KR20050020987A - Method of treating ischemia reperfusion injury using adenosine receptor antagonists - Google Patents

Abstract

The present invention relates to methods useful for preventing, limiting or treating ischemia reperfusion injury in mammals. More specifically, the present invention relates to a method of preventing, limiting or treating ischemic reperfusion injury by administering an A _2b adenosine receptor antagonist.

Description

METHODS OF TREATING ISCHEMIA REPERFUSION INJURY USING ADENOSINE RECEPTOR ANTAGONISTS}

The present invention relates to cardiology, medical chemistry and pharmacology. More specifically, it relates to the prevention or treatment of A _2b adenosine receptor antagonists and ischemic reperfusion injury.

Disruption of blood flow and oxygen delivery to tissues leads to conditions such as ischemia. Substantial reduction of oxygen delivery leads to a condition known as hypoxia. Continued ischemia and hypoxia can lead to tissue loss and even cell death. There are a number of conditions that are both natural and clinical, causing ischemia and hypoxia, including but not limited to obstructive vascular disease, coronary thrombosis, cerebrovascular thrombosis, aneurysm rupture, systemic bleeding, crush injury, sepsis, severe skin burns, Vascular occlusion surgery methods (eg, spinal cord ischemia in thoracoabdominal aneurysm surgery), cardiopulmonary bypass methods, organ transplantation, cardiopulmonary collapse (acute heart death), and asphyxia.

Conventional treatment for ischemia and hypoxia is to restore blood flow and oxygen delivery to normal levels by increasing systemic oxygenation or eliminating the cause of vascular blockage. Recovery of blood flow yields improved results compared to situations where ischemia or hypoxia is maintained for a long time. However, it is well known that repair of blood flow and oxygen delivery can lead to further cell death and loss of function regardless of the damage caused by ischemia or hypoxia. Additional damage caused by repair of blood flow and oxygen delivery is known as reperfusion injury. Paradoxical tissue damage caused by reperfusion injury appears to be similar to acute inflammatory conditions resulting from the attachment of inflammatory cells to reperfused tissues, the activation of these inflammatory cells and the subsequent formation of free radicals [Granger et al. Ann. Rev. Physiol., 57, 311-332, (1995)]. The production of free radicals and other cytotoxic biomolecules in reperfused tissue can lead to cell death by necrosis or activation of apoptosis pathways.

Adenosine is an intracellular and extracellular messenger that all cells produce in the body. Adenosine may occur extracellularly by enzymatic conversion. Ischemic and hypoxic tissues form increased amounts of adenosine through the degradation of adenosine triphosphate (ATP) during energy expenditure. These adenosine receptors are classified into four subtypes (ie A ₁ , A _2a , A _2b and A ₃ ) based on the relative affinity for various adenosine receptor ligands and the sequence analysis of the genes encoding these receptors. The activation of each subtype is unique but sometimes leads to the opposite effect.

Three of the four adenosine receptor subtypes are known to affect the function of inflammatory cells during reperfusion injury. Activation of the A _2a adenosine receptor is known to inhibit the release of oxygen free radicals from stimulated neutrophils, reduce the adhesion of neutrophils to vascular endothelial cells, and inhibit neutrophil release of TNF and LTB ₄ (eg, Cronstein et al. al., J. Immunology, 148, pp. 2201-2206 (1992); Thiel et al., (1995) J. Lab. Clin. Med., 126, pp. 275-282; Krump et al., J. Exp. Med., 186, pp. 1401-6 (1997))

In contrast to the anti-inflammatory effect of A _2a adenosine receptor activation, activation of the A ₁ receptor promotes chemotaxis and phagocytosis by stimulated neutrophils (eg, Cronstein et al. (1992), supra; Salmon et al., J. Immunology 145, pp. 2235-2240. (1990)), which promote differentiation of monocytes into multinuclear giant cells (Merrill et al., Arth. Rheum., 40, pp. 1308-1315 (1997)). It has been confirmed that. In addition, activation of A ₁ receptors in vascular endothelial cells promotes inflammation and tissue damage in a model of reperfusion injury of the heart (Becker et al., Pharm. Pharmacol. Letters, 2, pp. 8-11 (1992); Schwartz et al., J. Mol. Cell.Cardio., 25, pp. 927-938 (1993); Zahler et al., Cardiovascular Res., 28, pp. 1366-1372 (1994); and Forman et al., J. Pharmacol.Exp. Ther., 292 (3), pp. 929-38 (2000).

Activation of the A _2b receptor increased production of IL-6 (Sitaraman et al., J. Clin. Invest., 107, pp. 861-9 (2001)) and mast cell degranulation (Linden et al.) Characterized by local inflammation. , Life Sci., 62, pp. 1519-24 (1998); and Auchampach et al., Mol. Pharmacol., 52, 846-60 (1997)). In addition, activation of the A _2b receptor in vascular smooth muscle cells induces cell loss through direct stimulation of apoptosis (Peyot et al., Circ. Res., 86, pp. 76-85 (2000)).

The current treatment for ischemia-reperfusion injury is only adequate treatment of ischemic injury with blood flow repair and oxygenation. However, the damage caused by reperfusion injury is generally under-treated. Ischemia-reperfusion treatment by the study includes the use of adenosine and adenosine analogs as well as the inhibition of sodium-calcium exchange pumps in ischemic muscle cells. However, these therapies are not adequate enough. For example, the use of adenosine and adenosine analogs is burdensome due to depressor activity and undesirable effects of bradycardia. Similarly, inhibition of the sodium-calcium exchange pump on ischemic myocytes is inappropriate because it does not prevent or treat inflammatory conditions or direct stimulation of apoptosis. Accordingly, there is a need for new pharmaceutically acceptable compounds and compositions for preventing, limiting or treating ischemic reperfusion injury.

1 shows data representing myocardial infarction size obtained from Protocol I (see Example 2). Panel A shows the size of the risk area in the four experimental groups, expressed as a percentage of the left ventricle. Panel B shows infarct size as a percentage of risk area. Panel C shows infarct size expressed as a percentage of the left ventricle. Panel D shows a plot of infarct size and penetrating bypass blood flow, expressed as a percentage of the risk area measured 30 minutes after coronary artery occlusion.

2 shows myocardial infarct size data obtained from Protocol II (see Example 3). Panel A depicts the risk area size in four experimental groups expressed as percentage of left ventricle. For comparison purposes, a control of Protocol I was also included. Panel B shows infarct size as a percentage of risk area. Panel C shows infarct size expressed as a percentage of the left ventricle. Panel D shows a plot of infarct size and penetrating bypass blood flow, expressed as a percentage of the risk area measured 30 minutes after coronary artery occlusion.

3 shows myocardial infarct size data obtained from Protocol III. Panel A depicts the risk area size in four experimental groups expressed as percentage of left ventricle. Panel B shows infarct size as a percentage of risk area. Panel C shows infarct size expressed as a percentage of the left ventricle. Panel D shows a plot of infarct size and penetrating bypass blood flow, expressed as a percentage of the risk area measured 30 minutes after coronary artery occlusion.

4 depicts competitive binding of BG9928 to recombinant human A ₁ adenosine receptors. Membrane prepared with HEK 293 cells stably expressing human A ₁ adenosine receptor (50 μg membrane protein), 0.92 nM radioligand [ ³ H] -DPCPX and various concentrations of BG9928 in 0.1 ml buffer HE + 2 units / ml adenosine Incubation was carried out in diaminase in triplicate at 21 ° C. for 2.5 h. Nonspecific binding was measured in the presence of 10 μM NECA. The binding assay was terminated by filtration. (N = 1).

5 depicts the competitive binding of BG9928 on recombinant human A _2a adenosine receptors. Membrane prepared with HEK 293 cells stably expressing human A _2a adenosine receptor (50 μg membrane protein), 1.16 nM radioligand [ ³ H] -ZM241385 and various concentrations of BG9928 in 0.1 ml buffer HE + 2 units / ml adenosine Incubation was carried out in diaminase in triplicate at 21 ° C. for 2.5 h. Nonspecific binding was measured in the presence of 10 μM XAC. The binding assay was terminated by filtration. (N = 1).

6 depicts the competitive binding of BG9928 on recombinant human A _2b adenosine receptors. 0.1 mL buffer HE + membranes prepared from HEK 293 cells stably expressing recombinant human A _2b adenosine receptor (40-70 μg membrane protein), 30-40 nM radioligand [ ³ H] -ZM241385 and various concentrations of BG9928 Incubated in 2 units / ml adenosine deaminase for 3 hours at 21 ° C. in triplicate. Nonspecific binding was measured in the presence of 10 μM NECA. The binding assay was terminated by filtration. (N = 3).

7 depicts one-point binding of BG9928 on recombinant human A ₃ adenosine receptors. Membrane (50 μg membrane protein) and 0.12 nM radioligand [ ¹²⁵ I] -AB-MECA prepared from HEK 293 cells stably expressing either 10 μM IB-MECA or 10 μM BG9928 and the human A ₃ adenosine receptor Incubated in 0.1 ml buffer HE + 2 units / ml adenosine deaminase for 3 hours at 21 ° C. in triplicate. The binding assay was terminated by filtration. (N = 2).

FIG. 8 shows FLIPR analysis of BG9928 with recombinant human A ₁ adenosine receptor stably expressed in CHO-K1 cells. The FLIPR assay measures the response of CHO-K1 cells expressing recombinant human A ₁ adenosine receptors with increasing concentrations of agonist (CPA) (top graph), and then measures IC ₅₀ (the concentration at which 50% of the response is obtained). Using the null method, the K _B value for the antagonist BG9928 is measured at a fixed agent concentration (200 nM CPA) (bottom graph).

9 shows FLIPR analysis of BG9928 using recombinant human A _2b adenosine receptor stably expressed in HEK-293 cells. FLIPR assay measures the response of HEK-293 cells stably expressing the recombinant human A _2b adenosine receptor with increasing concentration of agonist (NECA) (top graph) and IC ₅₀ (the concentration at which 50% of the response is obtained). The Knud method is then used to determine the K _B value for antagonist BG9928 at fixed agonist concentration (5 μM NECA) (bottom graph).

10 depicts FLIPR analysis of BG9928 with recombinant human A _2b adenosine receptor stably expressed in HEK-293 cells. FLIPR is an assay that measures the fraction of control responses observed with 10, 100 and 300 nM BG9928 in HEK-293 cells expressing rat A _2b adenosine receptors with increasing concentrations of agonist (NECA) (top graph). The lower graph is the Schild analysis of the data presented in the upper graph.

Detailed description of the invention

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those disclosed herein can be used in the practice or testing of the present invention, suitable materials and methods are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to limit the invention.

Throughout the specification, it will be understood that variations thereof, such as the term "comprises" or "comprising", are meant to include the entirety or group of entities mentioned, and are not intended to exclude any other completeness or group of entities. .

The term "alkyl" group, as used herein, is a saturated aliphatic hydrocarbon group. Alkyl groups may be straight or branched, for example having from 1 to 6 carbon atoms in the chain. Examples of linear alkyl groups include, but are not limited to, ethyl and butyl. Alkyl groups include, but are not limited to, branched alkyl groups such as isopropyl and t-butyl. Alkyl groups may be optionally substituted with one or more substituents such as alkoxy, amino, nitro, carboxy, carboalkoxy, cyano, halo, hydroxy, mercaptyl, trihalomethyl, sulfoxy or carbamoyl.

As used herein, an "alkenyl" group is an aliphatic carbon group having one or more double bonds. Alkenyl groups may be straight or branched chains, for example having 3 to 6 carbon atoms and 1 or 2 double bonds in the chain. Examples of alkenyl groups include, but are not limited to, allyl and isoprenyl. Alkenyl groups may be optionally substituted with one or more substituents, such as alkoxy, amino, nitro, carboxy, carboalkoxy, cyano, halo, hydroxy, mercaptyl, trihalomethyl, sulfoxy, or carbamoyl.

As used herein, "alkynyl" is an aliphatic carbon group having one or more triple bonds. Alkenyl groups may be straight or branched chains, for example having from 3 to 6 carbon atoms and 1 or 2 triple bonds in the chain. Examples of alkynyl groups include, but are not limited to propargyl and butynyl. Alkynyl groups may be optionally substituted with one or more substituents, such as alkoxy, amino, nitro, carboxy, carboalkoxy, cyano, halo, hydroxy, mercaptyl, trihalomethyl, sulfoxy or carbamoyl.

As used herein, an "aryl" group is a phenyl or naphthyl group, or derivative thereof. "Substituted aryl" groups are alkyl, alkoxy, amino, nitro, carboxy, carboalkoxy, cyano, alkylamino, dialkylamino, halo, hydroxy, hydroxyalkyl, mercaptyl, alkylmercaptyl, trihaloalkyl , An aryl group substituted with one or more substituents such as carboxyalkyl, sulfoxy, or carbamoyl.

As used herein, an "aralkyl" group is an alkyl group substituted with an aryl group. An example of an aralkyl group is benzyl.

As used herein, a "cycloalkyl" group is, for example, an aliphatic ring of 3 to 8 carbon atoms. Examples of cycloalkyl groups include cyclopropyl and cyclohexyl.

As used herein, an "acyl" group is a straight or branched chain alkyl-C (= 0)-or formyl group. Examples of acyl groups include alkanoyl groups (eg, having 1 to 6 carbon atoms in the alkyl group). Acetyl and pivaloyl are examples of acyl groups. Acyl groups may be substituted or unsubstituted.

As used herein, a "carbamoyl" group is a group having the structure H ₂ N-CO _2- . "Alkylcarbamoyl" and "dialkylcarbamoyl" refer to carbamoyl groups, each having one or two alkyl groups in which nitrogen is bonded instead of hydrogen. Similarly, "arylcarbamoyl" and "arylalkylcarbamoyl" groups include aryl groups instead of one of hydrogen, and in the case of arylalkylcarbamoyl groups, include alkyl groups instead of second hydrogen.

As used herein, a "carboxyl" group is a -COOH group.

As used herein, an "alkoxy" group is an alkyl-O- group in which "alkyl" has already been disclosed.

As used herein, an "alkoxyalkyl" group is an alkyl group as previously disclosed in which a hydrogen atom is substituted with an alkoxy group, as already disclosed.

As used herein, a "halogen" or "halo" group is fluorine, chlorine, bromine or iodine.

As used herein, a "heterocyclyl" group is a 5 to about 10 membered ring structure wherein at least one of the atoms in the ring is an element other than carbon (eg, N, O, S). Heterocyclyl groups can be aromatic or non-aromatic, ie, saturated or partially or fully unsaturated. Aromatic heterocyclyl groups may be referred to as "heteroaryl" groups. Examples of heterocyclyl groups are pyridyl, imidazolyl, furanyl, thienyl, thiazolyl, tetrahydrofuranyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, indolyl, indolinyl, isoindole Nil, piperidinyl, pyrimidinyl, piperazinyl, isoxazolyl, isoxazolidinyl, tetrazolyl and benzimidazolyl and the like.

As used herein, a "substituted heterocyclyl" group wherein at least one hydrogen is alkoxy, alkylamino, dialkylamino, carvalkoxy, carbamoyl, carboxyl, cyano, halo, trihalomethyl, Heterocyclyl group substituted with a substituent such as hydroxy, carbonyl, thiocarbonyl, hydroxyalkyl or nitro.

As used herein, "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group.

As used herein, a "sulfamoyl" group has the structure -S (O) ₂ NH ₂ . "Alkylsulfamoyl" and "dialkylsulfamoyl" refer to sulfamoyl groups each having one or two alkyl groups in which nitrogen is bonded instead of a hydrogen atom. Similarly, "arylsulfamoyl" and "arylalkylsulfamoyl" groups include aryl groups instead of one of hydrogen, and in the case of arylalkylsulfamoyl groups, include alkyl groups instead of second hydrogen.

As used herein, an "antagonist" is a molecule that binds to a receptor without activating the receptor. Antagonists compete with the endogenous ligand for the binding site, thereby reducing the ability of the endogenous ligand to stimulate the receptor.

As used herein, a "selective antagonist" is an antagonist that binds to a particular adenosine receptor subtype with a higher affinity than for other subtypes. As used herein, “A _2b selective antagonist” is an antagonist with high affinity for the A _2b receptor, and (a) nanomolar binding affinity for the A _2b receptor subtype and (b) A _2a and A _It has a 10-fold higher, more preferably 50-fold, most preferably 100-fold higher affinity for the A _2b subtype than for the ₃ receptor subtype. A _2b selective antagonists optionally have affinity for the A ₁ receptor subtype, and (a) nanomolar binding affinity for the A ₁ receptor subtype and (b) for A _2a and A ₃ receptor subtypes. It has a high affinity of at least 10 times, more preferably at least 50 times and most preferably at least 100 times relative to the A ₁ subtype.

As used herein, “infarction” refers to local necrosis resulting from impairment of blood supply to tissues (eg, myocardium).

As used herein, "ischemic" means that the blood supply (circulation) to the area is inadequate due to vascular blockage into the local area (ie, organ or tissue). Ischemia includes a complete interruption of blood flow and oxygen delivery to tissues, as well as a substantial reduction of oxygen delivery to tissues due to hypoxia.

As used herein, “reperfusion” means recovery of blood flow to an organ or tissue.

As used herein, "ischemic reperfusion injury" refers to damage to tissue caused by ischemia and subsequent reperfusion.

As used herein, “pharmaceutically acceptable” means an amount effective for treating or preventing a condition characterized by an increase in adenosine concentration and / or an increase in sensitivity to adenosine.

As used herein, the term "patient" refers to animals, including mammals (eg, humans).

As used herein, "pharmaceutically acceptable carrier or adjuvant" means a non-toxic carrier or adjuvant that can be administered to an animal with a compound of the present invention and does not destroy the pharmacological activity of the compound of the present invention. .

Pharmaceutically acceptable anionic salts include methanesulfonic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, benzoic acid, citric acid, tartaric acid, fumaric acid, maleic acid, CH _3- (CH ₂ ) _n -COOH [where n is 0-4], and salts of HOOC- (CH ₂ ) _n -COOH, wherein n is as described above.

If solvent pairs are used, the ratio of solvents used is volume / volume (v / v).

If solubility of solids in the solvent is used, the ratio of solids to solvent is weight / volume (wt / v).

In addition, the following abbreviations are used throughout the specification:

BCA means non-shinconinic acid.

BG9928 is 3- [4- (2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl) -bicyclo [2.2.2] oct -1-yl] -propionic acid.

(Ca ²⁺ ) i means intracellular calcium.

CCD means a charge coupled device.

CPA means N6-cyclopentyladenosine.

CPM means count per minute.

DPM means collapse per minute.

DR is the ratio of concentrations, i.e., the concentration of the agent that produces a predetermined response (but not necessarily the maximum of 50%) in the presence of the antagonist divided by the concentration that leads to the same response in the absence of the antagonist.

EDTA means ethylenediaminetetraacetic acid.

FLIPR stands for Fluorescence Imaging Plate Reader.

[ ³ H] -BG9928 means BG9928 labeled with tritium.

[ ³ H] -DPCPX means 8-cyclopentyl-1,3-dipropylxanthine labeled with tritium, a competitive substrate for A ₁ and A _2b adenosine receptors.

[ ³ H] -ZM241385 is a 4- (2- [7-amino-2- (furyl) (1,2,4) triazolo (2,3) -labeled tritium, a competitive substrate for the A _2a adenosine receptor 3-a) (1,3,5) triazin-5-ylaminoethyl) phenol.

[I] means the concentration of free radioligand.

[ ¹²⁵ I] AB-MECA means [ ¹²⁵ Iodine] -labeled N 6-(4-aminobenzyl) -9- (5- (methylcarbonyl) -β-D-ribofuranosyl) adenine.

IB-MECA is 1-deoxy-1- [6-[[(3-iodophenyl) methyl] amino] -9H-purin-9-yl] -N-methyl-βN6- (4-aminobenzyl)- 9- (5- (methylcarbonyl) -β-D-ribofuranuronamide.

IC ₅₀ refers to the concentration of the agent that inhibits 50% of the measured activity.

K _B means the antagonist dissociation constant.

K _D means the dissociation constant for the radiolabeled drug as measured by saturation analysis.

K _i means the inhibition constant for the drug; Concentration of ligands competing in a competitive assay that occupies 50% of receptors when no radioligand is present.

AB-MECA means N6- (4-aminobenzyl) -9- (5- (methylcarbonyl) -β-D-ribofuranosyl) adenine.

N means the number of observations.

NECA means 5'N-ethylcarboxamidoadenosine.

pA ₂ means a logarithmic measure of the efficacy of the antagonist; Negative logarithm of the concentration of the antagonist, which may indicate a twofold shift in the concentration-response curve.

PMSF means phenylmethyl sulfonyl fluoride.

RFU means relative fluorescence unit.

³ HR-PIA means [ ³ H] -RN ⁶ -phenylisopropyladenosine (radioligand for A3 adenosine receptor).

The Schild plot shows a graph of log (concentration ratio-1), ie log (DR-1) versus log (antagonist concentration). The intercept of the log concentration axis is equal to the pA ₂ value and the slope provides information on the nature of the antagonism.

SD stands for standard deviation.

SEM means standard error of the mean.

XAC stands for xanthine amino congener.

In general, the invention features a highly effective selective antagonist of the A _2b adenosine receptor. In certain embodiments, compounds of the invention may optionally be selective antagonists of A ₁ adenosine receptors.

Synthesis of Adenosine Antagonist Compounds

Compounds useful in the present invention can be prepared by conventional methods known in the art. For example, the synthesis of the compounds of formula (I) is disclosed in WO 01/34604 and WO 01/34610.

Two general methods are disclosed herein. Each of these uses 1,3-disubstituted-5,6-diaminouracil (Compound VI), which is a common starting material, as shown in the two schemes below. Cyanoacetic acid may be used to treat 1,3-disubstituted-5,6-diaminouracil by treatment with the corresponding symmetrical or asymmetrically substituted urea, followed by nitration and reduction (e.g. Org.Chem. 16, 1879, 1951; Can J. Chem. 46, 3413, 1968). Asymmetrically substituted xanthines can be assessed by Mueller's method (J. Med. Chem. 36, 3341, 1993, incorporated by reference). In this method, 6-aminouracil in N3 of uracil is specifically monoalkylated under Vorbruggen conditions. Alternatively, unsubstituted N1 or N3 positions can be functionalized (eg alkylation) at the end of the synthesis.

In the first general method, a ring closure reaction is first performed on 1,3-disubstituted-5,6-diaminouracil (Compound VI) to form an unsubstituted xanthine intermediate at the 8-position. This intermediate can be coupled with the precursor compound of the ZR ₃ moiety to form the desired 8-substituted xanthine. Referring to Scheme 1 below, the starting material 1,3-disubstituted-5,6-diaminouracil (Compound VI) is first reacted with HC (OEt) ₃ to undergo a ring closure reaction, thereby unsubstituted at the 8-position. Produces xanthine intermediates (ie, compound A). After protection with an amino protecting group (eg, using THP or BOM at the N7 position), the presence of a strong base (e.g., n-butyl-lithium (nBuLi) or lithium di-isopropyl-amide (LDA)) for this intermediate The coupling reaction with the precursor compound (ie aldehyde or ketone) of the ZR ₃ moiety is carried out to form an alcohol (ie compound C). The hydroxyl groups of the alcohols can then be reacted by methods known to those skilled in the art to convert the alcohols to amines, mercaptans, ethers, lactones (eg Compound E) or other functionalized compounds. The N7 protection can be removed to obtain a deprotected product (ie Compound F), which product can be further functionalized to produce the compounds of the present invention.

In a second general method, the starting material 1,3-disubstituted-5,6-diaminouracil is reacted with a precursor compound of the ZR ₃ moiety (e.g. aldehyde or carboxylic acid or carboxylic acid chloride) to give 6- The compound of the present invention can be prepared by forming an amide substituted uracil intermediate and proceeding the ring closure reaction to obtain the desired xanthine compound. Referring to Scheme 2 below, the starting material 1,3-disubstituted-5,6-diaminouracil (i.e. compound VI) was first added to the ZR ₃ moiety of the dicarboxyl / ester-substituted precursor compound, HOOC-ZR. ₃ -COOR _a (ie compound G; R _a represents H, C _1-5 alkyl or benzyl, and the phenyl ring is optionally one to _three selected from the group consisting of halo, hydroxyl or C _1-3 alkoxy Optionally substituted with a substituent) and reactions known to those skilled in the art (e.g., benzotriazol-1-yloxytris (dimethylamino) -phosphonium hexafluorophosphate (BOP), O-benzo-triazole-1 -Yl-N, N, N ', N'-tetramethyluronium hexafluorophosphate (HBTU) or O- (7-azabenzotriazol-1-yl) -N, N, N', N'- Coupling using a coupling reagent such as tetramethyluronium hexafluorophosphate (HATU) to form a 6-amide substituted uracil intermediate (ie, Compound H). Examples of compound G include bicyclo [3.2.1] octane-1,5-dicarboxylic acid monomethyl ester and bicyclo [2.2.2] octane-1,4-dicarboxylic acid monoethyl ether. The uracil intermediate is subjected to a ring closure reaction under basic conditions (such as with KOH and isopropyl alcohol) to form a xanthine compound (ie, compound J), which is further functionalized to produce the various compounds of the present invention. Can be.

Desired aldehydes, ketones, carboxylic acids and carboxylic acid chlorides are from materials commercially available (eg, available from Aldrich Chemical Company, Milwaukee, WI, Inc.), or commercially available by known synthetic methods. It can be manufactured easily. Such synthetic methods include, but are not limited to, oxidation, reduction, hydrolysis, alkylation and Wittig homologation reactions. Regarding the preparation of the bicycloalkane carboxylic acid of the present invention (eg, Compound III, which is an example of Compound G), for example, Aust. J. Chem. 38, 1705, 1985; Aust J. Chem. 39, 2061, 1986; J. Am. Chem. Soc. 75, 637, 1953; J. Am. Chem. Soc. 86, 5183, 1964; J. Am. Chem. Soc. 102, 6862, 1980; J. Org. Chem. 46, 4795, 1981; And J. Org. Chem. 60, 6873, 1995.

There are several ways to further functionalize compound J comprising a carboxylic acid or ester attached to the R ₃ moiety. For example, compound J can be converted to the corresponding acrylic acid derivative. One method involves first hydrolyzing the ester group of compound J (where R _a is not H) to give the corresponding carboxylic acid, reducing the carboxylic acid to the corresponding alcohol, and oxidizing the alcohol to the corresponding aldehyde. Next, Wadsworth-Horner-Emmons or Witting reactions are carried out to form the corresponding acrylic acid derivatives. Compound J can be converted directly to the corresponding alcohol. A different variant is to directly convert Compound J to the corresponding aldehyde. A further variant is the conversion of the ester containing compound J to the corresponding carboxylic acid followed by the aldehyde direct conversion. Alternatively, the precursor of the ZR ₃ moiety prior to coupling to 1,3-disubstituted-8-unsubstituted xanthine in Scheme 1 or 1,3-disubstituted-5,6-diaminouracil in Scheme 2 Compounds can be functionalized. In addition, the compounds of the present invention can be prepared on a solid support (eg, Wang resin).

3- [4- (2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl) -bicyclo [2.2.2] oct-1 Synthesis of -yl] -propionic acid (BG9928) is as disclosed in International Publication No. WO 01/34610.

In certain embodiments, the compound may exist in the form of an achiral compound, an optically active compound, a pure diastereomer, a mixture of diastereomers, a prodrug or a pharmacologically acceptable salt thereof.

In some embodiments of the invention, the compound of formula I exhibits an affinity for the A _2b adenosine receptor at least 10 times greater than the affinity for the A _2a adenosine receptor or the A ₃ adenosine receptor. In another embodiment, the compound of formula I exhibits an affinity for the A _2b adenosine receptor at least 50 times greater than the affinity for the A _2a adenosine receptor or the A ₃ adenosine receptor. In another embodiment, compounds of Formula (I) exhibit affinity for A _2b adenosine receptors at least 100 times greater than affinity for A _2a adenosine receptors or A ₃ adenosine receptors. In some embodiments, compounds of Formula I of the present invention optionally exhibit affinity for A ₁ adenosine receptor, in addition to affinity for A _2b adenosine receptor.

In some embodiments of the invention, the compound of formula I has a K _i value for the A _2b adenosine receptor of less than 500 nM. In another embodiment of the invention, the compound of formula I has a K _i value for the A _2b adenosine receptor of less than 200 nM. In another embodiment of the invention, the compound of formula I has a K _i value for the A _2b adenosine receptor of less than 10 nM.

A _2b  Preparation of Adenosine Receptor Antibodies

The present invention encompasses the use of antibodies formed against A _2b adenosine receptors as antagonists of receptors. Such antibodies block the ligand (eg adenosine) binding site on the A _2b adenosine receptor or prevent the ligand (eg adenosine) from binding to the receptor.

A _2b adenosine receptors can be used to elicit polyclonal or monoclonal antibodies that bind to the A _2b adenosine receptor using a variety of methods known to those skilled in the art. Alternatively, peptides corresponding to specific regions of the A _2b adenosine receptor can be synthesized and used to form immunological reagents according to known methods.

Cloning the human A _2b adenosine receptor and determining the protein sequence for the receptor as well as the DNA sequence encoding the receptor (Rivkee et al., Mol. Endocrinol., 6, pp. 1598-1604 (1992); Pierce et al. , Biochem.Biophys.Res.Commun., 187, pp. 86-93 (1992); Reppert et al., US Pat. No. 5,516,894).

Antibodies directed against the A _2b adenosine receptor of the invention are immunoglobulin molecules or portions thereof that immunologically react with the A _2b adenosine receptor of the invention. More preferably, the antibodies used in the methods of the invention are immunologically reactive with the ligand binding domain of the A _2b adenosine receptor.

Antibodies directed against A _2b adenosine receptors can be formed by immunization of appropriate hosts. Such antibodies can be polyclonal or monoclonal. Preferably, it is a monoclonal. The production of polyclonal and monoclonal antibodies is known to those skilled in the art. For a review of methods useful in the practice of the present invention see, eg, Harlow and Lane (1988), Antibodies, A Laboratory Manual, Yelton, DE et al. (1981); Ann. Rev. of Biochem., 50, pp. 657-80., And Ausubel et al. (1989); See Current Protocols in Molecular Biology (New York: John Wiley & Sons), updated annually. Immunoreactivity with A _2b adenosine receptors can be determined by any of several methods known in the art, such as immunoblot analysis and ELISA.

Monoclonal antibodies having an affinity of at least 10 ⁻⁸ M ⁻¹ or preferably at least 10 ⁻⁹ to 10 ⁻¹⁰ M ⁻¹ are typically standard procedures as disclosed, eg, in Harlow and Lane, (1988, supra). To manufacture. In summary, the appropriate animal is selected and the desired immunization protocol is as follows. After the appropriate period of time has elapsed, the spleens of the animals are excised and each spleen cell is typically fused to immortalized myeloma under appropriate selection conditions. Cells were then separated by clones, and the supernatants of each clone were tested for the production of appropriate antibodies specific for a given region of antigen.

Other suitable techniques include in vitro exposure of lymphocytes to antigenic A _2b adenosine receptors, or alternatively to selection of antibody libraries in phage or similar vectors. Huse et al., Science, 246, pp . 1275-81 (1989). Antibodies useful in the present invention may be used without modification, or may be used without modification. Antigens (in this case, A _2b adenosine receptors) and antibodies can be labeled by covalently or non-covalently binding a substance that provides a detectable signal. Various labeling and conjugation techniques are known in the art and can be used for the practice of the present invention. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles, and the like. Patents teaching the use of such labels include US Pat. No. 3,817,837; 3,850,752; 3,850,752; 3,939,350; 3,939,350; 3,996,345; 3,996,345; No. 4,277,437; No. 4,275,149; And 4,366,241. In addition, recombinant immunoglobulins can also be prepared (see US Pat. No. 4,816,567).

In addition, the antibodies of the invention can be hybrid molecules formed of immunoglobulin sequences from different species (eg, mouse and human) or immunoglobulin light and heavy chain sequences from the same species. The antibody may be a single chain antibody or a humanized antibody. It may be a molecule having multiple binding specificities, for example a bifunctional antibody, which may be prepared using any of a number of techniques known to those skilled in the art, examples of which include hybrid hybridomas, Disulfide exchange, chemical crosslinking, addition of a peptide linker between two monoclonal antibodies, introduction of two sets of immunoglobulin heavy and light chains into specific cell lines, and the like. In addition, the antibodies of the present invention may be human monoclonal antibodies, for example those produced by endogenous human cells, SCID-hu mice or other non-human animals capable of producing “human” antibodies, or cloning It may be prepared by the expression of a human immunoglobulin gene. The preparation of humanized antibodies is taught in US Pat. Nos. 5,777,085 and 5,789,554.

Collectively, the disclosure herein allows a person skilled in the art to utilize the stability or half-life, immunogenicity, toxicity, affinity, or the like of a given antibody molecule using various available methods that can be used to alter the biological properties of the antibodies of the invention. The yield may be increased or decreased, or its properties may be altered in other ways to better suit the particular purpose.

A _2b  Uses of Adenosine Receptor Antagonists

The methods and compositions of the invention can be used to prevent, limit or treat a person who has experienced ischemia or a patient who is about to have ischemia. Ischemia includes, for example, acute coronary syndrome (including myocardial infarction), seizures, organ transplants, kidney ischemia, shock and organ transplant surgery. In some embodiments, the ischemia is myocardial infarction.

In some embodiments of the invention, the A _2b adenosine receptor antagonist is administered 10 days before ischemia or 10 days after ischemia. In another embodiment of the invention, the A _2b adenosine receptor antagonist is administered within 5 days before ischemia or within 5 days after ischemia. In another embodiment of the invention, the A _2b adenosine receptor antagonist is administered 2 days before ischemia or 2 days after ischemia. In another embodiment, A _2b Adenosine receptor antagonists are administered within 2 days after ischemia.

In addition, the present invention A _2b A _2b by administering a pharmaceutically effective or prophylactically effective amount of an adenosine receptor antagonist to a mammal in need thereof. Provided are methods for treating a disease or condition mediated by activation of adenosine receptors.

Ischemia often results in necrosis of affected tissue. The present invention also provides a method for limiting tissue necrosis due to ischemia, which method comprises identifying a mammal experiencing ischemia or imminent ischemia and A _2b of the present invention. Administering a therapeutically effective or prophylactically effective amount of an adenosine receptor antagonist. In some embodiments, A _2b Adenosine receptor antagonists are administered 10 days before ischemia or 10 days after ischemia. In another embodiment of the invention, the A _2b adenosine receptor antagonist is administered within 5 days before ischemia or within 5 days after ischemia. In another embodiment of the invention, the A _2b adenosine receptor antagonist is administered 2 days before ischemia or 2 days after ischemia.

Myocardial infarction is the occurrence of myocardial necrosis caused by an imbalance between the oxygen supply and the oxygen demand of the myocardium, resulting in myocardial necrosis. Often myocardial infarction is caused by rupture of plaques with thrombus formation in the coronary vessels, resulting in a sharp decrease in the blood supply to a portion of the myocardium. It can cause partial occlusion or complete occlusion of blood vessels and subsequent myocardial ischemia. Complete occlusion of coronary vessels for several hours (eg, 4-6 hours) results in irreversible myocardial necrosis. However, reperfusion within this period can save myocardium and reduce morbidity and mortality. Accordingly, the present invention provides a method for identifying a patient having a myocardial infarction or a mammal having an impending myocardial infarction following myocardial infarction, and then limiting the size of the infarction by administering a therapeutically effective or prophylactically effective amount of the A _2b adenosine receptor antagonist of the invention. to provide. In some embodiments, A _2b Adenosine receptor antagonists are administered 10 days before ischemia or 10 days after ischemia. In another embodiment of the invention, the A _2b adenosine receptor antagonist is administered within 5 days before ischemia or within 5 days after ischemia. In another embodiment of the invention, the A _2b adenosine receptor antagonist is administered 2 days before ischemia or 2 days after ischemia.

Pharmaceutical composition

A _2b adenosine receptor antagonists can be formulated into pharmaceutical compositions for administration to animals, including humans. These pharmaceutical compositions preferably comprise A _2b adenosine receptor antagonists and pharmaceutically acceptable carriers that are effective in treating, limiting or preventing ischemic reperfusion injury.

Pharmaceutically acceptable carriers useful in these pharmaceutical compositions include, for example, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as serum albumin, buffers such as phosphate, glycine, sorbic acid, Potassium sorbate, partial glyceride mixtures of labeled vegetable fatty acids, water, salts or electrolytes, for example protamine sulfate, dihydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pi Rollidone, cellulose based materials, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block copolymers, polyethylene glycols and wool fats.

The compositions of the present invention can be administered by parenteral administration, oral administration, inhalation spray administration, topical administration, rectal administration, nasal administration, sublingual administration, vaginal administration or implanted containers. The term “parenteral” as used herein is meant to include subcutaneous, intravenous, intramuscular, intraarticular, intravitreal, intrasternal, intramedullary, intrahepatic, intralesional and intracranial injection or infusion techniques. The composition is preferably administered orally, intraperitoneally or intravenously.

Sterile injectable forms of the compositions of the invention may be aqueous or oily suspensions. These suspensions can be formulated using techniques known in the art using suitable dispersing or wetting agents and suspending agents. In addition, sterile injectable preparations may be sterile injectable solutions or suspensions in nontoxic parenterally acceptable diluents or solvents, for example solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used are water, Ringer's solution, and sodium chloride isotonic solution. Sterile fixed oils are also those commonly used as solvents or suspending media. For this purpose any bland fixed oil can be employed including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful for the preparation of injectable preparations, for example, in pharmaceutically acceptable natural oils such as olive oil or castor oil, especially in their polyoxyethylated forms. These oil solutions or suspensions contain carboxymethyl cellulose or similar dispersants conventionally used in the formulation of pharmaceutically acceptable formulations, including long chain alcohol diluents or dispersants, for example emulsions and suspensions. Other commonly used surfactants such as tween, span and other emulsifiers or bioavailability increasing agents conventionally used in the preparation of pharmaceutically acceptable solids, liquids or other formulations may be used for formulation purposes.

Parenteral preparations may be single ring administration, infusion or loading ring administration with maintenance administration. These compositions may be administered once daily or as needed.

The pharmaceutical compositions of the present invention may be administered orally in any acceptable oral dosage form, including capsules, tablets, aqueous suspensions or aqueous solutions. In the case of oral tablets, carriers commonly used include lactose and corn starch. Lubricants such as magnesium stearate may also typically be added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. If an aqueous suspension is required for oral use, the active ingredient is combined with emulsifiers and suspending agents. If desired, any sweetening, flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of the present invention may be administered in the form of suppositories for rectal administration. They can be prepared by mixing the formulation with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical composition of the present invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the pharmaceutical formulating art, and are solutions in saline using benzyl alcohol or other suitable preservatives, absorption accelerators to increase bioavailability, fluorocarbons and / or other conventional solubilizers or dispersants. It can be prepared by.

The amount of A _2b adenosine receptor antagonist that can be mixed with the carrier material to produce a single formulation will vary depending upon the host to be treated and the particular mode of administration. The compositions may be formulated so that 0.01-100 mg of A _2b adenosine receptor antagonist per kg body weight can be administered to patients receiving these compositions. In some embodiments of the invention, the dosage is 0.1-10 mg per kg of body weight. The composition may be administered in a single dose, multiple doses or infusion over a defined period of time.

The particular dosing and treatment regimen for a particular patient will depend on a number of factors, including examples of specific A _2b adenosine receptor antagonists, the patient's age, weight, general health status, sex and diet, and time of administration, Secretion rates, drug combinations, and the severity of the particular disease being treated. The determination of these factors by practitioners is within the scope of those skilled in the art. In addition, the amount of antagonist depends on the individual patient to be treated, the route of administration, the type of formulation, the nature of the compound used, the severity of the disease and the desired effect. The amount of antagonist can be determined according to pharmacological or pharmacokinetic principles that are well known in the art.

According to some embodiments, the present invention provides a method of preventing, limiting or treating ischemia reperfusion injury, including administering one of the pharmaceutical compositions.

The present invention has been described above, and the present invention will be further described by the following embodiments. The embodiments described below are for illustrative purposes only and are not intended to limit the invention to the few embodiments that point out the technical spirit of the present invention.

Applicants solved this problem by discovering that A _2a adenosine receptor antagonists can prevent, limit or treat ischemic reperfusion injury. The present invention relates to a method for preventing, limiting or treating ischemic reperfusion injury in a mammal experiencing an ischemic event or having an ischemic event imminent using an A _2b adenosine receptor antagonist. Compounds useful in the methods of the present invention specifically antagonize or block the A _2b adenosine receptor to achieve the desired effect.

In some embodiments, the methods of the present invention comprise administering to the patient a therapeutically effective or prophylactically effective amount of A _2b adenosine receptor within 10 days before or after an ischemic event.

In some embodiments of the invention, the A _2b adenosine receptor antagonist is a compound of formula (I) or a pharmaceutically acceptable salt or N-oxide thereof.

Where

R ₁ , R ₂ , and R ₃ are each independently

a) hydrogen;

b) C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl is unsubstituted or hydroxy, alkoxy, amino, monoalkylamino Substituted with one or more substituents selected from the group consisting of, dialkylamino, cycloalkyl, aryl, heterocyclyl, aralkyl, heterocyclylalkyl, acylamino, alkylaminocarbonyl, alkylsulfonylamino, and alkylaminosulfonyl being];

c) substituted or unsubstituted aryl; or

d) substituted or unsubstituted heterocyclyl;

R ₄ is a single _{bond, -0-, - (CH 2)} 1-3 -, -0 (CH 2) 1-2 -, -CH 2 OCH 2 -, - (CH 2) 1-2 0-, - CH = CHCH _2- , -CH = CH-, or -CH ₂ CH = CH-;

R ₅ is

(a) phenyl, or

(b) A bicyclic or tricyclic group selected from the group consisting of wherein the phenyl, bicyclic or tricyclic group is unsubstituted, or

(a) C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl group is unsubstituted or amino, monoalkylamino, dialkylamino , Substituted or unsubstituted heterocyclylaminocarbonyl, (amino) (R _b ) acylhydrazinylcarbonyl-, (amino) (R _b ) acyloxycarboxy-, (hydroxy) (carbonalkoxy) alkylcarbon Barmoyl, acyloxy, aldehyde, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylaminoalkylamino, dialkylaminoalkylamino, alkylphosphono, alkylsulfonylamino, carbamoyl, R _b- , R _b -Alkoxy-, R _b -alkylamino-, cyano, cyanoalkylcarbamoyl, cycloalkylamino, dialkylphosphono, haloalkylsulfonylamino, heterocyclylalkylamino, heterocyclylcarbamoyl, hydroxide Hydroxy, hydroxyalkylsulfonylamino, oxymino, phosphono, substituted or unsubstituted aral One selected from the group consisting of kelpamino, substituted or unsubstituted arylcarboxyalkoxycarbonyl, substituted or unsubstituted heteroarylsulfonylamino, substituted or unsubstituted heterocyclyl, thiocarbamoyl, and trifluoromethyl Substituted with one or more substituents); And

(b) (alkoxycarbonyl) aralkylcarbamoyl, aldehyde, alkenoxy, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylcarbamoyl, alkoxycarbonylamino, alkoxycarbonylalkylamino, alkylsulfonyl Amino, alkylsulfonyloxy, amino, aminoalkylaralkylcarbamoyl, aminoalkylcarbamoyl, aminoalkylheterocyclylalkylcarbamoyl, aminocycloalkylalkylcycloalkylcarbamoyl, aminocycloalkylcarbamoyl, They are alkoxycarbonylamino, aryl, heterocyclyl, aryloxy, aryl-sulfonylamino, aryl sulfonyloxy, carbamoyl, carbonyl, R _b -, R _b - alkoxy -, R _b - alkylthio -, R _b -Alkyl (alkyl) amino-, R _b -alkyl (alkyl) carbamoyl-, R _b -alkylamino-, R _b -alkylcarbamoyl-, R _b -alkylsulfonyl-, R _b -alkylsulfonyl Amino, R _b -alkylthio, R _b -heterocyclylcarbonyl, aminoalkylaminocarbo Nyl, dialkylaminoalkylamino, alkylaminoalkylamino, cyano, cycloalkylamino, dialkylaminoalkylcarbamoyl, halogen, heterocyclylalkylamino, hydroxy, oxymino, phosphate, substituted or unsubstituted Alkylamino, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylsulfonylamino, sulfoxyacylamino, and thiocarbamoyl

Is substituted with one or more R _a groups selected from the group consisting of:

In this case, R _b is -COOH, -C (CF ₃ ) ₂ 0H, -CONHNHSO ₂ CF ₃ , -CONHOR _c , -CONHS0 ₂ R _c , -CONHSO ₂ NHR _c , -C (OH) R _c PO ₃ H ₂ , -NHCOCF ₃ , -NHCONHSO ₂ R _c , -NHPO ₃ H ₂ , -NHSO ₂ R _c , -NHSO ₂ NHCOR _c , -OPO ₃ H ₂ , -OS0 ₃ H, -PO (OH) R _c , -PO ₃ H ₂ , -SO ₃ H, -SO ₂ NHR _c , -SO ₃ NHCOR _c , -SO ₃ NHCONHCO ₂ R _c , and the following compounds

Selected from the group consisting of;

R _c is selected from the group consisting of hydrogen, —C _1-4 alkyl, —C _1-4 alkyl-CO ₂ H, and phenyl, wherein —C _1-4 alkyl, —C _1-4 alkyl-CO ₂ H, and phenyl group are unsubstituted or consist of halogen, -OH, -OMe, -NH ₂ , -NO ₂ , unsubstituted benzyl, and halogen, -OH, -OMe, -NH ₂ , and -NO ₂ Substituted with 1 to 3 substituents selected from the group consisting of benzyl substituted with 1 to 3 substituents selected from the group;

X ₁ and X ₂ are independently selected from the group consisting of O and S;

X ₃ is N or CR _d , where R _d is

a) hydrogen;

b) C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl is unsubstituted or hydroxy, alkoxy, amino, monoalkylamino Substituted with one or more substituents selected from the group consisting of, dialkylamino, cycloalkyl, aryl, heterocyclyl, aralkyl, heterocyclylalkyl, acylamino, alkylaminocarbonyl, alkylsulfonylamino, and alkylaminosulfonyl being];

c) substituted or unsubstituted aryl; And

d) substituted or unsubstituted heterocyclyl

It is selected from the group consisting of.

In some embodiments of the invention, R ₁ is C _1-6 alkyl. In some embodiments, R ₂ is C _1-6 alkyl. In some embodiments, R ₃ is hydrogen. In some embodiments, R ₄ is a single bond.

In some embodiments of the invention, R ₅ is substituted phenyl. In other embodiments, R ₅ is Substituted bicyclic or tricyclic group selected from the group consisting of:

In another embodiment, R ₅ is Wherein R ₅ is unsubstituted, or

(a) C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl groups are each unsubstituted or amino, monoalkylamino, dialkyl Amino, substituted or unsubstituted heterocyclylaminocarbonyl, (amino) (R _b ) acylhydrazinylcarbonyl-, (amino) (R _b ) acyloxycarboxy-, (hydroxy) (carbonalkoxy) alkyl Carbamoyl, acyloxy, aldehyde, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylaminoalkylamino, dialkylaminoalkylamino, alkylphosphono, alkylsulfonylamino, carbamoyl, R _b- , R _b -alkoxy-, R _b -alkylamino-, cyano, cyanoalkylcarbamoyl, cycloalkylamino, dialkylphosphono, haloalkylsulfonylamino, heterocyclylalkylamino, heterocyclylcarbamoyl, Hydroxy, hydroxyalkylsulfonylamino, oxymino, phosphono, substituted or unsubstituted Selected from the group consisting of lealkylamino, substituted or unsubstituted arylcarboxyalkoxycarbonyl, substituted or unsubstituted heteroarylsulfonylamino, substituted or unsubstituted heterocyclyl, thiocarbamoyl, and trifluoromethyl Substituted with one or more substituents; And

(b) (alkoxycarbonyl) aralkylcarbamoyl, aldehyde, alkenoxy, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylcarbamoyl, alkoxycarbonylamino, alkoxycarbonylalkylamino, alkylsulfonyl Amino, alkylsulfonyloxy, amino, aminoalkylaralkylcarbamoyl, aminoalkylcarbamoyl, aminoalkylheterocyclylalkylcarbamoyl, aminocycloalkylalkylcycloalkylcarbamoyl, aminocycloalkylcarbamoyl, They are alkoxycarbonylamino, aryl, heterocyclyl, aryloxy, aryl-sulfonylamino, aryl sulfonyloxy, carbamoyl, carbonyl, R _b -, R _b - alkoxy -, R _b - alkylthio -, R _b -Alkyl (alkyl) amino-, R _b -alkyl (alkyl) carbamoyl-, R _b -alkylamino-, R _b -alkylcarbamoyl-, R _b -alkylsulfonyl-, R _b -alkylsulfonyl Amino, R _b -alkylthio, R _b -heterocyclylcarbonyl, aminoalkylaminocarbo Nyl, dialkylaminoalkylamino, alkylaminoalkylamino, cyano, cycloalkylamino, dialkylaminoalkylcarbamoyl, halogen, heterocyclylalkylamino, hydroxy, oxymino, phosphate, substituted or unsubstituted Alkylamino, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylsulfonylamino, sulfoxyacylamino, and thiocarbamoyl

It is substituted with at least one R _a group selected from the group consisting of.

In some embodiments of the invention, R _a is

(a) C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl groups are each unsubstituted or amino, monoalkylamino, dialkyl Amino, substituted or unsubstituted heterocyclylaminocarbonyl, (amino) (R _b ) acylhydrazinylcarbonyl-, (amino) (R _b ) acyloxycarboxy-, (hydroxy) (carbonalkoxy) alkyl Carbamoyl, acyloxy, aldehyde, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylaminoalkylamino, alkylphosphono, alkylsulfonylamino, carbamoyl, R _b- , R _b -alkoxy-, R _b -alkylamino-, cyano, cyanoalkylcarbamoyl, cycloalkylamino, dialkylaminoalkylamino, dialkylphosphono, haloalkylsulfonylamino, heterocyclylalkylamino, heterocyclylcarbamoyl, Hydroxy, hydroxyalkylsulfonylamino, oxymino, phosphono, substituted aralkylami , Substituted aryl carboxy alkoxycarbonyl, substituted heteroaryl-sulfonylamino, substituted heterocyclyl, alkylthio carbamoyl, and tri substituted with one or more substituents selected from the group consisting of fluoromethyl; And

(b) (alkoxycarbonyl) aralkylcarbamoyl, aldehyde, alkenoxy, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylcarbamoyl, alkoxycarbonylamino, alkoxycarbonylalkylamino, alkylsulfonyl Amino, alkylsulfonyloxy, amino, aminoalkylaralkylcarbamoyl, aminoalkylcarbamoyl, aminoalkylheterocyclylalkylcarbamoyl, aminocycloalkylalkylcycloalkylcarbamoyl, aminocycloalkylcarbamoyl, are alkoxycarbonylamino, aryl, heterocyclyl, aryloxy, aryl-sulfonylamino, aryl sulfonyloxy, carbamoyl, carbonyl, R _b -, R _b - alkoxy -, R _b - alkyl (alkyl) amino- , R _b -alkyl (alkyl) carbamoyl-, R _b -alkylamino-, R _b -alkylcarbamoyl-, R _b -alkylsulfonyl-, R _b -alkylsulfonylamino, R _b -alkylthio , R _b - heterocyclyl-carbonyl, cyano, cycloalkylamino, dialkyl amino Alkylcarbamoyl, halogen, heterocyclylalkylamino, hydroxy, oxymino, phosphate, substituted aralkylamino, substituted heterocyclyl, substituted heterocyclylsulfonylamino, sulfoxylamino, and thiocarba It is selected from the group consisting of moles.

In other embodiments of the invention, R _a is

(a) C _1-6 alkyl or C _2-6 alkenyl, each of which is unsubstituted or amino, monoalkylamino, dialkylamino, substituted or unsubstituted heterocyclylaminocarbonyl, R _b- , R _b -alkoxy- and at least one substituent selected from the group consisting of substituted or unsubstituted heterocyclyl; And

(b) alkoxycarbonylalkylamino, cyano, and hydroxy

It is selected from the group consisting of.

In some embodiments of the invention, X ₁ is O. In some embodiments, X ₂ is O. In some embodiments, X ₃ is N.

In some embodiments of the invention, each R ₁ and R ₂ is C _2-4 alkyl; R ₃ is hydrogen; R ₄ is a single bond; Each X ₁ and X ₂ is O; X ₃ is N. In another embodiment of the invention, each R ₁ and R ₂ is independently C _2-4 alkyl; R ₃ is hydrogen; R ₄ is a single bond; Each X ₁ and X ₂ is O; X ₃ is N; R ₅ is phenyl substituted with R _a .

In other embodiments of the invention, each R ₁ and R ₂ is C _2-4 alkyl; R ₃ is hydrogen; R ₄ is a single bond; Each X ₁ and X ₂ is O; X ₃ is N; R ₅ is phenyl substituted with R _a ; R _a is

(a) C _1-6 alkyl or C _2-6 alkenyl, each unsubstituted or amino, monoalkylamino, dialkylamino, substituted or unsubstituted heterocyclylaminocarbonyl, substituted or unsubstituted Substituted with one or more substituents selected from the group consisting of heterocyclyl, R _b- , and R _b -alkoxy-; And

(b) alkoxycarbonylalkylamino, R _b -alkoxy-, cyano, substituted or unsubstituted heterocyclyl, and hydroxy

It is selected from the group consisting of.

In another embodiment of the invention, each R ₁ and R ₂ is C _2-4 alkyl; R ₃ is hydrogen; R ₄ is a single bond; Each X ₁ and X ₂ is O; X ₃ is N; R ₅ is phenyl substituted with R _a ; R _a is cyano.

In some embodiments of the invention, each R ₁ and R ₂ is independently C _2-4 alkyl; R ₃ is hydrogen; R ₄ is a single bond; Each X ₁ and X ₂ is O; X ₃ is N; R ₅ is Wherein R ₅ is unsubstituted, or

(a) C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl groups are each unsubstituted or amino, monoalkylamino, dialkyl Amino, substituted or unsubstituted heterocyclylaminocarbonyl, (amino) (R _b ) acylhydrazinylcarbonyl-, (amino) (R _b ) acyloxycarboxy-, (hydroxy) (carbonalkoxy) alkyl Carbamoyl, acyloxy, aldehyde, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylaminoalkylamino, dialkylaminoalkylamino, alkylphosphono, alkylsulfonylamino, carbamoyl, R _b- , R _b -alkoxy-, R _b -alkylamino-, cyano, cyanoalkylcarbamoyl, cycloalkylamino, dialkylaminoalkylamino, dialkylphosphono, haloalkylsulfonylamino, heterocyclylalkylamino, hetero Cyclylcarbamoyl, hydroxy, hydroxyalkylsulfonylamino, oxymino, phosph Furnace, substituted or unsubstituted aralkylamino, substituted or unsubstituted arylcarboxyalkoxycarbonyl, substituted or unsubstituted heteroarylsulfonylamino, substituted or unsubstituted heterocyclyl, thiocarbamoyl, and trifluor Substituted with one or more substituents selected from the group consisting of rommethyl; And

(b) (alkoxycarbonyl) aralkylcarbamoyl, aldehyde, alkenoxy, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylcarbamoyl, alkoxycarbonylamino, alkoxycarbonylalkylamino, alkylsulfonyl Amino, alkylsulfonyloxy, amino, aminoalkylaralkylcarbamoyl, aminoalkylcarbamoyl, aminoalkylheterocyclylalkylcarbamoyl, aminocycloalkylalkylcycloalkylcarbamoyl, aminocycloalkylcarbamoyl, They are alkoxycarbonylamino, aryl, heterocyclyl, aryloxy, aryl-sulfonylamino, aryl sulfonyloxy, carbamoyl, carbonyl, R _b -, R _b - alkoxy -, R _b - alkylthio -, R _b -Alkyl (alkyl) amino-, R _b -alkyl (alkyl) carbamoyl-, R _b -alkylamino-, R _b -alkylcarbamoyl-, R _b -alkylsulfonyl-, R _b -alkylsulfonyl Amino, R _b -alkylthio, R _b -heterocyclylcarbonyl, aminoalkylaminocarbo Nyl, dialkylaminoalkylamino, alkylaminoalkylamino, cyano, cycloalkylamino, dialkylaminoalkylcarbamoyl, halogen, heterocyclylalkylamino, hydroxy, oxymino, phosphate, substituted or unsubstituted Substituted with one or more R _a groups selected from the group consisting of alkylamino, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylsulfonylamino, sulfoxyacylamino, and thiocarbamoyl.

In another embodiment of the invention, each R ₁ and R ₂ is C _2-4 alkyl; R ₃ is hydrogen; R ₄ is a single bond; Each X ₁ and X ₂ is O; X ₃ is N; R ₅ is Is; Wherein R ₅ is unsubstituted, or

(a) C _1-6 alkyl or C _2-6 alkenyl, each unsubstituted or amino, monoalkylamino, dialkylamino, substituted or unsubstituted heterocyclylaminocarbonyl, substituted or unsubstituted Substituted with one or more substituents selected from the group consisting of heterocyclyl, R _b- , and R _b -alkoxy-; And

(b) alkoxycarbonylalkylamino, R _b -alkoxy-, cyano, substituted or unsubstituted heterocyclyl, and hydroxy.

It is substituted with one or more R _a selected from the group consisting of.

In another embodiment, each R ₁ and R ₂ is C _2-4 alkyl; R ₃ is hydrogen; R ₄ is a single bond; Each X ₁ and X ₂ is O; X ₃ is N; R ₅ is Is; Wherein R ₅ is unsubstituted or substituted with one or more R _a groups selected from the group consisting of C _2-5 alkyl substituted with one or more substituents selected from the group consisting of amino, monoalkylamino, and dialkylamino.

In some embodiments of the invention, each R ₁ and R ₂ is C _2-4 alkyl; R ₃ is hydrogen; R ₄ is a single bond; Each X ₁ and X ₂ is O; X ₃ is N; R ₅ is Is; Wherein R ₅ is unsubstituted, or

(a) C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl groups are each unsubstituted or amino, monoalkylamino, dialkyl Amino, substituted or unsubstituted heterocyclylaminocarbonyl, (amino) (R _b ) acylhydrazinylcarbonyl-, (amino) (R _b ) acyloxycarboxy-, (hydroxy) (carbonalkoxy) alkyl Carbamoyl, acyloxy, aldehyde, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylaminoalkylamino, dialkylaminoalkylamino, alkylphosphono, alkylsulfonylamino, carbamoyl, R _b- , R _b -alkoxy-, R _b -alkylamino-, cyano, cyanoalkylcarbamoyl, cycloalkylamino, dialkylphosphono, haloalkylsulfonylamino, heterocyclylalkylamino, heterocyclylcarbamoyl, Hydroxy, hydroxyalkylsulfonylamino, oxymino, phosphono, substituted or unsubstituted Selected from the group consisting of lealkylamino, substituted or unsubstituted arylcarboxyalkoxycarbonyl, substituted or unsubstituted heteroarylsulfonylamino, substituted or unsubstituted heterocyclyl, thiocarbamoyl, and trifluoromethyl Substituted with one or more substituents;

(b) (alkoxycarbonyl) aralkylcarbamoyl, aldehyde, alkenoxy, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylcarbamoyl, alkoxycarbonylamino, alkoxycarbonylalkylamino, alkylsulfonyl Amino, alkylsulfonyloxy, amino, aminoalkylaralkylcarbamoyl, aminoalkylcarbamoyl, aminoalkylheterocyclylalkylcarbamoyl, aminocycloalkylalkylcycloalkylcarbamoyl, aminocycloalkylcarbamoyl, They are alkoxycarbonylamino, aryl, heterocyclyl, aryloxy, aryl-sulfonylamino, aryl sulfonyloxy, carbamoyl, carbonyl, R _b -, R _b - alkoxy -, R _b - alkylthio -, R _b -Alkyl (alkyl) amino-, R _b -alkyl (alkyl) carbamoyl-, R _b -alkylamino-, R _b -alkylcarbamoyl-, R _b -alkylsulfonyl-, R _b -alkylsulfonyl Amino, R _b -alkylthio, R _b -heterocyclylcarbonyl, aminoalkylaminocarbo Nyl, dialkylaminoalkylamino, alkylaminoalkylamino, cyano, cycloalkylamino, dialkylaminoalkylcarbamoyl, halogen, heterocyclylalkylamino, hydroxy, oxymino, phosphate, substituted or unsubstituted Alkylamino, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylsulfonylamino, sulfoxyacylamino, and thiocarbamoyl

It is substituted with at least one R _a group selected from the group consisting of.

In another embodiment of the invention, each R ₁ and R ₂ is C _2-4 alkyl; R ₃ is hydrogen; R ₄ is a single bond; Each X ₁ and X ₂ is O; X ₃ is N; R ₅ is Is; Wherein R ₅ is unsubstituted, or

(a) C _1-6 alkyl or C _2-6 alkenyl [unsubstituted or amino, monoalkylamino, dialkylamino, substituted or unsubstituted heterocyclylaminocarbonyl, substituted or unsubstituted hetero, respectively Substituted with one or more substituents selected from the group consisting of cyclyl, R _b- , and R _b -alkoxy-; And

(b) alkoxycarbonylalkylamino, R _b -alkoxy-, cyano, substituted or unsubstituted heterocyclyl, and hydroxy

It is substituted with at least one R _a group selected from the group consisting of.

In another embodiment of the invention, each R ₁ and R ₂ is C _2-4 alkyl; R ₃ is hydrogen; R ₄ is a single bond; Each X ₁ and X ₂ is O; X ₃ is N; R ₅ is Is; Wherein R ₅ is unsubstituted, or

(a) C _1-4 alkyl or C _2-4 alkenyl, each unsubstituted or amino, monoalkylamino, dialkylamino, substituted or unsubstituted heterocyclylaminocarbonyl, substituted or unsubstituted Substituted with one or more substituents selected from the group consisting of heterocyclyl, and R _b- ; And

(b) R _b -alkoxy- and substituted heterocyclyl

It is substituted with at least one R _a group selected from the group consisting of.

In some embodiments of the invention, each R ₁ and R ₂ is propyl; R ₃ is hydrogen; R ₄ is a single bond; R ₅ is one or more R _a groups, Phenyl substituted with wherein the bicyclic or tricyclic group is unsubstituted or substituted with one or more R _a groups;

R _a is

(a) C _1-6 alkyl or C _2-6 alkenyl, each unsubstituted or amino, monoalkylamino, dialkylamino, substituted or unsubstituted heterocyclylaminocarbonyl, R _b- , R _b -alkoxy- and at least one substituent selected from the group consisting of substituted or unsubstituted heterocyclyl); And

(b) alkoxycarbonylalkylamino, cyano, and hydroxy

Selected from the group consisting of;

Each X ₁ and X ₂ is O; X ₃ is N.

In a preferred embodiment, the compound of formula (I) used in the process of the invention is 3- [4- (2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H- Furin-8-yl) -bicyclo [2.2.2] oct-1-yl] -propionic acid.

In some embodiments, the A _2b adenosine receptor antagonist is administered to a human.

In some embodiments, the A _2b adenosine receptor antagonist used in the methods of the invention is combined with a pharmaceutically acceptable carrier into a pharmaceutically acceptable composition.

The present invention is useful for the treatment of patients who experience or have an ischemic event imminent. Examples of ischemic events include acute coronary syndromes (eg myocardial infarction), seizures, organ transplants, kidney ischemia, shock and organ transplant surgery.

In some embodiments, the methods of the present invention comprise administering an A _2b adenosine receptor antagonist within 2 days before or after an ischemic event. In another embodiment, the method comprises administering an A _2b adenosine receptor antagonist within two days after the ischemic event.

In some embodiments, the compounds used in the methods of the invention exhibit an affinity for the A _2b adenosine receptor at least 10 times greater than the affinity for the A _2a adenosine receptor or the A ₃ adenosine receptor. In another embodiment, the compounds used in the methods of the invention also exhibit an affinity for the A ₁ adenosine receptor at least 10 times greater than the affinity for the A _2a adenosine receptor or the A ₃ adenosine receptor.

In some embodiments, the compounds used in the methods of the present invention have a K _i value for the A _2b adenosine receptor of less than 500 nM. In another embodiment, the compounds used in the methods of the invention have a K _i value for the A _2b adenosine receptor of less than 200 nM.

In some embodiments, the invention is the activation of, A _2b adenosine receptor, which comprises administering to the mammal an active in the treatment of a disease or disorder mediated by of an effective amount of a compound of formula I as described above A _2b adenosine receptors required A method of treating a disease or disorder mediated by

In some embodiments, the invention relates to a method of experiencing an ischemic event using an A _2b adenosine receptor antagonist or to limiting tissue necrosis due to an ischemic event in a mammal in which the ischemic event is imminent.

In some embodiments, the present invention relates to a method of experiencing myocardial infarction using an A _2b adenosine receptor antagonist or to limiting infarct size after myocardial infarction in a mammal that is imminent.

1. Animal Models and General Procedures

This study was performed in a barbital anesthetized dog with a chest opening equipped with equipment for measuring heart rate, blood pressure, left ventricular pressure, and local myocardial blood flow (radioactive microspheres). Mechanical occlusion was placed around the front end of the left anterior descending coronary artery to produce ischemia and reperfusion. At the end of the experiment, infarct size was measured by histochemical staining (patent blue dye and triphenyltetrazolium) and expressed as a percentage of the total left ventricle or the area of risk.

2. Pretreatment Experiment Protocol

In the pretreatment protocol (see FIG. 1, Protocol I), dogs were subjected to 60 minutes of coronary artery occlusion and 3 hours of reperfusion, followed by cardiac extraction and infarct size evaluation. Four groups of dogs were randomly assigned to CPX (8-cyclopentyl-1,3-dipropyl-3,7-dihydro-purine-2,6-dione), BG 9719 (8- (2S-5,6-exo). -Epoxyendo-norbor-2-en-yl) -1,3-dipropyl-3,7-dihydro-purine-2,6-dione), or BG 9928 (3- [4- (2, 6-dioxo-1,3-dipropyl-2,3,6,7-terahydro-1H-purin-8-yl) -bicyclo [2.2.2] oct-1-yl] -propionic acid) Administration was performed 10 minutes before occlusion. All antagonists are i.v. It was administered at 1 mg / kg as a pill and then continued infusion at 10 μg / kg / min until just before reperfusion (70 min total).

There were no significant differences in systemic hemodynamic parameters (heart rate and blood pressure), maximum left ventricular dP / dt, or local myocardial blood flow between the four groups (see Tables 1, 4 and 5), indicating that hemodynamic variables were influenced by antagonists. It is to prove that you do not receive. In addition, there was no difference in the portion of the left ventricle that had ischemia during coronary obstruction (hazard area size; However, infarct size, expressed as a percentage of the risk area (FIG. 1B) or the percentage of the left ventricle (FIG. 1C), was significantly smaller in two groups of dogs treated with CPX (51% reduction) or BG 9928 (49% reduction). All. Infarct size in the group of dogs to be treated with BG 9928 was similar to the infarct size of the control group. If infarct size, expressed as a percentage of the risk area, is plotted against the transverse lateral blood flow (FIG. 1D), the inverse relationship is evident by linear regression analysis. In the CPX-treated group and the BG 9928-treated group, this relationship shifted downward relative to the control, which means that the infarct size is smaller in these two groups at any given lateral blood flow. The relationship between infarct size and lateral blood flow was similar between the control and BG 9719-treated groups. Thus, treatment with CPX or BG 9928 (but not BG 9719) prior to occlusion resulted in a significant reduction in infarct size independent of systemic hemodynamic parameters or changes in local lateral blood flow.

TABLE 1

Hemodynamic Parameters of Protocol I (Pretreatment)

HR = heart rate; MBP = mean arterial blood pressure; LVdP / dt = maximum left ventricular dP / dt.

3. Preconditioning Experiment Protocol

In the preconditioning protocol (FIG. 2, Protocol II), all dogs underwent 60 minutes of coronary artery occlusion and subsequent 3 hours of reperfusion. Preconditioning was performed by four 5 minute occlusion / 5 minute reperfusion cycles prior to 60 minute occlusion 10 minutes. Four groups of dogs were randomly administered vehicle, CPX, BG 9719 or BG 9928 for the first 10 minutes prior to initial preconditioning occlusion. Antagonist 1 mg / kg i.v. The dose of the pill was administered, followed by injection at 10 μg / kg / min for 1 hour (115 minutes total).

Similar to the pretreatment group, no significant difference in systemic hemodynamics, local myocardial blood flow or risk area size was found between the four groups in the preconditioning protocol (Tables 2, 4 and 5, FIG. 2A). Four five-minute occlusion / 5-minute reperfusion cycles prior to 60-minute occlusion significantly reduced infarct size (˜65% reduction) compared to the non-conditioned control in Protocol I (FIGS. 2B and 2C). In the group of dogs treated with adenosine receptor antagonists, the mean infarct size (expressed as the risk area or the left ventricle) was significantly smaller than the non-preconditioned control, similar or slightly smaller than the preconditioned control (FIG. 2B). And 2C). Preconditioning shifted down the relationship between infarct size and lateral blood flow compared to the non-preconditioned control (FIG. 2D). This relationship shifted further down in the group of dogs treated with CPX or BG 9928, but this phenomenon was not observed by BG 9719. As can be seen from these results, treatment with CPX, BG 9719 or BG 9928 did not block the protective effect of ischemic preconditioning caused by multiple occlusion / reperfusion cycles. The results also suggest that treatment with CPX or BG 9928 (but not BG 9719) assists in the protective effect of ischemic preconditioning.

TABLE 2

Hemodynamic Variables in Protocol II (Preconditioning)

HR = heart rate; MBP = mean arterial blood pressure; LVdP / dt = maximum left ventricular dP / dt.

4. Reperfusion Experimental Protocol

In the reperfusion protocol (see Figure 3, Protocol III), the dogs were subjected to coronary artery occlusion for 60 minutes followed by reperfusion for 3 hours. Four groups of dogs were randomly administered vehicle, CPX, BG 9719 or BG 9928 for the first 10 minutes prior to alleviation of occlusion. Antagonist 1 mg / kg i.v. It was administered at a dose of the pill and then injected at 10 μg / kg / min for 1 hour.

No significant difference in hemodynamic parameters, local myocardial blood flow or risk zone size was found between the four groups of dogs in the experimental protocol (Tables 3-5 and FIG. 3A). Infarct size, expressed as a percentage of the risk area, was significantly reduced by administration of CPX or BG 9928 during initial reperfusion (FIG. 3B). However, no protective effect was found upon administration of BG 9719. The relationship between infarct size and lateral blood flow shifted downward in two groups of dogs treated with CPX or BG 9928 compared to the control (FIG. 3C). The reduction in infarct size produced by CPX and BG 9928 in this protocol was smaller in size compared to Protocol I, where they were administered prior to ischemia (42% and 44%, respectively), indicating data as a percentage of the overall left ventricle. No significant reduction in case infarct size was observed (FIG. 3D), probably because a few animals were studied. As can be seen from these data, CPX and BG 9928 (but not BG 9719) reduced infarct size when administered upon reperfusion.

TABLE 3

Hemodynamic Parameters of Protocol III (Reperfusion)

HR = heart rate; MBP = mean arterial blood pressure; LVdP / dt = maximum left ventricular dP / dt.

TABLE 4

Local myocardial blood flow data (ml / min / gm) of protocols I, II and III in the non-ischemic region (area perfused by left line coronary artery)

epi = outer pericardium; mid = central membrane; endo = intima; trans (mural) = transverse wall

TABLE 5

Local myocardial blood flow data (ml / min / gm) of protocols I, II, and III in ischemic reperfused regions (area perfused by left anterior descending coronary arteries)

epi = outer pericardium; mid = central membrane; endo = intima; trans (mural) = transverse wall

TABLE 6

Dissociation Constants of Antagonists for Recombinant Dog A ₁ , A _2a and A ₃ Adenosine Receptors Measured by Radioligand Binding Assay

compound A ₁ A _2a A ₃ CPX 18.1 ± 4.4 162 ± 22 1960 ± 420 BG 9719 35.8 ± 4.0 2820 ± 268 19070 ± 540 BG 9928 28.9 ± 4.1 4307 ± 1230 37670 ± 9030

Ki values obtained from competition binding experiments using membranes of transfected HEK 293 cells using ³ H-CPX, ³ H-ZM 241385 and ³ R-PIA as radioligands for A ₁ , A _2a and A ₃ receptors (nM ± SEM; n = 3)

5. Membrane Manufacturing

HEK 293 (human embryonic kidney) membranes expressing human A _2b adenosine receptors were purchased from Receptor Biology; HEK 293 cell membranes expressing human A _2a receptor were purchased from Perkin Elmer (Boston, Mass.); CHO-K1 cell membranes expressing human A ₁ receptor and HEK 293 cell membranes expressing human A ₃ receptor were prepared from corresponding stably transfected cells established in Korea.

6. Radioligand binding assay

Membranes (40-70 μg membrane protein), radioligands and variable concentrations of competing ligands were incubated three times for 2.5 hours in 0.1 ml of buffer HE + 2 units / ml of adenosine deaminase and at 21 ° C. Radioligands used for competitive binding assays are as follows: [ ³ H] -8-cyclopentyl-1,3-dipropylxanthine ([ ³ H] -DPCPX) (NEM) for A ₁ and A _2b adenosine receptors , Boston, MA), a _2a adenosine receptor ^{[3 H] -4- (2- [} 7- amino-2- (furyl) (1,2,4) on-triazole (2,3-a) ( 1,3,5) triazine-5-ylaminoethylyl) phenol ([ ³ H] ZM241385) (Tocris, Bristol, UK) and [ ¹²⁵ Iodine] -labeled N6- (4- for A ₃ adenosine receptors Aminobenzyl) -9- (5- (methylcarbonyl) -β-D-ribofuranosyl) adenine ([ ¹²⁵ I] -AB-MECA) or [ ³ H] -R-N6-phenylisopropyladenosine ([ ³ H] -R-PIA) (both NEM, Boston, MA). Non-specific binding is 10 μM of 5'N-ethylcarboxamidoadenosine (NECA; RBI-Sigma, Natick, MA) for A ₁ and A _2b receptors or 10 μM xanthine amino conjugate (XAC, RBI for A _2a receptors) Measured in the presence of Sigma, Natrick, MA). Binding assays were terminated by filtration through Whatman GF / C glass fiber filters using a BRANDEL cell collector (Gaithersburg, MD). The filter was washed with 3-4 ml of ice cold 10 mM Tris-HCl, pH 7.4 and 5 mM magnesium chloride (MgCl ₂ ) at 4 ° C. and counted on Wallac β-counter (Perkin Elmer, Boston, Mass.).

TABLE 7

K _I value (nM) or percent inhibition (%) at 10 μM antagonist during radioligand competition binding assay

K _I value (nM) or percent inhibition (%) at 10 μM antagonist during radioligand competition binding assay Adenosine receptors A ₁ A _2a A _2b A ₃ BG 9928 12.2 4059 88.53 ± 21.03 ^a 30% ^b DPCPX 5.3 156 ^c 56 262 BG9719 10.3 9152 853 ± 270 ^a 40.6%

ND: Do not perform

a: N = 3

b: inhibition at 10 μM BG 9928

c: J. Linden, Annu. Rev. Pharmacol. Toxicol. , 41, pp. See 775-787 (2001).

In competitive binding assays using recombinant human A ₁ adenosine receptor and [ ³ H] -DPCPX as radioligand, K _I for BG 9928, DPCPX and BG 9717 were 12.2 nM, 5.3 nM and 10.3 nM, respectively (see Table 7 and 4). In competitive binding assays using recombinant human A _2a adenosine receptor and [ ³ H] -ZM241385 as radioligand, the K _I for BG 9928, DPCPX and BG 9717 were 4059 nM, 156 nM and 9152 nM, respectively (see Table 7, 5). In competitive binding assays using recombinant human A _2b adenosine receptor and [ ³ H] -ZM241385 as radioligand, K _I for BG 9928, DPCPX and BG 9717 was 88.53 ± 21.03 nM (N = 3), 56 nM and 853, respectively. ± 270 nM (N = 3) (see Table 7, FIG. 6).

One-point binding assays were performed to determine the effect of 10 μM BG 9928 on binding of [ ¹²⁵ I] -AB-MECA to recombinant human A ₃ adenosine receptor membrane. In a single point binding assay using recombinant human A ₃ adenosine receptors, 10 μM BG 9928 resulted in 30% inhibition of [ ³ H] -ZM241385 binding (FIG. 7).

7. Radioligand binding assay

Membranes (50 μg membrane protein), radioligands and various concentrations of competing ligands were incubated three times for two hours at 21 ° C. and in 0.1 ml of buffer HE + 2 units / ml of adenosine deaminase. Radioligands used for competitive binding assays for human A _2b adenosine receptors were [ ³ H] -8-cyclopentyl-1,3-dihydroxyxanthine ([ ³ H] -DPCPX, 30-40 nM) (NEN, Boston, MA). Nonspecific binding was measured in the presence of 10 μM of 5′N-ethylcarboxymidoadenosine (NECA; RBI-Sigma, Natick, MA). Binding assays were terminated by filtration through Whatman GF / C glass fiber filters using a BRANDEL cell collector (Gaithersburg, MD). The filter was washed with 3-4 ml of ice cold 10 mM Tris-HCl, pH 7.4 and 5 mM magnesium chloride (MgCl ₂ ) at 4 ° C. and counted on Wallac β-counter (Perkin Elmer, Boston, Mass.).

Competition binding data was fitted to a single site binding model and plotted using Prizm GraphPad. K _I values were calculated from IC ₅₀ values using the Cheng-Prusoff equation K _I = IC ₅₀ / (l + [I] / K _D ), where K _I is the affinity constant for the competing ligand, [ I] is the concentration of free radioligand and KD is the affinity constant for the radioligand (Cheng and Prusoff 1973). K _I values of some compounds of the invention are provided in Table 8.

TABLE 8

K _I (nM) in Radioligand Competitive Binding Assay

8. Fluorescence Imaging Plate Reader (FLIPR) Functional Analysis

Fluorescence Imaging Plate Reader (FLIPR) Assay for Calcium Calculation in Human and Rat A _2b HEK293 cells showing stable expression of adenosine receptors and CHO-K1 cells showing stable expression of recombinant human A ₁ adenosine receptors were performed. Cells were seeded into 96 well tissue culture plates (with black walls and transparent bottoms) and incubated to 80-90% confluent monolayers. Without removing the medium, the same volume of dye (calcium assay kit purchased from Molecular Devices) was added. Cell plates were incubated for 1 hour at 37 ° C. and then transferred to FLIPR units (Molecular Devices).

For analysis of the recombinant human A ₁ adenosine receptor, CHO-K1 cells were incubated with increasing dose of agonist (N6-cyclopentyladenosine, CPA) to determine the concentration of agent that produced 50% of the maximal response. This concentration of agonist (200 nM CPA) was then incubated with increasing concentrations of antagonist BG 9928 (10 ⁻¹² to 10 ⁻¹⁵ M). For analysis of recombinant human and rat A _2b adenosine receptors, HEK-293 cells were incubated with increasing doses of the agent (5'N-ethylcarboxamidoadenosine, NECA) to produce 50% of the maximal response. The concentration of agent was determined. This concentration of agonist 5 μM NECA for human A _2b receptor) or various variable concentrations (for rat A _2b receptor) was incubated with increasing concentrations of antagonist BG 9928 (for human A _2b receptor). 10 ^-12 M to 5 x 10 ^-6 M and 10, 1100 or 300 nM for rat A ^2b receptor).

FLIPR was integrated with a detection system using an argon laser excitation source, a 96-well pipette and a charge coupled device (CCD) imaging camera. Fluorescence emission from the 96 wells was simultaneously monitored at excitation wavelengths and emission wavelengths at 488 nm and 520 nm, respectively. Fluorescence data was collected at 1 second intervals before and after rapid simultaneous addition of compounds to 96 well plates. The results were read in relative fluorescence units (RFU).

FLIPR function analysis was performed using BG 9928 using recombinant human A ₁ adenosine receptor stably expressed in CHO-K1 cells. The antagonist dissociation constants (K _B ) for BG 9928 and BG9719 were 0.60 nM and 0.46 nM, respectively, as determined by the null method at recombinant human A1 adenosine receptors (Table 9 and FIG. 8).

FLIPR function analysis was performed with BG 9928 using recombinant human A2b adenosine receptor stably expressed in HEK293 cells. The antagonists K _B for BG 9928, BG9719 and DPCPX were 3.36 nM, 182 nM and 23.6 nM, respectively, as determined by zero method at the recombinant human A _2b adenosine receptor (see Tables 9 and 9).

FLIPR function analysis was performed using BG 9928 using recombinant rat A _2b adenosine receptor stably expressed in HEK293 cells. The antagonist K _B against BG 9928 was 257 nM as measured by zero method and 6.59 as measured by Schild analysis (see Table 9 and FIG. 10).

TABLE 9

Summary of K _B (nM) values for antagonists in the FLIPR function analysis (human receptor subtype)

Bell K _B (nM) values for antagonists in LIPR functional analysis Adenosine receptors A ₁ A _2a A _2b A ₃ BG 9928 0.60 ND 3.36 ND BG9719 0.16 ND 182 ND DPCPX ND ND 23.6 ND

ND: not measured

9. Data Analysis

Data are expressed as mean ± standard error of the mean (SEM) or standard deviation (SD). Saturation data were analyzed using Marquardt's nonlinear least-squares method and plotted using Prizm GraphPad. Competitive binding data was fitted to a single quartile binding model and plotted using Prizm GraphPad. K _I values were calculated from IC ₅₀ values using the Cheng-Prusoff equation K _I = IC ₅₀ / (l + [I] / K _D ), where K _I is the affinity constant for the competing ligand, [ I] is the concentration of free radioligand and K _D is the affinity constant for the radioligand (Cheng and Prusoff 1973).

In the FLIPR function analysis, the agent concentration-response curves were fitted to logarithmic equations using a nonlinear regression program on the Prizm GraphPad. Antagonist dissociation constants (K _B ) were estimated by the zero method developed by Lazareno and Roberts (1987). Schild analysis was performed to estimate the compound's ability as an antagonist (pA ₂ ). pA ₂ is the negative logarithm of the concentration of the antagonist which can exhibit a two-fold shift in the concentration-response curve (response defined as 50% of the maximum response).

Claims (37)

Identifying a mammal that has experienced an ischemic event or a mammal that is near to an ischemic event; And Administering to said mammal a therapeutically effective amount or a prophylactically effective amount of an A _2b adenosine receptor antagonist within 10 days before or after an ischemic event. As a method for preventing, limiting or treating ischemia reperfusion injury of a mammal comprising: A _2b adenosine receptor antagonist is a compound of formula (I) or a pharmaceutically acceptable salt or N-oxide thereof. Formula I Where R ₁ , R ₂ , and R ₃ are each independently a) hydrogen; b) C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl is unsubstituted or hydroxy, alkoxy, amino, monoalkylamino Substituted with one or more substituents selected from the group consisting of, dialkylamino, cycloalkyl, aryl, heterocyclyl, aralkyl, heterocyclylalkyl, acylamino, alkylaminocarbonyl, alkylsulfonylamino, and alkylaminosulfonyl being]; c) substituted or unsubstituted aryl; or d) substituted or unsubstituted heterocyclyl; R ₄ is a single _{bond, -0-, - (CH 2)} 1-3 -, -0 (CH 2) 1-2 -, -CH 2 OCH 2 -, - (CH 2) 1-2 0-, - CH = CHCH _2- , -CH = CH-, or -CH ₂ CH = CH-; R ₅ is (a) phenyl, or (b) A bicyclic or tricyclic group selected from the group consisting of wherein the phenyl, bicyclic or tricyclic group is unsubstituted, or (a) C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl groups are each unsubstituted or amino, monoalkylamino, dialkyl Amino, substituted or unsubstituted heterocyclylaminocarbonyl, (amino) (R _b ) acylhydrazinylcarbonyl-, (amino) (R _b ) acyloxycarboxy-, (hydroxy) (carbonalkoxy) alkyl Carbamoyl, acyloxy, aldehyde, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylaminoalkylamino, dialkylaminoalkylamino, alkylphosphono, alkylsulfonylamino, carbamoyl, R _b- , R _b -alkoxy-, R _b -alkylamino-, cyano, cyanoalkylcarbamoyl, cycloalkylamino, dialkylphosphono, haloalkylsulfonylamino, heterocyclylalkylamino, heterocyclylcarbamoyl, Hydroxy, hydroxyalkylsulfonylamino, oxymino, phosphono, substituted or unsubstituted Selected from the group consisting of lealkylamino, substituted or unsubstituted arylcarboxyalkoxycarbonyl, substituted or unsubstituted heteroarylsulfonylamino, substituted or unsubstituted heterocyclyl, thiocarbamoyl, and trifluoromethyl Substituted with one or more substituents); And (b) (alkoxycarbonyl) aralkylcarbamoyl, aldehyde, alkenoxy, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylcarbamoyl, alkoxycarbonylamino, alkoxycarbonylalkylamino, alkylsulfonyl Amino, alkylsulfonyloxy, amino, aminoalkylaralkylcarbamoyl, aminoalkylcarbamoyl, aminoalkylheterocyclylalkylcarbamoyl, aminocycloalkylalkylcycloalkylcarbamoyl, aminocycloalkylcarbamoyl, They are alkoxycarbonylamino, aryl, heterocyclyl, aryloxy, aryl-sulfonylamino, aryl sulfonyloxy, carbamoyl, carbonyl, R _b -, R _b - alkoxy -, R _b - alkylthio -, R _b -Alkyl (alkyl) amino-, R _b -alkyl (alkyl) carbamoyl-, R _b -alkylamino-, R _b -alkylcarbamoyl-, R _b -alkylsulfonyl-, R _b -alkylsulfonyl Amino, R _b -alkylthio, R _b -heterocyclylcarbonyl, aminoalkylaminocarbo Nyl, dialkylaminoalkylamino, alkylaminoalkylamino, cyano, cycloalkylamino, dialkylaminoalkylcarbamoyl, halogen, heterocyclylalkylamino, hydroxy, oxymino, phosphate, substituted or unsubstituted Alkylamino, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylsulfonylamino, sulfoxyacylamino, and thiocarbamoyl Is substituted with one or more R _a groups selected from the group consisting of: In this case, R _b is -COOH, -C (CF ₃ ) ₂ 0H, -CONHNHSO ₂ CF ₃ , -CONHOR _c , -CONHS0 ₂ R _c , -CONHSO ₂ NHR _c , -C (OH) R _c PO ₃ H ₂ , -NHCOCF ₃ , -NHCONHSO ₂ R _c , -NHPO ₃ H ₂ , -NHSO ₂ R _c , -NHSO ₂ NHCOR _c , -OPO ₃ H ₂ , -OS0 ₃ H, -PO (OH) R _c , -PO ₃ H ₂ , -SO ₃ H, -SO ₂ NHR _c , -SO ₃ NHCOR _c , -SO ₃ NHCONHCO ₂ R _c , and the following compounds Selected from the group consisting of; R _c is selected from the group consisting of hydrogen, —C _1-4 alkyl, —C _1-4 alkyl-CO ₂ H, and phenyl, wherein —C _1-4 alkyl, —C _1-4 alkyl-CO ₂ H, and phenyl group are unsubstituted or consist of halogen, -OH, -OMe, -NH ₂ , -NO ₂ , unsubstituted benzyl, and halogen, -OH, -OMe, -NH ₂ , and -NO ₂ Substituted with 1 to 3 substituents selected from the group consisting of benzyl substituted with 1 to 3 substituents selected from the group; X ₁ and X ₂ are independently selected from the group consisting of O and S; X ₃ is N or CR _d , where R _d is a) hydrogen; b) C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl is unsubstituted or hydroxy, alkoxy, amino, monoalkylamino Substituted with one or more substituents selected from the group consisting of, dialkylamino, cycloalkyl, aryl, heterocyclyl, aralkyl, heterocyclylalkyl, acylamino, alkylaminocarbonyl, alkylsulfonylamino, and alkylaminosulfonyl being]; c) substituted or unsubstituted aryl; And d) substituted or unsubstituted heterocyclyl It is selected from the group consisting of. The method of claim 1, wherein R ₁ is C _1-6 alkyl. The method of claim 1, wherein R ₂ is C _1-6 alkyl. The method of claim 1, wherein R ₃ is hydrogen. The method of claim 1, wherein R ₄ is a single bond. The method of claim 1, wherein R ₅ is phenyl substituted with R _a . The compound of claim 1, wherein R ₅ is And a substituted bicyclic or tricyclic group selected from the group consisting of: The method of claim 1, R ₅ is Wherein R ₅ is unsubstituted, or (a) C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl groups are each unsubstituted or amino, monoalkylamino, dialkyl Amino, substituted or unsubstituted heterocyclylaminocarbonyl, (amino) (R _b ) acylhydrazinylcarbonyl-, (amino) (R _b ) acyloxycarboxy-, (hydroxy) (carbonalkoxy) alkyl Carbamoyl, acyloxy, aldehyde, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylaminoalkylamino, dialkylaminoalkylamino, alkylphosphono, alkylsulfonylamino, carbamoyl, R _b- , R _b -alkoxy-, R _b -alkylamino-, cyano, cyanoalkylcarbamoyl, cycloalkylamino, dialkylphosphono, haloalkylsulfonylamino, heterocyclylalkylamino, heterocyclylcarbamoyl, Hydroxy, hydroxyalkylsulfonylamino, oxymino, phosphono, substituted or unsubstituted Selected from the group consisting of lealkylamino, substituted or unsubstituted arylcarboxyalkoxycarbonyl, substituted or unsubstituted heteroarylsulfonylamino, substituted or unsubstituted heterocyclyl, thiocarbamoyl, and trifluoromethyl Substituted with one or more substituents; And (b) (alkoxycarbonyl) aralkylcarbamoyl, aldehyde, alkenoxy, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylcarbamoyl, alkoxycarbonylamino, alkoxycarbonylalkylamino, alkylsulfonyl Amino, alkylsulfonyloxy, amino, aminoalkylaralkylcarbamoyl, aminoalkylcarbamoyl, aminoalkylheterocyclylalkylcarbamoyl, aminocycloalkylalkylcycloalkylcarbamoyl, aminocycloalkylcarbamoyl, They are alkoxycarbonylamino, aryl, heterocyclyl, aryloxy, aryl-sulfonylamino, aryl sulfonyloxy, carbamoyl, carbonyl, R _b -, R _b - alkoxy -, R _b - alkylthio -, R _b -Alkyl (alkyl) amino-, R _b -alkyl (alkyl) carbamoyl-, R _b -alkylamino-, R _b -alkylcarbamoyl-, R _b -alkylsulfonyl-, R _b -alkylsulfonyl Amino, R _b -alkylthio, R _b -heterocyclylcarbonyl, aminoalkylaminocarbo Nyl, dialkylaminoalkylamino, alkylaminoalkylamino, cyano, cycloalkylamino, dialkylaminoalkylcarbamoyl, halogen, heterocyclylalkylamino, hydroxy, oxymino, phosphate, substituted or unsubstituted Alkylamino, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylsulfonylamino, sulfoxyacylamino, and thiocarbamoyl And at least one R _a group selected from the group consisting of: The compound of claim 1, wherein R _a is (a) C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl groups are each unsubstituted or amino, monoalkylamino, dialkyl Amino, substituted or unsubstituted heterocyclylaminocarbonyl, (amino) (R _b ) acylhydrazinylcarbonyl-, (amino) (R _b ) acyloxycarboxy-, (hydroxy) (carbonalkoxy) alkyl Carbamoyl, acyloxy, aldehyde, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylaminoalkylamino, dialkylaminoalkylamino, alkylphosphono, alkylsulfonylamino, carbamoyl, R _b- , R _b -alkoxy-, R _b -alkylamino-, cyano, cyanoalkylcarbamoyl, cycloalkylamino, dialkylphosphono, haloalkylsulfonylamino, heterocyclylalkylamino, heterocyclylcarbamoyl, Hydroxy, hydroxyalkylsulfonylamino, oxymino, phosphono, substituted aralkylami , Substituted aryl carboxy alkoxycarbonyl, substituted heteroaryl-sulfonylamino, substituted heterocyclyl, alkylthio carbamoyl, and tri substituted with one or more substituents selected from the group consisting of fluoromethyl; And (b) (alkoxycarbonyl) aralkylcarbamoyl, aldehyde, alkenoxy, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylcarbamoyl, alkoxycarbonylamino, alkoxycarbonylalkylamino, alkylsulfonyl Amino, alkylsulfonyloxy, amino, aminoalkylaralkylcarbamoyl, aminoalkylcarbamoyl, aminoalkylheterocyclylalkylcarbamoyl, aminocycloalkylalkylcycloalkylcarbamoyl, aminocycloalkylcarbamoyl, are alkoxycarbonylamino, aryl, heterocyclyl, aryloxy, aryl-sulfonylamino, aryl sulfonyloxy, carbamoyl, carbonyl, R _b -, R _b - alkoxy -, R _b - alkyl (alkyl) amino- , R _b -alkyl (alkyl) carbamoyl-, R _b -alkylamino-, R _b -alkylcarbamoyl-, R _b -alkylsulfonyl-, R _b -alkylsulfonylamino, R _b -alkylthio , R _b - heterocyclyl-carbonyl, cyano, cycloalkylamino, dialkyl amino Alkyl-carbamoyl, halogen, heterocyclyl-alkyl, amino, hydroxy, oxy Mino, phosphate, substituted aralkyl, amino, substituted heterocyclyl, substituted heterocyclyl rilseol -5-, sulfonic Foxy acylamino and alkylthio carbamoyl The method is selected from the group consisting of. The compound of claim 1, wherein R _a is (a) C _1-6 alkyl or C _2-6 alkenyl, each unsubstituted or amino, monoalkylamino, dialkylamino, substituted or unsubstituted heterocyclylaminocarbonyl, R _b- , R _b -alkoxy- and at least one substituent selected from the group consisting of substituted or unsubstituted heterocyclyl; (b) alkoxycarbonylalkylamino, cyano, and hydroxy The method is selected from the group consisting of. The method of claim 1, wherein X ₁ is O. The method of claim 1, wherein X ₂ is O. The method of claim 1, wherein X ₃ is N. The compound of claim 1, wherein each R ₁ and R ₂ is C _2-4 alkyl; R ₃ is hydrogen; R ₄ is a single bond; Each X ₁ and X ₂ is O; X ₃ is N. The method of claim 14, wherein R ₅ is phenyl substituted with R _a . The compound of claim 15, wherein R _a is (a) C _1-6 alkyl or C _2-6 alkenyl, each unsubstituted or amino, monoalkylamino, dialkylamino, substituted or unsubstituted heterocyclylaminocarbonyl, substituted or unsubstituted Substituted with one or more substituents selected from the group consisting of heterocyclyl, R _b- , and R _b -alkoxy-; And (b) alkoxycarbonylalkylamino, R _b -alkoxy-, cyano, substituted or unsubstituted heterocyclyl, and hydroxy The method is selected from the group consisting of. The method of claim 16, wherein R _a is cyano. The method of claim 14, R ₅ is Wherein R ₅ is unsubstituted, or (a) C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl groups are each unsubstituted or amino, monoalkylamino, dialkyl Amino, substituted or unsubstituted heterocyclylaminocarbonyl, (amino) (R _b ) acylhydrazinylcarbonyl-, (amino) (R _b ) acyloxycarboxy-, (hydroxy) (carbonalkoxy) alkyl Carbamoyl, acyloxy, aldehyde, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylaminoalkylamino, dialkylaminoalkylamino, alkylphosphono, alkylsulfonylamino, carbamoyl, R _b- , R _b -alkoxy-, R _b -alkylamino-, cyano, cyanoalkylcarbamoyl, cycloalkylamino, dialkylphosphono, haloalkylsulfonylamino, heterocyclylalkylamino, heterocyclylcarbamoyl, Hydroxy, hydroxyalkylsulfonylamino, oxymino, phosphono, substituted or unsubstituted Selected from the group consisting of lealkylamino, substituted or unsubstituted arylcarboxyalkoxycarbonyl, substituted or unsubstituted heteroarylsulfonylamino, substituted or unsubstituted heterocyclyl, thiocarbamoyl, and trifluoromethyl Substituted with one or more substituents; And (b) (alkoxycarbonyl) aralkylcarbamoyl, aldehyde, alkenoxy, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylcarbamoyl, alkoxycarbonylamino, alkoxycarbonylalkylamino, alkylsulfonyl Amino, alkylsulfonyloxy, amino, aminoalkylaralkylcarbamoyl, aminoalkylcarbamoyl, aminoalkylheterocyclylalkylcarbamoyl, aminocycloalkylalkylcycloalkylcarbamoyl, aminocycloalkylcarbamoyl, They are alkoxycarbonylamino, aryl, heterocyclyl, aryloxy, aryl-sulfonylamino, aryl sulfonyloxy, carbamoyl, carbonyl, R _b -, R _b - alkoxy -, R _b - alkylthio -, R _b -Alkyl (alkyl) amino-, R _b -alkyl (alkyl) carbamoyl-, R _b -alkylamino-, R _b -alkylcarbamoyl-, R _b -alkylsulfonyl-, R _b -alkylsulfonyl Amino, R _b -alkylthio, R _b -heterocyclylcarbonyl, aminoalkylaminocarbo Nyl, dialkylaminoalkylamino, alkylaminoalkylamino, cyano, cycloalkylamino, dialkylaminoalkylcarbamoyl, halogen, heterocyclylalkylamino, hydroxy, oxymino, phosphate, substituted or unsubstituted Alkylamino, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylsulfonylamino, sulfoxyacylamino, and thiocarbamoyl And at least one R _a group selected from the group consisting of: The compound of claim 18, wherein R _a is (a) C _1-6 alkyl or C _2-6 alkenyl, each unsubstituted or amino, monoalkylamino, dialkylamino, substituted or unsubstituted heterocyclylaminocarbonyl, substituted or unsubstituted Substituted with one or more substituents selected from the group consisting of heterocyclyl, R _b- , and R _b -alkoxy-; And (b) alkoxycarbonylalkylamino, R _b -alkoxy-, cyano, substituted or unsubstituted heterocyclyl, and hydroxy The method is selected from the group consisting of. 20. The method of claim 19, wherein R _a is C _2-5 alkyl substituted with one or more substituents selected from the group consisting of amino, monoalkylamino, and dialkylamino. The method of claim 14, R ₅ is Is; Wherein R ₅ is unsubstituted, or (a) C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl, wherein the alkyl, alkenyl, or alkynyl groups are each unsubstituted or amino, monoalkylamino, dialkyl Amino, substituted or unsubstituted heterocyclylaminocarbonyl, (amino) (R _b ) acylhydrazinylcarbonyl-, (amino) (R _b ) acyloxycarboxy-, (hydroxy) (carbonalkoxy) alkyl Carbamoyl, acyloxy, aldehyde, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylaminoalkylamino, dialkylaminoalkylamino, alkylphosphono, alkylsulfonylamino, carbamoyl, R _b- , R _b -alkoxy-, R _b -alkylamino-, cyano, cyanoalkylcarbamoyl, cycloalkylamino, dialkylphosphono, haloalkylsulfonylamino, heterocyclylalkylamino, heterocyclylcarbamoyl, Hydroxy, hydroxyalkylsulfonylamino, oxymino, phosphono, substituted or unsubstituted Selected from the group consisting of lealkylamino, substituted or unsubstituted arylcarboxyalkoxycarbonyl, substituted or unsubstituted heteroarylsulfonylamino, substituted or unsubstituted heterocyclyl, thiocarbamoyl, and trifluoromethyl Substituted with one or more substituents; And (b) (alkoxycarbonyl) aralkylcarbamoyl, aldehyde, alkenoxy, alkenylsulfonylamino, alkoxy, alkoxycarbonyl, alkylcarbamoyl, alkoxycarbonylamino, alkoxycarbonylalkylamino, alkylsulfonyl Amino, alkylsulfonyloxy, amino, aminoalkylaralkylcarbamoyl, aminoalkylcarbamoyl, aminoalkylheterocyclylalkylcarbamoyl, aminocycloalkylalkylcycloalkylcarbamoyl, aminocycloalkylcarbamoyl, They are alkoxycarbonylamino, aryl, heterocyclyl, aryloxy, aryl-sulfonylamino, aryl sulfonyloxy, carbamoyl, carbonyl, R _b -, R _b - alkoxy -, R _b - alkylthio -, R _b -Alkyl (alkyl) amino-, R _b -alkyl (alkyl) carbamoyl-, R _b -alkylamino-, R _b -alkylcarbamoyl-, R _b -alkylsulfonyl-, R _b -alkylsulfonyl Amino, R _b -alkylthio, R _b -heterocyclylcarbonyl, aminoalkylaminocarbo Nyl, dialkylaminoalkylamino, alkylaminoalkylamino, cyano, cycloalkylamino, dialkylaminoalkylcarbamoyl, halogen, heterocyclylalkylamino, hydroxy, oxymino, phosphate, substituted or unsubstituted Alkylamino, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylsulfonylamino, sulfoxyacylamino, and thiocarbamoyl And at least one R _a group selected from the group consisting of: The method of claim 21, wherein R _a is (a) C _1-6 alkyl or C _2-6 alkenyl, each of which is unsubstituted or amino, monoalkylamino, dialkylamino, substituted or unsubstituted heterocyclylaminocarbonyl, substituted or unsubstituted Substituted with one or more substituents selected from the group consisting of heterocyclyl, R _b- , and R _b -alkoxy-; And (b) alkoxycarbonylalkylamino, R _b -alkoxy-, cyano, substituted or unsubstituted heterocyclyl, and hydroxy The method is selected from the group consisting of. The method of claim 21, wherein R _a is (a) C _1-4 alkyl or C _2-4 alkenyl, each unsubstituted or amino, monoalkylamino, dialkylamino, substituted or unsubstituted heterocyclylaminocarbonyl, substituted or unsubstituted Substituted with one or more substituents selected from the group consisting of heterocyclyl, and R _b- ; And (b) R _b -alkoxy- and substituted heterocyclyl The method is selected from the group consisting of. The method of claim 1, Each R ₁ and R ₂ is propyl; R ₃ is hydrogen; R ₄ is a single bond; R ₅ is R _a , Phenyl substituted with a bicyclic or tricyclic group optionally substituted with _a R _a group; R _a is (a) C _1-6 alkyl or C _2-6 alkenyl, each unsubstituted or amino, monoalkylamino, dialkylamino, substituted or unsubstituted heterocyclylaminocarbonyl, R _b- , R _b -alkoxy- and at least one substituent selected from the group consisting of substituted or unsubstituted heterocyclyl; And (b) alkoxycarbonylalkylamino, cyano, and hydroxy Is selected from the group consisting of; Each X ₁ and X ₂ is O; X ₃ is N. The compound of claim 1, wherein the compound of formula I is 3- [4- (2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl) -Bicyclo [2.2.2] oct-1-yl] -propionic acid. The method of claim 1, wherein the ischemic event is selected from the group consisting of acute coronary syndrome, seizures, organ transplants, kidney ischemia, shock and organ transplant surgery. The method of claim 26, wherein the acute coronary syndrome is myocardial infarction. The method of claim 1, wherein the A _2b adenosine receptor antagonist is administered within 2 days before or after an ischemic event. The method of claim 28, wherein the A _2b adenosine receptor antagonist is administered within two days after the ischemic event. The method of claim 1, wherein the mammal is a human. The method of claim 1, wherein the compound of formula I exhibits affinity for the A _2b adenosine receptor at least 10 times greater than the affinity for the A _2a adenosine receptor or the A ₃ adenosine receptor. 32. The method of claim 31, wherein the compound of formula (I) further exhibits an affinity for the A ₁ adenosine receptor at least 10 times greater than the affinity for the A _2a adenosine receptor or the A ₃ adenosine receptor. The method of claim 1, wherein the compound of Formula I has a K _i value for A _2b adenosine receptor less than 500 nM. The method of claim 1, wherein the compound of formula I has a K _i value for an A _2b adenosine receptor of less than 200 nM. The, A _2b treatment of a disease or disorder mediated by activation of the adenosine receptor, which comprises the treatment of a disease or disorder mediated by an effective amount of claim 1. The compounds of formula (I) to the activation of the A _2b adenosine receptor administered to the required mammalian Way. Identifying a mammal that has experienced an ischemic event or a mammal that is near to an ischemic event; And Administering to said mammal a therapeutically effective amount or a prophylactically effective amount of an A _2b adenosine receptor antagonist within 10 days before or after an ischemic event. As a method of limiting tissue necrosis caused by an ischemic event comprising: A _2b adenosine receptor antagonist is a compound of formula I of claim 1. Identifying a mammal that has experienced myocardial infarction or a mammal that is imminent in myocardial infarction; And Administering to said mammal a therapeutically effective or prophylactically effective amount of an A _2b adenosine receptor antagonist within 10 days before or after myocardial infarction. As a method of limiting infarct size after myocardial infarction, comprising: A _2b adenosine receptor antagonist is a compound of formula I of claim 1.