JP2003522759A - 2-carboxamide-benzimidazoles - Google Patents

Abstract

(57) [Summary] The present invention relates to a compound having the following structure. Embedded image Wherein: (a) R1 is selected from the group consisting of alkyl, aryl, alkoxy and aryloxy; preferred alkyl and aryl moieties of R1 have from 1 to about 14 carbon atoms; (b) R3 and R4 are Independently selected from the group consisting of hydrogen, halo, alkyl, alkoxy, alkyl-aryloxy, alkylthio, amino, and mono- or di-alkylamino, with the preferred alkyl portions of R3 and R4 being from 1 to about 8 carbon atoms. R3 and R4 are not both hydrogen; (c) each R5 is independently hydrogen, halo, cyano, alkyl, hydroxy, alkoxy, thio, alkylthio, amino, and mono- or di-alkylamino Wherein the alkyl portion of R5 is a compound having from 1 to about 8 carbon atoms; . The subject invention further relates to compositions containing these compounds. The subject compounds are useful for preventing or treating reperfusion injury of various tissues.

Description

Detailed Description of the Invention

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of US Provisional Application No. 60 / 181,234, 7945P, filed February 9, 2000.

TECHNICAL FIELD The subject invention is useful for treating or preventing ischemia-reperfusion injury of myocardium and other tissues, and other cardiovascular or inflammatory diseases and disorders.
-Carboxamide-benzimidazole.

[0003]     (Outline of the invention)   The subject invention includes compounds having the structure:

[0004]

[Chemical 2]

Wherein: (a) R 1 is selected from the group consisting of alkyl, aryl, alkoxy and aryloxy, preferred alkyl and aryl moieties of R 1 have from 1 to about 14 carbon atoms; (b) R 3 And R4 are each hydrogen, halo, alkyl, alkoxy, alkyl-
Selected from the group consisting of aryloxy, alkylthio, amino, and mono- or di-alkylamino, preferred alkyl moieties of R3 and R4 have from 1 to 8 carbon atoms; both R3 and R4 are not hydrogen (C) each R5 is independently hydrogen, halo, cyano, alkyl, hydroxy,
A preferred R5 alkyl moiety selected from the group consisting of alkoxy, thio, alkylthio, amino, and mono- or di-alkylamino, has 1 to 8 carbon atoms; and optical isomers, diastereomers or enantiomers Alternatively, a mixture thereof; a compound which is a pharmaceutically acceptable salt, hydrate or biologically hydrolyzable ester, amide or imide thereof.

The invention further relates to pharmaceutical compositions containing such compounds; and methods of using such compounds to treat or prevent tissue reperfusion injury. Each year in the United States, approximately 1.1 million people suffer from myocardial infarction and 350,000 deaths. Myocardial infarction (MI), or heart attack, occurs when the blood vessels supplying the heart are completely or partially occluded. Restoring blood flow with thrombolytic agents (reperfusion) is often the primary treatment strategy. However, the benefits of reperfusion carry the risk of an acute inflammatory response associated with it, resulting in a syndrome called reperfusion injury. Inflammation generally plays a self-defense role. For example, at the site of bacterial infection,
Bacterial endotoxin promotes the production of inflammatory cytokines, which induce circulating leukocytes, including neutrophils and monocytes, to destroy bacteria. Inflammation subsides when infection disappears. However, there are persistent inflammatory conditions (rheumatoid arthritis) and unnecessarily severe conditions (ischemia-reperfusion injury).

An essential property of inflammation is the migration of neutrophils (PMNs) from the blood into tissues. This migration occurs as a result of a cascade mediated by adhesion molecules. Adhesion of PMNs to vascular endothelial cells requires the interaction of adhesion molecules on the surface of both cells. These molecules belong to three different families: the selectins, integrins and the immunoglobulin superfamily. Neutrophils first migrate along the endothelial cells, a process mediated by selectins. At the site of inflammation, tight adhesion is expressed on 2 integrins on PMNs and endothelial cells and ICAM-1 (
It is mediated by the interaction of intercellular adhesion molecules-1). Ultimately, the migration of PMNs across the endothelium into tissue leads to tissue damage. Compounds that can protect the adhesion of neutrophils to the endothelium include, but are not limited to, myocardial infarction, coronary artery bypass graft, angiogenesis, angina, stroke, peripheral vascular disease, inflammatory bowel disease, Ulcerative colitis, burns, frostbite, adult respiratory distress syndrome, asthma, tissue and organ transplantation, general surgery, reimplantation, acute renal failure, rheumatoid arthritis, psoriasis, hepatitis,
It may be useful in treating a variety of conditions including ischemia-reperfusion injury including pancreatitis, sunburn, radiation, ulcers and shock. (See recent reviews: C. Cornejo, J. Harlan, R. Winn, Health & Disease Adhesion Molecules, L. Paul and T. Isekts (L. Paul and T. Issekutz), edited by Marcel Dekker, 1997, chapter 18; J. Prince, C. Ballantyne, The Emergence of Therapeutic Purposes, 199.
9 years, 263-177)

Glossary of Terms Unless otherwise specified, the following terms have the indicated meanings when used in this application. The term "alkyl" means a hydrocarbon chain which may be straight, branched or cyclic, saturated or unsaturated (other than aromatic), substituted or unsubstituted. The term may be used alone or as part of another term, which may be abbreviated as "alk" (eg, alkoxy, alkylamino). Preferred straight chain alkyls are from about 1 to about 20. It has carbon atoms, more preferably 1 to about 8 carbon atoms, even more preferably 1 to about 4 carbon atoms; most preferably methyl or ethyl. Preferred cyclic and branched chain alkyls have 3 to 20 carbon atoms, more preferably 3 to about 10 carbon atoms, and even more preferably 3 to about 6 carbon atoms. Preferred cyclic alkyls have one hydrocarbon ring, but may have two, three or more and may have fused or spiro cyclic hydrocarbon rings. Alkyl has only one or more double bonds (alkenyl) (
No triple bond), preferably having one, two, or three double bonds,
More preferably, it may be an unsaturated type having one double bond. The alkyl may be unsaturated with one or more triple bonds (“alkynyl”), preferably unsaturated with one triple bond. More preferably alkyl is saturated ("alkanyl"). The term "alkylene" means an alkyl bound to two or more moieties. Preferred substituents for alkyl include alkyl, aryl, halo, hydroxy, alkoxy, aryloxy, amino, alkylamino, arylamino, thio, alkylthio, arylthio, acyl, alkylacyl, arylacyl, carboxy, alkyl ester, aryl ester, Examples thereof include amino, alkylamino, arylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, nitro, cyano and heterocycle. Preferred alkyl is unsubstituted.

The term “aryl” means a substituted or unsubstituted aromatic hydrocarbon ring. The terms may be used alone or as part of another term (eg, aryloxy, arylamino). Preferred aryls have 6 to about 14 carbon atoms in the aromatic ring, and have a total of about 6 to about 20, preferably up to about 12 carbon atoms.
Preferred aryl is phenyl or naphthyl; most preferred is phenyl. The term "arylene" means aryl bound to two or more other moieties. Preferred substituents for aryl are alkyl, aryl, halo, hydroxy,
Alkoxy, aryloxy, amino, alkylamino, arylamino, thio, alkylthio, arylthio, acyl, alkylacyl, arylacyl, carboxy, alkyl ester, aryl ester, amino, alkylamino, arylamino, sulfonyl, alkylsulfonyl, arylsulfonyl. , Nitro, cyano, and heterocycle. The term "heteroatom" means a nitrogen, oxygen, or sulfur atom. The term "heterocycle" or "heterocycle" means a cyclic alkyl or aryl having one or more heteroatoms substituted on a ring carbon atom, preferably 1, 2 or 3 heteroatoms in the ring. Preferred substituents on the heterocycle are the same as the alkyl substituents. The term "heteroaryl" refers to a subset of heterocycles including aromatic rings. Preferred heteroaryls have from 5 to about 14, more preferably up to about 10, even more preferably 5 or 6 carbons and heteroatoms in the ring and a total of 5 to about 20, more preferably about 10. It has up to 12 carbon and heteroatoms.

The term "safe and effective amount" is within the medical criteria and is sufficient to elicit a clear effect in the condition to be treated, but sufficiently low to avoid serious side effects (reasonable amount). Amount of pharmaceutically active compound) (in terms of relative benefit / risk ratio). A safe and effective amount of the compound is determined by the particular condition in need of treatment, the size and age and physical condition of the patient, the severity of the condition, the duration of treatment, the nature of the current treatment, the particular pharmaceutically acceptable use It will depend on the exact career, knowledge of the treating physician and other factors within the experience. The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient"
Means one or more compatible solid or liquid excipients suitable for human or lower animal applications. The term "compatible" refers to a pharmacologically active compound or excipient which is admixable with the compound in such a way that there is no interaction which essentially diminishes the pharmaceutical effect of the composition under normal use conditions. means. Prosthetic agents do not have intrinsic pharmaceutical activity per se, but include, for example, diluents, lubricants, degradation enhancers, dissolution enhancers, encapsulants, preservatives, colorants, fragrances and others. May function as. The pharmaceutically acceptable excipient must be of markedly high purity and of markedly low toxicity to be suitable for application to the human or lower animal to be treated.

The term “dosage unit” means a composition comprising an amount of a pharmacologically active compound when administered to a human or lower animal in a single dose according to good medical practice. A "biologically hydrolysable ester" is an ester of the present invention, including 2-carboxamidobenzimidazole, which does not interfere with the activity of 2-carboxamidobenzimidazole and which is readily activated by animals by the active form 2 of the invention. -Converted to carboxamide benzimidazole. Such esters include lower alkyl esters, lower acyloxy-alkyl esters (acetoxymethyl, acetoxyethyl, aminocarbonyloxymethyl, pivaloyloxymethyl and pivaloyloxyethyl esters, etc.), lactonyl esters (phthalidyl). And thiophthalidyl esters), lower alkoxyacyloxyalkyl esters (eg methoxycarbonyloxymethyl, ethoxycarbonyloxyethyl and isopropoxycarbonyloxyethyl esters), alkoxyalkyl esters, choline esters and alkylacylaminoalkyl esters Examples thereof include acetamide methyl esters.

Compounds The subject invention comprises 2-carboxamidobenzimidazole compounds having the structure:

[0013]

[Chemical 3]

In Structural Formula (I), R1 is selected from alkyl, aryl, alkoxy and aryloxy. Preferred alkyl and aryl moieties of the R1 moiety have 1 to about 14 carbon atoms. More preferred R1 is selected from unsubstituted or substituted alkyl having 1 to about 12 carbon atoms and unsubstituted or substituted phenyl or naphthyl. More preferred alkyl R1 includes unsubstituted or substituted straight chain alkyl having about 2 to about 8 carbon atoms, even more preferably about 3 to about 6 carbon atoms; and even more preferably n. -Propyl or n-butyl or n-pentyl. More preferred R1 includes branched chain alkyl having from about 3 to about 8 carbon atoms, even more preferably from about 3 to about 6 carbon atoms; and even more preferably isobutyl or isopentyl. More preferable R1 is about 3 to about 8 carbon atoms, and even more preferably 3 to about 6 carbon atoms.
Included are cyclic alkyls having 4 carbon atoms. Preferred substituents for such linear, branched or cyclic alkyl include halo, hydroxy, alkoxy,
Amino, mono- and di-alkylamino, thio, alkylthio, aryl (especially phenyl), and heterocycles are mentioned; more preferably such alkyls are unsubstituted. Preferred R1 is a straight chain type, a branched chain type or a cyclic alkyl which is a saturated type or an unsaturated type having one or more double bonds; and more preferably a saturated type.

More preferred aryl R1 is unsubstituted or substituted phenyl.
Preferred substituents on such phenyl include halo, hydroxy, alkoxy, amino, mono- and di-alkylamino; and preferred are such unsubstituted phenyl. More preferable aralkyl R1 includes unsubstituted or substituted benzyl. Preferred substituents on such benzyl include halo, hydroxy, alkoxy, amino, mono- and di-alkylamino; also preferred are unsubstituted benzyl. In Structural Formula (I), R3 is R3 except that R3 and R4 are not hydrogen.
And R4 are each selected from the group consisting of hydrogen, halo, alkyl, alkoxy, alkyl-aryloxy, alkylthio, amino, and mono- or di-alkylamino. Preferred alkyl moieties of the R3 and R4 moieties have 1 to about 8 carbon atoms. Preferably, R3 and R4 are not hydrogen, except for R
3 and R4 are each selected from the group consisting of hydrogen, halo, alkyl, alkoxy, alkylthio, and mono- or di-alkylamino.

Preferred R3 and R4 are hydrogen, alkoxy having 1 to about 6, preferably up to 3 carbon atoms; alkylthio having 1 to about 6, preferably up to 3 carbon atoms; 1 to about Mention is made of monoalkylamino or di-alkylamino having up to 6 and preferably up to 3 carbon atoms; alkyl with 1 to up to 6 and preferably up to 3 carbon atoms. Preferred substituents on the alkyl of such moieties include halo, hydroxy, alkoxy, amino, mono- or di-alkylamino, thio, alkylthio; more preferably such R3 and R4.
The part is unsubstituted. Even more preferred is at least 1 of R3 and R4
Is ethoxy or especially methoxy. Preferably one of R3 and R4 is hydrogen; more preferably R3 is hydrogen. In structural formula (I), R5 is located at sites 4 and 7 of the benzimidazole ring. Each R5 is independently selected from hydrogen, halo, cyano, alkyl, hydroxy, alkoxy, thio, alkylthio, amino, and mono- or di-alkylamino. Preferred alkyl moieties of the R5 moiety have 1 to about 8 carbon atoms.

Preferred R5 is hydrogen, halo, alkyl having 1 to about 6, preferably up to 3 carbon atoms, alkoxy having 1 to about 6, preferably up to 3 carbon atoms, 1 to about Mention may be made of monoalkyl or dialkylamino having up to 6 and preferably up to 3 carbon atoms, and alkylthio having from 1 to about 6 and preferably up to 3 carbon atoms. Preferred substituents on the alkyl of such R5 moieties include alkoxy, amino, and alkyl; more preferably the alkyl of such moieties is unsubstituted. More preferably each R5 is selected from hydrogen, halo, and unsubstituted alkyl having 1 to about 3 carbon atoms. More preferably, there is no more than one R5 other than hydrogen. Most preferably both R5 are hydrogen.

Preferred compounds of the subject invention include structural formula (I) and the following examples having the indicated substituents.

[0019]

[Table 1]

[0020]

[Table 2]

The subject invention compounds include all active optical isomers, diastereomers and enantiomers of the above compounds and mixtures thereof. The subject invention compounds include pharmaceutically acceptable salts, hydrates and biohydrolyzable esters, amides and imides of such compounds.

Synthesis of Compounds The following provides a general scheme for the preparation of compounds of the subject invention and specific methods of synthesizing preferred subject invention compounds. Unless otherwise noted, all available reagents are used without purification. Reactions are generally under an inert atmosphere (argon or nitrogen)
Done in. Concentrated salt water means a saturated sodium chloride aqueous solution. The remaining solvent is at room temperature (rt
) Under vacuum (about 0.03 mm Hg).

The structure of the synthesized compound is confirmed using the following analytical methods. Proton NMR spectrum is GEQE-300 (300MHz) spectrometer, Bruker A
C-300 (300 MHz) 4 nuclear probe system or Varian Unityplus (300 MHz). All chemical shifts are (
CH ₃₎ are recorded on a scale from ₄ parts per million sent from Si (ppm). CDC
The spectrum found in l ₃ is (CH ₃ ) ₄ Si or residual CHCl ₃ (7.24
ppm). The spectrum found in D ₂ O is HOD (4.8
0 ppm), those in (CO ₃ ) ₂ CO remained (CH ₃ ) ₂ CO (
2.04 ppm), those in the CD ₃ OD remained CH ₃ OH (3.
30 ppm) and those in (CD ₃ ) ₂ SO remained (CH ₃
) ₂ SO (2.49 ppm). Carbon-13 spectrum is GE QE-
300 (75MHz) spectrometer or Bruker AC-300 (75
MHz) 4 nuclear probe system. Spectra in CDCl ₃ refer to solvent (78 ppm), those in CD ₃ OD refer to solvent (49 ppm), (CD ₃ ) ₂ SO refer to solvent (39.7 ppm), and (CD ₃
) ₂ CO refers to solvent (206.5, 29.8 ppm). Mass spectra are determined on a Fision's Trio 2000 equipped with a robotic probe or a Fisons Platform II mass spectrometer. Chemical ionization spectra are obtained using methane and / or ammonia as reagent gases. ESI compound analysis as elution solvent methanol, 0
． Performed on a Hewlett Packard 1050 HPLC autosampler using 2% formic acid and 0.2 mM ammonium acetate. Thin layer chromatography is performed on plates precoated with silica gel 60-F254. Flash chromatography is performed using silica gel 60 (Merck, 230-400 mesh). Melting point electrothermal 1A9
200 or MelTemp II Capillary Melting Point apparatus, uncorrected.

Scheme I is a general scheme useful in the synthesis of many subject invention compounds: Scheme I

[0025]

[Chemical 4]

A: NaNO ₂ , propionic acid; b: HNO ₃ ; c: Tf ₂ O / Et ₃ N, toluene; d: R1-NH ₂ ; e: HCOOH, 10% Pd / C; f: n-BuLi , E
_{CF; g: NH 3, MeOH} , KCN.

5-methoxy-2-nitrophenol (R4 = methoxy and R3 and both R
5) B of scheme I where H = 2: 2 with mechanical stirrer and additional funnel
Propionic acid (300 mL) and 3-methoxyphenol (37.2 g, 0.3 mol) are added to a liter round bottom flask. The resulting mixture is cooled to 0 ° C. and sodium nitrate (21 g, 0.304 mol) in water (50 ml) is added slowly. After stirring at 0 ° C. for 1 hour, fuming nitric acid (40 mL) is slowly added. The resulting mud is stirred at 0 ° C. for 1 hour and then warmed to room temperature for 2 hours or more. Water (250
ml) at room temperature, the resulting solid is filtered and washed with 300 mL 50% aqueous propionic acid solution and dried to give solid 5-methoxy-2-nitrophenol.

N-alkyl-5-methoxy-2-nitrianiline (R1 = alkyl, R4 =
Methoxy and R3 and C in Scheme I where both R5 = H): Toluene (300 mL), 5-methoxy-2-nitrophenol (in a 1 liter round bottom flask).
5.0 g, 0.03 mol) and triethylamine (6.68 g, 0.066 mo)
l) is added. The resulting solution is then cooled to 0 ° C. and trifluoroacetic anhydride (Tf ₂ O) is added slowly via syringe. (9.3 g, 0.033 mol) The reaction mixture is stirred at 0 ° C. for 5 minutes; amine (R1-NH ₂ ) (0.12 mol) is added and the reaction mixture is refluxed for 5.5 hours. After cooling to room temperature, the reaction contents are filtered through a plug of silica gel (eluted with 90:10 hexane: ethyl acetate) and concentrated on a rotary evaporator to give crude N-alkyl-5-methoxy-2-nitroaniline. This material is used in the next synthetic step.

N1-alkyl-6-methoxybenzimidazole (R1 = alkyl, R4 =
Methoxy, and R3 and D of Scheme I where both R5 = H): To a 250 mL round bottom flask, add 88% formic acid (50 mL) and N-alkyl-5-methoxy-2-nitroaniline (0.02 mol). . To the homogeneous mixture was added 10% Pd-C ethyl acetate slurry (600 mg). The resulting heterogeneous mixture is heated to 100 ° C. for 1 hour, cooled to room temperature and filtered through Celite. (Elution with water) was then added 28% NH ₄ OH in filtrate to produce a salt, and washed with ethyl acetate (3 × 10
The 0 mL) The combined organics were dried (MgSO _4), filtered to give a residue is concentrated in a rotary evaporator. Chromatograph the residue (SiO ₂ , 50: 5
0 hexane: ethyl acetate) N1-alkyl-6-methoxybenzimidazole is obtained.

Ethyl 1-alkyl-6-methoxy-1H-benzimidazole-2-carboxylate (R1 = alkyl, R4 = methoxy, and E of Scheme I where R3 and both R5 = H): under argon atmosphere. A 250 mL round bottom flask was charged with N1-alkyl-6-methoxybenzimidazole (2.9 g, 0.014 mol) and TH.
Add F (100 mL). This was cooled to −78 ° C. and n-butyllithium (1
2.3 mL, 0.019 mol in hexane) is added dropwise to give a yellow mixture.
Stirring is continued at −78 ° C. for 30 minutes, after which ethyl chloroformate (ECF) is added (2.13 g, 0.019 mol). The reaction mixture is warmed to room temperature and stirred for 30 minutes. Water (100 mL) is added and the resulting mixture is extracted with ethyl acetate (3x
100 mL). The combined organics dried (MgSO _4), filtered, and concentrated to obtain a residue of yellow. The residue is then chromatographed (flash, 70:30 hexane: ethyl acetate) ethyl 1-alkyl-6-methoxy-1H-benzimidazole-2-carboxylate.

N1-alkyl-6-methoxybenzimidazole-2-carboxamide (R
Scheme I where 1 = alkyl, R4 = methoxy, and R3 and both R5 = H
F ): Ethyl 1-alkyl-6-methoxy-1H-benzimidazole-2-carboxylate (1) in an ACE GLASS pressure tube equipped with stir bar.
． 8 mmol), ammonia methanol (10 mL, 2.0 M), and sodium or potassium cyanide (8.8 mg, 0.18 mmol). Then the reaction vessel is sealed and heated at 80 ° C. for 3 hours and then cooled to room temperature. The resulting reaction mixture is concentrated by rotary evaporator and the residue is chromatographed (flash, 60:40 hexane: ethyl acetate) to give N1-alkyl-6-methoxybenzimidazole-2-carboxamide.

Scheme II is another general scheme useful in the synthesis of many subject invention compounds.

Scheme II

[0034]

[Chemical 5]

H: (CH ₂ O) _n , KCN, HOAc, ZnCl ₂ ; j: EtOH, KOH; k
: R2-OH, DEAD, Ph 3 P, THF; m: KOH, t-BuOH N- ( cyanomethyl) -4-methoxy-2-nitroaniline (R4 = methoxy and R3 and both R5 = H a is Scheme II H ): 4-methoxy-2-nitroaniline (8.4 g, 0.05 mol), paraformaldehyde in a 3-neck 1 liter round bottom flask equipped with mechanical stirrer, reflux condenser and additional funnel. (4.5 g, 0.15 mol), potassium cyanide (9.75 g, 0.15 mo)
1) and zinc chloride (25 g, 0.18 mol). To this vigorously stirred mixture is slowly added a mixture of H ₂ SO ₄ (4 drops) and glacial acetic acid (250 mL). The resulting reaction mixture is heated at 50 ° C. for 8 hours and then cooled to room temperature. The reaction contents were poured into ice / water, the solid formed was collected by suction filtration, washed with water, dried, recrystallized (extract with EtOH) and N- (cyanomethyl) -4-methoxy-2-.
Nitroaniline is obtained as needle crystals.

2-Cyano-6-methoxybenzimidazole-N1-oxide ( J in Scheme II where R4 = methoxy and R3 and both R5 = H): N- (cyanomethyl) -4- in a 500 mL round bottom flask. Methoxy-2-nitroaniline (2.5
g, 0.012 mol) and 95% ethanol (130 mL). The reaction mixture is heated to 60 ° C., solid potassium carbonate (1.77 g, 0.013 mmol) is added, the resulting mixture is then heated to reflux for 4.5 h, cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in water and then acidified (conc.HCl) to form a white solid which was collected by filtration, dried and recrystallized (DMF) to give solid 2-cyano-6-methoxybenzimidazole-N. -Oxide is obtained.

[0037]   2-Cyano-1-isopropyl-6-methoxybenzimidazole (R1 = I
Sopropyl R4 = methoxy, and R3 and both R5 = H K ): 2-Cyano-6-methoxybenzimidazole in a 100 mL round bottom flask.
-N-oxide (457 mg, 2.41 mmol), tetrahydrofuran (TH
F) (50 mL), isopropyl alcohol (145 mg, 2.41 mmol)
, Triphenylphosphine (Ph₃P) (632 mg, 2.41 mmol) and
Finally diethyl azodicarboxylate (DEAD) (444mg, 2.55m
mol) is added. The resulting mixture was stirred at room temperature for 18 hours, then concentrated under reduced pressure.
Contract. The residue was chromatographed (80:20 hexane: ethyl acetate) and solidified.
The body of 2-cyano-1-isopropyl-6-methoxybenzimidazole is obtained.

1-isopropyl-6-methoxybenzimidazole-2-carboxamide (
R1 = isopropyl, R4 = methoxy and R3 and both R5 = H; Scheme II L ): 2-cyano-1-isopropyl 6-methoxybenzimidazole (100 mg, 0.43 mmol), t- in a 25 mL round bottom flask. Butyl alcohol (5 ml) and potassium hydroxide (200 mg, 3.57 mmol) are added. The mixture is refluxed for 1 hour, cooled to room temperature, poured into concentrated brine (50 ml) and extracted with ethyl acetate (3 x 50 mL). Dry the organic fractions were washed with (MgSO ₄₎
Filter and concentrate under reduced pressure to give a yellow oil. Chromatograph this oil (7
0:30 hexane: ethyl acetate), solid 1-isopropyl-6-methoxybenzimidazole-2-carboxamide is obtained.

Scheme III is another general scheme useful in the synthesis of many subject invention compounds: Scheme III

[0040]

[Chemical 6]

N: PhCHO, HNO ₃ , H ₂ SO ₄ ; p: (Boc) ₂ CO, CH ₂ Cl ₂ ; q:
_{R1-NH 2, CH 3 CN} ; r: 10% Pd / C, EtOH; s: (CHO-CO
OEt) _n / toluene, I ₂ / EtOH; t: CF ₃ COOH, CH ₂ Cl ₂ ; u:
NH ₄ OH, THF 3- fluoro-4-nitroaniline (R4 = amino, and R3 and both R5
= N, Scheme III, where H): 3-fluoroaniline (5 g; 1 eq) is reacted with benzaldehyde (5 g; 1.05 eq). Mix 1/2 at 25 ° C
Stir for a period of time and gently reflux. The mixture is dissolved in concentrated sulfuric acid (20 ml) and cooled in an ice bath to <5 ° C. A mixture of concentrated nitric acid (3 ml) and concentrated sulfuric acid (10 ml) is added slowly and the mixture is stirred at 25 ° C. for 11/2 hours. The reaction is terminated by pouring it into water (200 ml); then washing with ethyl acetate. The combined ethyl acetate layers are dried over magnesium sulphate and dried by evaporation. The product is chromatographed on a silica gel column using 20% ethyl acetate-hexane as eluent to give the title compound and other regioisomers.

(3-Fluoro-4-nitro-phenyl) -carbamic acid tert-butyl ester (R4 = Boc-NH and R3 and both R5 = H P in Scheme III)
: Ditertiary butyl dicarbonate (1.4 g; 1 eq) is reacted with 3-fluoro-4-nitroaniline (1 g; 1 eq) in dichloromethane (30 ml). When the mixture was stirred at 25 ° C. for 10 minutes, dimethylaminopyridine (0.08 g; 0
． 1 equivalent) is added. The mixture is stirred at 25 ° C. for 24 hours. The mixture is diluted with ethyl acetate and washed with 1 mol of aqueous citric acid solution; the mixture is then washed twice with water. The ethyl acetate layer is dried over magnesium sulfate and dried by evaporation. The product is chromatographed on a silica gel column using 30% ethyl acetate-hexane as eluent to give a mixture of the title compound (and bis-Boc protected compound).

(3-isobutylamino-4-nitro-phenyl) -carbamic acid tert-butyl ester (R1 = isobutyl, R4 = Boc-NH and R3 and both R5 = H
Scheme III Q) which is: isobutylamine (0.29 g; 2 eq.) (3
-Fluoro-4-nitro-phenyl) -carbamic acid tertiary butyl ester (0.
5 g; 1 eq) is slowly added to a solution of acetonitrile (20 ml). The mixture is stirred at 25 ° C. for 24 hours. Then the mixture is diluted with dichloromethane and 2 times with water.
Wash once. The dichloromethane phase is dried over sodium sulphate and evaporated to dryness to give the title compound.

(4-Amino-3-isobutylamino-phenyl) -carbamic acid tert-butyl ester (R1 = isobutyl, R4 = Boc-NH and R3 and both R5 = H
R in Scheme III, which is: (3-isobutylamino-4-nitro-phenyl) -carbamic acid tert-butyl ester (0.51 g; 1 eq) in ethanol (
The solution (15 ml) is reacted with a suspension of palladium (10 mass%) (0.1 g; 2 equivalents) on carbon in ethanol (15 ml) under nitrogen atmosphere with stirring. The nitrogen was then replaced with a positive pressure of hydrogen and the mixture was heated at 25 ° C under a hydrogen atmosphere at 16 ° C.
Stir for hours. The mixture is then filtered through Celite and the precipitate washed with dichloromethane. The filtrate is evaporated to dryness to give the title compound.

6-tertiary butoxycarbonylamino-1-isobutyl-1H-benzimidazole-2-carboxylic acid ethyl ester (R1 = isobutyl, R4 = Boc-NH
And S in Scheme III where R3 and R5 = H): a solution of ethyl glyoxylate polymer (0.5 g; 1.5 eq.) In 50% toluene (4-amino-3-isobutylamino-phenyl) -carbamic acid. Tertiary butyl ester (0.46 g;
1 equivalent, assuming 100% yield from the pre-reaction stage) is reacted with a solution of ethanol (3 ml). The mixture is stirred in air for 1/2 hour. Iodine (0.21 g;
A solution of 0.5 eq) in ethanol (1.5 ml) is added and the mixture is then stirred at 25 ° C. for 8 hours in air. Sodium thiosulfate (0.41 g; 1 equivalent) in water (
2 ml) and the mixture is stirred at 25 ° C. for 1/2 hour. The mixture is diluted with ethyl acetate and washed with water and concentrated brine. The ethyl acetate layer was dried over magnesium sulfate,
Dry by evaporation. The product is chromatographed on a silica gel column using 50% ethyl acetate-hexane as eluent to give the title compound.

6-Amino-1-isobutyl-1H-benzimidazole-2-carboxylic acid ethyl ester (R1 = isobutyl, R4 = Boc-NH and R3 and both R5
= T in Scheme III where H =: trifluoroacetic acid (0.5 ml) is added to 6-tertiary butoxycarbonylamino-1-isobutyl-1H-benzimidazole-2-
Slowly add to a solution of carboxylic acid ethyl ester (0.12g) in dichloromethane (4.5ml). The mixture is stirred at 25 ° C for 3 hours. The mixture is then evaporated to dryness and the residue is dissolved in methanol. Evaporate the methanol solution to dryness;
This is dissolved in dichloromethane and washed with 2M aqueous potassium carbonate solution. The dichloromethane phase is dried over sodium sulphate and evaporated to dryness to give the title compound.

6-Amino-1-isobutyl-1H-benzimidazole-2-carboxylic acid amide (R 1 = isobutyl, R 4 = Boc-NH and R 3 and U in Scheme III where both R 5 = H): Ammonia (5 ml 25% aqueous solution of 1) is added to a tetrahydrofuran (0.5 ml) solution of 6-amino-1-isobutyl-1H-benzimidazole-2-carboxylic acid ethyl ester (0.02 g) with stirring.

The mixture is stirred at 25 ° C. for 72 hours. Then the mixture is diluted with ethyl acetate and washed with water. The ethyl acetate fraction is dried over magnesium sulfate and evaporated to dryness to give the title compound.

Scheme IV is another general scheme useful in synthesizing many subject compounds: Scheme IV

[0050]

[Chemical 7]

V: R6-COCl, CH ₂ Cl ₂ ; w: B ₂ H ₆ , THF; x: 10% Pd / C,
EtOH; y: (CHO-COOEt ) n, I 2 / EtOH, z: NH 4 OH, T
HF N- (4-methoxy-2-nitrophenyl) -isobutyramide (R3 = methoxy, R4 and both R5 = H and R6 = W in Scheme IV where iPr): 4
-Methoxy-2-nitroaniline (1.0 g; 1 eq) is reacted with isobutyryl chloride (0.634 g; 1 eq) in pyridine (60 ml) and stirred for 4 hours. The mixture is concentrated under reduced pressure and the residue is dichloromethane (DCM) (70 ml).
Dissolve in water, wash twice with 1N HCl (aq), saturated Na ₂ CO ₃ (aq), water and concentrated brine and dry over MgSO ₄ . The solvent was removed under reduced pressure and the title compound 1.
35 g are obtained.

Isobutyl- (4-methoxy-2-nitrophenyl) -amine (R3 = methoxy, R4 and both R5 = H and R6 = iPr in Scheme IV X ): isobutyl- (4-methoxy-2- Nitrophenyl) -amine (0.1 g; 1 eq)
In THF (2 mL) is reacted with a 1 M borane-tetrahydrofuran complex solution in THF (1.25 mL; 3 eq) and stirred for 18 hours. Methanol (1
(mL) is slowly added and the solution is heated at 50 ° C. for 2 hours. The solvent is removed under reduced pressure and the residue is chromatographed (silica gel, 0-30% EtOAc / hexane) to give the title compound.

[0053]   N1-isobutyl-4-methoxy-benzene-1,2-diamine (R3 = meth
Xy, R4 and both R5 = H and R6 = iPrY): N ¹ -Isobutyl-4-methoxy-benzene-1,2-diamine (0.054g;
1 equivalent) was dissolved in ethanol (2 mL) and water (0.1 mL), and carbon was used as a carrier.
Add another 10% palladium (50% mass) (0.011 g). Hydrogen gas suspension
Stir for 2 hours under a nitrogen atmosphere and then replace the flask with nitrogen. Celite suspension
Filtered through a pad of N, washed with DCM and the reacted organic phase dried under reduced pressure to give the title compound.
Get the compound.

1-isobutyl-5-methoxy-1H-benzimidazole-2-carboxylic acid ethyl ester (R3 = methoxy, R4 and both R5 = H and R6 = Z in Scheme IV where iPr): 1-isobutyl- 5-Methoxy-1H-benzimidazole-2-carboxylic acid ethyl ester (0.034 g; ethanol (0.5 m
L. 1) and a 50% solution of ethylglyoxalate polymer in toluene (0.
(051 mL; 1.5 eq.) And stir for 0.5 h. Iodine (0.022
g; 0.5 eq) in ethanol (0.5 mL) is added and the reaction is stirred for a further 20 hours. Sodium thiosulfate (in water (0.5 mL)
0.044 g) solution was added and the mixture was stirred for 1 h, then EtOAc (20 m
Dilute with L). The mixture was washed with water (× 3) and concentrated brine, dried over MgSO _4, the solvent removed under reduced pressure. The residue is purified by chromatography (silica gel, 5% methanol in DMC) to give the title compound.

1-isobutyl-5-methoxy-1H-benzimidazole-2-carboxylic acid amide ( AA of Scheme IV with R3 = methoxy, R4 and both R5 = H and R6 = iPr): ammonia (1 mL) A 25% aqueous solution is added to a solution of 1-isobutyl-5-methoxy-1H-benzimidazole-2-carboxylic acid ethyl ester (0.0036 g) in THF (1 mL) with stirring. The mixture is stirred for 50 hours, then diluted with EtOAc and washed with water. The organic phase is dried over MgSO ₄ and evaporated under reduced pressure to give the title compound.

Schemes V and VI are two general schemes useful in the synthesis of many subject invention compounds: Scheme V

[0057]

[Chemical 8]

Aa: R7PhCHO / AcOH, NaCNBH ₃ , MeOH 6-[[(4-methoxy-phenyl) -methyl] amino] -1-isobutyl-
1-H-benzimidazole-2-carboxylic acid amide (R1 = isobutyl, R3
And AB of Scheme V where both R5 = H and R7 = methoxy): 200 mL
Dissolve 6-amino-1-isobutyl-1H-benzimidazole-carboxylic acid amide (U; 0.1 g) in anhydrous MeOH (10 mL) in a round bottom flask. Reagent grade acetic acid (0.1 mL) and 4-methoxybenzaldehyde (0.15 g) are added and the reaction is refluxed until complete conversion of the starting material. At this point the reaction mixture is cooled to room temperature and sodium cyanoborohydride hydrate (0.1 g) is added in portions. The reaction mixture is stirred at room temperature for 4 hours. Then the mixture is evaporated to dryness,
The residue is dissolved in ethyl acetate and washed with 5% aqueous sodium bicarbonate solution, water and concentrated brine. The organic layer is dried over sodium sulfate and evaporated to dryness. The residue is chromatographed on a silica gel column using 25% ethyl acetate-hexane as eluent to give the title compound.

Scheme VI

[0060]

[Chemical 9]

Ab: R8-CH (NHR9) COOH, DIEA, EDC, DMAP, HOB
T, DCM; ac: 1) H 2, 10% Pd / C EtOH; 2) 2NHCH 6 - {[(2S) -2- (N- benzyloxycarbonyl) amino-1-oxo-3-phenylpropyl] amino } -1-Isobutyl-1H-benzimidazole-2-carboxamide (R1 = isobutyl, R3 and both R5 = H and R
9 = Z, AC in Scheme VI): N-benzyloxycarbonyl-L-phenylalanine (64.4 mg) with dry dichloromethane (1.5 mL) and N, N-.
Dissolve in diisopropylethylamine (41.2 L). 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide (45.4 mg) and 4-dimethylaminopyridine (catalyst) are added and the mixture is stirred at room temperature for 10 minutes. 1-Hydroxybenzotriazole (32.0 mg) is then added and stirring is continued for another 5 minutes. Finally, 6-amino-1-isobutyl-1H-benzimidazole-2-carboxamide is added and the resulting mixture is allowed to stir at room temperature for 5 days. The reaction mixture is diluted with dichloromethane and washed with saturated sodium bicarbonate (x1), water (x1) and concentrated brine (1x). The organic layer is dried and concentrated to a solid. Trituration with methanol gives the title compound as a solid.

6-{[(2S) -2-amino-1-oxo-3-phenylpropyl] amino} -1-isobutyl-1H-benzimidazole-2-carboxamide hydrochloride (
R1 = isobutyl, R3, both R5 and R9 = H AD of scheme VI)
: 6-{[(2R) -2- (N-benzyloxycarbonyl) amino-1-oxo-3-phenylpropyl] amino} -1-isobutyl-1H-benzimidazole-2-carboxamide (84.7 mg) 10% Pd / C (17.0 mg)
In a flask. The argon line is replaced by a hydrogen balloon in dry ethanol (3.5 mL). After stirring at room temperature overnight, hydrogen was purged from the system with argon,
The reaction mixture is filtered through Celite® with 1: 1 ethanol / water (100 ml). Concentrate the filtrate to a solid residue. The obtained solid is dissolved in a minimum amount of methanol and added dropwise to excess ether to obtain a mud. The solid title compound is obtained by filtration.

Compositions The subject invention includes herein a pharmaceutical composition comprising a safe and effective amount of a 2-carboxamide benzimidazole compound as described above and a pharmaceutically acceptable excipient. The composition optionally comprises other pharmacologically active compounds, especially those having activities such as thrombolysis (eg reteplase, streptokinase or tissue plasminogen activator), anticoagulants (eg heparin), beta. Blockers (eg carvedilol, propanalol), calcium channel blockers (eg verapamil, nifedipine) and antiplatelet compounds (eg aspirin, abciximab) may also be included. Some examples of pharmaceutically acceptable carriers or their components are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; celluloses such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; Derivatives; powdered tragacanth; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa oil; propylene. Polyols such as glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as TWEEN (registered trademark); wetting agents such as sodium lauryl sulfate; coloring agents; fragrances Excipient; tableting; stabilizers; antioxidants; preservatives; water, pyrogen-free; isotonic saline; and phosphate buffers.

The choice of pharmaceutically acceptable carrier for use with a compound is essentially dictated by the method by which the compound is administered. The compounds and compositions of this invention can also be administered systemically. The route of administration is topical or transdermal (patch,
Ointments, creams, powders, etc.); oral; parenteral such as subcutaneous, intramuscular or intravenous injection; topical; rectal; colon; intraperitoneal; intraocular, sublingual; cheek; inhalation; and / or nasal cavity Raru The preferred route of administration is subcutaneous, especially daily or intravenously as needed. Appropriate amounts of the compound to be used will be determined by repeated experiments with animal models. Such models include, but are not limited to, ferrets, dogs and non-human primate models. Generally 0.01 g / day of body weight
Amounts from kg to 100 mg / kg will be administered depending on the effectiveness of the compound or composition used.

Preferred dosage unit units for injection include sterile solutions of water, physiological saline or mixtures thereof. Subcutaneous dosage unit compositions may be in the form of solutions prepared for injection, or may be dry (eg, lyophilized) compositions that are reconstituted with water or saline solution prior to injection. Good. The pH of the solution should be adjusted to about 7.4. Suitable carriers for injection or surgical implantation include hydrogels, controlled- or sustained-release devices, polylactic acid and collagen matrices. Other suitable carriers for injection include dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate and the like.

The compositions of the present invention are preferably provided in dosage form unit. Dosage unit compositions are disclosed herein, preferably about 50 mg or more, more preferably 200 mg or more, and preferably about 500 mg or more, preferably up to about 2000 mg, more preferably up to 1000 mg, and preferably up to about 500 mg. 2-carboxamide benzimidazole compound. The subject compositions may be in various forms suitable for (for example) oral, topical or parenteral administration. Depending on the particular route of administration desired, pharmaceutically acceptable carriers well known in the art can be used. These carriers include solid or liquid fillers, diluents, hydrotropes, surfactants and encapsulating substances. The amount of carrier component used in association with the active compounds of this invention is sufficient to provide a substantial quantity of the substance for administration per unit dose of the active compound of this invention. Techniques and compositions for making the subject dosage form units are described in the following references: Modern Pharmaceutics, Volume 7, Sections 9 and 10, Banker and Rhodes, (1979). ); Lieberman
) Et al 7, Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Edition, (197).
6).

The preferred dosage forms of the subject invention are intended for parenteral administration. Preferred pharmaceutically acceptable excipients for such compositions include sterile, pyrogen-free water and saline solutions. Subcutaneous dosage unit compositions may be in the form of solutions prepared for injection or may be dry (eg lyophilized) compositions which are reconstituted with water or saline solution prior to injection. . Preferred compositions of the subject invention include those intended for oral administration such as tablets, capsules, powders and liquids. Suitable pharmaceutically acceptable excipients for such compositions and sugars as antiplatelet compounds (eg aspirin, avicixima),
Examples include starch, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, magnesium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffers, emulsifiers, alcohols and water.

Methods of Using the Compounds The 2-carboxamide-benzimidazole compounds of the subject invention are useful in treating ischemic reperfusion injury. Without being limited to any particular mechanism, it is believed that the compounds act via regulation of adhesion molecule metabolism. Therefore, the subject compound is a cardiovascular disease (myocardial ischemia, angina, cardiac arrhythmia, heart failure, hypertension)
Treatment of ischemic reperfusion injury including; treatment to reduce neurotoxic damage associated with subsequent stroke, cardiac arrest, or perinatal asphyxia such as anoxia or ischemia; Treatment to reduce perfusion disorders; treatment of frostbite, inflammatory valve disease, psoriasis, asthma, adult respiratory distress syndrome; treatment of chronic inflammatory lung diseases including emphysema, bronchitis; and fibrosis, urticaria, Potentially useful in the treatment of edema, angiopathy, migraine, rheumatoid arthritis, gout and allergies.

Various processes are known to be involved in reperfusion injury, inflammation and related processes. Without being limited to theory, the following mechanisms are of interest in connection with the subject invention. The key to the reperfusion injury process is the induction, expression, and activation of intracellular adhesion molecule-1 (IIDCM-1) in endothelial cells. ICAM-1 then interacts with neutrophils causing the migration of neutrophils into tissues, releasing subsequent deleterious enzymes and destructive reactive oxygen molecules. Thus, compounds capable of interfering with the induction, expression or activation of ICAM-1 would have beneficial effects on ischemic reperfusion. . These compounds have effective doses of ICAM-1
Administered by oral, intravascular, subcutaneous, intramuscular, intranasal, intraocular, sublingual / buccal, inhalation and topical (patch, ointment, powder, or cream) routes You can The 2-carboxamide-benzimidazoles of the subject invention significantly reduce ICAM-1 induction, expression or activation. In other tests, the subject compounds exhibit cardioprotective-related activities. The following are useful test methods for determining such activity of compounds.

Tissue for inhibition of ICAM-1 expression Tissue: Expression of molecular adhesion (especially ICAM-1) was analyzed by Clonetic (Clon).
etics Corp.) (Catalog # CC2519) Human umbilical vein vascular endothelial cells (HUVEC) obtained from San Diego, Calif. Endpoint: Surface ICA of HUVECs induced with TNF-alpha 300 U / ml.
The concentration of a substance that inhibits the expression of M-1 by 50% (IC ₅₀ ).

Method: One vial of frozen HUVEC was rapidly lysed at 37 ° C. for ˜2 minutes (5 × 10 ⁵ -1 × 10 ⁶ cells in 1 ml medium) and the cells were pre-warmed in a 225 cm ² flask. Transfer culture medium (EGM for HUVEC) (2500-5000 cells / cm ² ) and place in a humidified 37 ° C. incubator with 5% CO ₂ . After 24-30 hours the medium is changed (remove dead cells and cytoprotective agents) and then every 2-3 days-the medium becomes confluent with 5-7 days of growth. Trypsinized to recover the cells from the flask - centrifugation (200 g, 5 min) cells were pelleted and the cells were resuspended in medium -50ml (~1-2 × 10 ⁵ cells / ml is desired), Incubate 100 μl of cell suspension in 96-well collagen-coated plate (~ 1-2 x 104 cells / well) -grow to confluence (1-2
Day). Remove old media (discard) and add 90 μl fresh media containing TNF-alpha (or other desired concentration of ICAM-1 stimulator) or 90 μl media alone (for unstimulated control wells). Compound to be tested (10
× 10 μl of medium containing the desired concentration (PBS or PBS, 1% DM
SO) or even 10 μl of medium (PBS or PBS, 1% DMSO)-(final compound concentration = 1 ×; final DMSO concentration = 0.1% if used) for control wells. Addition and incubate at 37 ° C. for 4 hours. The medium is removed (discarded) and the cells are fixed with 200 μl of 80% acetone: 20% H ₂ O (at −20 ° C.) for 20 minutes. Acetone: H ₂ O is removed (discarded), plates are air dried and stored in desiccator overnight (-20 ° C). Wash the plate with PBS (
5 × 250 μl), then 200 μl of BLOTTO solution is added to block non-specific binding—incubate for 1 hour at room temperature. Remove BLOTTO (
Discard), wash the plate with PBS (2 × 250 μl), then 100 μl of I
Add CAM-1 antiserum (in BLOTTO) -incubate for 1 hour at room temperature,
ICAM-1 antiserum was removed (discarded) and the plate washed with PBS (5 x 250
μl), then 100 ml of goat anti-mouse HRP antiserum conjugate (in BLOTTO) is added and incubated for -1 hour at room temperature. Remove the antiserum-HRP conjugate (
Discard), wash the plate with PBS (5 × 250 μl) and then with citrate buffer (1 × 300 (l; discard). Add 100 μl of HRP substrate and incubate at room temperature for 5-20 minutes (hours). May change; see color development), 50μ
11. Add 1NH ₂ SO ₄ to the wells to stop the reaction. Read the plate at 490 nm on a plate reader.

Solution for ICAM-1 ELISA: (1) Stored in 80% Acetone: 20% H ₂ O (v: v)-(-20 ° C). (2)) (1 ×) Dulbecco's phosphate buffer (DPBS), w /
oCa ⁺⁺ or Mg ⁺⁺ , pH 7.5- (Sigma D-5652, 1x)
Powder or Sigma D-1408, 10x liquid, diluted 1:10 before use). (3) BLOTTO-5% (w: v) non-fat dry milk (carnation or other) DPBS solution. (4) Mouse-anti-human ICAM-1 monoclonal antiserum (Research Diagnostics; Catalog # RD-CBL450-1
X: anti-CD54-clone 15.2) -stock solution (1 mg / ml) -B before use
Diluted 1: 1000 in LOTTO (1 mg / ml final antiserum concentration). (5) Goat anti-mouse IgG-horseradish peroxidase conjugate (IgG-
HRP, DAKO; Catalog # P0447) -stock solution (1 mg / ml) -diluted 1: 1000 in BLOTTO 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 citrate. Acid (anhydrous, no acid; MW = 192.1; 6.67 g / l) -check pH (5.0) and store at 4 ° C. (7) HRP substrate-o-phenylenediamine dihydrochloride (OPD; Sigma, P6912; 5 mg OPD / tablet) -1 tablet / 10 ml citrate buffer (room temperature), then 4 ml 30% H ₂ immediately before use. O ₂ (Sigma, H1
009) / 10 ml substrate solution-final concentration = 0.5 mg OPD / ml and 0.012% H ₂ O ₂ . (8) Human tumor necrosis factor alpha (TNF-alpha, Boehringer-Manheim; Catalog # 1371843) -10 mg / vial (in 1 ml) -10 ⁸ U activity / mg = 10 ⁶ U / 10 mg-20 ml endothelium. Diluted in cell basal medium (EBM) (50000 U / ml; 500 ng / ml) 150 ml (
Dispense 7500 U; 75 ng) into an Eppendorf tube (x133) -store at 20 ° C-in each experiment, add one sentence to 25 ml EBM-final concentration = 300
U / ml or 3 ng / ml TNF-alpha.

Inhibition of Human Umbilical Vein Endothelial Cells (HUVEC) / Test for Neutrophil Adhesion Tissue: Adhesion is performed on human umbilical vein endothelial cells (HUVEC) obtained from Clonetics (Catalog # CC2519). . End point: HUVECs adjusted with 300 U / ml TNF alpha (IC ₂₀ ).
Concentration of substances that inhibit PMN adhesion by 20%. Method: I. Cells: A. Endothelial cells: Human umbilical vein vascular endothelial cells (HUVEC) are purchased as frozen cells in a 1 ml aliquot (Clonetics Corporation, San Diego, Calif.). Endothelial growth medium-umbilical cord blood vessels (EGM-UV), Barrett's kit additives, trypsinization agents (trypsin neutralizing solution and HEPES buffer) are purchased from Clonetics. Flask is 5% CO ₂ +9
Place in an incubator at 37 ° C with 5% air, 100% humidity. Thaw 1 vial of liquid N ₂ frozen cells in a 37 ° C. water bath, transfer all vials to a T-275 flask containing 50 ml of fresh medium and place in a CO ₂ incubator. 24 medium
-Replace after 48 hours. It becomes confluent within 4-5 days. The medium is changed at least once during that period. After washing the monolayers with Hanks balanced salt solution (HBSS), the monolayers in the flask are separated using trypsin solution. The trypsinized cells are centrifuged at 200 × Gs for 5 minutes and resuspended in about 150 ml of medium. 100 ul of suspension is placed in each well of a 96-well plate pre-coated with collagen. The monolayer of the plate becomes confluent within 48 hours.

B. Neutrophil Separation: Peripheral blood polynuclear neutrophils (PMNs) are separated by established methods. (1) Human blood is obtained from the elbow by conventional venipuncture by a qualified phlebotomist. Blood is collected in a vacutainer containing heparin (Vacutainer # 648
9. Green cap, 15 ml for blood collection, VWR) 30 ml of blood is used for each test. Blood that has been prevented from clotting with heparin is diluted with approximately 1/2 volume of phosphate buffered saline containing 0.2% glucose (PBS-G). Discontinuous gradient histo pack (3 ml histo pack at the bottom-1119 and 3 ml histo pack at the top-
1077) (Sigma, St. Louis, MO) are prepared in 6 15 ml conical centrifuge tubes. Carefully layer the diluted blood on Histopack-1077. Centrifuge the tubes at 800 x G for 30 minutes at room temperature. After the centrifugation step, PMNs are removed by suction from the area between Histopack-1077 / Histopack-1119 and from the top of the pelleted blood cells. Collect PMNs from all tubes, further dilute to a total volume of 30 ml and centrifuge at 600 x G for 15 minutes. Discard the supernatant and pellet (PMNs and some red blood cells (RBC)
) Are treated with 6 ml cold water for 30 seconds to dissolve the contaminating RBCs. Normal osmolality is restored by adding 3 ml of 2.7% saline. PMNs to P
Wash twice more with BS-G. The viability and number of PMNs is determined using the trypan blue exclusion test in a hemocytometer chamber. Occasionally, small aliquots of PMNs suspension are used for cytofuge preparation. Cytofuge slides are stained with Wright's blood stain (Sigma Chemical Co.) and run in various fractions to assess the percentage of PMNs in the preparation.

II. Induction of Endothelial Cells: Monolayer of endothelial cells in 96-well plate with 300 U / ml tumor necrosis factor (TNF
Boehringer-Mannheim, catalog # 1371-843). TNF
And compound is added to each well 4 hours before addition of PMNs.

III. Fluorescent labeling of neutrophils: The neutrophil pellet was finally washed and then resuspended in 5 ml PBS-G (approximately 1
-3 × 10 ⁶ ml) 5 (and 6) Carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes, Eugene, Oreg.). Add 20 mM CFSE stock solution to 2.24 ml DMSO (MW 557.5).
Prepare by dissolving 5 mg. 5 μl of stock solution is added to 5 ml PMNs suspension to give a final concentration of 2 μM. Incubate the mixture for 20 minutes in the refrigerator. Finally PM
Ns is washed 4 times with PBS-G. After the final wash, the PMNs were completely washed with EGM-
Resuspend to the desired concentration in UV medium (typically 0.6-1.2 x 10 ^{5 in} each well of a 96-well plate).

IV. Addition of test compound: 100 μl medium containing compound with TNF is replaced with medium in wells containing a monolayer of endothelial cells 4 hours before addition of PMNs.

V. Evaluation of Adhesion: A. Data Collection: HUVE CFSE labeled neutrophils (0.7-1.5 × 10 ⁵ ) in 10 μl volume.
C Add to monolayer. Plate is 5% CO ₂ + 95% air, 100% humidity, 37 ° C
Incubate for 30 minutes in the incubator. Non-adherent cells are removed by centrifugation according to the following protocol: (1) After culturing at 37 ° C., recording on a cytofloor 2400. This record is considered as 100% of all cells added. (2) Fill a slightly convex shape at the top of the well with warm (cultured) medium (
Usually 260 μl (100 μl or in addition to the amount already in the well). (3) Adhesive sealing film for microplates (Rainin)
, Catalog # 96-SP-100). (4) Place the lid on the plate and record the size and position of the bubbles on the lid with a marker. (5) Remove the lid, fold in four, and place the cut paper towel on top of the sealing film. Then place the lid of the plate on top of the paper towel and invert the plate.

(6) Place the plate in the plate holder of the centrifuge (Sorvall RT6000D), rotate at a low speed of 500 RPMs, and then turn on the knob of the timer of the centrifuge. The speed control adjusts the tachometer to 1100 RPM (which equals 200 Gs) and starts the separation timer the moment the speed reaches 1100 RPM. Stop the motor when the centrifuge timer has been exactly 2 minutes. Stop the plate without braking. (7) Remove plate and record empty wells. Remove the lid and the folded paper towel (the plate remains upside down), remove the sealing film into the biological material treatment bottle, and shake off the medium. Place the plate on a paper towel and aspirate excess liquid. (8) Return the top and bottom of the plate to their original positions and inspect with a microscope. (9) Perform the second recording with Cytofluor. This second recording is used to determine the percentage of PMNs remaining adhered to the single layer. (10) Information from the CSV file of Cytofluor was downloaded and the data was processed in an EXCEL spreadsheet, where the background (PMNs adhesion to non-induced endothelial cells) was removed and the percent inhibition of adhesion was below. Is determined as:

[0081]

[Equation 1]

(11) The EXCEL spreadsheet has a) percent PMNs adhesion to monolayer, b) percent PMNs adhesion to monolayer minus background (adhesion of PMNs to unactivated endothelial cells) and c. ) Calculate percent adhesion inhibition, taking into account wells receiving TNF alone with 0% inhibition.

B. Statistics, data handling and storage: Statistics are carried out in EXCEL using a two-sided t-test of equal variance and the results are recorded as P-values.

VI. Additional information: A. Coat wells with collagen: 25 mg of acid-soluble rat tail collagen is dissolved in 446 ml of water and acidified with a few drops of HCl. Sterilize by filtration. 50 μl is added to each well of a 96-well plate (.28 cm ² ) to make 10 μg / well. Incubate overnight at 37 ° C in an incubator. Aspirate all liquid and store in refrigerator until use. B. Preparation of PBS + 0.2% glucose (dextrose): Prepare 2 liters of PBS (Sigma) and add 2 grams of glucose. Sterilize with a filter and store.

Model of Rat Myocardial Infarction / Reperfusion Injury Surgical Preparation of Rats: Male, Sprague-Dawley rats, 1.25 g / urethane.
Anesthetize with kg. The carotid artery and jugular vein are exposed externally and PE-50 tubing is inserted to record blood pressure and facilitate intravenous administration of dye or drug. Perform a tracheotomy. Animals are connected to a Harvard Rodent ventilator (Type 683, Harvard Apparatus, South Natick, Mass.) And ventilated at 50 pulse / min, 1.5 ml / 100 g body weight. Lead I for needle electrode
I Set up for ECG. Animals are maintained at 37 ° C. by means of an electric heating pad to adjust to the desired temperature and controlled with a rectal thermistor probe and controller. The heart is carefully separated by a left thoracotomy in the fifth intercostal space to locate the left anterior descending coronary artery (LAD). A 6-0 silk ligature is placed around the LAD and terminating through a short PE-320 to facilitate acute occlusion and arterial reperfusion. LAD is sewn and clamped, 25mm Schwarz
Aneurysm clips are used to tightly tub and close the opposite side of the heart surface. Closure is continued for 90 minutes and reperfusion is performed for 3.0-4.5 hours. Animals are dosed 10 minutes prior to reperfusion with drug or vehicle by intravenous delivery via the jugular vein to the area affected by the heart. Sham-operated rats are not affected by ischemia or reperfusion. At the end of the experiment, the LAD was permanently reoccluded with 10 m of Evans Blue dye.
The area affected by ischemia, such as the risk area (AAR), is identified by administering the g / ml solution via the coronary vein. The stained hearts are rapidly dissected and stored in 0.9% saline at 4 ° C until measurement of creatine phosphokinase activity (CPK). Measurement of creatine phosphokinase activity: The left ventricle free wall (LVFW) is cut from the heart and weighed. Unstained partial AAR is cut from the LVFW and weighed. AAR in 4 ml of 0.25M sucrose containing 1 mM EDTA and 10 mM mercaptoethanol at 4 ° C.
Homogenize for 5 seconds. Homogenate the product at 1000 xg at 4 ° C for 30
Centrifuge for minutes. Supernatants are removed for CPK activity determination, pellets are separated and frozen and stored for testing myeloperxidase activity. CPK activity is tested spectrophotometrically at a wavelength of 340 nm at 24-26 ° C. in a commercially available substrate, CPK test Vial® (Sigma Diagnostics).

Measurement of myeloperoxidase activity: Myeloperoxidase (MPO) is separated from frozen pellets after CPK preparation. Pellets were mixed with 0.5% hexadecyltrimethylammonium bromide (HTA
Suspend at approximately 10% concentration in 50 mM phosphate buffer, pH 6 containing B), sonicate for 10 seconds, and freeze on dry ice. Three freeze-thaw cycles are performed with 10 seconds of sonication between cycles. Samples are chilled on ice for 30 minutes, followed by centrifugation at 12,500 xg for 15 minutes at 4 ° C. 0.167 mg
/ Ml o-dianisidine dihydrochloride and 50 mM sodium phosphate buffer, pH 6, containing 0.113 ml hydrogen peroxide in 100 ml buffer, pH 6, the MPO activity of the aliquot of the supernatant was measured spectrophotometrically at 24-26. ℃, 460nm
At the wavelength of.

Calculations and Statistical Analysis: Results are recorded as mean ± SEM. CPK and MPO activities are expressed as units / g tissue, where CPK activity is 1. It is the amount of CPK that consumes mol phosphocreatine, and MPO activity is defined as the amount that decomposes 1 μmol hydrogen peroxide per minute. AAR is measured as a percentage of LVFW on a weight basis. Mean arterial blood pressure (MABP) is calculated as one-third of the difference between systolic pressure and diastolic pressure applied to diastolic pressure. Data are analyzed for statistical significance of treatment effect with 95% confidence by pooled t-test or one-way analysis of variance.

The subject invention treats or prevents any of the diseases and disorders described above, particularly reperfusion injury, by administering a safe and effective amount of a 2-carboxamide benzimidazole compound disclosed above. Including a method. The method of treatment can include administration of such compounds parenterally, orally, or by topical dosage unit. Parenteral administration includes intravenous, intramuscular, subcutaneous, peritoneal, or other injectable dosage forms. Oral administration includes ingestion of the dosage form and absorption of the active substance from the gastrointestinal system. Topical administration includes, but is not limited to, dosage forms contacting the surface of skin or mucosal tissues, including the digestive tract and respiratory system. The amount of 2-carboxamide benzimidazole compound typically administered in parenteral administration is preferably about 2 mg / kg or more, more preferably about 5 mg / k.
It is at least g, preferably up to 2 mg / kg, more preferably up to 10 mg / kg. The frequency of such administration is typically once or twice daily. The treatment regimen is typically a single dose or lasts about 1 day or longer, preferably 5 days or longer, up to about 30 days, preferably up to about 15 days. The amount of the 2-carboxamide benzimidazole compound typically administered in oral administration is preferably about 5 mg / kg or more, more preferably about 10 mg / k.
It is at least g, preferably up to 25 mg / kg, more preferably up to 15 mg / kg. The frequency of such administration is typically 1 to 4 times daily. The treatment regimen typically lasts about 1 day or longer, preferably 5 days or longer, up to about 30 days, preferably up to about 15 days.

Compositions and Methods of Examples The following non-limiting examples are illustrative of the subject invention. The following examples of compositions and methods are not limiting of the invention, but provide guidance to those of ordinary skill in the art in preparing and using the compounds, compositions, and methods of the invention. In each example, other compounds of the invention other than those already mentioned are substitutions of the compounds of that example and show similar results.

[0090]

【Example】

Example A A pharmaceutical composition in the form of an intravenous administration solution is prepared by conventional methods, such as by mixing the following:

[0091]

[Table 3]

¹ The compound of Example I can be replaced by any of Examples 2-14.

Immediately before or after tissue damage (aneurysm treatment, coronary artery bypass surgery, transplant surgery, traumatic bleeding, organ ischemia due to incomplete reperfusion, sepsis, etc.), 1 ml of the above composition is intravenously administered. When administered to, tissue damage is avoided or reduced.

Example B A pharmaceutical composition in liquid form is prepared by conventional methods as formulated below. :

[0095]

[Table 4]

¹ The compound of Example I can be replaced by any of Examples 2-14.

Immediately before or after tissue damage (aneurysm treatment, coronary artery bypass surgery, transplant surgery, traumatic bleeding, organ ischemia due to incomplete reperfusion, sepsis, etc.), 1 ml of the above composition is subcutaneously administered. When administered, tissue damage is avoided or reduced.

While particular embodiments of the present invention have been described, it will be appreciated that various changes and modifications may be made to the compositions disclosed herein without departing from the spirit and scope of the invention. It will be obvious to the trader. For all such modifications within the scope of the invention,
It is intended to be covered by the appended claims.

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Claims (10)

[Claims] 1. A compound having the following structure: Wherein: (a) R1 is selected from the group consisting of alkyl, aryl, alkoxy and aryloxy, wherein the alkyl and aryl moieties of R1 have 1 to 14 carbon atoms; (b) R3 and R4 are each Are independently selected from the group consisting of hydrogen, halo, alkyl, alkoxy, alkyl-aryloxy, alkylthio, amino, and mono- or di-alkylamino, wherein the alkyl moiety of R3 and R4 is 1-8 carbons. R3 and R4 are not both hydrogen; (c) each R5 is independently hydrogen, halo, cyano, alkyl, hydroxy, alkoxy, thio, alkylthio, amino, and mono- or Selected from the group consisting of di-alkylamino, wherein the alkyl moiety of R5 has 1 to 8 carbon atoms; and Optical isomers, diastereomers or enantiomers or mixtures thereof; pharmaceutically acceptable salts, hydrates or biologically hydrolysable esters, amides or imides thereof. 2. R1 is alkyl or aryl having 2 to 8 carbon atoms; preferably R1 is alkyl having 3 to 8 carbon atoms, alkyl being unsubstituted or halo, hydroxy, C ₁ To C ₃ alkoxy, thio, C _{1 to} C ₃ alkylthio, amino, C _{1 to} C ₃ mono- or di-alkylamino, phenyl and a substituent selected from the group consisting of heterocycles having 5 or 6 ring atoms. A substituted type, more preferably R1 is a saturated type or an unsaturated type with one double bond, and a linear or branched chain type or cyclic alkyl having 3 to 6 carbon atoms. Compound. 3. R3 and R4 are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, alkylthio, amino, and mono- or di-alkylamino, wherein the alkyl moiety of R4 is 1 to about 6 The compound of claim 2 having a carbon atom. 4. Each R5 is independently selected from the group consisting of hydrogen, halo, alkyl, alkoxy, alkylthio, and mono- or di-alkylamino, wherein the alkyl moiety of R5 has 1 to 6 carbon atoms. The compound of claim 3 having: 5. The alkyl moieties of the R3, R4 and R5 moieties each independently have 1 to 3 carbon atoms and are unsubstituted or halo, hydroxy, C ₁ -C ₃ alkoxy, thio, C _1- . A compound according to claim 4, which is substituted with C ₃ alkylthio, amino, and C ₁ -C ₃ mono- or di-alkylamino, preferably the alkyl moiety of R 3, R 4 and R 5 is unsubstituted. 6. R1 is phenyl or benzyl, unsubstituted or halo,
C ₁ -C ₃ alkyl, hydroxy, C ₁ -C ₃ alkoxy, amino, and C ₁ -C ₃ mono- - or di - substituted type with a substituent selected from the group consisting of alkylamino, preferably R1 Is a saturated or unsubstituted type, more preferably R3 is methoxy or ethoxy. 7. R4 is alkoxy, preferably methoxy or ethoxy,
A compound of claim 6 more preferably methoxy and R3 and both R5 are hydrogen. 8. The compound of claim 7 wherein R3 is alkoxy and R4 and both R5 are hydrogen. 9. A pharmaceutical composition comprising:       (A) a safe and effective amount of the compound of any of claims 1 or 6; and       (B) A pharmaceutical composition comprising a pharmaceutically acceptable excipient. 10. A method for preventing or treating ischemia-reperfusion injury, which comprises administering a safe and effective amount of the compound of claim 1 or 6 to a human or lower animal in need thereof. .