Hong Lim

Cardioselective anti-ischemic ATP-sensitive potassium channel (KATP) openers: Benzopyranyl indoline and indole analogues

This paper describes the design, syntheses, and biological evaluations of novel ATP-sensitive potassium channel (K(ATP)) openers, benzopyranyl indoline and indole derivatives. Among those, two enantiomers of indoline-2-carboxylic ethyl esters (14, 18) showed the best cardioprotective activities both in vitro and in vivo, while their vasorelaxation potencies were very low (concentration for 50% inhibition of vasorelaxation >30 microM). The cardioprotective effect of 14 was completely reversed by 5-hydroxydecanoate, a selective mitochondrial K(ATP) blocker, indicating its provable protective mechanism through the mitochondrial K(ATP) opening. In addition, we performed conformational analyses using 2D-NMR, X-ray crystallography and molecular modeling to study the structure-activity relationships in this series of compounds.

Protective effect of KR-31378 on oxidative stress in cardiac myocytes.

In this study, we investigated whether a novel anti-ischemic KATP opener KR-31378 [(2S,3S,4R)-N”-cyano-N-(6-amino-3,4-dihydro-3-hydroxy-2-methly-2-dimethoxymethly-2H-benzopyran-4-yl)-N’-benzylguanidine] has protective effect against oxidative stress-induced death in heart-derived H9c2 cells. Cell death was induced by BSO, butionine sulfoximine, which inhibits GSH synthesis and subsequently increases reactive oxygen species (ROS) level. Cell death was quantitatively determined by measuring lactate dehydrogenase (LDH) activity and stained by Hoechst 33258. BSO-induced ROS production and mitochondrial membrane potential (MMP) were measured using 2’,7’-dichlorofluorescein diacetate oxidation and rhodamine 123, respectively. Both the LDH release and the ROS elevation induced by treatment of H9c2 cells with 10 mM BSO, were significantly decreased by KR-31378. These protective effect and antioxidant effect of KR-31378 appeared to be independent on KATP channel opening. Cells exposed to BSO showed an early reduction inMMP, and this reduction inMMP was significantly reversed by treatment with KR-31378. Caspase-3 activity in BSO treated H9c2 cells was remarkably increased, and this increased caspase-3 activity was significantly reversed by KR-31378. In conclusion, our results suggest that KR-31378 can produce cardioprotective effect against oxidative stress-induced cell death through antioxidant mechanism.

Metabolism of a new neuroprotective agent for ischemia-rep-erfusion damage, KR-31543 in the rats using liquid chroma-tography/electrospray mass spectrometry

KR-31543, (2S,3R,4S)-6-amino-4-[N-(4-chlorophenyl)-N-(2-methyl-2H-tetrazol-5-ylmethyl)amino]-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-2H-1-benzopyran is a new neuroprotetive agent for ischemia-reperfusion damage. Thein vitro andin vivo metabolism of KR-31543 in rats has been studied by LC-electrospray mass spectrometry. Rat liver microsomal incubation of KR-31543 in the presence of NADPH resulted in the formation of a metabolite M1. M1 was identified as N-(4-chlorophenyl)-N-(2-methyl-2H-tetrazol-5-ylmethyl)amine on the basis of LC-MS/MS analysis with the synthesized authentic standard. Rat CYP3A1 and 3A2 are the major CYP isozymes involved in the formation of M1.

In Vitro Metabolism of a New Neuroprotective Agent, KR-31543 in the Human Liver Microsomes: Identification of Human Cytochrome P450

KR-31543, (2S,3R,4S)-6-amino-4-[N-(4-chlorophenyl)-N-(2-methyl-2H-tetrazol-5-ylmethyl) amino]-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-2H-1-benzopyran, is a new neuroprotective agent for preventing ischemia-reperfusion damage. This study was performed to identify the metabolic pathway of KR-31543 in human liver microsomes and to characterize cytochrome P450 (CYP) enzymes that are involved in the metabolism of KR-31543. Human liver microsomal incubation of KR-31543 in the presence of NADPH resulted in the formation of two metabolites, M1 and M2. M1 was identified asN-(4-chlorophenyl)-N-(2-methyl-2H-tetra-zol-5-ylmethyl)amine on the basis of LC/MS/MS analysis with a synthesized authentic standard, and M2 was suggested to be hydroxy-KR-31543. Correlation analysis between the known CYP enzyme activities and the rates of the formation of M1 and M2 in the 12 human liver microsomes have showed significant correlations with testosterone 6β-hydroxylase activity (a marker of CYP3A4). Ketoconazole, a selective inhibitor of CYP3A4, and anti-CYP3A4 monoclonal antibodies potently inhibited bothN-hydrolysis and hydroxylation of KR-31543 in human liver microsomes. These results provide evidence that CYP3A4 is the major isozyme responsible for the metabolism of KR-31543 to M1 and M2.

LC-MS/MS identification of in vitro metabolites of a new H+/K+ ATPase inhibitor, KR-60436 produced by rat and human liver microsomes

The metabolism of a new H+/K+ ATPase inhibitor, KR-60436 [1-(4-methoxy-2-methyl-phenyl)-4-[(2-hydroxyethyl)amino]-6-trifluoromethoxy-2,3-dihydropyrrolo[3,2-c]quinoline] has been studied by LC-electrospray mass spectrometry. In vitro incubation of KR-60436 with rat and human liver microsomes in the presence of NADPH produced seven metabolites (M1-M7). M3-M6 were identified as O-demethyl-KR-60436, O-demethyl-pyrrole-KR-60436, N-dehydroxyethyl-KR-60436 and pyrrole-KR-60436, respectively, based on LC/MS/MS analysis with authentic standards. M1, M2 and M7 were tentatively identified as monohydroxylated-KR-60436, monohydroxylated-pyrrole-KR-60436 and N-dehydroxyethyl-pyrrole-KR-60436, respectively. The four principal metabolic pathways are characterized in KR-60436 metabolism: oxidation of dihydropyrrole ring to pyrrole, O-demethylation of methoxy group, hydroxylation of quinoline moiety and N-dealkylation of hydroxyethylamino group.

HPLC analysis of a new reversible proton pump inhibitor, a dihydropyrroloquinoline derivative, in plasma, urine, and tissue homogenates

ABSTRACT A high-performance liquid chromatographic (HPLC) method was developed for the determination of a new reversible proton pump inhibitor, KR-60436, in human plasma and urine and in rat tissue homogenates. The method involved deproteinization of the biological samples with three volumes of acetonitrile. A 100 μL aliquot of the supernatant was injected onto a reversed-phase (C18) column. The mobile phase, 0.02 M phosphate buffer (pH 5) : acetonitrile:methanol (30 : 60 : 10, v/v/v), was run at a flow rate of 0.7 mL/min and the column effluent was monitored by a fluorescence detector set at an excitation wavelength of 340 nm and an emission wavelength of 484 nm. The retention time of KR-60436 was approximately 7.5 min. The coefficients of variation (within-day and between-day) were low (below 10.5%) for human plasma and urine and rat tissue homogenates. No interferences from endogenous substances were found.

Simultaneous determination of the novel neuroprotective agent KR-31378 and its metabolite KR-31612 using high performance liquid chromatography with tandem mass spectrometry in human plasma.

An LC/MS/MS method for the simultaneous determination of a neuroprotective agent for ischemia-reperfusion damage, KR-31378 and its N-acetyl metabolite KR-31612 in human plasma was developed. KR-31378, KR-31612 and the internal standard, KR-31543 were extracted from human plasma by liquid-liquid extraction. A reverse-phase HPLC separation was performed on Luna phenylhexyl column with the mixture of acetonitrile-5 mM ammonium formate (55:45, v/v) as mobile phase. The detection of analytes was performed using an electrospray ionization tandem mass spectrometry in the multiple reaction monitoring mode. The lower limits of quantification for KR-31378 and KR-31612 were 2.0 ng/ml. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity.

Effects of a new neuroprotective agent KR-31378 on liver cytochrome P450s in male sprague dawley rats

The effects of KR-31378, a neuroprotective agent for ischemia-reperfusion damage, on liver microsomal cytochrome P450s (CYPs) were investigated in male Sprague Dawley rats. When rats were treated orally with KR-31378 for 7 consecutive days, CYP3A-selective erythromycinN-demethylase (ERDM) activity was significantly induced in a dose-dependent manner. In Western immunoblotting, CYP 3A proteins were clearly induced by treatment with KR-31378. Within 24 h after treatment with 80 mg/kg of KR-31378, ERDM activity was induced in liver microsomes in accompanied by induction of the level of CYP 3A proteins. The present results suggest that KR-31378 might modulate the expression of CYP 3A enzymes in humans.

Determination of a new reversible proton pump inhibitor, DBM-819, in human plasma and urine, and rat tissue homogenates by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method was developed for the determination of a new proton pump inhibitor, DBM-819, in human plasma and urine and rat tissue homogenates using KR-60461 as an internal standard. A 100-microl aliquot of acetonitrile (containing 0.5 microg/ml of the internal standard) and a 200-microl aliquot of 0.1 M Na(2)HPO(4) (adjusted pH 11 with 1 N NaOH) were added to a 100-microl aliquot of biological sample. After vortex-mixing, the mixture was extracted with 1 ml of ethylacetate. After centrifugation at 12000 x g for 3 min, the organic layer was collected and evaporated under nitrogen gas. The residue was then reconstituted with a 100-microl aliquot of mobile phase, and a 40-microl aliquot was injected onto the HPLC column. The mobile phase, 0.02 M phosphate buffer (pH 5): acetonitrile: methanol (46:44:10, v/v/v), was run at a flow rate of 0.5 ml/min and the column effluent was monitored by the fluorescence detector set at an excitation wavelength of 340 nm and an emission wavelength of 470 nm. The retention times for DBM-819 and the internal standard were approximately 10.5 and 12 min, respectively. The detection limits of DBM-819 in human plasma and urine, and rat tissue homogenates were 0.01, 0.02 and 0.02 (or 0.05) microg/ml. respectively. The coefficients of variation (CV) of the assay were below 11% for human plasma and urine, and rat tissue homogenates. No interferences from endogenous substances were found.

HPLC ANALYSIS OF A NEW NEUROPROTECTIVE AGENT FOR ISCHEMIA-REPERFUSION DAMAGE, A BENZOPYRAN DERIVATIVE, IN PLASMA AND URINE

ABSTRACT A high-performance liquid chromatographic (HPLC) method was developed for the determination of a neuroprotective agent for ischemia-reperfusion damage, KR-31543, in human plasma and urine. The method involved deproteinization of the biological samples with two volumes of acetonitrile. A 80 μL aliquot of the supernatant was injected onto a reversed-phase (C18) column. The mobile phase, 50 mM triethylamine acetate:acetonitrile (15 : 85, v/v), was run at a flow rate of 1.0 mL/min. The column effluent was monitored by an ultraviolet detector set at 310 nm. The retention time of KR-31543 was approximately 3.5 min. The detection limits of KR-31543 in human plasma and urine were 0.2 and 0.5 μg/mL, respectively. The coefficients of variation (within-day and between-day) were low (below 4.86%) for human plasma and urine. No interferences from endogenous substances were found.