NO20023752L - 2-carboxamide benzimidazoles useful in the treatment and prevention of ischemic reperfusion injury - Google Patents

Description

Cross reference to related application

The present invention claims priority from US Provisional Application Serial No. 60/181,234, 7945P, filed Feb. 9, 2000.

Field of the invention

The present invention relates to 2-carboxamide-benzimidazoles which are useful in the treatment or prevention of ischemic reperfusion injury of myocardial or other tissues or other cardiovascular and inflammatory diseases and disorders.

Summary of the invention

The present invention includes compounds with the general structure:

in which:

(a) R1 is selected from the group consisting of alkyl, aryl,

alkoxy and aryloxy, wherein alkyl and aryl in the preferred RI contain 1-14 carbon atoms,

(b) R 3 and R 4 are independently selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, alkyl-aryloxy, alkylthio, amino and mono- or dialkylamino, the alkyl moieties of the preferred R 3 and R 4

has 1-8 carbon atoms, except when R3 and R4 are both hydrogen,

(c) each R 5 is independently selected from the group consisting of hydrogen, halogen, cyano, alkyl, hydroxy, alkoxy, thio, alkylthio, amino and mono- or dialkylamino, the alkyl moieties of preferred R 5 having 1-8 carbon atoms, and

an optical isomer, diastereomer or enantiomer or mixture thereof, a pharmaceutically acceptable salt, hydrate or biohydrolyzable ester, amide or imide thereof.

The present invention also relates to pharmaceutical mixtures containing such compounds, as well as methods for using such compounds to treat or prevent tissue reperfusion damage.

Detailed description of the invention

It is approx. 1.1 million myocardial infarctions in the United States each year and 350,000 people die. Myocardial infarction (MI) or heart attack occurs when blood vessels that supply blood to the heart become completely or partially occluded. Treatment with thrombolytic agents to restore blood flow (reperfusion) is the first treatment in many cases. However, the benefit of reperfusion is accompanied by the acute inflammatory response associated with it, leading to a syndrome called reperfusion injury.

The inflammation generally serves a protective role. For example, positions of bacterial infection, bacterial endotoxins will induce the production of inflammatory cytokines that attract circulating leukocytes, including neutrophils and monocytes to destroy the bacteria. As soon as the infection is stopped, the inflammation will subside. However, there are conditions where the inflammatory signal is maintained (rheumatoid arthritis) or it is unnecessarily severe (ischemia-reperfusion injury).

An essential feature of inflammation is the migration of neurophils (PMNs) from blood into tissue. This migration precedes a cascade of events caused by adhesion molecules. Adhesion of PMNs to vascular endothelial cells requires interaction of the adhesion molecules and the surface of both cell types. These molecules belong to three distinct families: the selectin, integrin and immunoglobulin superfamilies. Neurophils will first "roll" along the endothelial cells, a process caused by selectins. At the sites of inflammation, strong adhesion will be caused by the interaction of 02 integrins on PMNs and ICAM-1 (intercellular adhesion molecule-1) expressed on the endothelial cells. Finally, transendothelial migration of PMNs into the tissues will lead to tissue destruction. Compounds capable of blocking the adhesion of neutrophils to endothelium will be useful in the treatment of a number of conditions that include ischemia-reperfusion injury including but not necessarily limited to myocardial infarction, coronary artery bypass grafting, angioplasty, angina, stroke, peripheral vascular disease, inflammatory bowel disease, ulcerative colitis, burns, frostbite, respiratory stress syndrome in adults, asthma, tissue and organ transplants, general surgery, replantation, acute renal failure, rheumatoid arthritis, psoriasis, hepatitis, pancreatitis, sunburn, radiation, tumor and shock.

(For new reviews see: C.Cornejo, J. Harlan, R. Winn, in Adhesion Molecules in Health&Disease, L. Paul and T. Issekutz, Eds., Marcel Dekker, 1997, chapter 18; J. Prince, C. Ballantyne , Emerging Therapeutic Targets, 1999, 263-277.)

Glossary of terms

If not stated otherwise, the subsequent designations have the indicated meaning used in the present description. The term "alkyl" means a hydrocarbon chain which is linear, branched or cyclic, saturated or unsaturated (non-aromatic), substituted or unsubstituted. The term can be used alone or as part of another word where it can be abbreviated to "alk" (for example alkoxy, alkylamino). Preferred linear alkyl has 1-20 carbon atoms, more preferably 1-8 carbon atoms and most preferably 1-4 carbon atoms and the most preferred is methyl or ethyl. Preferred cyclic and branched alkyl has 3-20 carbon atoms, more preferably 3-10 carbon atoms and most preferably 3-6 carbon atoms. Preferred cyclic alkyl has one hydrocarbon ring, but may have two, three or more fused or spirocyclic hydrocarbon rings. Alkyl may be unsaturated with only one or more double bonds ("alkenyl") (no triple bonds), preferably with one, two or three double bonds, more preferably with one double bond. Alkyl may be unsaturated with one or more triple bonds ("alkynyl"), preferably with a triple bond. More preferred is saturated ("alkanyl"). The term "alkylene" means an alkyl which is attached to 2 or more groups. Preferred substituents on alkyl include alkyl, aryl, halo, hydroxy, alkoxy, aryloxy, amino, alkylamino, arylamino, thio, alkylthio, arylthio, acyl, alkylacyl, arylacyl, carboxy, alkylester, arylester, amino, alkylamino, arylamino, sulfonyl, alkylsulfonyl , arylsulfonyl, nitro, cyano, heterocycle. Preferred alkyl is unsubstituted.

The term "aryl" means an aromatic hydrocarbon ring which is substituted or unsubstituted. The term can be used alone or as part of another word (aryloxy, arylamino). Preferred aryl has 6-14 carbon atoms in the aromatic ring or rings, and a total of 6-20, preferably approx. 12 carbon atoms. Preferred aryl is phenyl or naphthyl, most preferred is phenyl. The term "arylene" means aryl which is attached to two or more groups. Preferred substituents on aryl include alkyl, aryl, halo, hydroxy, alkoxy, aryloxy, amino, alkylamino, arylamino, thio, alkylthio, arylthio, acyl, alkylacyl, arylacyl, carboxy, alkylester, arylester, amino, alkylamino, arylamino, sulfonyl, alkylsulfonyl , arylsulfonyl, nitro, cyano, heterocycle.

The term "heteroatom" means a nitrogen, oxygen or sulfur atom.

The term "heterocycle" or "heterocyclyl" means a cyclic alkyl or aryl in which one or more carbon atoms are replaced by heteroatoms in the ring or rings, preferably one, two or three heteroatoms in the ring or rings. Preferred heterocyclic substituents are the same as for alkyl. The term "heteroaryl" refers to a subgroup of heterocyclic compounds comprising an aromatic ring. Preferred heteroaryl has 5-14, more preferably 10 and most preferably 5 or 6 carbons plus heteroatoms in the ring (e) and a total of 5-20, more preferably approx. 12 carbons plus heteroatoms.

The term "safe and effective amount" means an amount of a pharmacologically active compound sufficient to substantially induce a positive modification of the condition to be treated, but sufficiently low to avoid serious side effects (a reasonable benefit/risk ratio) within the range of sound medical judgment. A safe and effective amount of a compound will vary with the particular condition being treated, the size and age and physical condition of the patient, the severity of the condition, the duration of treatment, the type of concomitant therapy, the particular pharmaceutically acceptable carrier employed, and similar factors determined by the attending physician based on his knowledge and expertise.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipients" means one or more compatible solid or liquid excipients suitable for administration to humans and lower animals. The term "compatible" means that the excipients are capable of being mixed with the pharmacologically active compound or compounds and with each other, in such a way that there is no interaction which substantially reduces the pharmaceutical effect of the mixture under normal situations of use. The excipients themselves have no significant pharmacological activity, but can, for example, function as diluents, lubricants, disintegration enhancers, dissolution enhancers, encapsulation materials, preservatives, dyes, flavorings and the like. Pharmaceutically acceptable excipients must be of sufficiently high purity and sufficiently low toxicity to make them suitable for administration to humans and lower animals being treated.

The term "unit dosage form" means that the composition comprises an amount of a pharmacologically active compound suitable for administration to a human or a lower animal in a single dosage form, according to good medical practice.

A "biohydrolyzable ester" is an ester of a

carboxylic acid-containing 2-carboxamide benzimidazole according to the present invention which does not affect the activity of the 2-carboxamide benzimidazoles or which is easily converted in an animal to give an active 2-carboxamide benzimidazole according to the invention. Such esters include lower alkyl esters, lower cycloalkyl esters (such as acetoxymethyl, acetoxyethyl, aminocarbonyloxymethyl, pivaloyloxymethyl and pivaloyloxyethyl esters), lactonyl esters (such as phthalidyl and thiophthalidyl esters).

The connections

The present invention includes 2-carboxamide benzimidazole compounds with the structure:

In formula (I), R 1 is selected from alkyl, aryl, alkoxy and aryloxy. The alkyl and aryl moieties in preferred R1 groups contain 1-14 carbon atoms.

More preferably, RI is selected from unsubstituted or substituted alkyl with 1-12 carbon atoms and unsubstituted or substituted phenyl or naphthyl. More preferred alkyl RI includes unsubstituted or substituted linear alkyl with 2-8 carbon atoms, more preferably 3-6 carbon atoms and further preferred is n-propyl or n-butyl or n-pentyl. More preferred RI includes branched alkyl with 3-8 carbon atoms, more preferably 3-6 carbon atoms and even more preferred is isomethyl or isopentyl. More preferably R1 includes cyclic alkyl of 3-8 carbon atoms, more preferably 3-6 carbon atoms. Preferred substituents for such linear, branched or cyclic alkyl include halo, hydroxy, alkoxy, amino, mono- and dialkylamino, thio, alkylthio, aryl (especially phenyl) and heterocycle; more preferably such alkyl is unsubstituted. Preferred RI which is linear, branched or cyclic alkyl, is saturated or unsaturated with one or more double bonds, more preferably is saturated.

More preferred aryl R 1 includes substituted or unsubstituted phenyl. Preferred substituents for such phenyl include halo, hydroxy, alkoxy, amino, mono- and dialkylamino, also preferred for such phenyl is that it is unsubstituted.

More preferred aralkyl R 1 includes substituted or unsubstituted phenyl. Preferred substituents for such benzyl include halo, hydroxy, alkoxy, amino, mono- and dialkylamino, also preferred for such benzyl is that it is unsaturated.

In formula (I), R 3 and R 4 are independently selected from the group consisting of hydrogen, halo, alkyl, alkoxy, alkyl-aryloxy, alkylthio, amino and mono- or dialkylamino, except that R 3 and R 4 are not both hydrogen. The alkyl parts of preferred R3 and R4 groups can have 1-8 carbon atoms. Preferably, R 3 and R 4 are independently selected from the group consisting of hydrogen, haloalkylalkoxy, alkylthio and mono- or dialkylamino, except that R 3 and R 4 are not both hydrogen.

Preferred R 3 and R 4 include hydrogen, alkoxy of 1-6, preferably up to about 3 carbon atoms, alkylthio of 1-6, preferably up to about 3 carbon atoms, monoalkylamino or dialkylamino where each alkyl has 1-6, preferably up to about 3 carbon atoms and alkyl of 1-6, preferably up to about 3 carbon atoms. Preferred substituents on alkyl of such groups include halo, hydroxy, alkoxy, amino, mono- or dialkylamino, thio, alkylthio, it is more preferred for such R3 and R4 groups that they are unsubstituted. Even more preferably, at least one of R3 and R4 is ethoxy and especially methoxy. Preferably one of R3 and R4 is hydrogen, more preferably R3 is hydrogen.

In formula (I), R5 denotes groups in positions 4 and 7 of the benzimidazole rings. Each R 5 is independently selected from hydrogen, halo, cyano, alkyl, hydroxy, alkoxy, thio, alkylthio, amino and mono- or dialkylamino. The alkyl parts in preferred R5 groups contain 1-8 carbon atoms.

Preferred R5 includes hydrogen, halo, alkyl of 1-6, preferably up to about 3 carbon atoms, alkoxy of 1-6, preferably up to about 3 carbon atoms, monoalkyl- or dialkylamino where each alkyl contains 1-6, preferably up to about 3 carbon atoms and alkylthio with 1-6, preferably up to 3 carbon atoms. Preferred substituents for alkyl in such R5 groups include alkoxy, amino and alkyl, it is more preferred that alkyl in such groups is unsubstituted.

More preferably, each R 5 is independently selected from hydrogen, halo and unsubstituted alkyl with 1-3 carbon atoms. It is more preferred that no more than one R5 is hydrogen. Most preferably, both R5 are hydrogen.

Preferred compounds according to the invention include the following examples with the structure (I) and with the indicated substituents:

The compound according to the invention includes all active optical isomers, diastereomers and enantiomers and mixtures thereof of the above compounds. The compound of the invention also includes pharmaceutically acceptable salts, hydrates and biohydrolyzable esters, amides and imides of such compounds.

Preparation of the compounds

In the following, general schemes for the preparation of the present compounds and special methods for the preparation of preferred compounds according to the invention are given. Unless otherwise stated, all commercially available reactants are used without further purification. The reactions are generally carried out under an inert atmosphere (argon or nitrogen). Brine refers to saturated aqueous sodium chloride. Residual solvent is removed under vacuum (approx. 0.03 mm hg) at room temperature (rt).

The structures for the compounds produced are substantiated using the following analytical tools. Proton NMR spectra were taken with a "GE QE-300" (300 MHz) spectrometer, a "Bruker AC-300" (300 MHz) quad core probe system or more a "Varian Unityplus" (300 MHz). All chemical shifts are reported in small 8-scale as ppm, expressed from (CH3)4Si. Spectra taken with CDC13 are denoted either as (CH3)4Si or as residual CHCl3 (7.24 ppm). Spectra captured in D20 are referred to as HOD (4.80 ppm), those captured in (CH3)2CO are designated as residue (CH3)2CO (2.04 ppm), those in CD30D are referenced as residue CH3OH (3, 30 ppm) and those in (CD3)2SO are referred to residual (CH3)2SO (2.49 ppm). Carbon-13 spectra are recorded with "GE QE-300" (75 MHz) spectrometer or "Bruker AC-300" (75 MHz) quad core probe system. Spectra taken in CDD13 are referenced to solvent (78 ppm), those in CD3OD are referenced to solvent (49 ppm), those in (CD3)2SO are referenced to solvent (39.7 ppm) and those in (CD3)2CO are referenced to solvent (206.5, 29.8 ppm). Mass spectra are determined on "Fision's Trio 2000" equipped with a robotic probe or a "Fision's Platform II Mass Spectrometer". Chemical ionization spectra were obtained using methane and/or ammonia as reactant gas. ESI compound entry was via "Hewlett Packard 1050 HPLC" autosampler using methanol, 0.2% formic acid and 0.2 mM ammonium acetate as elution solvent. Thin-layer chromatography is performed on silica gel 60-F254 precoated plates. Flash chromatography is performed using silica gel 60 (Merck, 230-400 mesh). Melting points were obtained with an "Electrothermal 1A9200" or a "MelTemp II" capillary melting point apparatus and are uncorrected.

Scheme I is a general scheme useful for the preparation of many compounds according to the invention:

5-Methoxy-2-nitrophenol (B from scheme I with R4 = methoxy and R3 and both R5 = H): To a 2 L round-bottom flask, equipped with a mechanical stirrer and addition funnel were added propionic acid (300 ml) and 3-methoxyphenol (37 .2 g, 0.3 mol) added. The resulting mixture was cooled to 0°C and a solution of sodium nitrite (21 g, 0.304 mol) in water (50 mL) was slowly added. After stirring for 1 hour at 0°C, fuming nitric acid (40 ml) was slowly added. The resulting slurry was stirred at 0°C for one hour and then warmed to room temperature over 2 hours. Water (250 ml) was added dropwise at room temperature and the resulting solid was filtered and washed with 300 ml of 50% aqueous propionic acid to give, after drying, 5-methoxy-2-nitrophenol as a solid.

N-Alkyl-5-methoxy-2-nitroaniline (C from Scheme I with R1 = alkyl, R4 = methoxy and R3 and both R5 = H). To a 1 L round bottom flask was added toluene (300 mL), 5-methoxy-2-nitrophenol (5.0 g, 0.03 mol) and triethylamine (6.68 g, 0.066 mol). The resulting solution was cooled to 0°C and trifluoromethanesulfonic anhydride (Tf 2 O) was slowly added via a pipette (9.3 g, 0.033 mol). The reaction mixture was stirred at 0°C for 5 min, the amine (R 1 -NH 2 ) (0.12 mol) was added and the reaction mixture was heated to reflux for 5.5 h. After cooling to room temperature, the reaction contents were filtered through a plug of silica gel (eluted with 90:10 hexane:ethyl acetate) and concentrated via a rotary evaporator to give crude N-alkyl-5-methoxy-2-nitroaniline. The material was used in the next synthesis step.

N1-Alkyl-6-methoxybenzimidazole (D from Scheme I with RI = alkyl, R4 = methoxy and R3 and both R5 = H): To a 250 ml round bottom flask was added 88% formic acid (50 ml) and N-alkyl- 5-Methoxy-2-nitroaniline (0.02 mol). To this homogeneous mixture was added an ethyl acetate slurry of 10% Pd-C (600 mg). The resulting heterogeneous reaction mixture was heated to 100°C for 1 hour, cooled to room temperature and filtered through Celite (eluted with water). The filtrate was made basic by the addition of 28% NH 4 OH solution and then washed with ethyl acetate (3 x 100 ml). The combined organic phases were dried (MgSO 4 ), filtered and concentrated via a rotary evaporator to give a residue. The residue was chromatographed (SiO 2 , 50:50 hexane:ethyl acetate) to give N1-alkyl-6-methoxybenzimidazole.

Ethyl 1-alkyl-6-methoxy-1H-benzimidazole-2-carboxylate (E from Scheme I with R1 = alkyl, R4 = methoxy and R3 and both R5 = H): to a 250 mL round bottom flask under argon, was added N1-alkyl-6-methoxybenzimidazole (2.9 g, 0.014 mol) and THF (100 mL). This was cooled to -78°C and n-butyl lithium (12.3 mL, 0.019 mol, in hexanes) was added dropwise, resulting in a yellow mixture. Stirring was continued at -78°C for 30 min, then ethyl chloroformate (ECF) was added neat (2.13 g, 0.019 mol). The reaction mixture was warmed to room temperature and stirred for 30 min. Water (100 mL) was added and the resulting mixture was extracted with ethyl acetate (3 x 100 mL). The combined organic phases were dried (MgSCM, filtered and concentrated to give a yellow residue. This residue was then chromatographed (flash, 70:30 hexane:ethyl acetate) to give ethyl 1-alkyl-6-methoxy-1H-benzimidazole- 2-carboxylate.

N1-Alkyl-6-methoxybenzimidazole-2-carboxamide (F from scheme I with RI = alkyl, RA - methoxy and R3 and both R5 = H): To an ACE GLASS pressure tube with stirrer was added ethyl 1-alkyl-6- methoxy-1H-benzimidazole-2-carboxylate (1.8 mmol), methanolic ammonia (10 mL, 2.0 M) and sodium or calcium cyanide (8.8 mg, 0.18 mmol). The reaction vessel was then sealed, heated to 80°C for 3 hours and then cooled to room temperature. The resulting reaction mixture was concentrated by rotary evaporation and the residue was chromatographed (flash, 60:40 hexane:ethyl acetate) to give N1-alkyl-6-methoxybenzimidazole-2-carboxamide.

Scheme II is another general scheme useful for the preparation of many compounds according to the invention. N-(Cyanomethyl)-4-methoxy-2-nitroaniline (H from Scheme II with R4 = methoxy and R3 and both R5 = H): to a 3-necked 1 1 round-bottomed flask fitted with a mechanical stirrer, reflux condenser and a loading funnel 4-methoxy-2-nitroaniline (8.4 g, 0.05 mol), paraformaldehyde (4.5 g, 0.15 mol), potassium cyanide (9.75 g, 0.15 mol) and zinc chloride (25 g, 0.18 mol). To this vigorously stirred mixture was slowly added a mixture of H 2 SO 4 (4 drops) and glacial acetic acid (250 ml). The resulting reaction mixture was heated to 50°C for 8 hours and then cooled to room temperature. The reaction contents were poured over ice/water and the solid formed was collected via suction filtration, washed with water, dried and recrystallized (abs. EtOH) to give N-(cyanomethyl)-4-methoxy-2-nitroaniline as needles.

2-Cyano-6-methoxybenzimidazole-Nl-oxide (J from Scheme II with R4 = methoxy and R3 and both R5 = H): To a 500 ml round-bottom flask was added N-(cyanomethyl)-4-methoxy-2- nitroaniline (2.5 g, 0.012 mol) and 95% ethanol (130 mL). The reaction mixture was heated to 60°C, solid potassium carbonate (1.77 g, 0.013 mol) was added and the resulting mixture was then heated to reflux for 4.5 hours, cooled to room temperature and concentrated in vacuo. The residue was dissolved in water and then acidified (conc. HCl) to form a white solid which was collected via filtration, dried and recrystallized (DMF) to give 2-cyano-6-methoxybenzimidazole-N-oxide as a solid.

2-cyano-1-isopropoxy-6-methoxybenzimidazole (K from scheme II with RI = isopropyl, R4 = methoxy and R3 and both R5 = H): To a 100 ml round-bottom flask was added 2-cyano-6-methoxybenzimidazole- N -oxide (457 mg, 2.41 mmol), tetrahydrofuran (THF) (50 mL), isopropyl alcohol (145 mg, 2.41 mmol), triphenylphosphine (Ph3P) (632 mg, 2.41 mmol) and finally diethyl azodicarboxylate ( DEAD)(444 mg, 2.55 mmol). The resulting mixture was stirred at room temperature for 18 h, then concentrated in vacuo. The residue was chromatographed (80:20 hexane:ethyl acetate) to give 2-cyano-1-isopropoxy-6-methoxybenzimidazole as a solid. l-Isopropoxy-6-methoxybenzimidazole-2-carboxamide (L from Scheme II with RI = isopropyl, R4 = methoxy and R3 and both R5 = H): To a 25 ml round-bottom flask was added 2-cyano-l-isopropoxy- 6-Methoxybenzimidazole (100 mg, 0.43 mmol), t-butyl alcohol (5 mL) and potassium hydroxide (200 mg, 3.57 mmol). This mixture was refluxed for 1 hour, cooled to room temperature, poured into hydrochloric acid (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic fractions were dried (MgSO 4 ), filtered and concentrated in vacuo to give a yellow oil. This oil was chromatographed (70:30 hexane:ethyl acetate) to give 1-isopropoxy-6-methoxybenzimidazole-2-carboxamide as a solid).

Scheme III is yet another general scheme useful for the preparation of many compounds according to the invention:

3-Fluoro-4-nitroaniline (N from Scheme III with R4 = amino and R3 and both R5 = H): 3-Fluoroaniline (5g; 1 equiv) was combined with benzaldehyde (5g; 1.05 equiv). The mixture was stirred at 25°C for ½ hour to achieve a gentle reflux.

The mixture was dissolved in concentrated sulfuric acid (20 mL) and cooled to <5°C with an ice bath. A mixture of concentrated nitric acid (3 ml) and concentrated sulfuric acid (10 ml) was slowly added and the mixture was stirred at 25°C for 1 hour. The reaction was quenched by pouring over water (200 mL); it was then washed with ethyl acetate. The combined ethyl acetate layers were dried over magnesium sulfate and evaporated to dryness. The product was chromatographed on a silica gel column using 20% ethyl acetate-hexane as eluent to give the title compound and a mixture of other regioisomers.

(3-Fluoro-4-nitro-phenyl)-carbamic acid tert-butyl ester (P from Scheme III with R4 = Boc-NH and R3 and both R5 = H): Ditert-butyl dicarbonate (1.4 g; 1 equiv) was combined with 3 -fluoro-4-nitroaniline (1 g; 1 equiv) in dichloromethane (30 mL). The mixture was stirred at 25°C for 10 min. and dimethylaminopyridine (0.08 g; 0.1 equivalent) was added. The mixture was stirred at 25°C for 24 hours. The mixture was diluted in ethyl acetate and washed with 1 molar aqueous citric acid; the mixture was then washed twice with water. The ethyl acetate layer was dried over magnesium sulfate and evaporated to dryness. The product was chromatographed on a silica gel column using 30% ethyl acetate-hexane as eluent to give a mixture of the title compound (and the bis Boc protected compound).

(3-Isobutylamino-4-nitro-phenyl)-carbamic acid tert-butyl ester (Q from Scheme III with Ri = isobutyl, R4 = Boc-NH and R3 and both R5 = H): Isobutylamine (0.29 g; 2 equivalents) was added slowly to a solution of (3-fluoro-4-nitrophenyl)-carbamic acid tert-butyl ester (0.5 g; 1 equivalent) in acetonitrile (20 mL). The mixture was stirred at 25°C for 24 hours. The mixture was then diluted in dichloromethane and washed twice with water. The dichloromethane layer was dried over sodium sulfate and evaporated to dryness to give the title compound.

(4-Amino-3-isobutylamino-phenyl)-carbamic acid tert-butyl ester (R from Scheme III with RI = isobutyl, R4 = Boc-NH and R3 and both R5 = H): A solution of (3-Isobutylamino-4- nitro-phenyl)-carbamic acid tert-butyl ester (0.51 g; 1 equiv) in ethanol (15 mL) was combined with a suspension of palladium on carbon (10 wt%) (0.1 g; 2 equiv) in ethanol ( 15 ml) while stirring under a nitrogen atmosphere. Nitrogen was then replaced with a positive pressure of hydrogen and the mixture was stirred for 16 hours at 25°C under a hydrogen atmosphere. The mixture was then filtered through diatomaceous earth and the precipitate was washed with dichloromethane. The filtrate was evaporated to dryness to give the title compound.

6-tert-Butoxycarbonylamino-1-isobutyl-1H-benzimidazole-2-carboxylic acid ethyl ester (S from Scheme III with RI = isobutyl, R4 = Boc-NH and R3 and both R5 = H): A 50% solution of ethyl glyoxylate polymer in toluene (0 .5 g; 1.5 equivalents) was combined with a solution of (4-amino-3-isobutylamino-phenyl)-carbamic acid tert-butyl ester (0.46 g; 1 equivalent, assuming 100% yield from previous step) in ethanol ( 3 ml). The mixture was stirred uncovered for H hour. A solution of iodine (0.21 g; 0.5 equivalents) in ethanol (1.5 mL) was added and the mixture was then stirred uncovered at 25°C for 8 hours. Sodium thiosulfate (0.41 g; 1 equivalent) in water (2 mL) was added and the mixture was stirred for 1 h at 25°C. The mixture was diluted in ethyl acetate and washed with water and brine. The ethyl acetate layer was dried over magnesium sulfate and evaporated to dryness. The product was chromatographed on a silica gel column using 50% ethyl acetate-hexane as the extractant to give the title compound.

6-Amino-1-isobutyl-1H-benzimidazole-2-carboxylic acid ethyl ester (T from Scheme III with RI = isobutyl, R4 = Boc-NH and R3 and both R5 = H): Trifluoroacetic acid (0.5 mL) was added slowly to a solution of 6-tert-butoxycarbonylamino-1-isobutyl-1H-benzoimidazole-2-carboxylic acid ethyl ester (0.12 g)

in dichloromethane (4.5 mL). The mixture was stirred at 25°C for 3 hours. The mixture was then evaporated to dryness and the residue was dissolved in methanol. The solution in methanol was evaporated to dryness; this was then dissolved in dichloromethane and washed with 2 M aqueous potassium carbonate. The dichloromethane layer was dried over sodium sulfate and evaporated to dryness to give the title compound.

6-Amino-1-isobutyl-1H-benzimidazole-2-carboxylic acid amide (U from Scheme III with RI = isobutyl, R4 = Boc-NH and R3 and both R5 = H): A 25% aqueous solution of ammonia (5 mL) was added to a solution of 6-amino-1-isobutyl-1H-benzoimidazole-2-carboxylic acid ethyl ester (0.02g) in tetrahydrofuran (0.5ml) with stirring. The mixture was stirred at 25°C for 72 hours. The mixture was then diluted in ethyl acetate and washed with water. The ethyl acetate fraction was dried over magnesium sulfate and evaporated to dryness to give the title compound.

Scheme IV is yet another general scheme useful for the preparation of many compounds according to the invention:

N-(4-Methoxy-2-nitrophenyl)-isobutyramide (W from Scheme IV with R3 = methoxy, R4 and both R5 = H and R6 = iPr): 4-methoxy-2-nitroaniline (1.0 g; 1 equiv ) and isobutyryl chloride (0.634 g; 1 equivalent) were combined in pyridine (60 mL) and stirred for 4 hours. The mixture was concentrated in vacuo to a residue and dissolved in dichloromethane (DCM) (70 mL), washed twice with 1N HCl (aq), saturated Na 2 CO 3 (aq), water and brine and dried over MgSO 4 . The solvent was removed in vacuo to give 1.35 g of the title compound.

Isobutyl-(4-methoxy-2-nitrophenyl)-amine (X from Scheme IV with R3 = methoxy, R4 and both R5 = H and R6 = iPr) : Isobutyl-(4-methoxy-2-nitrophenyl)-amine (0 .1 g; 1 equiv) was dissolved in THF (2 mL) and combined with a 1 M boranetetrahydrofuran complex solution in THF (1.25 mL; 3 equiv) and stirred for 18 h. Methanol (1 mL) was slowly added and the solution was heated to 50°C for 2 hours. The solvent was removed in vacuo and the residue chromatographed (silica gel, 0 to 30% EtOAc/hexanes) to give the title compound.

1^-isobutyl-4-methoxy-benzene-1,2-diamine (Y from Scheme IV with R3 = methoxy, R4 and both R5 = H and R6 = iPr) : N<1->isobutyl-4-methoxy-benzene -1,2-diamine (0.054 g; 1 equivalent) was dissolved in ethanol (2 mL) and water (0.1 mL), 10% palladium on carbon (50% wet) (0.011 g) was added. The suspension was stirred under an atmosphere of hydrogen gas for 2 hours and then the bottle was purged with nitrogen. The suspension was filtered through a diatomaceous earth pad, washed thoroughly with DCM and the combined organic phases were dried in vacuo to give the title compound.

l-Isobutyl-5-methoxy-1H-benzoimidazole-2-carboxylic acid ethyl ester { Z from Scheme IV with R3 = methoxy, R4 and both R5=H and R6 = iPr): l-isobutyl-5-methoxy-1H-benzimidazol- 2-carboxylic acid ethyl ester (0.034 g; 1 equivalent in ethanol (0.5 mL)) and a 50% solution of ethyl glyoxalate polymer in toluene (0.051 mL; 1.5 equivalent) were combined and stirred for 1 h. A solution of iodine (0.022 g; 0.5 equivalent) in ethanol (0.5 mL) was added and the reaction was further stirred for 20 hours. A solution of sodium thiosulfate (0.044 g) in water (0.5 mL) was added and the mixture was stirred for 1 h, then diluted with EtOAc (20 mL). The mixture was washed with water (x 3) and brine, dried with MgSO 4 and the solvents were removed in vacuo. The residue was purified by chromatography (silica gel, 5% methanol in DMC) to give the title compound. l-Isobutyl-5-methoxy-1H-benzoimidazole-2-carboxylic acid amide (AA from Scheme IV with R3 = methoxy and R4 and both R5 = H and R6 = iPr): A 25% aqueous solution of ammonia (1 mL) was added with stirring to a solution of 1-isobutyl-5-methoxy-1H-benzoimidazole-2-carboxylic acid ethyl ester (0.0036 g) in THF (1 mL). The organic phase was dried with MgSO 4 and evaporated in vacuo to give the title compound.

Schemes V and VI are two general schemes useful in the preparation of many compounds according to the invention: 6-[[(4-Methoxy-phenyl)-methyl]amino]-1-isobutyl-1-H-benzimidazole-2-carboxylic acid amide (AB from Scheme RI = isobutyl, R3 and both R5 = H and R7 = methoxy): To a 200 mL round bottom flask 6-amino-1-isobutyl-1H-benzimidazole carboxylic acid amide (U; 0.1 g) was dissolved in anhydrous MeOH (10 mL ). Reagent acetic acid (0.1 mL) and 4-methoxybenzaldehyde (0.15 g) were added and the reaction was refluxed until complete conversion of the starting material. At this point the reaction mixture was cooled to room temperature and sodium cyanoborohydrate (0.1 g) was added portionwise. The reaction mixture was stirred at room temperature for 4 hours. The mixture was then evaporated to dryness and the residue was dissolved in ethyl acetate and washed with 5% aqueous sodium bicarbonate, water and brine. The organic layer was dried over sodium sulfate and evaporated to dryness. The residue was chromatographed on a silica gel column using 25% ethyl acetate-hexane as the extractant to give the title compound.

6-{[(2S)-2-(ft-Benzyloxycarbonyl)amino-1-oxo-3-phenylpropyl]amino}-1-isobutyl-1H-benzimidazole-2-carboxamide ( AC from Scheme VI with RI - isobutyl, R3 and both R5 = H and R9 = Z): N-Benzyloxycarbonyl-L-phenylalanine (64.4 mg) was dissolved in dry dichloromethane (1.5 ml) and N,N-diisosporpylethylamine (41.2 µl). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (45.4 mg) and 4-dimethylaminopyridine (cat.) were added and the mixture was stirred for 10 minutes at room temperature. 1-Hydroxybenzotriazole (32.0 mg) was then added and stirring continued for 5 minutes. Finally, 6-amino-1-isobutyl-1#-benzimidazole-2-carboxamide was added and the resulting mixture was stirred at room temperature for 5 days. The reaction mixture was diluted with dichloromethane and washed with saturated sodium bicarbonate solution (1x), water (1x) and brine (1x). The organic layer was dried and concentrated to a solid. Trituration with methanol gave the title compound as a solid.

6-{[(2S)-2-amino-l-oxo-3-phenylpropyl]amino}-l-isobutyl-lff-benzimidazole-2-carboxamide hydrochloride (AD from scheme with RI = isobutyl, R3, both R5 and R9 - H). 6-{[(2S)-2-( N -Benzyloxycarbonyl)amino-1-oxo-3-phenylpropyl]amino}-1-isobutyl-1H-benzimidazole-2-carboxamide (84.7 mg) and 10% Pd/ C (17.0 mg) was placed in a bottle. In dry ethanol (3.5 mL), the argon supply was replaced by an air balloon. After stirring at room temperature overnight, hydrogen was flushed from the system with argon and the reaction mixture was filtered through a plug of Celite® with 1:1 ethanol/water (100 mL). The filtrate was concentrated through a solid residue. The resulting solid was dissolved in a minimum of methanol and dropped into excess ether to give a slurry. Filtration gave the title compound as a solid.

Compositions

The present invention includes pharmaceutical compositions comprising a safe and effective amount of a 2-carboxamide benzimidazole compound described above and pharmaceutically acceptable excipients. The compositions may also optionally include other pharmacologically active compounds, especially those with activity as thrombolytics (for example reteplase, streptokinase or tissue plasminogen activators), anticoagulants (for example heparin), beta blockers (for example carvedilol, propanalol), calcium channel blockers (for example verapamil, nifedipine) and anti-platelet compounds (eg aspirin, abciximab).

Some examples of pharmaceutically acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate; finely ground tragacanth; malt, gelatin, talc, solid lubricants, such as stearic acid, magnesium stearate or calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and tea brome oil; polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Tweens®; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; excipients; tablet excipients; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline and phosphate buffer solutions.

The choice of a pharmaceutically acceptable carrier for use with a compound is essentially determined by the manner in which the compound is to be administered. The compounds and compositions of the present invention can be administered systematically. Routes of administration include topical or transdermal (patch, ointment, cream, powder, etc), oral, parenteral, including subcutaneous, intramuscular or intravenous injection, topical, rectal, colonic, intraperitoneal, intraocular, sublingual, buccal, inhalation and/or intranasal. The preferred route of administration is parenteral, particularly intravenous injection on a daily or as needed basis.

The appropriate amount of compound to be used can be determined by routine experimentation with animal models. Such models include, but are not limited to, ferret, rabbit, and non-human primate models. Generally, an amount between 0.01 (jg/kg to 100 mg/kg body weight per day is administered depending on the potency of the compound or compositions used.

Preferred unit dosage forms for injection include sterile aqueous solutions, physiological saline, or mixtures thereof. Parenteral unit dosage form compositions may be in the form of ready-to-inject solutions or dry (eg, lyophilized) compositions that have been reconstituted with water or saline prior to injection. The solutions' pH should be adjusted to around 7.4. Suitable carriers for injection or surgical implants include hydrogels, controlled or continuous release devices, polyacetic acid and collagen matrices. Other suitable carriers for injection include dextrose, mannitol, lactose, lecithin, albumin, monosodium glutamate and the like.

Compositions of the present invention are preferably provided in unit dosage forms. One

unit dosage form composition preferably contains from about 50 mg, more preferably from about 200 mg, also preferably from about 500 mg, preferably to about 2000 mg, more preferably to about 1000 mg, also preferably to about 500 mg of a 2-carboxamide benzimidazole compound described above.

The present compositions may be in a variety of suitable forms (for example) for oral, topical or parenteral administration. Depending on the particular route of administration desired, a variety of pharmaceutically acceptable carriers well known in the art may be used. These include solid or liquid fillers, diluents, hydrotopes, surfactants and encapsulants. The amount of carrier components used together with the active compound is sufficient to provide a practical amount of the material for administration per unit dose of the active compound. Techniques and compositions for preparing unit dosage forms according to the invention are described in the following references: Modern Pharmaceutics, Volume 7, Chapters 9 and 10, Banker and Rhodes, editors, 1979; Lieberman et al., Pharmaceutical Dosage Forms: Tablets, 1981 and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd ed., 1976.

A preferred dosage form of the present invention is intended for parenteral administration. Preferred pharmaceutically acceptable carriers for such compositions include sterile, pyrogen-free water and physiological saline solutions. Parenteral unit dosage form compositions may be in the form of ready-to-inject solutions or dry (eg, lyophilized) compositions that have been reconstituted with water or saline prior to injection.

Preferred compositions of the present invention include those intended for oral administration, such as tablets, capsules, powders and liquids. Suitable pharmaceutically acceptable excipients and anti-platelet compounds (eg, aspirin, abciximab)nts for such compositions include sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, algal acid, phosphate buffers, emulsifiers, alcohols and water.

Method of application of the compounds

The 2-carboxamide-benzimidazole compounds of the present invention can be used in the treatment of ischemia-reperfusion injury. Although not limited to any specific mechanism, the compounds are believed to act via modulation of adhesion molecule metabolism. The compounds are therefore potentially useful for the treatment of ischemia-reperfusion injury including: cardiovascular disorder (myocardial ischemia, angina, cardiac arrhythmia, heart failure, hypertension); for treatment to reduce neurotic damage associated with anoxia or ischemia typically following stroke, cardiac arrest or perinatal asphyxia, for treatment to reduce reperfusion injury after organ transplantation, for treatment of frostbite, inflammatory valvular heart disease, psoriasis, asthma, respiratory distress syndrome in adults, for treatment of chronic inflammatory lung disease including emphysema, bronchitis and for the treatment of fibrosis, urticaria, angioedema, vasculitis, migraine, rheumatoid arthritis, gout and allergy.

It is known that a multitude of processes are involved in reperfusion injury, inflammation and related processes. Not being bound by theory, the following mechanism is interesting for the present invention. An important event in the reperfusion injury process is the upregulation, expression, activation of intracellular adhesion molecule-1 (ICAM-1) on endothelial cells. ICAM-1 can then interact with neutrophils resulting in the transmigration of neutrophils into the tissue with subsequent release of harmful enzymes and destructive reactive oxygen molecules. Thereby, compounds that can disrupt the up-regulation, expression, activation of ICAM-1 probably have a beneficial effect on ischemic reperfusion events. These compounds can be administered orally, intra-vascularly, subcutaneously, intramuscularly, intra-nasally, intra-rectally, intra-ocularly, sublingually/buccally, by inhalation and topically (patch, ointment, powder or cream), as long as an effective dose is delivered to the ICAM-1 source.

2-carboxamide-benzimidazoles according to the invention significantly reduce ICAM-1 up-regulation, expression or activation. In other tests, the compounds show activity consistent with protection of the heart. Following are test methods applicable to determining the activities of such compounds.

Assay for inhibition of ICAM-1 expression

Tissue: The expression of molecular adhesion (specifically ICAM-1) was performed on human umbilical cord endothelial cells (HUVEC) obtained from Clonetics Corp. (Ca# CC2519), San Diego, CA.

End point: Concentration of material that inhibits 50% of ICAM-1 expression on the surface of HUVECs upregulated with 300 U/ml TNF-alpha (IC50).

Method: Thaw 1 vial of frozen HUVEC (rapidly at 37°C for ~2 min (5 x 10<5->1 x 10<6>) cells in 1 ml medium), then transfer cells to 45 ml prewarmed growth medium (EGM for HUVEC) in a 225 cm<2> flask (seeded at 2500-5000 cells/cm<2>) and place this in a humidified 37°C incubator with 5% C02. Change medium after 24-30 hours (to remove dead cells and cytopreservatives) and then change every 2-3 days - cells should float together after 5-7 days of growth. Trypsinize cells to remove them from the flask - spin (200 g, 5 min) to pellet cells - resuspend cells in 50 ml medium (need~l-2 x 10<5> cells/ml) and plate 100 ml cell suspension in 96-well , collagen-coated plates (use ~l-2 x. 10<4>cells/well) - let grow together (1-2 days). Remove old nutrient medium (waste) and add 90 ml of fresh medium containing TNF-alpha (or other ICAM-1 stimulator at the desired concentration) or 90 ml of medium alone (for unstimulated control wells). Add 10 ml medium (or PBS or PBS, 1% DMSO) containing compound to be tested (at 10 x desired concentration) or additionally 10 ml medium alone (or PBS or PBS, 1% DMSO) for control wells - (final compound concentration = 1 x; final DMSO concentration = 0.1%, if used) - incubate 4 hours at 37°C. Remove medium (waste) and fix cells in 200 ml 80% acetone: 20% H2 for 20 min at (-20°C). Remove acetone:H20 (waste) and allow plates to dry - and store at (-20°C) overnight in a desiccator. Wash plates with PBS (5 x 250 ml), then add 200 ml of BLOTTO solution to inhibit non-specific binding - incubate for 1 hour at room temperature and remove ICAM-1 antiserum (debris) and wash plates with PBS (5 x 250 ml ), then add 100 ml goat anti-mouse-HRP antiserum conjugate (in BLOTTO) - culture 1 at room temperature. Remove antiserum-HRP conjugate (waste) and wash plates with PBS (5 x 250 ml), then wash with citrate buffer (1 x 300 (ul; waste)). Add 100 ml HRP substrate and allow to grow for 5-20 min at room temperature (time may vary, see color development), add 50 mL IN H2SO4 to wells to stop reaction Read plates at 490 nm on plate reader.

Solutions for ICAM-1 ELISA:

(1) 80% acetone: 20% H20 (wt:wt) - store at (-20°C). (2) (lx) Dulbecco's phosphate buffer solution (DPBS), w/o Ca<++>or Mg<++>, pH 7.5 - (Sigma D-5652, lx powder or Sigma D-1408, 10 x liquid, dilute 1:10 before use). (3) BLOTTO - 5% (w/v) skimmed dry milk (Carnation or other) in DPBS. (4) Mouse anti-human ICAM-1 monoclonal antiserum (Research Diagnostics; catalog #RDI-CBL450-lx, anti CD54 clone 15.2) - stock solution (1 mg/ml) - dilute 1:1000 in BLOTTO dish before use (1 mg/ml final antiserum concentration). (5) Goat anti-mouse IgG-horseradish peroxidase conjugate (IgG-HRP, DAKO Corp, catalog # P0447) stock solution (1 mg/ml) - dilute 1:1000 in BLOTTO immediately before use (1 mg/ml final concentration). (6) Citrate buffer, pH 5.0 - 65.3 mM sodium phosphate (dibasic, 12-hydrate, MW = 358.4, 23.4 g/l) and 34.7 mM citric acid (anhydrous, acid-free, MW = 192, 1, 6.67 g/l) - check pH (5.0), store at 4°C. (7) HRP substrate - o-phenylenediamine dihydrochloride (OPD, Sigma, P6912, 5 mg OPD/tablet) - add 1

tablet/10 ml citrate buffer (at room temperature), then add 4 ml 30% H202 (Sigma, H1009)/10 ml substrate solution immediately before use - final concentration = 0.5 mg OPD/ml and 0.012% H202.

(8) Human tumor necrosis factor alpha (TNF-alpha, Boehringer-Manheim, catalog # 1371843) - 10 mg/ampoule (in 1 ml) - 10<8>U activity/mg = IO<6>U/10 mg - diluted to 20 ml endothelial cells basal medium (EBM) (50000 U/ml, 500 ng/ml) - aliquot 150 ml (7500 U, 75 ng) in eppendorf tubes (x 133) - store at 20°C - for each experiment, add an aliquot to 25 ml EBM - final concentration = 300 U/ml or 3 ng/ml TNF-alpha.

Human umbilical cord (vein) endothelial cell (HUVEC)/neutrophil adhesion inhibition assay

Tissue: Adhesion is performed on human umbilical cord endothelial cells (HUVEC) obtained from Clonetics (Ca# CC2519).

Endpoint: Concentration of material that inhibits 20% of PMN adhesion to HUVECs upregulated with 300 U/ml of TNFalpha (IC20)

Method:

I. Cells:

A. Endothelial cells

Human umbilical cord endothelial cells (HUVEC) are purchased as frozen cells in 1 ml aliquots (Clonetic Corporation, San Diego, CA). Endotelia growth media umbilical cord (EGM-UV), "bullet kit" additives, trypsinization reagents (trypsin neutralization solution and HEPES buffer) are also purchased from Clonetics. The bottle is placed at 37°C in a 5% C02+ 95% air, 100% humidity incubator.

A vial of liquid N2 frozen cell is thawed in the 37°C water bath, the entire vial is placed in a T-275 bottle with 50 ml of fresh media and placed in the CO2 incubator. The media will be replaced 24-48 hours later. Confluence should take place within 4-5 days. The media is changed at least once during this period. The monolayer in the flask is separated using the trypsin solution after the monolayer is washed with Hank's Balanced Salt Solution (HBSS). The tysinized cells are centrifuged at 200 x Gs for 5 min and resuspended in about 150 ml of media. 100 µl aliquots are placed in each well of a 96-cm plate that was previously coated with collagen. The monolayer in the plate should flow together within 48 hours.

B. Neutrophil isolation

Peripheral blood polymorphonuclear neutrophils (PMNs) are isolated by established methodology (1). Human blood is obtained from the cubital vein by venipuncture performed by a qualified phlebotomist. The blood is collected in a heparinized "vacutainer" (Vacutainer #6489, green lid, 15 ml stretch, VWR). Thirty ml of blood is used for each test. The heparinized blood is diluted with about ^ volume of phosphate-buffered saline containing 0.2% glucose (PBS-G). A discontinuous gradient of Histopaque (3 ml Histopaque-1119 on the bottom and 3 ml Histopaque-1077 on the top) (Sigma Chemical Co., St. Louis, MO) is prepared in 6.15 ml conical centrifuge tubes. The diluted blood is carefully placed on top of the Histopaque-1077. The tubes are centrifuged at 800 x G for 30 min at room temperature. After centrifugation, the PMNs are removed by aspiration from the area between the Histopaque-1077/Histopaque-1119 interlayer and the top of the pelleted red blood cells. The PMNs are pooled from all tubes, then diluted to a total volume of 30 ml and centrifuged at 600 x G for 15 min. The supernatant is discarded and the pellet (containing PMNs and some red blood cells) is treated with 6 ml of cold water for 30 seconds to clear contaminating RBCs. Normal osmolarity is restored by adding 3 ml of 2.7% saline. The PMNs are washed twice more with PBS-G. Viability and number of the PMNs are determined by using the trypan blue exclusion test in a hemocytometer counting room. Every now and then a small aliquot of the PMN suspension is used for a Cytofuge preparation. The cytofuge slide is stained with Wright's blood stain (Sigma Chemical Co.) and a differential court is performed to evaluate the percentage of PMNs in the preparation.

II. Upregulation of endothelial cells:

The monolayers of endothelial cells in the 96-cm plates are upregulated with 300 U/ml tumor necrosis factor (TNF, Boehinger-Manheim catalog # 1371-843). TNF and the compound are added to each vessel 4 hours prior to the addition of PMNs PMNs.

III. Fluorescent labeling of neutrophils:

After the final wash, the neutrophil pellet is resuspended in 5 ml of PBS-G (approximately 1-3 x 10 ml). 5 (and 6) carboxyfluorescent diacetate succinimidyl ester (CFSE, Molecular Probes, Eugene, OR). A 20 mM stock of CFSE is prepared by dissolving 25 mg in 2.24 ml (MW557.5) DMSO. 5 µl of the stock is added to 5 ml suspension of PMNs for a final concentration of 2 µM. The mixture is incubated in the refrigerator for 20 minutes. At the end of this period, the PMNs were washed four times with PBS-G. After the last wash, the PMNs were resuspended in complete EGM-UV media to the desired concentration (typically each well of a 96-well plate receives 0.6-1.2 x IO<5>PMNs).

IV. Addition of test compounds

One hundred and ten media containing the TNF compounds are used to replace the media in the wells containing the monolayers of endothelial cells 4 hours before the addition of PMNs.

V. Adhesion test:

A. Collection of data:

CFSE-labeled neutrophils (0.7 to 1.5 x 10<5>) in 10 µl volumes (see III) are added to the HUVEC monolayers. The plates are incubated at 37°C in a 5% C02+ 95% air, 100% humidity incubator for 30 min. Non-adherent cells are removed by centrifugation according to the following protocol: (1) A reading is taken in Cytofluor 2400 after incubation at 37°C. This reading is considered as 100% of cells added. (2 ) The wells are filled with heated (incubator) media on top of the well with a slightly convex meniscus (typically 260 µl (in addition to the 100 µl or so already in the wells)).(3) The wells are sealed using adhesive sealing film for microplates (Rainin catalog # 96-SP-100). (4) The lid is replaced on the plate and the size and position of any bubble is recorded on the lids with a marker. (5) The lids are removed and folded (4 folds) and pieces cut of paper towel is placed on top of the sealed film.The plate lid is then replaced on top of the paper towel leet and the plate is turned over. (6) The plates are placed on the plate holders of the centrifuge (Sorvall Model ROOM TEMPERATURE 6000D) and centrifuged by turning the speed down to around 500 RPM, then the centrifuge is turned on with the time button. The cruise control is adjusted until the tachometer reads 1100 rpm (this is the equivalent of 200 Gs). When a speed of 1100 rpm is reached a separate timer is started. After exactly 2 minutes, the centrifuge's timer is reset to zero to stop the motor. The plates are allowed to come to rest without braking. (7) The plates are removed and-any empty sumps are recorded. The lid and the folded paper towel are removed (the plates are kept upside down). The sealing film is then removed from the biological waste container and the media is shaken out. The plate is then blotted onto the paper towel and any excess fluid is suctioned off. (8) The plate is turned to an upright position and studied under a microscope. (9) A second reading is performed on Cytofluor. This second reading is used to determine the proportion of PMNs that remained adhered to the monolayer. (10) The information from the CSV files of Cytofluor is downloaded and the data is processed in an EXCEL spreadsheet where background (PMNs adhered to non-upregulated endothelium) is subtracted and the proportion of adhesion inhibition is determined as follows:

(11) The EXCEL spreadsheet calculates a) the percentage of PMNs that adhere to the monolayer, b) the percentage of PMNs that adhere to the monolayer minus background (PMNs that adhere to unstimulated endothelial cells) and

c) the percentage of adhesion inhibition for the basins receiving TFN alone to 0% inhibition (negative

number indicates increase in adhesion).

B. Statistics, data processing and storage: Statistics are performed by applying the two-sided t-test with equal variance in EXCEL, and the results recorded as the P-value.

IV. Additional information:

A. To dress basins with collagen:

Dissolve 25 mg of acid-soluble rat tail collagen in 446 ml of water acidified with a few drops of HC1. Sterilize by filtration. Add 50 µl to each well in 96-well plate (.28 cm<2>) for 10 µg/well. Let this incubate overnight in the 37°C incubator. Blow away the liquid and store in the refrigerator until use.

B. To prepare PBS+0.2% glucose (dextrose): Prepare 2 liters of PBS (Sigma) and add 2 g of glucose. Sterilize filter and store.

Rat myocardial infarction/reperfusion injury model

Male Sprague-Dawley rats are anesthetized with urethane, 1.25 g/kg ip. A carotid artery and vein are externalized and a cannula with PE-50 tubing is inserted to record blood thickness and to facilitate intravenous administration of dye or drug. A tracheotomy is performed. Animals are attached to a Harvard Roden Ventilator (Model 683, Harvard Apparatus, South Natick, MA) and ventilated at 1.5 ml/100 g body weight at 50 bpm. Needle electrodes are placed for a lead II electrocardiogram. The animals are kept at 37°C using electric heating pads adjusted to the desired temperature and controlled via rectal thermistor probe and controller. The heart is carefully isolated by a left thoracotomy and at the fifth intercostal space, and the left external descending coronary artery (LAD) is located. A ligature of 6-0 silk is placed around the LAD with the end threaded through a short length of PE-320 tubing to facilitate rapid occlusion and reperfusion of the artery. The LAD is occluded by clamping the suture and clamped tightly around the cardiac surface using a 25 mm Schwarz aneurysm clamp. Occlusion lasts for 90 min and is followed by reperfusion for 3.0-4.5 hours. Animals are dosed with drug or agents 10 min before reperfusion of the affected area of the heart by intravenous delivery via the jugular vein. Sham-operated rats are not subjected to ischemia or reperfusion. At the end of the experiment, the LAD is permanently re-occluded and a 10 mg/ml solution of Evans Blue Stain is administered via the carotid cannula to identify the area attacked by ischemia, i.e. the area at risk (AAR). The damaged heart is quickly removed and placed in 0.9% saline at 4°C before

creatine phosphokinase (CPK) activity is terminated.

Determination of creatine phosphokinase activity:

The left ventricular free wall (LVFW) is dissected free from the heart and weighed. The AAR, as defined by the absence of defects, is dissected from the LVFW and also weighed. AAR is homogenized for 5 seconds in 4 ml of 0.25 M sucrose containing 1 g for 30 min at 4°C. The supernatant is decanted to determine CPK activity and the pellet is stored frozen for isolation and testing of myeloperoxidase activity. CPK activity is assayed spectrophotometrically with a commercially supplied substrate, the CPK Assay Vial® (Sigma Diagnostics) at a wavelength of 340 nm at 24-26°C.

Determination of myeloperoxidase activity:

Myeloperoskidase (MPO) is isolated from the frozen pellet after preparation of CPK. The pellet is suspended in 50 mM phosphate buffer, pH6, containing 0.5% hexadcyltrimethylammonium bromide (HTAB) to a concentration of about 10%, sonicated for 10 seconds and frozen on dry ice. Three freeze-thaw cycles are done with 10 seconds of sonication between cycles. The samples are cooled on ice for 30 min. followed by centrifugation at 12,500 x g for 15 min at 4°C. An aliquot of the supernatant is assayed spectrometrically for MPO activity in 50 mM sodium phosphate buffer, pH6, containing 0.167 mg/ml o-dianisidine dihydrochloride and 0.113 ml hydrogen peroxide per 100 ml buffer at a wavelength of 460 nm at 24-26°C.

Calculation and statistical analysis:

The results are reported as the mean ± SEM. CPK and MPO activity are expressed as units/g of tissue, where 1 unit of CPK activity is defined as the amount of CPK using 1 ujnol phosphocreatine per minute. AAR is quantified as wt% of LVFW. Mean arterial blood pressure (MABP) is calculated as one third of the difference between systolic and diastolic blood pressure added to diastolic blood pressure. Data are analyzed for statistical significance of the treatment effects at the 95% confidence level by a coordinated t-test or by one-way analysis of variance.

The invention relates to methods of treating or preventing any disorder or disorder as shown above, particularly reperfusion injury, by administering a safe and effective amount of a 2-carboxamide benzimidazole compound as described above. Such methods of treatment may involve administering a unit dosage form of such compounds parenterally, orally, or topically. Parenteral administration includes intravenous, intramuscular, subcutaneous, intraperitoneal or other injection of the dosage form. Oral administration involves swallowing the dosage form and absorption of the active from the gastrointestinal tract. Topical administration involves contacting the dosage form with the surface of skin or mucosal tissue, including but not limited to those of the digestive tract and respiratory system.

For parenteral administration, the amount of 2-carboxamide benzimidazole compound typically administered is preferably from about 2 mg/kg, more preferably from about 5 mg/kg, preferably to 2 mg/kg, more preferably to about 10 mg/kg. The frequency of such administration is typically once or twice a day. A course of treatment is typically a single dose or lasts from about 1 day, preferably from about 5 days, to about 30 days, preferably to about 15 days.

For oral administration, the amount of 2-carboxamide benzimidazole compound typically administered is preferably from about 5 mg/kg, more preferably from about 10 mg/kg, preferably to 25 mg/kg, more preferably to about 15 mg/kg. The frequency of such administration is typically from one to four times a day. A course of treatment is typically a single dose or lasts from about 1 day, preferably from about 5 days, to about 30 days, preferably to about 15 days.

Composition and method examples

The following non-limiting examples illustrate the invention. The following composition and method examples do not limit the invention, but are guidelines for the person skilled in the art for the preparation and use of the compounds, compositions and methods according to the invention. In each case, other compounds within the invention can be substituted for the example compound shown below with the same results.

Example A

Pharmaceutical compositions in the form of an intravenous solution are prepared by conventional methods, such as by mixing the following:

When 1 ml of the composition above is administered intravenously, either immediately before or immediately after a tissue injury (aneurysm repair, coronary bypass, transplant, bleeding, organic anemia due to hypoperfusion, sepsis, etc.), tissue injury is avoided or reduced.

Example B

Pharmaceutical compositions in liquid form are prepared by conventional methods, formulated as follows:

When 1.0 ml of the above composition is administered intravenously, either immediately before or immediately after a tissue injury (aneurysm repair, coronary bypass, transplantation,<1>Compound according to Example 1 can be substituted for any of Examples 2-14.< 2>Compound according to Example 1 can be replaced by any of Examples 2-14.

bleeding, organaemia due to hypoperfusion, sepsis, etc), tissue damage is avoided or reduced.

While certain embodiments of the invention have been described, it is clear to those skilled in the art that a number of changes and modifications to the compositions included herein can be made without departing from the spirit and scope of the invention.

Claims (10)

1. Connection with the structure: in which: (a) RI is selected from the group consisting of alkyl, aryl, alkoxy and aryloxy, wherein alkyl and aryl in the preferred RI contain 1-14 carbon atoms, (b) R 3 and R 4 are independently selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, alkyl-aryloxy, alkylthio, amino and mono- or dialkylamino, the alkyl moieties of the preferred R 3 and R 4 having 1-8 carbon atoms, except when R3 and R4 are both hydrogen, (c) each R 5 is independently selected from the group consisting of hydrogen, halogen, cyano, alkyl, hydroxy, alkoxy, thio, alkylthio, amino and mono- or dialkylamino, the alkyl moieties of preferred R 5 having 1-8 carbon atoms, and an optical isomer, diastereomer or enantiomer or mixture thereof, a pharmaceutically acceptable salt, hydrate or biohydrolyzable ester, amide or imide thereof. 2. Compound according to claim 1, in which RI is alkyl or aryl with from 2 to 8 carbon atoms, preferably RI is alkyl with from 3 to 8 carbon atoms, the alkyl is unsubstituted or substituted with substituents selected from the group consisting of halo, hydroxy, C1 -C3 alkoxy, thio, C1 -C3 alkylthio, amino, C1 -C3 mono- or dialkylamino, phenyl and heterocycle with 5 or 6 ring atoms, more preferably RI is saturated or unsaturated with a double bond, linear or branched or cyclic alkyl with from 3 to 6 carbon atoms. 3. Compound according to claim 2, in which R3 and R4 are independently selected from the group consisting of hydrogenalkyl, alkoxy, alkylthio, amino and mono- or dialkylamino, the alkyl parts of such R4 having from 1 to 6 carbon atoms. 4. Compound according to claim 3, in which each R5 is independently selected from the group consisting of hydrogen, halo, alkyl, alkoxy, alkylthio and mono- or dialkylamino, the alkyl parts of such R5 having from 1 to 6 carbon atoms. 5. Compound according to claim 4, wherein the alkyl parts of R3, R4 and R5 parts independently have from 1 to 3 carbon atoms, unsubstituted or substituted with halo, hydroxy, C1 -C3 alkoxy, thio, C1 -C3 alkylthio, amino and C1 -C3 mono - or dialkylamino, preferably the alkyl parts of R3/R4, R5 are unsubstituted. 6. Compound according to claim 5, wherein RI is phenyl or benzyl, unsubstituted or substituted with substituents selected from the group consisting of halo, C1 -C3 alkyl, hydroxy, C1 -C3 alkoxy, amino and C1 -C3 mono- or dialkylamino, preferred is R is saturated or unsaturated and preferably R 3 is methoxy or ethoxy. 7. Compound according to claim 6, in which R 4 is alkoxy, preferably methoxy or ethoxy, more preferably methoxy and R 3 and both R 5 are hydrogen. 8. A compound according to claim 7, wherein R3 is alkoxy and R4 and both R5 are hydrogen. 9. Pharmaceutical composition comprising: (a) a safe and effective amount of a compound of any one of claims 1 or 6; and (b) pharmaceutically acceptable excipients. 10. A method of preventing or treating an ischemia-reperfusion injury comprising administering a safe and effective amount of a compound according to any one of claims 1 or 6 to a human or lower animal in need thereof.