Blockade of ERK phosphorylation and Ca²⁺ mobilization by an isoquinolinone-based melatoninergic antagonist.

<p>(A) CHO cells expressing MT₁ or MT₂ were treated with the indicated concentrations of <b>7e</b> or luzindole in the absence or presence of a fixed concentration of melatonin (MLT) (for both MT₁ or MT₂) or <b>7b</b> (for MT₂ only). Other experimental details were as to the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113638#pone-0113638-g005" target="_blank">Figure 5</a>. Data shown were representative blots of three separate trials. (B) CHO cells expressing MT₂ were treated with increasing concentrations (1 ρM – 1 µM) of melatonin in the absence or presence of 10 µM or 1 µM of <b>7e</b> or <b>7f</b>. Other experimental details were as to the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113638#pone-0113638-g004" target="_blank">Figure 4</a>.</p

Phosphorylation of ERK induced by isoquinolinone derivatives.

<p>CHO cells expressing MT₁ or MT₂ were serum-starved before treating with the indicated concentrations of melatonin or individual tested compounds. Resolved proteins were electrotransferred for immunodetection using phosphorylated ERK-specific antibody. Total amount of ERK was also detected similarly and no observable change of their expression levels has been found for all the treatments (not shown). Three individual trails yielded similar results as the representative blots shown in the figure.</p

Synthesis of isoquinolinones 14a–d and 15a–d.

<p>a) DIC, <b>2</b>, CH₂Cl₂, r.t. 84–91%; b) BnBr, K₂CO₃, DMF, 91% for two steps; c) TFAA, CH₂Cl₂, 0°C; d) TsOH, toluene, heat, yields for two steps: 37% for <b>12</b>, 24% for <b>13</b>. e) RX, K₂CO₃, DMF, r.t., ∼95%.</p

Synthesis of isoquinolinones 7a–g.

<p>a) MeNH₂, THF, r.t., ∼75%; b) DIC, <b>2</b>, CH₂Cl₂, r.t., 84–91%; c) Ac₂O, reflux, ∼100%; d) TsOH, toluene, heat, 71% for two steps; e) RX, K₂CO₃, DMF, r.t., ∼95%.</p

Synthesis and Functional Characterization of Substituted Isoquinolinones as MT₂-Selective Melatoninergic Ligands

<div><p>A series of substituted isoquinolinones were synthesized and their binding affinities and functional activities towards human melatonin MT₁ and MT₂ receptors were evaluated. Structure-activity relationship analysis revealed that substituted isoquinolinones bearing a 3-methoxybenzyloxyl group at C5, C6 or C7 position respectively (C5>C6>C7 in terms of their potency) conferred effective binding and selectivity toward the MT₂ receptor, with <b>15b</b> as the most potent compound. Most of the tested compounds were MT₂-selective agonists as revealed in receptor-mediated cAMP inhibition, intracellular Ca²⁺ mobilization and phosphorylation of extracellular signal-regulated protein kinases. Intriguingly, compounds <b>7e</b> and <b>7f</b> bearing a 4-methoxybenzyloxyl group or 4-methylbenzyloxyl at C6 behaved as weak MT₂-selective antagonists. These results suggest that substituted isoquinolinones represent a novel family of MT₂-selective melatonin ligands. The position of the substituted benzyloxyl group, and the substituents on the benzyl ring appeared to dictate the functional characteristics of these compounds.</p></div

Competitive receptor binding curves of selective isoquinolinone derivatives.

<p>Intact CHO cells expressing MT₁ or MT₂ were incubated with [³H]melatonin with or without different concentrations of selected tested compounds or unlabeled melatonin. Data represented mean ± SEM of at least 3 different trials performed in duplicates, and normalized to the maximal binding values (in the absence of tested compound). Estimation of maximal displacement and IC₅₀ and K_i were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113638#pone-0113638-t001" target="_blank">Table 1</a>.</p

Isoquinolinone-induced inhibition of cAMP production in MT₁-CHO and MT₂-CHO cells.

<p>The pIC₅₀ and %Inh_max were mean ± SEM of 2 trials done in duplicates. The corresponding IC₅₀ values were calculated using the mean pIC₅₀ values.</p>a<p>%Inh_max – Maximal percentage inhibition of the forskolin-elevated cAMP level.</p>b<p>NSR – no significant response for up to 10 µM of tested compounds in the assay buffer.</p>c<p>Extrapolated %Inh_max from non-linear regression.</p><p>Isoquinolinone-induced inhibition of cAMP production in MT₁-CHO and MT₂-CHO cells.</p

Isoquinolinone derivative-induced inhibition of forskolin-stimulated cAMP production.

<p>CHO cells expressing MT₁ or MT₂ were treated with 50 µM forskolin and increasing concentrations of individual tested compounds as indicated at the lower left corner of each plot. All the responses were expressed as the percentage of that induced by forskolin alone (as 100%). Estimation of maximal inhibition and IC₅₀ were tabulated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113638#pone-0113638-t003" target="_blank">Table 3</a>.</p