Figure 1.
Synthesis of isoquinolinones 7a–g.
a) MeNH2, THF, r.t., ∼75%; b) DIC, 2, CH2Cl2, r.t., 84–91%; c) Ac2O, reflux, ∼100%; d) TsOH, toluene, heat, 71% for two steps; e) RX, K2CO3, DMF, r.t., ∼95%.
Figure 2.
Synthesis of isoquinolinones 14a–d and 15a–d.
a) DIC, 2, 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 12, 24% for 13. e) RX, K2CO3, DMF, r.t., ∼95%.
Table 1.
Binding affinities of the tested compounds towards human MT1 and MT2 expressed in CHO cells.
Figure 3.
Competitive receptor binding curves of selective isoquinolinone derivatives.
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 Ki were shown in Table 1.
Figure 4.
Stimulation of intracellular Ca2+ mobilization in CHO cells expressing MT1 or MT2 by isoquinolinone derivatives.
CHO cells expressing MT1 or MT2 were subjected to the treatment of increasing doses of selected tested compounds. Data were mean of peak fluorescence signals ± SEM of at least 3 different trials performed in triplicates, and normalized to the maximal response elicited by melatonin (as 100%) and the minimal response of vehicle-treated cells (as 0%). Estimation of maximal responses and EC50 were tabulated in Table 2.
Table 2.
Isoquinolinone-induced intracellular Ca2+ mobilization in MT1-CHO and MT2-CHO cells.
Figure 5.
Phosphorylation of ERK induced by isoquinolinone derivatives.
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.
Figure 6.
Isoquinolinone derivative-induced inhibition of forskolin-stimulated cAMP production.
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 Table 3.
Table 3.
Isoquinolinone-induced inhibition of cAMP production in MT1-CHO and MT2-CHO cells.
Figure 7.
Blockade of ERK phosphorylation and Ca2+ mobilization by an isoquinolinone-based melatoninergic antagonist.
(A) CHO cells expressing MT1 or MT2 were treated with the indicated concentrations of 7e or luzindole in the absence or presence of a fixed concentration of melatonin (MLT) (for both MT1 or MT2) or 7b (for MT2 only). Other experimental details were as to the legend of Figure 5. 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 7e or 7f. Other experimental details were as to the legend of Figure 4.