Molecular Determinants for the Activating/Blocking Actions of the 2H-1,4-Benzoxazine Derivatives, a Class of Potassium Channel Modulators Targeting the Skeletal Muscle K_ATP Channels

Abstract

The 2H-1,4-benzoxazine derivatives are modulators of the skeletal muscle ATP-sensitive-K ϩ channels (K ATP ), activating it in the presence of ATP but inhibiting it in the absence of nucleotide. To investigate the molecular determinants for the activating/blocking actions of these compounds, novel molecules with different alkyl or aryl-alkyl substitutes at position 2 of the 1,4-benzoxazine ring were prepared. The effects of the lengthening of the alkyl chain and of branched substitutes, as well as of the introduction of aliphatic/aromatic rings on the activity of the molecules, were investigated on the skeletal muscle K ATP channels of the rat, in excised-patch experiments, in the presence or absence of internal ATP (10 Ϫ4 M). In the presence of ATP, the 2-n-hexyl analog was the most potent activator (DE 50 ϭ 1.08 ϫ 10 Ϫ10 M), whereas the 2-phenylethyl was not effective. The rank order of efficacy of the openers was 2-n-hexyl Ն2-cyclohexylmethyl Ͼ2-isopropyl ϭ 2-n-butyl Ն 2-phenyl Ն 2-benzyl ϭ 2-isobutyl analogs. In the absence of ATP, the 2-phenyl analog was the most potent inhibitor (IC 50 ϭ 2.5 ϫ 10 Ϫ11 M); the rank order of efficacy of the blockers was 2-phenyl Ն 2-n-hexyl Ͼ 2-n-butyl Ͼ 2-cyclohexylmethyl, whereas the 2-phenylethyl, 2-benzyl, and 2-isobutyl 1,4-benzoxazine analogs were not effective; the 2-isopropyl analog activated the K ATP channel even in the absence of nucleotide. Therefore, distinct molecular determinants for the activating or blocking actions for these compounds can be found. For example, the replacement of the linear with the branched alkyl substitutes at the position 2 of the 1,4-benzoxazine nucleus determines the molecular switch from blockers to openers. These compounds were 100-fold more potent and effective as openers than other KCO against the muscle K ATP channels.Potassium channel openers (KCO) are chemically diverse compounds that belong to a number of structural classes, including benzopyrans (cromakalim, bimakalim), benzothiadiazines (diazoxide), cyanoguanidines (pinacidil), cyclobutenediones (WAY-151616), nicotinamides (nicorandil), pyrimidines (minoxidil), tertiary carbinoles (ZD-6169), thioformamides (aprikalim), and dihydropyridine-like structures (ZM-244085) (Mannhold, 2004;Jahangir and Terzic, 2005). Several derivatives of these compounds have been synthesized and tested against the ATP sensitive K ϩ -channel (K ATP ) subunits expressed in cell lines, which is the primary target for KCO action.These compounds show a broad spectrum of therapeutic applications, including asthma, urinary incontinence, hypertension, angina, hypoglycemia, neuromuscular disorders, and some forms of epilepsy (Andersson, 1992;Longman and Hamilton, 1992). KCO drugs exert their effects on pancreatic ␤ cells, neurons, and vascular/nonvascular smooth muscle and cardiac muscle by opening K ATP channels, thus shifting the membrane potential toward the reversal potential for potassium and reducing cellular electrical activity. In skeletal muscle, nicorandil is effective in restoring the depr...

Structural Nucleotide Analogs Are Potent Activators/Inhibitors of Pancreatic β Cell KATP Channels: An Emerging Mechanism Supporting Their Use as Antidiabetic Drugs

“…A conformational analysis showed that the planar area of the 2H-1,4-benzoxazines overlaps that of the adenine nucleotide tri-and diphosphates. The electronic distribution profile of this area is also similar to that of the adenine nucleotides, which were reached by electrons; this result suggests that these compounds share a common ATP/ADP interaction interface on the receptor sites (Tricarico et al, 2008). No data are available regarding the effects or possible dual actions of these nucleotide analogs on the pancreatic ␤ cell KATP channel or glucose disposal in normal mice.…”

“…A biphasic behavior was observed in the glucose disposal curves that were determined in vivo after the administration of increasing doses of the 2-nhexyl analog; these data can be explained by accounting for the opposite in vitro effects of this compound on pancreatic ␤ cells and skeletal muscle KATP channels (Fig. 9) (Tricarico et al, 2008).…”

“…The experiments are illustrated by the traces, means, or representative results that were obtained from at least three different cultures. The concentrationresponse data were fitted by an equation that described the interaction of the ligand with one inhibitory site, as in the case of the 2-hexyl analog data, or by the sum of two equations that described the interaction of the ligand with two sites that exerted opposite actions, as in the case of the 2-cyclic analog data (Tricarico et al, 2008). The stimulatory component can be described by the following term:…”

“…The drug experiments were performed using the patch-clamp methodology in the voltage-clamp mode (perforated whole-cell, conventional whole-cell, and inside-out configurations), and the current-clamp mode was performed using Clampex software (Molecular Devices, Sunnyvale, CA) (Rolland et al, 2006;Tricarico et al, 2008).…”

“…In the presence of ATP, these molecules display KCO activity, whereas in the absence of the nucleotide they display a blocking action (Tricarico et al, 2003(Tricarico et al, , 2008. These compounds have structural similarities with ATP and ADP molecules.…”