Discovery and Optimization of 1-Phenoxy-2-aminoindanes as Potent, Selective, and Orally Bioavailable Inhibitors of the Na+/H+ Exchanger Type 3 (NHE3).

The design, synthesis, and structure-activity relationship of 1-phenoxy-2-aminoindanes as inhibitors of the Na+/H+ exchanger type 3 (NHE3) are described based on a hit from high-throughput screening (HTS). The chemical optimization resulted in the discovery of potent, selective, and orally bioavailable NHE3 inhibitors with 13d as best compound, showing high in vitro permeability and lacking CYP2D6 inhibition as main optimization parameters. Aligning 1-phenoxy-2-aminoindanes onto the X-ray structure of 13d then provided 3D-QSAR models for NHE3 inhibition capturing guidelines for optimization. These models showed good correlation coefficients and allowed for activity estimation. In silico ADMET models for Caco-2 permeability and CYP2D6 inhibition were also successfully applied for this series. Moreover, docking into the CYP2D6 X-ray structure provided a reliable alignment for 3D-QSAR models. Finally 13d, renamed as SAR197, was characterized in vitro and by in vivo pharmacokinetic (PK) and pharmacological studies to unveil its potential for reduction of obstructive sleep apneas.

The ATP-regulated K+-channel inhibitor HMR-1372 affects synaptic plasticity in hippocampal slices.

Long-term potentiation (LTP) and long-term depression of synaptic transmission in the hippocampus are widely studied models of learning and memory processes. The role of ATP-regulated K+ channels (K(ATP)+ channels), which are abundant in the brain, has not yet been studied in long-term potentiation or long-term depression. We investigated whether K(ATP)+ channel inhibition by the highly selective K(ATP)+-channel blocker 1-[[5-[2-(5-tert-butyl-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea (HMR-1372), a novel putative class III antiarrhythmic, affects long-term potentiation or the long-term depression induced by 3,5-dihydroxyphenylglycine (30 microM) in submerged rat hippocampal slices. HMR-1372 (10 microM) did not affect basal synaptic transmission, paired pulse inhibition, long-term depression or long-term potentiation elicited by a weak (weak long-term potentiation) tetanus, but significantly amplified the long-term efficacy of long-term potentiation elicited by a strong tetanus (strong long-term potentiation). The K(ATP)+-channel inhibitor glibenclamide (20 microM) also ameliorated only strong long-term potentiation. Our data suggest that K(ATP)+ channels are activated during or after induction of long-term potentiation and play a role in controlling synaptic excitability.

Effects of a novel cardioselective ATP-sensitive potassium channel antagonist, 1-[[5-[2-(5-chloro-o-anisamido)ethyl]-beta-methoxyethoxyphenyl]sulfonyl]-3-methylthiourea, sodium salt (HMR 1402), on susceptibility to ventricular fibrillation induced by myocardial ischemia: in vitro and in vivo studies.

In the present study, a novel sulfonylthiourea, 1-[[5-[2-(5-chloro-o-anisamido)ethyl]-beta-methoxyethoxyphenyl]sulfonyl]-3-methylthiourea, sodium salt (HMR 1402), was investigated using in vitro and in vivo systems. HMR 1402 inhibited rilmakalim-induced currents in rat and guinea pig myocytes (IC(50) = 60 and 509 nM, respectively). Hypoxia-induced shortening of action potential duration at 90% repolarization was also significantly attenuated by HMR 1402 (68.1 +/- 3.9% of control at 0.3 microM). In contrast, HMR 1402 had a smaller effect on pancreatic beta-cells (rat insuloma cells, RINm5F) hyperpolarized with 100 microM diazoxide (IC(50) = 3.9 microM, compared with glibenclamide IC(50) = 9 nM). In a similar manner, hypoxia induced increases in coronary flow in isolated guinea pig hearts were only slightly reduced by HMR 1402. These data strongly suggest that HMR 1402 has pharmacological selectivity for cardiac myocytes and, therefore, may protect against ischemically induced ventricular fibrillation (VF) without the untoward effects of nonselective compounds. To test this hypothesis, VF was induced in 8 dogs with healed myocardial infarctions by a 2-min coronary occlusion during the last minute of exercise. On a subsequent day, the exercise plus ischemia test was repeated after HMR 1402 (3.0 mg/kg i.v., n = 4, infusion 4 microg/kg/min for 1 h before exercise, n = 4). This drug significantly reduced the incidence of VF protecting seven of eight animals (p = 0.0007) without altering plasma insulin, blood glucose, or the increases in mean coronary blood flow induced by either exercise or 15-s coronary occlusions. Thus, the ATP-sensitive potassium channel antagonist HMR 1402 can prevent ischemically induced VF without altering coronary blood flow or blood glucose.

Glucuronidation of HMR1098 in human microsomes: evidence for the involvement of UGT1A1 in the formation of S-glucuronides.

HMR1098, a novel KATP-blocking agent, is metabolized to form an S-glucuronide in rat and dog bile. Synthesis of the S-glucuronide metabolite was studied in human liver and kidney microsomes. Recombinant UPD-glucuronosyltransferases (UGTs) were screened for activity, and kinetic analysis was performed to identify the isoform or isoforms responsible for the formation of this novel S-glucuronide in humans. S-Glucuronidation is relatively rare, but from this study it appears that S-glucuronides are not generated exclusively by a single UGT isoform. From the panel of recombinant isoforms used, both UGT1A1 and UGT1A9 catalyzed the glucuronidation of HMR1098. The Vmax values in both instances were similar, but the Km for UGT1A1 was substantially lower than that measured for UGT1A9, 82 microM compared with 233 microM, respectively. Liver and kidney microsomes displayed similar Km values, but the Vmax in kidney was more than 20-fold less than in liver microsomes, which is suggestive of a significant role for the bilirubin UGT in catalysis of HMR1098, although other UGTs may play a secondary role.

Specificity of classical and putative Cl(-) transport inhibitors on membrane transport pathways in human erythrocytes.

The majority of anion transport inhibitors tend to be non-specific. This is problematic from a research point of view as caution is required when defining pathways purely based on pharmacology. Here we have tested a range of classical and putative Cl(-) transport inhibitors on three Cl(-) carrier systems (the KCl cotransporter (KCC), the NaK2Cl cotransporter (NKCC), and the Band 3 anion exchanger (AE)) found in human erythrocytes, using radiolabel tracer experiments. The study confirms the cross-reactivity of many anion transport inhibitors. However, two compounds, H25 and H156, were found to be both potent (IC(50) values < 0.1 mM) and specific (at least 1000-fold more effective against one carrier compared to the other two) inhibitors of NKCC and AE, respectively.

Blockers of the ATP-sensitive potassium channel SUR2A/Kir6.2: a new approach to prevent sudden cardiac death.

The cardiac ATP sensitive potassium channel (K(ATP) channel) SUR2A/Kir6.2 is an emerging target for antiarrhythmic intervention. This channel accounts for known electrophysiological derangements soon after the onset of myocardial ischemia. Consequently, blockers of this channel have the potential to prevent ischemic malignant arrhythmias and sudden cardiac death in humans. Since cardiac K(ATP) channels are closed at physiological intracellular ATP concentrations (ATP(i)) and open only when ATP(i) falls below a critical value, these agents do not affect the normal cardiac action potential and should be devoid of proarrhythmic side effects. Due to the existence of isoforms of this channel, mainly in vascular smooth muscle cells, pancreatic beta-cells and cardiac mitochondria, only specific blockers of SUR2A/Kir6.2 will offer a reasonable option for the treatment of cardiovascular patients at risk of sudden cardiac death. Presently known K(ATP) blockers are derived from diverse classes of compounds with antidiabetic sulfonylureas being their most prominent members. Retrospective evaluations of clinical studies with the sulfonylurea glibenclamide in diabetics revealed antifibrillatory activity to be an important additional effect of this class of compounds. However, for the safe treatment of arrhythmias nearly all presently known blockers lack sufficient selectivity, either within the target family or with respect to other ion channels modulating the cardiac action potential. The present article illustrates the new principle in terms of molecular biology and electrophysiology and summarizes all presently known K(ATP) blockers. As a highlight, first strategies to come to selective SUR2A/Kir6.2 blockers, such as HMR 1883, are reviewed.

Selective block of sarcolemmal IKATP in human cardiomyocytes using HMR 1098.

PURPOSE: Activation of the myocardial, ATP-dependent potassium current (IK(ATP)) during ischemia causes shortening of the action potential duration thereby increasing dispersion of repolarization between ischemic and non-ischemic myocardium and predisposing to reentrant arrhythmias. The IK(ATP) inhibitor HMR1098 allows selective block of the sarcolemmal myocardial K(ATP)-channel in various animal species. Therefore, we studied the concentration and pH-dependence of HMR1098 in human ventricular myocytes. METHODS: Human ventricular cardiomyocytes were isolated enzymatically. IK(ATP) was measured with the patch-clamp technique in whole cell configuration at 35 degrees C. Action potentials were recorded using Amphotericine B in perforated patch conditions. In voltage clamp experiments, the K(ATP)-channel was activated by application of 1 microM rilmakalim, a K(ATP)-channel opener. In action potential recordings, 0.1 microM rilmakalim was used. RESULTS: At physiological pH (pH = 7.3) half-maximal block of the rilmakalim-induced current occurred at 0.42 +/- 0.008 microM HMR1098 (at 0 mV membrane potential); under acidic conditions as can be expected to be present under ischemic conditions (pH = 6.5), half-maximal block was achieved at markedly lower concentrations (IC(50) = 0.24 +/- 0.009 microM). In current clamp experiments, block of IK(ATP) by HMR1098 was capable of reversing the action potential shortening induced by rilmakalim, and restored the action potential plateau. CONCLUSIONS: HMR1098 appears to be useful to prevent IK(ATP)-induced shortening of the action potential in human ventricular myocardium. More acidic conditions, as observed in ischemia, increase the sensitivity to HMR1098, indicating a more potent effect in ischemic myocardium. Thus, HMR1098 may be a useful agent to prevent action potential shortening and dispersion of repolarization during ischemia, which may protect against ischemia induced ventricular arrhythmias.

The stereoenantiomers of a pinacidil analog open or close cloned ATP-sensitive K+ channels.

ATP-dependent K(+) channels (K(ATP) channels) are composed of pore-forming subunits Kir6.x and sulfonylurea receptors (SURs). Cyanoguanidines such as pinacidil and P1075 bind to SUR and enhance MgATP binding to and hydrolysis by SUR, thereby opening K(ATP) channels. In the vasculature, openers of K(ATP) channels produce vasorelaxation. Some novel cyanoguanidines, however, selectively reverse opener-induced vasorelaxation, suggesting that they might be K(ATP) channel blockers. Here we have analyzed the interaction of the enantiomers of a racemic cyanoguanidine blocker, PNU-94750, with Kir6.2/SUR channels. In patch clamp experiments, the R-enantiomer (PNU-96293) inhibited Kir6.2/SUR2 channels (IC(50) approximately 50 nm in the whole cell configuration), whereas the S-enantiomer (PNU-96179) was a weak opener. Radioligand binding studies showed that the R-enantiomer was more potent and that it was negatively allosterically coupled to MgATP binding, whereas the S-enantiomer was weaker and positively coupled. Binding experiments also suggested that both enantiomers bound to the P1075 site of SUR. This is the first report to show that the enantiomers of a K(ATP) channel modulator affect channel activity and coupling to MgATP binding in opposite directions and that these opposite effects are apparently mediated by binding to the same (opener) site of SUR.