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Synthesis and functional characterization of substituted isoquinolinones as MT2-selective melatoninergic ligands.

Hu Y, Chan KH, He X, Ho MK, Wong YH - PLoS ONE (2014)

Bottom Line: 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 15b as the most potent compound.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Life Science and the Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.

ABSTRACT
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 15b 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 7e and 7f 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.

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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.
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pone-0113638-g005: 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.

Mentions: Melatonin can induce phosphorylation of extracellular signal-regulated protein kinases (ERK) in both MT1-CHO and MT2-CHO cells due to the presence of the 16z25 chimera. As shown in Figure 5, ERK phosphorylation became detectable when >1 nM of melatonin was added to either cell lines, indicating similar potencies of melatonin for both receptor subtypes. The total amount of ERK in the cell lysates loaded into the gels was monitored using a specific antibody and none of the treatment had any effect on the total amount of ERK (data not shown). Resembling the results in FLIPR assays, only 7b and 7c could induce weak ERK phosphorylation in MT1-CHO cells at a concentration of >1 µM, and all the other tested compounds were ineffective. In contrast, most of the tested compounds activated ERK phosphorylation in MT2-CHO cells in a concentration-dependent manner but with different potencies. The rankings of each group of tested compounds with the same position substituted with different modified benzyloxyl groups were generally very similar to those obtained in the FLIPR assay. Compounds bearing a 3-methoxybenzyloxyl substituent (7b, 14b, and 15b) were the most effective ones in each group, followed by compounds bearing a 3,5-dimethoxybenzyloxyl substituent (7c, 14c, and 15c). While the difference between 3-methoxybenzyloxyl (7b, 14b, and 15b) and 3,5-di-methoxybenzyloxyl (7c, 14c, 15c) derivatives were obvious, the difference of the ERK phosphorylation responses induced by 15b was only slightly better than 15c. These results further suggested that the 3,5-dimethoxybenzyloxyl substituent was better tolerated when located at C5 of the isoquinolinone scaffold. Compounds bearing a benzyloxyl (7a and 15a) or a 4-methylbenzyloxyl substituent (14d and 15d) at C6 or C5 could only stimulate ERK phosphorylation very weakly, and compounds (14a and 14d) bearing the same substituents at C7 were essentially inactive.


Synthesis and functional characterization of substituted isoquinolinones as MT2-selective melatoninergic ligands.

Hu Y, Chan KH, He X, Ho MK, Wong YH - PLoS ONE (2014)

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.
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pone-0113638-g005: 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.
Mentions: Melatonin can induce phosphorylation of extracellular signal-regulated protein kinases (ERK) in both MT1-CHO and MT2-CHO cells due to the presence of the 16z25 chimera. As shown in Figure 5, ERK phosphorylation became detectable when >1 nM of melatonin was added to either cell lines, indicating similar potencies of melatonin for both receptor subtypes. The total amount of ERK in the cell lysates loaded into the gels was monitored using a specific antibody and none of the treatment had any effect on the total amount of ERK (data not shown). Resembling the results in FLIPR assays, only 7b and 7c could induce weak ERK phosphorylation in MT1-CHO cells at a concentration of >1 µM, and all the other tested compounds were ineffective. In contrast, most of the tested compounds activated ERK phosphorylation in MT2-CHO cells in a concentration-dependent manner but with different potencies. The rankings of each group of tested compounds with the same position substituted with different modified benzyloxyl groups were generally very similar to those obtained in the FLIPR assay. Compounds bearing a 3-methoxybenzyloxyl substituent (7b, 14b, and 15b) were the most effective ones in each group, followed by compounds bearing a 3,5-dimethoxybenzyloxyl substituent (7c, 14c, and 15c). While the difference between 3-methoxybenzyloxyl (7b, 14b, and 15b) and 3,5-di-methoxybenzyloxyl (7c, 14c, 15c) derivatives were obvious, the difference of the ERK phosphorylation responses induced by 15b was only slightly better than 15c. These results further suggested that the 3,5-dimethoxybenzyloxyl substituent was better tolerated when located at C5 of the isoquinolinone scaffold. Compounds bearing a benzyloxyl (7a and 15a) or a 4-methylbenzyloxyl substituent (14d and 15d) at C6 or C5 could only stimulate ERK phosphorylation very weakly, and compounds (14a and 14d) bearing the same substituents at C7 were essentially inactive.

Bottom Line: 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 15b as the most potent compound.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Life Science and the Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.

ABSTRACT
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 15b 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 7e and 7f 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.

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