described above. To a solution of N, N-dimethylformamide (0.5 ml), acetonitrile (1 ml), triethylamine (0.25 ml) and N-ethyl-1'-deoxy-1 '- (amino-2-iodine-9H-purine -9-yl) -β-D-ribofuranuroamide (25 mg, 0.06 mol) add bis (triphenylphosphine) palladium dichloride (1 mg, 2 mol%) and copper (I) iodide (0.06 mg, 5 mol%).

Methyl 4- (2-propynyl) -1-cyclohexanecarboxylate (54 mg, 0.3 mmol) was added to the resulting mixture, and the reaction mixture was stirred under N ₂ for 16 hours. The solvent was removed in vacuo and the resulting residue was flash chromatographed in 20% methanol in chloroform (R _f = 0.45) to give 19 mg (not quite white solid, mp 125 ° C (decomposition)) methyl 4 - [3- (6-amino-9- (5 - [(ethylamino) carbonyl] -3,4-dihydroxytetrahydro-Z-furanyl) -9H-2-purinyl] -2-propynyl-1-cyclohexanecarboxylate (DWH-146e )

DWH-146e and JMR193 are much more effective as inhibitors in inflammatory model systems than the comparative compound, CGS21680 (2- [p- (carboxyethyl) phenylethylamino] -5'-N-ethylcarboxamidoadenosine). For example, DWH-146e is approximately 80 times stronger with respect to A _2A receptors and 40 times more selective with respect to A _2A compared with A ₃ receptors than CGS21680.

Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids forming a physiologically acceptable anion, for example, tosylate, methanesulfonate, malate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, α-ketoglutarate and α-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochlorides, sulfates, nitrates, bicarbonates and carbonates.

Pharmaceutically acceptable salts can be prepared using standard methods well known in the art, for example, by reacting a sufficiently basic compound, such as an amine, with a suitable acid to give a physiologically acceptable anion. Carboxylic acid salts of an alkali metal (e.g., sodium, potassium or lithium) or an alkaline earth metal (e.g., calcium) can also be prepared.

The compounds of formula I can be formulated into pharmaceutical compositions and administered to a mammalian host, such as a human, in various forms according to the chosen route of administration, i.e. oral or parenteral, intravenous, intramuscular, local or subcutaneous.

Thus, these compounds can be administered systemically, for example, by the oral route, in combination with a pharmaceutically acceptable excipient, such as an inert diluent or an assimilable edible carrier. They can be enclosed in capsules with hard or soft gelatin shells, can be compressed into tablets or directly mixed into the patient’s food. For oral therapeutic administration, the active compound can be mixed with one or more excipients and used in the form of tablets for swallowing, cheek tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of the active compound. Of course, the percentage of compositions and preparations may vary and is usually from about 2 to 60% by weight of a particular dosage form. The amount of active compound in these compositions for therapeutic use is such that it provides an effective dosage level.

Tablets, lozenges, pills, capsules, etc. may also contain the following substances: binders such as tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricating agent such as magnesium stearate and a sweetening agent such as sucrose, fructose, lactose or aspartame, or a flavoring agent such as peppermint, peanut butter or cherry flavor. If the dosage form is in the form of a capsule, it may contain, in addition to the materials of the above type, a liquid carrier such as vegetable oil or polyethylene glycol. Various other materials may be present in the form of shells or in some other way modify the physical form of the solid dosage form. For example, tablets, pills or capsules may be coated with gelatin, wax, shellac, sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propyl parabens as preservatives, a colorant and fragrance, such as a cherry or orange flavor. Of course, any material used in the preparation of any dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts used. In addition, the active compound may be formulated in sustained release preparations and devices.

The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts may be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions can also be obtained in glycerol, liquid polyethylene glycols, triacetin, mixtures thereof, as well as in oils. Under normal conditions of storage and use, these preparations contain a preservative that prevents the growth of microorganisms.

Pharmaceutical dosage forms intended for injection or infusion (infusion) may include sterile aqueous solutions, dispersions or sterile powders including the active ingredient, intended for the unplanned preparation of sterile solutions or dispersions for injection or infusion, optionally encapsulated in liposomes. In all cases, the final dosage form must be sterile, liquid and stable under the conditions of manufacture and storage. The liquid carrier or excipient may be a diluent or liquid dispersion medium, including, for example, water, ethanol, a polyol (e.g. glycerin, propylene glycol, liquid polyethylene glycols, etc.), vegetable oils, non-toxic glycerol esters, as well as their suitable mixtures. The desired fluidity can be achieved, for example, by the formation of liposomes, by using the required particle size when using dispersions, or by using surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, the inclusion of isotonic agents, for example sugars, buffers or sodium chloride, is preferred. Prolonged absorption of the compositions for injection can be ensured by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by injecting the active compound in the right amount into the appropriate solvent along with the various ingredients mentioned above, followed by filter sterilization, as required. When using sterile powders to form sterile injectable solutions, vacuum and freeze-drying are preferred methods to provide the active ingredient powder plus any additional desired ingredient present in solutions previously filtered sterilized.

When administered topically, these compounds can be applied in pure form, i.e. when they are in the form of liquids. However, it is usually desirable to apply them to the skin in the form of compositions or compositions in combination with a dermatologically acceptable carrier, which may be in solid or liquid form.

Suitable solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like. Suitable liquid carriers include water, alcohols or glycols, or water-alcohol / glycol mixtures in which these compounds can be effectively dissolved or dispersed, optionally with non-toxic surfactants. Adjuvants such as flavors and additional antimicrobial agents may be added to optimize properties for a particular purpose. The resulting liquid compositions can be applied using absorbent pads used to impregnate dressings and other dressings, or sprayed onto the affected area using pressure or aerosol dispensers.

Thickeners such as synthetic polymers, fatty acids, salts and esters of fatty acids, fatty alcohols, modified celluloses or modified mineral materials can also be used with liquid carriers to obtain applied pastes, gels, ointments, soaps, etc., applied directly onto the patient’s skin.

Examples of dermatological compositions that can be used to deliver the compounds of formula I to the skin are described by Jacquet et al. (US patent No. 4608392), Geria (US patent No. 4992478), Smith et al. (US patent No. 4,559157) and Wortzman (US patent No. 4820508).

Effective doses of the compounds of formula I can be determined by comparing their efficacy in vitro and in vivo in animal models. Methods of extrapolating effective doses in mice and other animals to humans are known in the art; see, for example, US Patent No. 4,938,949. Effective doses of PDE inhibitors, type IU, are known in the art. For example, see US Pat. No. 5,877,180, Col. 12.

In general, the concentration of the compound (s) of formula (I) in a liquid composition, such as a lotion, is about 0.1-25% by weight, preferably about 0.5-10% by weight. The concentration in a semi-solid or solid composition, such as a gel or powder, is about 0.1-5% wt., Preferably about 0.5-2.5% wt.

The amount of the compound or its active salt or derivative required for treatment varies depending not only on the particular salt selected, but also on the route of administration, the nature of the condition being treated, the age and condition of the patient, and is ultimately determined by the attending physician or clinician.

However, in general, a suitable dose is in the range of from about 0.5 to 100 μg / kg, for example from about 10 to 75 μg / kg of body weight per day, possibly from 3 to about 50 μg per kilogram of body weight of the recipient per day, preferably the range of 6 to 90 μg / kg / day, most preferably the range of 15 to 60 μg / kg / day.

This compound is preferably administered in the form of a dosage form, for example, containing from 5 to 1000 μg, suitably from 10 to 750 μg, most preferably from 50 to 500 μg of the active ingredient per dosage unit.

Ideally, the active ingredient should be administered in such a way as to achieve a maximum plasma concentration of the active compound of about 0.1 to 10 nM, preferably about 0.2 to 10 nM, most preferably about 0.5 to 5 nM . This can be achieved, for example, by intravenous injection of a 0.05-5% solution of the active ingredient, optionally in saline, or by oral administration in the form of a bolus containing approximately 1-100 μg of the active ingredient. The desired blood level can be maintained by continuous infusion, providing approximately 0.01-5.0 μg / kg / hour, or periodic infusion containing approximately 0.4-15 μg / kg of the active ingredient (s).

The desired dose may be administered as a single dose or as divided doses administered over appropriate time periods, for example, as two, three, four or more divided doses per day. In turn, the divided dose can also be divided, for example, into a series of discrete administrations (doses), such as repeated inhalations through an insufflator or instillation of a few drops into the eyes. For example, it is desirable to administer the composition data intravenously over a long period of time after the lesion causing the inflammation.

The ability of a compound of the present invention to act as an agonist (or antagonist) of the A _2A adenosine receptor can be determined using pharmacological models well known in the art or using the tests described below.

The present invention will now be described with reference to the following detailed examples, which are given to illustrate the invention and not to limit it. In the NMR spectra further, Hz is hertz, s is singlet, d is doublet, t is triplet, m is multiplet, and ears are broadened.

Example 1

trans- (1- [4- [Hydroxymethyl) cyclohexyl] methyl) -4-methyl-benzenesulfonate (5.2)

Sodium hydride (1.69 g, 70 mmol) was added to a solution of 10 g (70 mmol) of [4- (hydroxymethyl) cyclohexyl] methan-1-ol (5.1) in 700 ml of tetrahydrofuran, stirred for 1 hour, then p α-toluenesulfonyl chloride (13.3 g, 70 mmol) and the reaction mixture was refluxed for 5 hours. Then the reaction mixture is cooled to 0 ° C and slowly quenched with water until the reactive hydride disappears. After quenching the hydride, the reaction mixture was diluted with ether (700 ml) and extracted 2 times with 10% aqueous potassium carbonate (700 ml). The organic portion was dried using sodium sulfate, and the solvent was removed under reduced pressure. The product was purified by silica gel column chromatography, eluting with acetone-dichloromethane (5:95), to give compound 5.2 (35%).

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.75 (d, J = 8.3 Hz, 2H), 7.32 (d, J = 8.1 Hz, 2H), 3.79 (d, J = 6.35 Hz, 2H), 3.39 (d , J = 6.35 Hz, 2H), 2.42 (s, 3H), 1.75 (m, 4H), 1.59 (m, 1H), 1.37 (m, 1H), 0.9 (m, 4H). ¹³ C NMR (300 MHz, CDCl ₃ ) δ 145.3, 133.4, 130.3, 130.3, 128.3, 128.3, 75.8, 68.5, 40.6, 37.8, 28.9, 28.9, 28.9, 28.9, 22.1.

Example 2

(4-Prop-2-ynylcyclohexyl) methan-1-ol (5.3)

The lithium acetylide-ethylenediamine complex (90%) (6.4 g, 70 mmol) is slowly added to a solution of 5.2 (3 g, 10 mmol) in 40 ml of dimethyl sulfoxide. The reaction mixture was allowed to mix for 5 days and then quenched slowly at 0 ° C with water. The mixture was diluted with ether (300 ml) and extracted 3 times with saturated aqueous ammonium chloride (200 ml). The organics are dried with sodium sulfate. The solvent was removed under reduced pressure, and the product was purified by silica gel column chromatography, eluting with ethyl acetate-hexanes (20:80), to give compound 5.3 (85%).

¹ H NMR (300 MHz, CDCl ₃ ) δ 3.41 (d, J = 6.5 Hz, 2H), 2.07 (dd, J = 2.5, 6.5 Hz, 2H), 1.96-1.75 (m, 5H), 1.41 (m, 2H), 0.95 (m, 4). ¹³ C NMR (300 MHz, CDCl ₃ ) δ 83.8, 69.6, 68.9, 40.7, 37.7, 32.3, 32.3, 29.6, 29.6, 26.5.

Example 3

4-prop-2-ynylcyclohexanecarboxylic acid (5.4)

A solution of chromium trioxide (1.1 g, 11 mmol) in 1.5 M sulfuric acid (40 ml, 27 mmol) is maintained at a temperature of 0 ° C for more than 2 hours, during which compound 5.3 (0.46 g, 3 mmol) in 80 ml of acetone. Then the reaction mixture was stirred for another 2 hours at room temperature. The reaction mixture was diluted with ether (200 ml) and extracted 2 times with water. The organics are dried with sodium sulfate. The solvent was removed under reduced pressure and the product was purified by silica gel column chromatography, eluting with acetone-dichloromethane (70:30), to give compound 5.4 (75%).

¹ H NMR (300 MHz, CDCl ₃ ) δ 2.24 (dt, J = 3.66, 12.1 Hz, 1H), 2.10 (dd, J = 2.7, 6.5 Hz, 2H), 2.04-1.89 (m, 5H), 1.76 ( d, J = 2.3 Hz, 1H), 1.43 (dq, J = 3.28, 13.1 Hz, 2H), 1.03 (dq, J = 3.28, 13.1 Hz, 2H). ¹³ C NMR (300 MHz, CDCl ₃ ) δ 183.2, 83.3, 69.9, 43.4, 36.7, 31.8, 28.9, 26.3.

Example 4

Methyl 4-prop-2-ynylcyclohexanecarboxylate (5.5)

A solution of (trimethylsilyl) diazomethane (2.0 M) in hexanes (1 ml, 2 mmol) is added to a solution of compound 5.4 (0.34 g, 2 mmol) in 15 ml of a mixture of methanol: dichloromethane (3: 7). Solvents were removed under reduced pressure to give 100% conversion of the starting material to the product.

¹ H NMR (300 MHz, CDCl ₃ ) δ 2.24 (dt, J = 3.66, 12.1 Hz, 1H), 2.10 (dd, J = 2.7, 6.5 Hz, 2H), 2.06 (dd, J = 1.54, 6.54 Hz, 1H), 2.00-1.89 (m, 3H), 1.76 (d, J = 2.3 Hz, 1H). 1.43 (dq, J = 3.28, 13.1 Hz, 2H), 1.03 (dq, J = 3.28, 13.1 Hz, 2H). ¹³ C NMR (300 MHz, CDCl ₃ ) δ 176.8, 83.3, 69.8, 51.9, 43.4, 36.7, 31.9, 29.2, 26.3.

Example 5

[(2R, 3R, 4R, 5R) -3,4-Diacetyloxy-5- (2-amino-6-oxohydropurin-9-yl) oxolan-2-yl] methyl acetate (6.2)

A suspension of 113 g (0.4 mol) of dry guanosine (6.1), acetic anhydride (240 ml, 2.5 mol), dry pyridine (120 ml) and dry DMF (320 ml) is heated for 3.75 hours at 75 ° C, not allowing the temperature to exceed 80 ° C. The clear solution is then transferred to a 3 L Erlenmeyer flask, which is filled with 2-propanol. After cooling the solution to room temperature, crystallization is initiated and allowed to proceed at 4 ° C. overnight. The white solid precipitate was filtered, washed with 2-propanol and recrystallized from it to give compound 6.2 (96%).

¹ H NMR (300 MHz, CDCl ₃ ) δ 8.20 (s, 1H, H-8), 6.17 (d, J = 5.41 Hz, 1H, H-1 ') 5.75 (t, J = 5.39 Hz, 1H, H -2 '), 5.56 (t, J = 5.0, H-3'), 4.41 (m, 3H, H-4 ', 5'), 2.14 (s, 3H, Ac), 2.11 (s, 3H, Ac ), 2.10 (s, 3H, Ac). ¹³ C NMR (300 MHz, CD ₃ OD) δ 171.0, 170.3, 170.2, 157.7, 154.8, 152.4, 136.7, 117.7, 85.5, 80.4, 73.0, 71.3, 64.0, 31.3, 21.2, 21.0.

Example 6

[(2R, 3R, 4R, 5R) -3,4-Diacetyloxy-5- (2-amino-6-chloropurin-9-yl) oxolan-2-yl] methyl acetate (6.3)

80 g (0.195 mol) of [(2R, 3R, 4R, 5R) -3,4-diacetyloxy-5- (2-amino-6-oxohydropurin-9-yl) oxolan-2-yl] methyl acetate are placed in a 1000 ml flask (6.2), tetramethylammonium chloride (44 g, 0.4 mol), anhydrous acetonitrile (400 ml) and N, N-dimethylaniline (25 ml). The flask was placed in an ice salt bath and cooled to 2 ° C. POCl ₃ (107 ml, 1.15 mol) was added dropwise to this solution at a rate that kept the temperature below 5 ° C (45 minutes). Then the flask is removed from the ice bath, equipped with a condenser, placed in an oil bath and the mixture is allowed to reflux for 10 minutes, while the color of the solution changes to red-brown. Then, the solvent was removed under reduced pressure to obtain an oily residue, which was transferred to a beaker containing 1000 g of ice and 400 ml of CHCl ₃ , and the mixture was allowed to mix for 1.5 hours until the remaining POCl _{3 was} decomposed. Then the organic phase is removed, the aqueous phase is extracted with 3 × 50 ml of CHCl ₃ and combined with the organic phase. The combined organics are then back extracted with 50 ml of water, followed by stirring with 200 ml of saturated NaHCO ₃ . After that, the organic phase is extracted with NaHCO ₃ until the aqueous extract becomes neutral (2X). The organic phase is finally extracted with brine, and then dried over MgSO ₄ for 16 hours. 800 ml of 2-propanol was added to the solution, after which the solution was concentrated under reduced pressure. 200 ml of 2-propanol was added to the oily solid and the solution was refrigerated overnight. The crystalline product was filtered, washed and allowed to dry overnight to give compound 6.3 (77%).

¹ H NMR (300 MHz, CD ₃ OD) δ 8.31 (s, 1H, H-8), 7.00 (s, 2H, NH ₂ ) 6.06 (d, J = 5.8 Hz, 1H, H-1 '), 5.83 (t, J = 6.16 Hz, 1H, H-2 '), 5.67 (m, 1H, H-3'), 4.29 (m, 3H, H-4 ', 5'), 2.07 (s, 3H, Ac ), 1.99 (s, 3H, Ac), 1.98 (s, 3H, Ac). ¹³ C NMR (300 MHz, CD ₃ OD) δ 171.0, 170.4, 170.2, 160.8, 154.6, 150.8, 142.2, 124.5, 85.8, 80.6. 72.8, 71.2, 63.9, 21.4, 21.3, 21.1.

Example 7

[(2R, 3R, 4R, 5R) -3,4-Diacetyloxy-5- (6-chloro-2-iodpurin-9-yl) oxolan-2-yl] methyl acetate (6.4)

Isoamyl nitrite (5 ml, 37 mmol) was added to the solution 5.12 g (12 mmol) [(2R, 3R, 4R, 5R) -3,4-diacetyloxy-5- (2-amino-6-chloropurin-9-yl ) oxalan-2-yl] methyl acetate (6.3), I ₂ (3.04 g, 12 mmol), CH ₂ I ₂ (10 ml, 124 mmol) and Cul (2.4 g, 12.6 mmol) in THF (60 ml). The mixture is heated at the boil under reflux for 45 minutes, and then allow it to cool to room temperature. To this solution was added 100 ml of saturated Na ₂ S ₂ O ₃ , eliminating the reddish color caused by the presence of iodine. The aqueous solution was extracted with 3X chloroform, which was poured together, dried over MgSO ₄ and concentrated under reduced pressure. The product was then purified on a silica gel column using CHCl ₃ -MeOH (98: 2) to give [(2R, 3R, 4R, 5R) -3,4-diacetyloxy-5- (6-chloro-2-iodopurine-9- il) oxolan-2-yl] methyl acetate (6.4) (80% crystallizes from EtOH).

¹ H NMR (300 MHz, CDCl ₃ ) δ 8.20 (s, 1H H-8), 6.17 (d, J = 5.41 Hz, 1H, H-1 '), 5.75 (t, J = 5.39 Hz, 1H, H -2 '), 5.56 (t, J = 5.40 Hz, 1H, H-3'), 4.38 (m, 3H, H-4 ', 5'), 2.14 (s, 1H, Ac), 2.11 (s, 1 H, Ac), 2.10 (s, 1H, Ac).

Example 8

(4S, 2R, 3R, 5R) -2- (6-amino-2-iodpurin-9-yl) -5- (hydroxymethyl) oxolan-3,4-diol (6.5)

Into a flask containing 6.0 g (11.1 mmol) [(2R, 3R, 4R, 5R) -3,4-diacetyloxy-5- (6-chloro-2-iodpurin-9-yl) oxolan-2- il] methyl acetate (6.4), add 100 ml of liquid NH ₃ at -78 ° C and the solution is allowed to mix for 6 hours. After this, the solution was allowed to warm to room temperature overnight while evaporating NH ₃ to give a brown oil. The product is crystallized from hot isopropanol to give compound 6.5 (80%), mp. 143-145 ° C, rf = 0.6 in 20% MeOH / CHCl ₃ .

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.24 (s, 1H), 7.68 (s, 2H), 5.75 (d, J = 6.16, 1H), 5.42 (d, J = 5.40 Hz, 1H), 5.16 (d, J = 4.62 Hz, 1H), 4.99 (t, J = 5.39 Hz, 1H), 4.67 (d, J = 4.81 Hz, 1H), 4.06 (d, J = 3.37 Hz, 1H), 3.89 ( m, 1H); 3.54 (m, 2H).

Example 9

[(1R, 2R, 4R, 5R) -4- (6-amino-2-iodpurin-9-yl) -7,7-dimethyl-3,6,8-trioxabicyclo [3.3.0] oct-2-yl ] methan-1-ol (6.6)

To a solution of 2.0 g (5.08 mmol) (4S, 2R, 3R, 5R) -2- (6-amino-2-iodpurin-9-yl) -5- (hydroxymethyl) oxolan-3,4-diol (6.6) 9.6 g of p-toluenesulfonic acid and 5 ml of dimethoxypropane are added to 100 ml of acetone. The reaction mixture was stirred at room temperature for 1 hour, during which 15 g of NaHCO ₃ was added and then stirred for another 3 hours. The residue was filtered and washed with 2X EtOAc. Then the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography with MeOH-CHCl ₃ (1:99) to give compound 6.6 (72%) as a solid, mp. 185-187 ° C.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.22 (s, 1H, H-8), 7.69 (s, 2H, NH ₂ ), 6.00 (d, J = 2.70 Hz, 1H, H-1 ') , 5.21 (m, 1H, H-2 '), 5.07 (br, 1H, OH), 4.88 (m, 1H, H-3'), 4.13 (m, 1H, H-4 '), 3.47 (m, 2H, H-5 '), 1.49 and 1.28 (s, 3H, C (CH ₃ ) ₂ ).

Example 10

(2S, 1R, 4R, 5R) -4- {6-amino-2-iodpurin-9-yl) -7,7-dimethyl-3,6,8-trioxabicyclo [3.3.0] octane-2-carboxylic acid (6.7)

To a stirred solution of 1.6 g (3.7 mmol) [(1R, 2R, 4R, 5R) -4- (6-amino-2-iodpurin-9-yl) -7,7-dimethyl-3,6, 8-trioxabicyclo [3.3.0] oxy-2-yl] methan-1-ol (6.6) in 200 ml of H ₂ O add 0.60 g of KOH and, dropwise, a solution of 1.70 g (10.8 mmol) KMnO ₄ in 50 ml of H ₂ O. The mixture was placed in a dark place at room temperature for 225 hours. Then the reaction mixture is cooled to 5-10 ° C and decolorized with a solution of 4 ml of 30% H ₂ O ₂ in 16 ml of water, while maintaining the temperature below 10 ° C using an ice-salt bath. The mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to approximately 10 ml, then acidified to pH 4 2 N. HCl. The resulting precipitate was filtered off and washed with ether, yielding, after drying, compound 6.7 (70%) as a white solid, mp. 187-190 ° C. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.11 (s, 1H, H-8), 7.62 (s, 2H, NH ₂ ), 7.46 (s, 1H, COOH), 6.22 (s, 1H, H -1 '), 5.42 (d, J = 5.71 Hz, 1H, H-2'), 5.34 (d, J = 6.16 Hz, 1H, H-3 '), 4.63 (s, 1H, H-4') 1.46 and 1.30 (s, 3H, C (CH ₃ ) ₂ ).

Example 11

(2S, 3S, 4R, 5R) -5- (6-amino-2-iodpurin-9-yl) -3,4-dihydroxyoxolan-2-carboxylic acid (6.8)

Solution 1.72 g (3.85 mmol) (2S, 1R, 4R, 5R) -4- (6-amino-2-iodpurin-9-yl) -7,7-dimethyl-3,6,8-trioxabicyclo [3.3.0] octane-2-carboxylic acid (6.7) in 80 ml of 50% HCOOH was stirred at 80 ° C for 1.5 hours. The reaction mixture was evaporated under reduced pressure, dissolved in H ₂ O and the solution was again evaporated. This process is repeated until the residue loses the smell of formic acid. Recrystallization from water gives 1.33 g (85%) of compound 6.8 as a white solid, mp. 221-223 ° C, decomp.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.31 (s, 1H, H-8), 7.68 (s, 2H, NH ₂ ), 5.90 (d, J = 6.55 Hz, 1H, H-1 ') 4.42 (m, 1H, 4-2 '), 4.35 (d, J = 2.31 Hz, 1H, 11-4'), 4.22 (m, 1H, H-3 ').

Example 12

[2S, 3S, 4R, 5R) -5- (5-amino-2-iodpurin-9-yl) -3,4-dihydroxyoxolan-2-yl] -N-ethylcarboxamide (6.9)

To a cooled (5 ° C) and stirred solution, 1.29 g (3.17 mmol) (2S, 3S, 4R, 5R) -5- (6-amino-2-iodpurin-9-yl) -3,4- dihydroxyoxolane-2-carboxylic acid (6.8) in 150 ml of absolute ethanol was added dropwise 1.15 ml of ice-cold SOCl ₂ . The mixture was stirred at room temperature overnight, and then its pH was adjusted to 8 with saturated aqueous NaHCO ₃ . The mixture was filtered, then the filtrate was concentrated under reduced pressure to give a white solid, which was dried and then redissolved in 20 ml of dry ethylamine at -20 ° C for 3 hours and then at room temperature overnight. The reaction mixture was diluted with absolute ethanol, the precipitated product was filtered off and washed with dry ether to give 530 mg (72%) of 6.9 as a pure solid, mp. 232-234 ° C.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.34 (s, 1H, H-8), 8.12 (t, 1H, NH), 7.73 (s, 2H, NH ₂ ), 5.85, (d, J = 6.93 Hz, 1H, H-1 '), 4.54 (m, 1H, H-2'), 4.25 (d, J = 1.92 Hz, 1H, H-4 '), 4.13 (m, 1H, H-3' ), 3.28 (m, 2H, CH ₂ CH ₃ ), 1.00 (t, J = 7.2 Hz, 3H, CH ₂ CH ₃ ).

Example 13

Methyl 4- (3- {9 - [(4S, 5S, 2R, 3R) -5- (N-ethylcarbamoyl) -3,4-dihydroxyoxolan-2-yl] -6-aminopurin-2-yl)} prop- 2-ynyl) cyclohexanecarboxylate (DWH-146e)

To a degassed solution of 25 mg (0.063 mmol) [(2S, 3S, 4R, 5R) -5- (6-amino-2-iodopurin-9-yl) -3,4-dihydroxyoxolan-2-yl] -N-ethylcarboxamide (6.9), 16.9 mg (0.094 mmol) (5.5) and 0.75 Cul in 5 ml (each) of triethylamine (TEA) and acetonitrile add 15 mg of Pd (PPh ₃ ) ₄ . The solution was stirred for 24 hours at 70 ° C, after which it was filtered through celite and chromatographed on silica gel with MeOH-CHCl ₃ (5:95) to give DWH-146e (24%).

Example 14

(4-Prop-2-ynylcyclohexyl) methyl acetate (5.6)

Acetic anhydride (0.92 ml, 8.25 mmol) and pyridine (0.2 ml, 2.5 mmol) are added to a solution of compound 5.3 (250 mg, 1.65 mmol) in 25 ml of ether. The reaction mixture was allowed to stir at room temperature for 24 hours. Water was added to the reaction mixture, and the organic phase was further extracted with 10% NaHCO ₃ . The organic layer was dried with MgSO ₄ and evaporated. The residue was subjected to silica gel chromatography with EtOAc-hexanes (5:95) to give compound 5.6 (47%).

Example 15

[4- (3- {9- (4S, 5S, 2R, 3R) -5- (N-Ethylcarbamoyl) -3,4-dihydroxyoxolan-2-yl] -6-aminopurin-2-yl} prop-2- inyl) cyclohexyl] methyl acetate (JMR 193)

To a degassed solution 125 mg (0.29 mmol) [(2S, 3S, 4R, 5R) -5- (6-amino-2-iodpurin-9-yl) -3,4-dihydroxyoxolan-2-yl] -N ethyl carboxamide (6.9), 150 mg (0.77 mmol) of compound (5.6) and 1.0 mg of Cul in 1.3 ml of TEA and 4 ml of TMF add 25 mg of Pd (PPh ₃ ) ₄ . The solution was stirred for 72 hours at 60 ° C, after which it was filtered through celite and subjected to silica gel chromatography with MeOH-CHCl ₃ (5:95) to give JMR193 (10%).

Example 16

[(2S, 3S, 4R, 5R) -5- (6-amino-2- {3- [4- (hydroxymethyl) cyclohexyl] prop-2-ynyl} purin-9-yl) -3,4-dihydroxyoxolan- 2-yl] -N-ethylcarboxamide

A. (4-Prop-2-ynylcyclohexyl) methan-1-ol

The lithium acetylide-ethylenediamine complex (90%) (6.4 g, 70 mmol) is slowly added to a solution of trans- [4- (hydroxymethyl) cyclohexyl] methyl 4-methylbenzenesulfonate (3 g, 10 mmol) in 40 ml of dimethyl sulfoxide. The reaction mixture was allowed to mix for 5 days, and then it was slowly quenched at 0 ° C with water. This mixture was diluted with ether (300 ml) and washed 3 times with saturated aqueous ammonium chloride (200 ml). The organics are dried with sodium sulfate. The solvent is removed under reduced pressure. The product was purified by silica gel column chromatography, eluting with ethyl acetate-hexanes (20:80), to give the product (85%).

¹ H NMR (300 MHz, CDCl ₃ ) d 3.41 [d, J = 6.5 Hz, 2H), 2.07 (dd, J = 2.5, 6.5 Hz, 2H), 1.96-1.75 (m, 5H), 1.41 (m, 2H), 0.95 (m, 4). ¹³ C NMR (300 MHz, CDCl ₃ ) d 83.8, 69.6, 68.9, 40.7, 37.7, 32.3, 32.3, 29.6, 29.6, 26.5.

B. [(2S, 3S, 4R, 5R) -5- (6-amino-2- {3- [4- (hydroxymethyl) cyclohexyl] prop-1-ynyl} purin-9-yl) -3,4- dihydroxyoxolan-2-yl] -N-ethylcarboxamide (JMR2037)

Pd (PPh ₃ ) ₄ , 10 mg, 28 mg (0.065 mmol) [(2S, 3S, 4R, 5R) -5- (6-amino-2-iodopurin-9-yl) -3,4 are added to the degassed solution -dihydroxyoxolan-2-yl] -N-ethylcarboxamide, 30 mg (0.20 mmol) (4-prop-2-ynylcyclohexyl) methan-1-ol, 1.0 mg Cul in 1 ml of triethylamine (TEA), 1 ml DMF and 1 ml of acetonitrile. The solution was stirred for 60 hours at room temperature, after which it was filtered through celite and subjected to silica gel chromatography with MeOH-CHCl ₃ (7:93) to obtain 5 mg (17%) of compound JMR2037. The title compound was subjected to the binding tests described herein, which were found to bind to human recombinant A _2A receptors, with Ki being 694 ± 69 nM.

General Method 4: Preparation of 2-AAs (2-alkynyladenosines)

In a flame-dried 25 ml round bottom flask in a nitrogen atmosphere, 2 NECA iodine (40 mg) was charged and dissolved in 2 ml of DMF. Then add the corresponding alkin (about 0.1 ml), followed by the addition of 4 ml of acetonitrile and 0.1 ml of TEA. All three solvents are degassed with nitrogen for at least 24 hours. To this solution, 5 mol% of Pd (PPh ₃ ) ₄ and 6 mol% of copper iodide are added. The yellowish solution was stirred at room temperature for 24 hours or until completion of the reaction according to HPLC. If the reaction does not complete within this time, an additional catalyst, Cul and TEA are added. After completion of the reaction in vacuo, the solvents were removed and the red-black residue was transferred to a small amount of DMF. This solution was added to a preparative TLC silica gel plate (Analtech 1000 microns, 20 cm × 20 cm) and first eluted with 120 ml of a mixture of 40% hexane / CH ₂ Cl ₂ and then 40 ml of MeOH. A UV-active band (usually yellow) is collected in the middle of the plate, washed slowly with 4 × 25 ml of 20% MeOH / CH ₂ Cl ₂ and concentrated. This product is then purified by reverse phase HPLC.

Example 17

4- {3- [6-amino-9- (5-ethylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl) -9H-purin-2-yl] prop-2-ynyl} cyclohexanecarboxylic acid (ATL146a)

Reaction of ATL146e with five equivalents of LiOH in a THF / water mixture for 6 hours gives ATL146a (7 mg, 72%) as a white solid, which crystallizes from MeOH / water (0.1% TFA) after purification by reverse phase HPLC .

¹ H NMR (DMSO-d ₆ ) δ 8.70 (s, 1H), 8.41 (s, 1H), 7.62 (s, 2H), 5.89 (d, J = 7.25 Hz, 1H), 4.53 (m, 1H), 4.27 (s, 1H), 4.08 (d, J = 3.6 Hz, 1H), 2.29 (d, J = 6.4 Hz, 2H), 2.15-1.99 (m, 1H), 1.92-1.76 (m, 4H), 1.52-1.38 (m, 1H), 1.38-1.19 ( m, 2H), 1.02 (t, J = 6.3 Hz, 3H).

¹³ C NMR (DMSO-d ₆ ) 176.7, 169.2, 155.6, 148.9, 145.2, 141.6, 119.0, 87.7, 85.0, 84.6, 81 6, 73.1, 71.9, 43.2, 35.9, 33.3, 31.2, 28.3, 25.6, 15.0. MS is high. permission (bombardment. fast, atoms) m / z 474.2196 [(M + H) ⁺ calculated for C ₂₂ H ₂₉ N ₆ O ₆ 474.2182].

Example 18

5- {6-amino-2- [3- (4-hydroxymethylcyclohexyl) prop-1-ynyl] purin-9-yl} -3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR2037)

The interaction of (4-prop-2-ynylcyclohexyl) methan-1-ol (5.3) (30 mg, 0.2 mmol) with 2-NECA (28 mg, 0.07 mmol) under the general conditions described above gives JR2037 ( 7 mg, 24%) as a white solid.

¹ H NMR (CD ₃ OD) δ 8.22 (s, 1H), 5.92 (d, J = 7.7 Hz, 1H), 4.70-4.66 (dd, J = 7.7 Hz , 4.8 Hz, 1H), 4.40 (d, J = 1.2 Hz, 1H), 4.25-4.23 (dd, J = 4.8 Hz, 1.2 Hz, 1H), 3.51-3.37 (m, 2H), 3.31 (d, J = 6 Hz, 2H), 2.30 (d, J = 6.8 Hz, 2H), 1.94-1.89 (m, 2H), 1.83-1.78 (m, 2H), 1.64-1.42 (m, 2H), 1.12 (t, J = 7.3 Hz, 3H), 1, 09-0.91 (m, 4H).

¹³ C NMR (CD ₃ OD) δ 170.3, 155.4, 148.5, 146.0, 141.6, 118.8, 88.7, 85.5, 84.6, 80.6, 73 , 1, 71.3, 66.8, 39.6, 36.9, 33.3, 31.5, 28.6, 25.6, 13.5. MS is high. permission (bombard. fast, by atoms) m / z 459.2373 [(M + H) ⁺ calc. for C ₂₂ H ₃₁ N ₆ O ₅ 459.2356].

Example 19

4- {3- [6-amino-9- (3,4-dihydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -9H-purin-2-yl] prop-2-ynyl} cyclohexanecarboxylic acid methyl ester (JR2145)

JR2145 is synthesized according to general protocol 4 using alkin (5.5) and 10 mg (0.025 mmol) of 2-iodadenosine. The product was isolated as an off-white solid: 5.5 mg (48%) yield.

¹ H NMR (CD ₃ OD) δ 8.35 (s, 1H), 5.87 (d, J = 6.5 Hz, 1H), 4.70-4.66 (dd, J = 6.4 Hz , 5.6 Hz, 1H), 4.26 (dd, J = 5.0 Hz, 2.3 Hz, 1H), 3.85 (dd, J = 12.7 Hz, 2.3 Hz, 1H) 3.70 (dd, J = 12.3 Hz, 2.7 Hz, 1H), 3.59 (s, 3H), 2.31 (d, J = 6.6 Hz, 2H), 1.95 (m, 4H); 1.61-1.20 (m, 6H). APCI m / z (relative intense) 446.3 (MH ⁺ , 100) 314.5 (30). MS is high. permission (bombard. fast. atoms) m / z 446.2040 [(M + H) ⁺ calc. for C ₂₁ H ₂₉ N ₅ O ₆ 446.2040].

Example 20

4- {3- {6-amino-9- (3,4-dihydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -9H-purin-2-yl] prop-2-ynyl} acetic acid cyclohexyl methyl ester (JR2147)

The title compound, JR2147, was synthesized according to general protocol 4 using alkin (5.3) and 10 mg (0.025 mmol) of 2-iodadenosine. The product was isolated as an off-white solid: 4.8 mg (48%) yield.

¹ H NMR (CD ₃ OD) δ 8.25 (s, 1H), 5.75 (d, J = 6.5 Hz, 1H), 4.70-4.66 (dd, J = 8.1 Hz , 4.6 Hz, 1H), 4.40 (d, J = 1.2 Hz, 1H), 4.25-4.23 (dd, J = 4.6 Hz, 1.2 Hz, 1H), 3.83 (d, J = 6.5, 2H), 3.53-3.31 (m, 2H), 2.29 (d, J = 6.5 Hz, 2H), 1.97 (s, 3H), 1.93-1.89 (m, 2H), 1.79-1.75 (m, 2H), 1.64-1.42 (m, 2H), 1.12 (t, J = 7.3 Hz, 3H); 1.09-0.91 (m, 4H). APCI m / z (rel. Intense) 460.3 (MH ⁺ , 100), 328.5 (35). MS is high. permission (bombard. fast. by atoms) m / z 460.2193 [(M + H) ⁺ calc. for C ₂₂ H ₃₀ N ₅ O ₆ 460.2196].

Example 21

4- {3- [6-amino-9- (5-ethylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl) -9H-purin-2-yl] prop-2-ynyl} cyclohexanecarboxylic acid 2-amino-ester (JR3033 )

The title compound, JR3033, was synthesized by reacting pure trifluoroacetic acid for 2 hours with the reaction product of 2-iNECA and JR3115 in accordance with General Method 4. The product was isolated as a salt with TFA: 9 mg (98%). APCI m / z (rel. Intensive) 516.4 (MH ⁺ , 100), 343.3 (10).

Example 22

5- {6-amino-2- [3- (4-ethylcarbamoylcyclohexyl) prop-1-ynyl] purin-9-yl} -3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3037)

To a sealed tube containing 5 ml of freshly distilled ethylamine, 10 mg (0.02 mmol) of ATL146e was added. The flask is sealed and the contents are stirred at 60 ° C for 80 hours. After this time, the reaction proceeds by approximately 50% according to HPLC. The vessel was cooled to 0 ° C, opened, and ethylamine was removed in vacuo to give 4.5 mg (73%) of JR3037 as a white solid and isolated by regenerating 4.0 mg of the starting material after purification of the residue by reverse phase HPLC.

Spectra ¹ H NMR (CD ₃ CD-d ₄ ) δ, ¹³ C NMR (CD ₃ CD-d ₄ ) δ corresponded to the above structure. APCI m / z (relative intense) 500.8 (MH ⁺ , 100), 327.4 (3).

Example 23

5- {6-amino-2- [3- (4-carbamoylcyclohexyl) prop-1-ynyl] purin-9-yl} -3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JR3055)

5 mg (0.01 mmol) of ATL146e are added to a sealed tube containing 10 ml of a saturated MeOH / NH ₃ solution. The flask was closed and the contents were stirred at 25 ° C for 48 hours. The vessel was cooled to 0 ° C, opened and ammonia was removed, sparging with N ₂ for 1 hour. Then, the remaining solvent was removed in vacuo to afford 4.0 mg (83%) of JR3055 as a white solid after purification of the residue by reverse phase HPLC.

¹ H NMR (CD ₃ OD-d ₆ ) δ 8.41 (s, 1H), 5.98 (d, J = 7.2 Hz, 1H), 4.65 (dd, J = 7.3 Hz, 4.8 Hz, 1H), 4.41 (d, J = 2.0 Hz, 1H), 4.28 (dd, J = 4.6 Hz, 2.0 Hz, 1H), 3.35 (m , 2H), 2.37 (d, J = 6.4 Hz, 2H), 2.10 (m, 1H), 1.90 (m, _H), 1.53 (m, _H), 1.23 (m, _H), 1.12 (t, J = 7.3 Hz, 3H). ¹³ C NMR (CD ₃ OD-d ₄ ) δ corresponds to the above structure. APCI m / z (rel., Intense) 472.3 (MH ⁺ , 100), 299.4 (10).

Example 24

5- {6-amino-2- [3- (4-methylcarbamoylcyclohexyl) prop-1-ynyl] purin-9-yl} -3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (JB3065)

16.5 mg (0.03 mmol) of ATL146e are added to a sealed tube containing 10 ml of 2.0 M methylamine in methanol. The flask was closed and the contents were stirred at 70 ° C for 120 hours. The vessel was cooled to 0 ° C, opened and the solvent was removed in vacuo to give 8.0 mg (48%) of JR3065 as a white solid after purification of the residue by reverse phase HPLC. . APCI m / z (rel. Intense) 486.3 (MH ⁺ , 100), 313.4 (35).

Example 25

1- (4-Prop-2-ynylcyclohexyl) ethanone (JR3115)

840 mg (4.43 mmol) of Cul and 2 ml of anhydrous THF are added to a 50 ml flame-dried round bottom flask under nitrogen atmosphere and the solution is cooled to -40 ° C in an acetonitrile / dry ice bath. To this solution, 6.8 ml (9.50 mmol) of 1.4 M MeLi (in ether) was added in portions over 30 minutes and the contents were stirred for a total time of 2 hours. 4-prop-2-ynylcyclohexanecarboxylic acid chloride (88) was prepared in a separate flask, stirring 88 in 5 ml of SOCl ₂ at 50 ° C for 3 hours and then evaporating thionyl chloride under reduced pressure to obtain a yellow oily substance. This residue was taken up in 2 ml of THF and added dropwise to a solution of methyl cuprate at -60 ° C. The flask was washed with an additional 2 ml of THF and this solution was also added dropwise. The reaction mixture was stirred for 40 minutes and then quenched with methanol, resulting in the formation of bubbles, and the reaction mixture changed color from orange to yellow. The reaction mixture was stirred for an additional 30 minutes at room temperature, after which 5 ml of EtOAc and 3 ml of a saturated solution of NH ₄ Cl were added. After two minutes of stirring, 20 ml of water and 20 ml of EtOAc are added and the light green organic layer is separated from the blue aqueous layer. The aqueous layer was extracted with an additional 3 × 15 ml EtOAC, dried and concentrated to a residue, which was subjected to chromatography using a mixture of EtOAc / hexane. APCI m / z (rel. Intense) 165.1 (MH ⁺ , 100).

Example 26

5- (6-amino-2- {3- [4- (1-hydroxyethyl) cyclohexyl] prop-1-ynyl} purin-9-yl) -3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide

The title compound, JR3121, was synthesized by reacting NaBH ₄ in THF with the reaction product of 2-iNECA and JR3115 in accordance with general method 4. The product was isolated as an off-white solid. Yield 7 mg (52%). APCI m / z (rel. Intense) 473.4 (MH ⁺ , 100).

Example 27

Isopropyl 4-prop-2-ynylcyclohexanecarboxylate

1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (103 mg, 0.54 mmol) and dimethylaminopyridine (12 mg, 0.098 mmol) are added to a solution of 4-prop-2-ynylcyclohexanecarboxylic acid (78 mg, 0.47 mmol) in anhydrous methylene chloride (8.0 ml). After stirring at room temperature for 1.5 hours, isopropyl alcohol (2.0 ml, 26 mmol) was added and the mixture was stirred for 19 hours at room temperature. Water (25 ml) was added and the mixture was extracted with methylene chloride (3 × 15 ml). The combined extracts were washed with water (25 ml) and brine (20 ml), dried over Na ₂ SO ₄ , filtered and evaporated to dryness under reduced pressure. The crude product is purified by column chromatography, eluting with hexane / ether (95: 5). Yield 76 mg, 78%.

¹ H NMR (CDCI ₃ ) δ 0.96-1.12, 1.19-1.25, 1.33-1.52, 1.86-2.00 (4 x m, 10H, 9 x cyclohexyl, acetylene), 1.20 (d, 6H, -OCH (CH ₃ ) ₂ ), 2.09 (dd, 2H, -C ₆ H ₁₀ CH ₂ CCH), 2.16 (tt, 1H, 1 x cyclohexyl) 4.97 (m, 1H, -OCH (CH ₃ ) ₂ ).

Example 28

4- {3- [6-amino-9- (5-ethylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl) -9H-purin-2-yl] prop-2-ynyl} cyclohexanecarboxylic acid isopropyl ester

¹ H NMR (CD ₃ OD) δ 1.13-1.26, 1.36-1.50, 1.54-3.67, 1.94-2.03 (4 x m, 18H, cyclohexyl, 2 x -CH ₃ , -NHCH ₂ CH ₃ ), 2.36 (d, 2H, -C ₆ H ₁₀ CH ₂ CC-), 3.45 (m, 2H, -NHCH ₂ CH ₃ ), 4.29 ( dd, 1H, 3'-H), 4.45 (d, 1H, 4'-H), 4.72 (dd, 1H, 2'-H), 5.97 (d, 1H, 1'-H ), 8.28 (s, 1H, 8-H). APCI m / z (rel. Intense) 515.6 (MH ⁺ , 100).

Example 29

tert-butyl 4-prop-2-ynylcyclohexanecarboxylate

The title compound, AB1B, was synthesized as described for AB1A using tert-butyl alcohol instead of isopropyl alcohol. Yield 56 mg, 56%.

¹ H NMR (CDCl ₃ ) δ 0.97-1.12, 1.18-1.23, 1.32-1.52, 1.85-2.00 (4 x m, 10H, 9 x cyclohexyl, acetylene), 1.43 (s, 9H, 3 x-OCHS ₃ ), 2.05-2.16 (m, 3H, -C ₆ H ₁₀ CH ₂ CCH, 1 x cyclohexyl).

Example 30

4- {3- [6-amino-9- (5-ethylcarbamoyl-3, 4-dihydroxytetrahydrofuran-2-yl) -9H-purin-2-yl] prop-2-ynyl} cyclohexanecarboxylic acid tert-butyl ester

The title compound, ATL211, was obtained as the reaction product of 2 NECA 2-iodine and JR3115 in accordance with general method 4. Yield 27.5 mg, 63%.

¹ H NMR (CD ₃ OD) δ 1.08-1.23, 1.33-1.49, 1.54-1.67, 1.92-2.03 (4 x m, 10H, cyclohexyl), 1.17 (t, 3H, -NHCH ₂ CH ₃ ), 1.43 (s, 9H, 3 x -CH ₃ ), 2.41 (d, 2H, -C ₆ H ₁₀ CH ₂ CC-), 3 40 (m, 2H, -NHCH ₂ CH ₃ ), 4.32 (dd, 1H, 3'-H), 4.46 (d, 1H, 4'-H), 4.70 (dd, 1H, 2'-H), 6.03 (d, 1H, 1'-H), 8.46 (s, 1H, 8-H). APCI m / z (rel. Intense) 529.6 (MH ⁺ , 100).

Example 31

Radioligand binding studies

Binding to A _2A receptors is determined using radioligand ¹²⁵ I-ZM241385. Figure 2 shows the competition of selective agonists for binding to recombinant human Adenosine A _2A receptors. Compound DWH-146e is highly selective for the recombinant human A _2A subtype (hA2A). Selectivity for the A ₃ receptor (not shown) is less impressive, but nevertheless about 50 times higher. The DWH146e compound is approximately 5 and 50 times more effective than WRC0470 and CGS21680, respectively (Figure 1). An unexpected and interesting discovery is that the DWH-146e ester is also approximately 50 times more effective than the DWH-146a acid (Figure 1).

Example 31a

The effect of DWH-146e and JMR193 on the oxidative activity of neutrophils

A. Materials.

f-met-leu-phe (fMLP), luminol, superoxide dismutase, cytochrome C, fibrinogen, adenosine deaminase and trypan blue are obtained from Sigma Chemical. Hipac-ficoll is purchased from ICN (Aurora, OH), as well as Cardinal Scientific (Santa Fe, NM) and Accurate Chemicals, and Scientific (Westerbury, NY). Endotoxin (lipopolysaccharide; E. coli K235) is obtained from List Biologicals (Campbell, CA). Hanks Balanced Salt Solution (HBSS) and an amoebocyte lysate sample kit are available from Bio-Wittaker (Walkersville, MD). Human Serum Albumin (HSA) is obtained from Cutter Biological (Elkhart, IN). Recombinant human alpha necrosis tumor factor is provided by Dianippon Pharmaceutical Co. Ltd. (Osaka, Japan). ZM241385 (4- (2- [7-amino-2- (2-furyl) [1,2,4] triazolo [2,3-a] [1,3,5] triazin-5-yl-amino] ethyl ) phenol) was donated by Simon Poucher, Zeneca Pharmaceuticals, Cheshire, UK. Stock solutions are prepared (1 mM and 10 mM in DMSO) and stored at -20 ° C.

B. Obtaining human neutrophils.

Purified neutrophils (approximately 98% neutrophils and> 95% viable, as determined by trypan blue exclusion, containing <1 platelet per 5 neutrophils and <50 pg / ml endotoxin (sample with amoebocyte lysate), obtained from normal heparinized (10 units / ml) of venous blood as a result of a one-step procedure for the separation of hypacifol (A. Ferrante et al., J. Immunol. Meth., 36, 109 (1980)).

C. Release from inflammation of reactive oxygen from primated and stimulated human neutrophils.

Chemiluminescence

Chemiluminescence enhanced by luminol, a measure of the oxidative activity of neutrophils, depends on both the production of superoxide and the mobilization of myeloperoxidase, an enzyme of lysosomal granules. Light is emitted from unstable high-energy oxygen produced by activated neutrophils. Purified neutrophils (5-10 × 10 ⁵ / ml) are incubated in Hanks balanced salt solution containing 0.1% human serum albumin (1 ml) with or without DWH-146a, DWH-146e, CGS 21680 or JMR193, with rolipram with or without it, and with or without tumor necrosis factor alpha (1 unit / ml), for 30 minutes at 37 ° C in a shaken ice bath. Then, luminol-enhanced (1 × 10 ^-4 M), f-met-leu-phe (1 msM) stimulated chemiluminescence was determined on a Chronolog® photometer (Crono-log Corp., Havertown, PA) at 2 ° C for 2-4 minutes. Chemiluminescence is defined as the relative maximum of the emitted light (= curve height) compared with that in samples with tumor necrosis factor alpha and without DWH, JMR or rolipram.

D. Results

As shown in FIG. 2, both JMR193 and DWH-146e reduce the oxidative activity of human neutrophils stimulated by f-met-leu-phe and primed by tumor necrosis factor alpha, when measured with luminol-enhanced chemiluminescence, more efficiently than A _2A - agonist CGS21680 adenosine receptor. The horizontal axis shows the concentrations of CGS21680, DWH-146a, DWH-146e, or JMR193 (log nM). The vertical axis shows the resulting peak in human neutrophil activity as the relative amount of stimulated release of reactive oxygen, as measured by luminol enhanced chemiluminescence, relative to control samples that were not primed with tumor necrosis factor alpha. Average SEM values (n = 4-5 individual experiments).

The data under the horizontal axis in FIG. 2 shows the EC ₅₀ value for decreasing human neutrophil activity (based on the data of FIG. 2). Average SEM values (n = 4-5 individual experiments). * p <0.05, decrease in IC ₅₀ compared to CGS21680.

JMR193 and DWH-146e reduce the oxidative emission of stimulated neutrophils-EC ₅₀ less than 1 nm (0.8 and 0.3 nm, respectively). DWH-146a and CGS21680 free acid adenosine A _2A receptor agonists, in contrast, do not inhibit oxidative release so effectively (53 and 9 nM, respectively; FIG. 2). Inhibition of the oxidative release of stimulated neutrophils by DWH-146e was inhibited by the selective antagonist A _2A- AR ZM241385.

As shown in FIG. 3, JMR193 (1 nm) with rolipram (100 nM) synergistically reduces the stimulated release of reactive oxygen. Human neutrophils are primed with tumor necrosis factor alpha (1 U / ml) and stimulated with f-met-leu-phe (1 μM). The vertical axis shows the percentage inhibition of oxidative activity, as measured by luminol enhanced chemiluminescence. Average SEM values (n = 4) for individual experiments. * p <0.05 synergy between JMR193 and rolipram instead of additive activity.

As shown in FIG. 4, the highly selective antagonist of the A _2A adenosine receptor ZM241385 (100 nM) (ZM) counteracts the oxidative activity of human neutrophils inhibited by JMR193 (10 nM), as follows from the results of chemiluminescence enhanced luminol. SEM mean values of 4 separate experiments. * p = 0.0004 ZM241385 counteracts JMR193 inhibitory oxidative activity.

E. [cAMP] _i human neutrophils and adhesion of neutrophils to the biological surface.

A 24-well tissue culture plate was coated with human fibrinogen (5 mg / ml in 1.5% sodium bicarbonate; 0.5 ml / well; Sigma Chemical) overnight at 37 ° C in 5% CO ₂ .

Neutrophils (3-4 × 10 ⁶ / 0.5 ml / sample) are incubated in a well of a coated tablet for 45 minutes in 0.5 ml of HBSS containing 0.1% HSA and ADA (1 unit / ml) if present and absence of recombinant human TNF _α (10 units / ml), DWH-146e (3-300 nM), rolipram (300 nM), and / or ZM241385 (100 nM). After incubation, 0.5 ml of HCl (0.2 N) was added to the wells and incubated for another 45 minutes, extracting cAMP. Then the samples are centrifuged on a microfuge for 2 minutes, removing cell debris. Half-milliliter samples were frozen for analysis on cAMP (B. Brooker et al., Science, 194, 270 (1976)). The walls are washed twice with normal saline, and the remaining monolayer is hydrolyzed with 0.2 ml of 0.2 N. SDS-containing NaOH for 2 hours at room temperature. Protein samples are then frozen (-70 ° C) for further protein analysis to determine the comparative adhesion of PMN (K. P. Stowell et al., Anal. Biochem. 85, 572 (1978)).

results

DWH-146e (30-300 nM) separately and synergistically together with rolipram (300 nM) increases the content of cAMP in human neutrophils and together with rolipram synergistically reduces the adhesion of neutrophils to the fibrinogen-coated surface (Figure 5). The action of DWH-146e (300 nM) + rolipram (300 nM) on cAMP production by neutrophils and adhesion is counteracted by the selective antagonist of the A _2A adenosine receptor, ZM241385 (ZM; 100 nM). The average SEM of 5 individual experiments. * p <0.05 increased [cAMP] in neutrophils compared with the absence of DWH-146e; ** p <0.05 reduction in neutrophil adhesion compared to the absence of DWH-146e.

F. Oxidative activity of adhesive human neutrophils.

Ways

Using the modified methods from Section E, neutrophils (2 × 10 ⁶ / ml) obtained by separation on hypak-Ficoll are incubated for 15 minutes at 37 ° C in 0.45 ml of Hanks balanced saline solution containing 0.1% human serum albumin and adenosine deaminase (1 u / ml), rolipram (300 nM) and DWH-146e (3-300 nM). After incubation, cytochrome C (120 μM) and catalase (0.062 mg / ml) are added in the presence and absence of recombinant human tumor necrosis factor alpha (1 unit / ml), and 200 μl aliquots of the cell suspension are immediately transferred to paired cells of a 96-well mattress for tissue culture coated with human fibrinogen overnight. The optical density of the samples was determined at 500 nm against the corresponding superoxide dismutase samples (200 u / ml).

G. Results.

As shown in FIG. 6, inhibition of tumor necrosis factor-stimulated alpha release of superoxide by human adhesive neutrophils on a fibrinogen-coated surface provides rolipram (300 nM) and DWH-146e. DWH-146e reduces the oxidative emission of adhesive neutrophils and also synergistically reduces the oxidative emission in the presence of rolipram, which alone does not affect the oxidative activity of neutrophils. The horizontal axis shows the concentration of DWH-146e in nM, and the vertical axis shows the amount of superoxide released by neutrophils, determined by measuring the decrease in cytochrome C. There is a noticeable synergy with DWH-146e and a type IV PDE inhibitor, rolipram, in reducing the oxidative activity of adhesive neutrophils human stimulated tumor necrosis factor alpha. The average values of SEM repeats for 4-5 individual experiments. * p <0.05 reduction in superoxide release compared to when DWH-146e is absent; ** p <0.05 reduction in superoxide release compared with the presence of rolipram and the absence of DWH-146e.

Example 32

Treatment of ischemic / reperfusion damage to the kidneys using DWH-146e

To determine whether the activation of the A _2A adenosine A 2 receptor induced by DWH-146e reduces plasma creatinine 24 and 48 hours after ischemic / reperfusion injury in rats, rat kidneys are subjected to 45-minute ischemia and 24- or 48-hour reperfusion. DWH-146e (0.004 μg / kg / min) or excipient is continuously administered via a mini-pump 5 hours before ischemia / reperfusion. As shown in FIG. 7, DWH-146e significantly reduced plasma creatinine in 7/7 rats (P <0.05) and in 6/6 rats treated with DWH-146e (P <0.001) after 24 and 48 hours, respectively .

To determine whether the effect of DWH-146e on plasma creatinine reduction in rats subjected to ischemia / reperfusion is mediated by an A _2A receptor, rat kidneys are subjected to 45-minute ischemia and then 48-hour reperfusion. DWH-146e (0.004 μg / kg / min) is started continuously administered through a mini-pump 5 hours before ischemia. As shown in Fig. 8, the improvement in renal function was completely eliminated by antagonist A _2A ZM241385 (0.003 μg / kg / min - equimolar delivery rate compared to DWH-146e) (* P <0.001 in the case of a filler instead of DWH; ** p < 0.05 DWH vs DWH / ZM. N = 5 for filler, DW; N = 6 for DWH / ZM. ANOVA followed by Bonferroni correction).

DWH-146e, at a concentration that does not have a hemodynamic effect, prevents kidney swelling, necrosis and the concentration of red blood cells in the internal brain.

The protection against renal damage exerted by DWH-146e (0.01 μg / kg / min subcutaneously for 48 hours) correlates with a sharp inhibition of neutrophil adhesion to vascular endothelium. It is believed that inhibition of the interaction between neutrophils and vascular endothelium by DWH-146e is responsible, at least in part, for protecting the kidneys from damage.

In order to determine whether the activation of A _2A- AR reduces the number of neutrophils in the internal medulla of rats subjected to ischemia / reperfusion using Neuroludica®, the kidney is examined at a 100-fold increase and the entire kidney is drawn. The number of polymorphonuclear leukocytes is counted, considering sections of the kidney at a 250-fold increase. Optical frames examined under the microscope are superimposed on the renal sections and all polymorphonuclear leukocytes (PMNs) inside each frame are counted. Such a system ensures that each PMN will be counted only once. As shown in FIG. 9, the neutrophil density is 15.65 / mm ² when using a bulking agent and 3.02 / mm ² when treating with DWH-146e.

Example 33

Effect of DWH-146e on reperfusion injury to the lungs

A. Ways.

An isolated, perfused whole blood, ventilated rabbit lung model is used. Donor rabbits are removed after pulmonary arterial injection with PGEi ₁ and rinsed with Euro-Collins preservative, then the lungs are stored for 18 hours at 4 ° C. Group I lung (n = 9) serves as a control group. Group II lungs (n = 9) are reperfused with whole blood, which is first passed through a filter that removes white blood cells. In group III (n = 9), DWH-146e (25 μg / kg) was added to the blood reperfusate immediately after reperfusion and administered over the reperfusion period (1 μg / kg / min). All lungs were reperfused for 30 minutes and pulmonary artery pressure (PAP), pulmonary vascular resistance (PVR), air distensibility (CPL), and arterial oxygenation were recorded. Myeloperoxidase (MPO) activity is recorded to determine the amount of neutrophil sequestration, and a wet / dry weight ratio is measured to determine pulmonary edema.

B. Results.

Arterial oxygenation in groups II and III is significantly higher than the indicated oxygenation in group I after 30 minutes of reperfusion (514.27 ± 35.80 and 461.12 ± 43.77 versus 91.41 ± 20.58 mm Hg, p < 0.001. As shown in Figure 10, lung group III exhibits an increasing consumption of pO ₂ during reperfusion. The absence of leukocytes in lung group II improves arterial oxygenation at the beginning of reperfusion. * P = 0.004 (group II versus groups I and III); ** p <0.001 (groups II and III against group I).

As shown in FIG. 11, the average PVR in group II is significantly lower compared to control lungs (* p <0.001). PVR of lung group III is significantly lower than even in lungs reperfused with blood without leukocytes (** p <0.001 against groups I and II). Pulmonary vascular resistance is significantly lower in group III (22.783 ± 357 dyn · sec · cm ^-5 ) compared with both groups II and I (31.057 ± 1743 and 36.911 ± 2173 dyn · sec · cm ^-5 , p <0.001). Air elongation is better in groups II and III compared with group I (1.68 ± 0.08 and 1.68 ± 0.05 versus 1.36 ± 0.13, p = 0.03). The permeability of microvessels in group III was reduced to 106.82 ± 17.09 compared with 165.70 ± 21.83 ng of Evans blue dye / gm tissue in group I (p = 0.05). As shown in FIG. 12, the myeloperoxidase activity in group III is significantly lower than in group I (* p = 0.03). MPO = myeloperoxidase. The activity of myeloperoxidase in group III is 39.88 ± 4.87 compared with 88.70 ± 18.69 ΔOD / gm / min in group I (p = 0.03), and the activity of myeloperoxidase in group II is 56.06 ± 7.46.

C. Conclusions

DWH-146e reduces lung neutrophil sequestration and dramatically improves pulmonary transplant function. Neutrophils are important components of the inflammatory cascade of reperfusion injury, and their source can be both circulating blood and the pulmonary implant itself. Selective activation of A _2A adenosine stops the neutrophil-mediated inflammatory response and reduces reperfusion damage to the lungs after transplantation.

Light microscopy results show that the control lungs in group I have severe leukocyte infiltration and edema in the alveolar spaces after 18 hours of ischemic storage and 30 minutes of reperfusion. In group II (lungs that underwent reperfusion without leukocyte blood) and in group III (lungs that received DWH-146e during reperfusion), this infiltration was much weaker.

Example 34

Effect of DWH-146e on the formation of neointima after arterial damage

The activation of leukocytes during the release of inflammatory cytokines occurs after subcutaneous coronary intervention and may play some role in restenosis. In mice, the formation of a healthy neointima in the presence of an intact endothelial lining occurs after ligation of the common carotid artery. Using this model, C57 / BL6 mice are randomized during carotid artery ligation for a 7-day infusion through a DWH-146e osmotic pump (n = 7) or excipient (n = 8).

On the 14th day after ligation of the carotid artery, histomorphometry shows a significant decrease in neointimal area (0.005 ± 0.004 mm ² versus 0.021 ± 0.014 mm ² , p = 0.02), as well as the ratio of neointimal area to average area (0.13 ± 0, 07 versus 0.64 ± 0.44, p = 0.01) in treated animals compared with the control. The average area in both groups is almost the same (0.035 ± 0.007 mm ² versus 0.036 ± 0.009 mm ² , p = 0.81). This restriction of neointimal growth lasted 28 days. Fig. 13 summarizes the effect of DWH-146e on inhibition of neointimal growth in LCCA mouse models. These experiments show that in the mouse carotid artery ligation model, prolonged A _2A stimulation (7 days) using DWH-146e leads to a significant decrease in neointimal formation for at least 21 days, possibly due to its effect on the activation and function of leukocytes.

Example 35

Inhibition of Endotoxin-Stimulated TNFα Release of Human Monocytes

A. Materials.

Hipac-ficoll is purchased from ICN (Aurora, OH), Cardinal Scientific (Santa Fe, NM), Accurate Chemicals and Scientific (Westbury, NY). Endotoxin (lipopolysaccharide; E. coli 0111B4) is obtained from List Biologicals (Campbell, CA). Hanks Balanced Salt Solution (HBSS) and amebocyte lysate sample kit are available from BioWittaker (Walkersville, MD). Human Serum Albumin (HSA) is obtained from Cutter Biological (Elkhart, IN). ZM241385 (4- (2- [7-amino-2- (2-furyl) [1,2,4] triazole [2,3-a] [1,3,5] triazin-5-ylamino] ethyl] phenol ) was donated by Simon Poucher, Zeneca Pharmaceuticals, Cheshire, UK, stock solutions (1 mM and 10 mM in DMSO) are prepared and stored at -20 ° C.

B. Production of TNFα by Purified Adhesive Human Monocytes

Ways

A monocyte-rich monolayer (> 65% of monocytes) is obtained by incubating 1 ml of the mononuclear fraction of leukocytes (5 × 10 ⁵ / ml) obtained by separation on a hypakicoll (A. Ferrante et al., J. Immunol. Meth., 36 109 (1980)) in the wells of a 24-well tissue culture plate (1 hour; 37 ° C; 5% CO ₂ ). Unattached leukocytes are removed by washing and the culture medium (1 ml of Hanks balanced saline solution containing 0.1% human serum albumin, adenosine deaminase [5 u / ml] and 1% heat inactivated autogenous serum) is added to wells containing adhesive mononuclear cells. As indicated above, the following compounds are added: (1) endotoxin (100 ng / ml) and a selective antagonist of A _2A AR ZM241385 (100 nM), as well as (2) selective agonists of the A _2A adenosine receptor JMR193 (1-1000 nM), DWH-146e (1-1000 nM) and CGS21680 (10-1000 nM). Then the samples were incubated for 4 hours (37 ° C; 5% CO ₂ ) and the supernatants were collected. Suspended cells are removed by centrifugation, and cell-free samples are frozen (-70 ° C). TNFα was examined in cell-free supernatants (n = 6) using an ELISA kit (Coulter / Immunotech, Miami, FL).

C. Results.

As shown in FIGS. 10, 14, agonists of the A _2A adenosine receptor reduce the production of TNFα by endotoxin-stimulated adhesive monocytes. Selective antagonist A _2A AR ZM241385 (100 nM) resists the effect of JMR193 on the production of TNFα (Fig. 15).

Thus, DWH-146e and JMR193 reduce the LPS endotoxin-stimulated production of TNFα by human monocytes as a result of the action of a mechanism dependent on the binding of the agonist to A _2A adenosine receptors.

Example 36

The activity of DWH-146e in the model of peritonitis in mice

Preliminary studies of experimental peritonitis include the injection of zymosan (Zym) as a strong stimulus of inflammation (Y. Zhang et al., Eur. J. Pharmacol., 313, 237 (1996)). As shown in FIG. 16, after zymosan injection, the average leukocyte concentration determined by the neubauer hemocytometer is 7.325 ± 1.893 / mm ³ . An intraperitoneal injection of DWH-146e at a dose of 2.5 μg / kg an hour before zymosan inhibits the development of peritonitis, with an average mean leukocyte concentration ± SEM of 2.012 ± 374 / mm ³ 6 hours later (p <0.05). Thus, these studies show that A _2A AR is a tool in mediating the penetration of PMN into the peritoneum after administration of zymosan.

Example 37

Cardioprotective mediated by anti-inflammatory action of JMR193

Compounds in accordance with the present invention are investigated, inducing myocardial "stunning", a type of heart lesion occurring after repeated, temporary periods of termination of the coronary blood flow, by repeated occlusion of blood flow into the coronary artery.

A. The effect of four cycles of occlusion-reperfusion.

The left anterior descending (LAD) coronary artery in a group of dogs is isolated and enclosed in a loop obturator. The blood flow to the LAD artery of dogs is subjected to occlusion 4 times for 5 minutes. After each occlusion, blood flow is resumed for ten minutes. After each occlusion period, one group of six dogs was infused with a solution containing the acetate compound (JMR193) obtained in Example 15 (JMR193) (0.01 μg / kg / min). A second group of six dogs is infused with a solution containing a vehicle (vehicle). After the last cycle of occlusion-reperfusion, the cardiac function of animals is monitored for 3 hours. The results are presented in Figs. 17 and 18. Fig. 17 shows a systolic left ventricular (LV) thickening reaction in 6 control dogs. Cardiac thickening is reduced by approximately 50% 3 hours after the last occlusion. Fig. 18 shows the LV thickening response in 6 dogs receiving an intravenous infusion of the test compound, JMR193 (0.01 μg / kg / min.), Starting from the baseline period and throughout the experiment. Cardiac function as a result of the JMR193 infusion almost normalized 90 minutes after reperfusion.

B. The effect of ten cycles of occlusion-reperfusion.

Two additional groups of dogs are subjected to ten (and not 4) cycles of occlusion-reperfusion, with each occlusion lasting 5 minutes, interrupted by 5 minutes of reperfusion. In this example, a solution containing the acetate compound (JMR193) obtained in Example 15 (0.01 μg / kg / min) was infused into two animals after each occlusion period. The other three animals are infused with a solution containing a vehicle (vehicle). After the last cycle of occlusion-reperfusion, the cardiac function of animals is monitored for 3 hours.

The results are presented in Figs. 19 and 20. Fig. 19 shows a systolic left ventricular thickening reaction in 3 control dogs. This is a more severe heart lesion than in Example 23A, but the result of LV thickening is completely absent shortly after reperfusion and remains unchanged for 3 hours. Figure 20 shows LV thickening in 2 dogs that received an intravenous infusion of the test compound, JMR193 (0.01 μg / kg / min), starting from the baseline period and throughout the experiment. Compared to the control group, dogs treated with an JMR193 infusion showed a significant and noticeable improvement in cardiac function immediately after reperfusion lasting 3 hours.

C. Effect of acetate compound JMR193 on neutrophil uptake during occlusion-reperfusion.

Some animals are injected with neutrophils labeled with radioactive isotopes. Neutrophils are isolated from the blood of dogs, incubated with a compound containing ^99m Tc, and re-administered to dogs. Neutrophils labeled with ^99m Tc are administered intravenously as a marker to determine the degree of inflammation in the reperfusion zone after four cycles of ischemia-reperfusion. Inflammation caused by occlusion-reperfusion cycles causes adhesion of radioactive neutrophils, the number of which is counted using a gamma camera. JMR193 inhibits neutrophil adhesion. The results are shown in FIG. 21, where the localization of neutrophils labeled with ^99m Tc in dogs treated with vehicle only (solid bars) is higher than in dogs treated with JMR193 (shaded bars). Thus, a decrease in the number of neutrophils labeled with radioactive isotopes in the central ischemic zone caused by the infusion of JMR193 illustrates a decrease (*) in cardiac inflammation.

The studies described in examples 23A and 23B show that cardiac inflammation plays a significant negative role, causing myocardial damage. In addition, the administration of an adenosine A _2A receptor agonist, such as, for example, JMR193, either prevents slight damage (Figs. 17 and 18), or significantly alleviates myocardial dysfunction that accompanies a severe lesion (Figs. 19 and 20).

All publications, patents and patent documents are hereby incorporated by reference, as if they were separately listed as references. The present invention is described with reference to various specific and preferred options and methods for its implementation. However, it is understood that many variations and modifications are possible if they do not violate the nature and scope of the present invention.

Claims (56)

1. The compound of formula (I)

where (a) each of R independently represents hydrogen, C ₁ -C ₆ alkyl, C ₃ -C ₇ cycloalkyl, phenyl or phenyl (C ₁ -C ₃ ) alkyl; (b) X and X ′ each independently represents —CH ₂ OH, —CO ₂ R ² , —OC (O) R ² , —CH ₂ OC (O) R ^2, or C (O) NR ³ R ⁴ ; (c) each of R ² , R ³ and R ⁴ independently represents H, C _1-6 alkyl; or C _1-6 alkyl substituted with one to three groups of C _1-6 alkoxy, C _1-6 alkylthio, halogen, hydroxy, amino, mono (C _1-6 alkyl) amino, di (C _1-6 -alkyl) amino; (d) Z and Z 'independently represent (C ₁ -C ₆ ) alkyl, optionally interrupted by one to three S atoms or non-peroxide O, or absent, an is 1-3; or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, in which X 'is —CH ₂ OH or —C (O) NR ³ R ⁴ . 3. The compound according to claim 2, in which X 'represents C (O) NR ³ R ⁴ . 4. The compound according to claim 3, in which R ³ represents H, and R ⁴ represents (C ₁ -C ₄ ) alkyl. 5. The compound according to claim 1, in which each of R represents H or (C ₁ -C ₄ ) alkyl. 6. A compound according to claim 1, wherein Z 'is -CH ₂ - or -CH ₂ -CH _2. 7. The compound according to claim 6, in which Z is —CH ₂ - or —CH ₂ —CH ₂ -. 8. The compound according to claim 1, in which C ₃ -C ₁₀ cycloalkyl is cyclohexyl or cyclopentyl. 9. The compound of claim 8, in which X represents (C ₁ -C ₄ ) -alkoxycarbonyl, C (O) NR ³ R ⁴ or acetoxymethyl. 10. The compound of claim 8, in which XZ represents HO ₂ CZ. 11. The compound of claim 8, in which XZ and Z 'has a transconfiguration relative to C ₃ -C ₁₀ cycloalkyl. 12. The compound according to claim 1, in which R is H, X 'is ethylaminocarbonyl, and (X-Z-) _n [(C ₃ -C ₁₀ ) cycloalkyl] -Z'-C≡C- is 2- (4 -methoxycarbonylcyclohexylmethyl) ethynyl or 2- (4-carboxycyclohexylmethyl) ethynyl. 13. The compound according to claim 1, in which R represents H, X 'represents ethylaminocarbonyl, a (XZ-) _n [(C ₃ -C ₁₀ ) cycloalkyl] -Z'-C≡C- represents 2- (4-acetoxymethylcyclohexylmethyl ) ethynyl. 14. The compound according to claim 1, representing [4- (3- {9- (2R, 3R, 4S, 5S) -5- (N-ethylcarbamoyl) -3,4-dihydroxyoxolan-2-yl] -6- aminopurin-2-yl} prop-2-ynyl) cyclohexyl] methyl acetate or a pharmaceutically acceptable salt thereof. 15. The compound according to claim 1, representing [(2R, 3R, 4S, 5S) -5- (6-amino-2- {3- [4- (hydroxymethyl) cyclohexyl] prop-1-ynyl} purine - 9-yl) -3,4-dihydroxyoxolan-2-yl] -N-ethylcarboxamide or a pharmaceutically acceptable salt thereof. 16. The compound according to claim 1, which is methyl 4- (3- {9 - [(2R, 3R, 4S, 5S) -5- (N-ethylcarbamoyl) -3,4-dihydroxyoxolan-2-yl] -6 -aminopurin-2-yl)} prop-1-ynyl) cyclohexanecarboxylate or a pharmaceutically acceptable salt thereof. 17. The compound according to claim 1, which is 4- (3- {9 - [(2R, 3R, 4S, 5S) -5- (N-ethylcarbamoyl) -3,4-dihydroxyoxolan-2-yl] -6- aminopurin-2-yl)} prop-1-ynyl) cyclohexane-carboxylic acid or a pharmaceutically acceptable salt thereof. 18. The compound according to claims 1-17, as an agonist of the activity of A _2A adenosine receptors. 19. The compound of claim 18 as an active agent for suppressing an inflammatory reaction or treating inflammation. 20. The compound of claim 18, wherein 5'-X is CH ₂ OH or C (O) NR ³ R ⁴ . 21. The compound of claim 18, wherein X 'is C (O) NR ³ R ⁴ . 22. The compound according to item 21, in which R ³ represents H, and R ⁴ represents (C ₁ -C ₄ ) alkyl. 23. The compound of claim 18, wherein each R is H or (C ₁ -C ₄ ) alkyl. 24. The compound of claim 18, wherein Z 'is —CH ₂ - or —CH ₂ —CH ₂ -. 25. The compound of claim 18, wherein Z is —CH ₂ - or —CH ₂ —CH ₂ -. 26. The compound of claim 18, wherein the C ₃ -C ₁₀ cycloalkyl includes cyclohexyl or cyclopentyl. 27. The compound of claim 26, wherein X is (C ₁ -C ₄ ) alkoxycarbonyl or acetoxymethyl. 28. The compound of claim 26, wherein XZ is HO ₂ CZ-. 29. The compound of claim 26, wherein XZ and Z ′ are transconfiguration relative to C ₃ -C ₁₀ cycloalkyl. 30. The compound of claim 18, wherein R is H, X is ethylaminocarbonyl, and (X-Z-) _n [(C ₃ -C ₁₀ ) -cycloalkyl] -Z'-C≡C- is 2- (4 -methoxycarbonylcyclohexylmethyl) ethynyl or 2- (4-carboxycyclohexylmethyl) ethynyl. 31. The compound of claim 18, wherein R is H, X is ethylaminocarbonyl, and (X-Z-) _n [(C ₃ -C ₁₀ ) -cycloalkyl] -Z'-C≡C- is 2- (4 -acetoxymethylcyclohexylmethyl) ethynyl. 32. The compound of claim 18, which is [4- (3- {9- (2R, 3R, 4S, 5S) -5- (N-ethylcarbamoyl) -3,4-dihydroxyoxolan-2-yl] -6- aminopurin-2-yl} prop-1-vinyl) cyclohexyl] methyl acetate. 33. The compound of claim 18, which is [(2R, 3R, 4S, 5S) -5- (6-amino-2- {3- [4-hydroxymethyl) cyclohexyl] prop-1-ynyl} purine-9- yl) -3,4-dihydroxyoxolan-2-yl] -N-ethylcarboxamide. 34. The compound of claim 18, which is methyl 4- (3- {9 - [(2R, 3R, 4S, 5S) -5- (N-ethylcarbamoyl) -3,4-dihydroxyoxolan-2-yl] -6 -aminopurin-2-yl)} prop-1-ynyl) cyclohexanecarboxylate. 35. The compound of claim 18, which is 4- (3- {9 - [(2R, 3R, 4S, 5S) -5- (N-ethylcarbamoyl) -3,4-dihydroxyoxolan-2-yl] -6- aminopurin-2-yl)} prop-1-ynyl) cyclohexanecarboxylic acid. 36. The compound according to claim 19, wherein said treatment further includes the use of a type IV phosphodiesterase inhibitor. 37. The compound of claim 36, wherein the inhibitor is rolipram. 38. The compound according to claim 19, in which the inflammatory reaction is caused by ischemia. 39. The compound according to claim 19, in which the inflammatory reaction is associated with atherosclerosis. 40. The compound according to claim 19, in which the inflammatory reaction is caused by an autoimmune disease. 41. The compound according to claim 19, in which the inflammatory reaction is caused by ischemic / reperfusion injury. 42. The compound according to claim 19, in which the inflammatory reaction occurs in the heart, kidney or lung. 43. The compound according to claim 19, in which the inflammatory reaction is caused by a stroke, traumatic damage to the brain or spinal cord. 44. The compound according to claim 19, in which the inflammatory reaction is caused by a transplant of an organ, tissue or cell. 45. The compound according to claim 19, in which the inflammatory reaction is caused by infection. 46. The compound according to claim 19, in which the inflammatory reaction is associated with a skin disease. 47. The compound according to claim 19, in which the inflammatory reaction is caused by angioplasty, the introduction of a stent, a shunt or implantation. 48. The compound according to claim 19, in which the inflammatory reaction is caused by an allergic disease. 49. The compound according to claim 19, in which the inflammatory reaction is caused by a debilitating disease. 50. The compound according to claim 19, in which the inflammatory reaction is caused by immunosuppressive therapy. 51. The compound of claim 19, wherein the inflammatory response is caused by a pathological condition or symptom in a mammal, caused by the activity of A _2A adenosine receptors and agonism of such activity is desired. 52. The compounds of claim 18, as an active agent of a medicament for treating inflammation. 53. The compound according to paragraph 52, in which the drug includes a phosphodiesterase inhibitor, type IV. 54. The compound of claim 53, wherein the phosphodiesterase inhibitor is rolipram. 55. The compound of claim 53, wherein the preparation comprises a liquid carrier. 56. The compound of claim 53, wherein the drug is adapted for parenteral administration.

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