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

  • Yueqing Hu,

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

  • King H. Chan,

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

  • Xixin He,

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

  • Maurice K. C. Ho,

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

  • Yung H. Wong

    boyung@hkust.hk

    Affiliations Division of Life Science and the Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, State Key Laboratory of Molecular Neuroscience, and the Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

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

  • Yueqing Hu, 
  • King H. Chan, 
  • Xixin He, 
  • Maurice K. C. Ho, 
  • Yung H. Wong
PLOS
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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.

Introduction

Melatonin (N-acetyl-5-methoxytryptamine) is a versatile hormone which regulates circadian rhythm as well as many other biological functions [1][3]. It is secreted by the pineal gland and its biological effects are exerted through specific melatonin binding sites. Two of them belong to the family of seven-transmembrane-domain G protein-coupled receptors (GPCR) have been cloned (MT1 and MT2), and shown to be expressed in mammals [4][6]. They are believed to play different, or in some circumstances opposite, roles in biological systems [7]. MT1 receptors modulate neuronal firing, arterial vasoconstriction, cell proliferation, reproductive, and metabolic functions [8][12]. MT2 receptors are responsible for resetting the circadian rhythm of neuronal firing in the suprachiasmatic nucleus (phase-shifting), inhibiting dopamine release in retina, inducing vasodilation, and enhancing immune responses [9], [13][15]. While the melatonin receptor subtypes may work in concert to regulate various chronobiotic and homeostatic responses, the distinct roles of MT1 and MT2 spur the interest to develop subtype-specific pharmacological agents to pinpoint their individual roles in the regulation of circadian rhythmicity, or promoting sleep without phase-shifting the circadian clock.

The therapeutic potential of melatonin is limited by its non-subtype specific actions at multiple receptors as well as its unfavorable pharmacokinetic properties, such as high first-pass metabolism, short half-life and poor oral bioavailability [16], [17]. Novel melatoninergic compounds with different chemical scaffolds have therefore been synthesized and discovered, such as indoles, naphthlenes, benzoxazoles, pyrrolidines, and tetralins. Many of the compounds consist of an alkylamide with a terminal alkyl chain not longer than 3–4 carbons, which mainly governs the binding affinity towards the melatonin receptors. A number of early studies have also shown that the presence of an aromatic substituent on the C2 position of the melatonin indole ring can confer MT2 selectivity [18], [19], but none of these selective melatoninergic agents had been developed into clinical uses. To date, the area of subtype-selective therapeutic melatoninergics has not been thoroughly addressed.

Our research goals were to discover novel compounds that exhibit potent binding affinity and good subtype selectivity at MT1 and/or MT2 receptors. The compound 7-hydroxy-6-methoxy-2-methyl-2H-isoquinolin-1-one (compound 12) was identified as a modest melatoninergic agonist with selectivity towards MT2 in high-throughput drug screening assays (Figure S1). An obvious distinctive feature of this compound as compared to melatonin and many other melatoninergics is the lack of free alkylamide side chain but a N-methylamide moiety confined in the isoquinolinone scaffold, and there is no previous evidence showing any structural resemblance or functional equivalence of such N-methylamide moiety to the alkylamide chain of other known melatoninergics.

As isoquinolinone represents a novel chemical scaffold possessing melatoninergic activity, it prompted us to develop a series of more potent and selective isoquinolinone derivatives. In this study, a series of substituted isoquinolinones were synthesized. Different substituted benzyloxyl and methoxy substituents were incorporated at C5–C7 positions of the isoquinolinone ring to generate sufficient derivatives for structure-activity relationship analysis. The binding affinities of the tested compounds were evaluated in competitive receptor binding assays using radiolabeled melatonin as the probe on intact cells expressing each of the recombinant melatonin receptor subtypes. The abilities of the tested compounds to trigger receptor-mediated inhibition of cAMP production, intracellular Ca2+ mobilization and phosphorylation of extracellular signal-regulated protein kinases (ERK) were compared with melatonin-induced responses.

Chemistry

Isoquinolinone compounds were synthesized in 4–5 steps, using Lee's method [20] (Figure 1) or its modified version (Figure 2). Starting with commercially available phenylvinyl sulfoxide 1, 1,4-conjugate addition with methylamine formed the amine 2. Coupling of 4-hydroxy-3-methoxybenzoic acid 3 with amine 2 generated the amide 4. Pummerer rearrangement [21], [22] of 4 by refluxing with acetic anhydride converted the sulfoxide to masked “acetal” compound 5. Heating compound 5 in toluene under acidic conditions (p-toluenesulfonic acid, TsOH) completed the heterocyclic ring to give isoquinolinone 6 in 71% yield together with minor O-methyl migrated byproduct. Alkylation of the free hydroxyl group of 6 with different halides yielded a series of isoquinolinones 7a–7g with different substituents at C6 position.

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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%.

https://doi.org/10.1371/journal.pone.0113638.g001

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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%.

https://doi.org/10.1371/journal.pone.0113638.g002

For analogues with different substitutions at C7 (compounds 14a–d) or C5 (compounds 15a–d) positions, 3-hydroxy-4-methoxybenzoic acid 8 was used as the starting material. The Lee's method was initially attempted. Coupling of acid 8 with amine 2 formed the amide 9. Pummerer rearrangement of amide 9 in refluxing acetic anhydride followed by acidic cyclization provided a complex of products resulting from clockwise and anti-clockwise cyclizations and O-methyl group migrations. In order to simplify the product isolation, the hydroxyl group of compound 9 was protected as benzyl ether 10. Refluxing of 10 in acetic anhydride followed by acidic cyclization provided a similar complex of products without the benzyl protection group. The Pummerer rearrangement reaction was performed with trifluoroacetic anhydride (TFAA) in dichloromethane at 0°C. After acidic cyclization, isoquinolinones 12 and 13 were produced with satisfactory yields with low amount of O-methyl group migration byproducts. Alkylations of 12 or 13 with different halides gave the desired analogues 14a–d or 15a–d, respectively.

Results

Competitive binding assays

Table 1 shows the compound structures and their binding affinities toward human MT1 and MT2 receptors. Competitive receptor binding characteristics of melatonin and the 15 tested compounds towards human MT1 and MT2 stably expressed in Chinese hamster ovary (CHO) cells were determined using intact cell preparation pre-incubated with 1 nM [3H]melatonin. The Kd of melatonin for MT1 and MT2 receptors was 0.30 nM and 0.43 nM, respectively, as determined by saturation binding assays. All of the tested compounds showed very weak (Ki>1 µM) or basically no significant binding towards MT1 (Table 1). Contrarily, most of the tested compounds showed detectable binding at MT2. Among compounds 7a7g with a benzyloxyl or substituted benzyloxyl group at C6, 7b which bears a 3-methoxybenzyloxyl group displayed the most outstanding binding affinity, consistent with our previous report [23]. 7b was also the only compound among this group which completely displaced the pre-incubated [3H]melatonin.

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Table 1. Binding affinities of the tested compounds towards human MT1 and MT2 expressed in CHO cells.

https://doi.org/10.1371/journal.pone.0113638.t001

The next group of compounds 14a–d is 6-methoxy-isoquinolinones with a benzyloxyl or substituted benzyloxyl substituent at C7 position. They appeared to bind MT2 much weaker than the last group of substituted 7-methoxy-isoquinolinones 7a7g. The compound 14b containing the 3-methoxybenzyloxyl substituent as of 7b also exhibited the highest affinity toward MT2 receptor subtype among this group, but its affinity was significantly lower than 7b. 14b also completely displaced [3H]melatonin under the same experimental condition. The last group 15a–d is 6-methoxy-isoquinolinones with a different substituent at C5 position. Similar to the previous two groups, 15b bearing a 3-methoxybenzyloxyl substituent conferred the highest binding affinity toward MT2 among this group. 15b displayed high MT2 affinity (Ki = 1.7 nM), only approximately one order lower than melatonin as determined in the same condition, but 1800-fold selectivity over MT1. Among the compounds bearing a 3,5-dimethoxybenzyloxyl group (7c, 14c and 15c), 15c showed significantly higher affinity toward MT2 and almost complete displacement of [3H]melatonin. It also displayed 51-fold of selectivity toward MT2 than MT1. The order of compounds in terms of their MT2 binding affinity and selectivity in descending order was 15b>15c>15a (Figure 3). Overall, the results of competitive binding assays showed that the presence of a 3-methoxybenzyloxyl group increased the affinity toward MT2 but not MT1, and its attachment position on the isoquinolinone scaffold was important for the overall binding affinity, with 15b bearing a 3-methoxybenzyloxyl group at C5 being the most potent compound (Figure 3).

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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.

https://doi.org/10.1371/journal.pone.0113638.g003

Functional characterization of isoquinolinone-based melatoninergic ligands

Individual melatonin receptor subtypes were stably expressed in Chinese hamster ovary (CHO) cells together with a chimeric G protein α subunit 16z25; the 16z25 chimera channels receptor activation signals to the mobilization of intracellular Ca2+ for real-time detection in a FLIPR [24][26]. The estimated EC50 and percentage activation of the melatonin-induced responses as deduced from the concentration-dependent stimulation induced by the various isoquinolinone compounds are summarized in Table 2. In the positive controls, melatonin induced robust dose-dependent responses in both MT1 and MT2-expressing CHO cells (MT1-CHO and MT2-CHO) with very similar EC50 in the sub-nM range, and melatonin induced a slightly more potent response in MT1-expressing cells as expected. Compound 12 partially activated both MT1 and MT2 receptors at 10 µM with a maximal response of 36% and 53%, respectively (data not shown), but for most of the tested compounds, no significant response could be induced in MT1-CHO cells at the maximal concentration tested (10 µM; Table 2). Exceptions are 7b and 7c where concentration response curves could be constructed (Figure 4) with EC50 values of 31.1 and 266 nM, respectively. Both compounds have a meta-methoxybenzyloxyl substitutent at C6 position of the isoquinolinone scaffold. Similar analogs with a meta-methoxybenzyloxyl group at either C7 (14b and 14c) or C5 (15b and 15c) position could not produce any activity at MT1. Again, 7b outperformed 7c to activate MT1 with an EC50 lower by an order of magnitude, which correlated with the results in the binding assay.

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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.

https://doi.org/10.1371/journal.pone.0113638.g004

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Table 2. Isoquinolinone-induced intracellular Ca2+ mobilization in MT1-CHO and MT2-CHO cells.

https://doi.org/10.1371/journal.pone.0113638.t002

When assayed in MT2-CHO cells, most of the tested compounds elicited concentration-dependent stimulation of Ca2+ signals, and allowed for a detailed interpretation of their structure-function relationship. In the binding assay, among compounds 7a–7g with a benzyloxyl or substituted benzyloxyl group at C6, 7b showed the most outstanding binding to MT2. Expectedly, 7b induced robust dose-dependent Ca2+ signals with an EC50 of 0.3 nM and its maximal response was very close to that of melatonin (Table 2). Although the apparent Ki values of the other C6-benzyloxyl substituted compounds were very close to each other (Table 1), they behaved differently in triggering Ca2+ signals. Compound 7c with an additional methoxy group on the benzyloxyl branch was ∼70-fold less potent than 7b. Further reduction in potency was observed for 7a which was devoid of methoxy group on the benzyloxyl branch. Concentration response curves of 7a7c in both MT1 and MT2-expressing cells are shown in Figure 4. It is obvious that 7a could trigger Ca2+ signals exclusively in MT2-CHO cells at around 1 µM, whereas the concentration of 7c to induce exclusive MT2 activation was even lower (<10 nM).

Very different responses were observed for another two mono-methoxybenzyloxyl derivatives 7d and 7e. By simply moving the methoxy group on the benzyloxyl branch from meta to ortho position, the ability of 7d to trigger Ca2+ signal was reduced by 2 orders of magnitude (Table 2), indicating a stringent structural requirement for the MT2 selectivity and the importance of this particular methoxy group. The position effect was further manifested by the null response of 7e – the para-methoxybenzyloxyl derivative. 7f and 7g are methylbenzyloxyl derivatives, analogous to 7e and 7c for the number and position of the methyl substituent. In terms of EC50 and maximal response of Ca2+ signals, 7f and 7g behaved very similarly to 7e and 7c, respectively.

Compounds 14a–d with a benzyloxyl or substituted benzyloxyl group at C7 had the weakest binding affinities (Table 1), and so were their abilities to trigger Ca2+ signals. The best of this group, 14b with a 3-methoxybezyloxyl substituent similar to 7b showed an EC50>160- fold of 7b. Compound 14c was 11-fold less potent than 7c, and 14a and 14d were basically inactive. A comparison of the concentration-dependent responses shown in Figure 4 clearly showed the much weaker responses of C7-benzyloxyl substituted compounds. Although both 14b and 14c bound and activated MT2 receptor, 14c displayed only partial efficacy in FLIPR assay.

Compounds 15a–d represented the best group of isoquinolinones as they were all capable of activating MT2 exclusively with high potency. The 3,5-dimethoxybenzyloxyl derivative 15c was 5-fold more potent than 7c, whereas the potency of 15a was essentially indistinguishable from 7a. The 3-methoxybenzyloxyl derivative 15b possessed an EC50 comparable to that of melatonin toward MT2 and was totally inactive at MT1 (Table 2), further indicating that the importance of the presence of a single methoxy group on the meta position of the benzyloxyl branch in subtype selectivity. Its subtype selectivity was obviously better than 7b (Figure 4, middle column). Interestingly, 15d was the only 4-methylbenzyloxyl derivative (c.f. 7f and 14d) able to induce a receptor-mediated Ca2+ signal concentration-dependently, suggesting that MT2 receptor has a greater tolerance for substituent extending off the C5 position of the isoquinolinone scaffold.

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.

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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.

https://doi.org/10.1371/journal.pone.0113638.g005

Both MT1 and MT2 are classified as Gi-coupled receptors with their activation leading to inhibition of intracellular cAMP production. The ability of the tested compounds to activate the endogenously expressed Gi-mediated inhibition of cAMP production was therefore examined. Melatonin induced 60–70% inhibition of the cAMP level elevated by forskolin, a direct activator of adenylyl cyclase, in both MT1-CHO and MT2-CHO cells with sub-nM IC50's (Table 3), reflecting intact Gi-dependent regulatory pathways in both cell lines. Eight selected isoquinolinones were examined for their dose-dependent inhibition of cAMP accumulation (Figure 6). In MT1-CHO cells, only 7b and 7d showed observable inhibition of cAMP levels with estimated IC50 values in the single digit or sub-µM range. Other tested compounds were basically unable to cause any inhibition. Similar to the previous assays in MT2-CHO cells, derivatives bearing a 3-methoxybenzyloxyl substituent (7b, 14b and 15b) outperformed other subgroup members bearing another substituent at the same position of the isoquinolinone scaffold. Both 7b and 14b showed similar IC50's and their maximal percentage inhibition resembled that of melatonin, but 14b showed a better selectivity toward MT2. Compound 15b has an IC50 close to that of melatonin at MT2 but was completely inactive at MT1 (Table 3). The derivatives containing a 3,5-dimethoxybenzyloxyl substituent showed a progressive decrease of IC50 in the order of 7c, 14c and 15c, further manifesting the positional effect of the benzyloxyl substituent in which C5 appeared to be the most desirable position for a highly MT2-selective compound. Overall, the results in all three different functional assays indicated that a 3-methoxybenzyloxyl substitutent at C5 of the isoquinolinone scaffold yielded a novel MT2-selective melatoninergic agonist with single digit or sub-nM activities.

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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.

https://doi.org/10.1371/journal.pone.0113638.g006

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Table 3. Isoquinolinone-induced inhibition of cAMP production in MT1-CHO and MT2-CHO cells.

https://doi.org/10.1371/journal.pone.0113638.t003

Identification of potential MT2-selective antagonists

Among the low affinity tested compounds (7a, 7d, 7e, 7f and 7g), 7e and 7f did not show any activities in all three functional assays (Tables 2, 3, Figures 5, 6, and S2). This observation prompted us to explore if these para-substituted benzyloxyl derivatives possess antagonistic activity towards MT2. In the presence of 1 nM of melatonin (MLT), increasing doses of 7e suppressed the agonist-induced ERK phosphorylation in MT2-CHO but not in MT1-CHO cells (Figure 7A, left hand side, upper two blots). The level of ERK phosphorylation was reduced to almost the basal level when 10 µM of 7e was present (lane 5 versus 7). Moreover, ERK phosphorylation induced by the application of a structurally closely related agonist 7b (100 nM) was also significantly suppressed by 7e in MT2-CHO cells (at 10 µM; Figure 7A, lower left hand panel). Despite having a similar binding affinity as 7e, para-methylbenzyloxyl derivative 7f demonstrated a weaker antagonistic activity, with partial inhibition of the MT2 response at 10 µM (Figure S3). To confirm competitive antagonism, MT2-CHO cells were treated with melatonin in the absence or presence of 7e or 7f. A parallel right shift of the melatonin dose-response curve was observed in FLIPR assay upon preincubation of 7e or 7f (Figure 7B), displaying a competitive interaction between melatonin and the isoquinolinone antagonists. A typical MT2-selective antagonist luzindole was employed in the control experiment (Figure 7A, right hand side). Luzindole blocked the ERK phosphorylation induced by either MLT in both MT1-CHO and MT2-CHO cells or 7b in MT2-CHO cells in a more effective manner, which was consistent to the higher affinity of luzindole towards both MT1 (Ki = 102 nM) and MT2 (Ki = 2.9 nM) under our experimental conditions. In MT1-CHO cells, luzindole completely eliminated basal ERK phosphorylation at 1 µM. However, the observed basal ERK phosphorylation was constantly enhanced in MT2-CHO cells treated with luzindole alone. In fact, a weak but significant MT2-selective agonistic activity of luzindole was also detected in FLIPR assay (Figure S2). In conclusion, the results suggested that 7e and 7f represent novel competitive MT2-selective antagonists with relatively low affinity.

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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.

https://doi.org/10.1371/journal.pone.0113638.g007

Discussion

The results of the present study demonstrate that substituted isoquinolinones possess melatoninergic activities with selectivity toward MT2. A substituted benzyloxyl group on three carbons (C5>C6>C7 in descending order of binding affinity) of the isoquinolinone scaffold rendered differential affinity toward MT2, whereas most of the derivatives were basically unable to bind MT1. Compound 15b with a 3-methoxybenzyloxyl group at C5 position was the most potent MT2-selective agonist with a binding affinity of 1.66 nM toward MT2 and a selectivity of 1800-fold over MT1. Benzyloxyl group bearing one meta-methoxy outperformed two meta-methoxy substituents, suggesting the MT2 ligand binding pocket was very sensitive to steric hindrance. This was further demonstrated by the distinctive behaviors of 7d and 7e: while ortho-methoxy derivative 7d was tolerated in maintaining the agonist property, para-methoxybenzyloxyl group attached to C6 position generated an MT2-selective weak antagonist 7e.

The extended ethylamido chain is a key feature for most of the previously known melatoninergic compounds [27], [28]. Farce et al. suggested that both the carbonyl oxygen and the amine proton of the amide are engaged in hydrogen bonds with the serine residues in the putative binding site, thus governing the compounds' binding affinity toward both melatonin receptors [29]. The lack of such extended ethylamido chain in our tested compounds might cause a significant reduction of binding affinity when compared with melatonin, but at the same time the importance of other structural features corresponding to receptor binding affinity could become more apparent.

Our lead compound 12, which does not contain an alkyladimoethyl chain or an additional aromatic group, showed very weak activities toward both MT1 and MT2 receptors as shown in Ca2+ mobilization assay. By attaching an aromatic 3-methoxyphenyl ring through CH2-O chain to isoquinolinone at C7, compound 14b partially compensated the effect caused by the lack of an alkyladimoethyl chain and exhibited fair activity toward MT2. When the aromatic 3-methyoxylphenyl was switched from C7 to C6 or C5 position, the activities (compound 7a and 15b) towards MT2 were further enhanced. These results demonstrated the importance of the aromatic 3-methoxyphenyl group and its relative orientations on the isoquinolinone scaffold. When the aromatic 3-methoxyphenyl group is attached at C5 position of isoquinolinone through CH2-O chain, it (compound 15b) provided the most potent activity and highest selectivity towards MT2. Melatonin ligands bearing additional aromatic rings and no alkylamidoethyl chain have been identified previously. Karageorge et al developed a series of tetrahydroisoquinoline derivatives as melatonin MT2 receptor antagonists after following a lead compound identified by high throughput screening [30]. These compounds and our isoquinolinone derivatives may bind to melatonin MT2 at a similar site through interacting with the additional aromatic group.

The flexibility of the ethylamido chain allows diverse conformational arrangements in order to fit the ligand into the binding pockets of the two melatonin receptors in a slightly different manner. In previous mutagenesis studies, MT1 and MT2 receptors displayed distinct tolerance to conserved serine mutations, which hinted at structural divergence between the two subtypes. [31], [32]. It has been shown that a substituent at C2 position of melatonin (either halogen atom or phenyl group, which is either lying on the same plane of the indole ring or has only very limited degree of freedom) can enhance the binding affinity for both receptor subtypes [33]. However, a 2-benzyl substituent which lies away from the plane of the indole scaffold of melatonin specifically enhances MT2 binding only [34]. It means that an additional binding cavity, which is away from the plane of the indole ring and is able to accommodate the aromatic substituent, may be present in MT2 but not MT1. Such a cavity is most likely formed by a large group of hydrophobic amino acid side chains located at TM5 (Y200, V204, V205, H208), TM6 (W264, L267, Ile270) and TM7 (F290, Y294, Y298) [35]. This might explain why our isoquinolinone compounds with a 3-methoxybenzyloxyl substituent show modest affinity towards MT2 even in the absence of the ethylamido chain.

With the 2-benzyl moiety of luzindole resembling the benzyloxyl group of 7a, 14a and 15a, the partial agonist activity of luzindole, as illustrated in Ca2+ mobilization (Figure S2) and ERK phosphorylation assays (Figure 7A), further support the notion that additional aromatic ring may be important for MT2 binding. Luzindole has previously been suggested as a MT2 partial agonist in vivo based on its melatonin-like effect to depress the excitatory postsynaptic potentials evoked by mouse hippocampal neurons [36] and the firing of rat medial vestibular nucleus neurons [37]. The coexistence of an aromatic group and the alkylamidoethyl chain at juxtaposition on either a benzofuran [38] or simply phenyl scaffold [26] gives rise to very potent MT2-selective ligands, suggesting that the two key features bind to non-overlapping regions of MT2 and produce synergistic effect on receptor binding.

Conclusions

This study has provided new insights on the design of subtype selective melatonin receptor agonists and antagonists. Isoquinolinone derivatives are a novel category of melatoninergic ligands without an extended ethylamido side chain. Both agonists and antagonists selective to MT2 have been identified, and the results have refined our understanding on the importance of the aromatic substituent on the receptor subtype selectivity and functional characteristics of melatoninergic compounds. A 3-methoxybenzyloxyl substituent at C5 or C6 position of isoquinolinone scaffold rendered MT2 selectivity. The number and position of methoxy group on the benzyl ring dictates the binding affinity as well as functionality of the ligands. Further investigation on the stability and fine-tuning of the structure-activity relationship of isoquinolinone-based compounds may therefore represent a promising design strategy for melatonin receptor subtype-specific therapeutic agents.

Experimental section

Synthesis

All reagents and solvents were purchased from commercial sources and used as received unless specified. Dry methylene chloride was distilled from CaH2. Flash chromatography was performed with Merck silica gel 60 (230–400 mesh), and reaction progress was determined by thin-layer chromatography (TLC) on silica gel plates. Yields were based on purified compounds and were not optimized. Melting points were determined on Aldrich Mel-TEMP II capillary melting-point apparatus and uncorrected. NMR spectra were recorded on a Joel EX 400, or a Varian Mercury 300 spectrometer. Chemical shifts of the 1H-NMR were referenced to residual solvent (chloroform at 7.26 ppm) or TMS (0.00 ppm). Chemical shifts of 13C-NMR were referenced to CDCl3 at 77.00 ppm. ESI-MS was taken on a Thermo-Finnigan LCQ Classic ion trap mass spectrometer; only molecular ions (M+1) were given. HR-MS recorded on MALDI Micro MX Mass Spectrometer by Waters MICROMASS. Purity and characterization of compounds were established by both NMR and MS. The purity of the final compounds was >95% by NMR and HPLC analysis.

(2-Benzenesulfinylethyl)-methylamine (2).

Phenylvinyl sulfoxide (1, 3 ml, 22.4 mmol) was added to a solution of methylamine in THF (2.0 M, 16.8 ml, 33.7 mmol). The mixture was sealed and stirred at room temperature for 18 hours. The solvent was removed under vacuum after the reaction, and the crude product was purified by flash column chromatography on silica gel (MeOH∶CH2Cl2∶NH3 = 10∶90∶1) to yield the desired product 2 (2.9 g, 17.1 mmol, 76%), which was used directly without further purifications.

N-(2-Benzenesulfinylethyl)-4-hydroxy-3-methoxy-N-methylbenzamide (4).

Diisopropyl carbodiimide (2.6 ml, 20.6 mmol) was added dropwise to a solution of compound 2 (2.9 g, 17.1 mmol), 4-hydroxy-3-methoxybenzoic acid (3, 3.5 g, 20.6 mmol) and hydroxybenzotriazole (2.8 g, 20.6 mmol) dissolved in 80 ml CH2Cl2 under N2. The reaction was stirred at room temperature for 12 hours, and the white urea solid was removed by filtration. The filtrate was concentrated and the crude product was purified by flash column chromatography on silica gel (MeOH/CH2Cl2 = 1∶25) yielding 4 (4.8 g, 14.4 mmol, 84%) as a white solid. 1H-NMR (400 MHz, CDCl3) δ 7.64 (1H, dd, J = 8.4, 1.2 Hz), 7.56 (1H, d, J = 1.2 Hz), 7.30 (5H, m), 6.93 (1H, d, J = 8.4 Hz), 4.49 (2H, t, J = 5.6 Hz), 3.92 (3H, s), 3.31 (2H, t, J = 5.6 Hz), 2.94 (3H, s).

Acetic acid 4-[(2-acetoxy-2-phenylsulfanylethyl)-methylcarbamoyl]-2-methoxyphenyl ester (5).

Compound 4 (4.8 g, 14.4 mmol) was dissolved in 40 ml acetic anhydride and refluxed for 6 hours. The excessive acetic anhydride was then removed under reduced pressure. The resulting residue was treated with saturated NaHCO3, extracted with CH2Cl2, dried and concentrated. The crude product was purified by flash column chromatography on silica gel (ethyl acetate∶hexane = 1∶1) yielding 5 (6.1 g, 100%) as a brown oil, which was used in the next step without further purification. 1H-NMR (400 MHz, CDCl3): δ 7.53 (1H, m), 7.32 (4H, m), 7.01 (2H, m), 6.90 (1H, m), 6.50 (1H, s), 3.88 (2H, m), 3.81 (3H, s), 3.03 (3H, s), 2.32 (3H, s), 2.09 (3H, s).

6-Hydroxy-7-methoxy-2-methylisoquinolin-1(2H)-one (6).

p-Toluenesulfonic acid monohydrate (12.2 g, 64.1 mmol) was added to a solution of compound 5 (crude 6.1 g) dissolved in 70 ml toluene. The mixture was refluxed under N2 for 40 min, and the solvent was then removed under reduced pressure. The resulting residue was neutralized with saturated NaHCO3, and extracted with CH2Cl2 for several times until TLC of the aqueous phase did not show the desired product. The combined CH2Cl2 extract was dried and concentrated. The crude product was purified by flash column chromatography on silica gel (MeOH∶CH2Cl2 = 1∶25) yielding 6 (2.1 g, 10.2 mmol, 71%) as a white solid. 1H-NMR (400 MHz, CDCl3): δ 7.82 (1H, s), 6.99 (1H, s), 6.98 (1H, d, J = 7.2 Hz), 6.38 (1H, d, J = 7.2 Hz), 3.99 (3H, s), 3.60 (3H, s). 13C-NMR (100 MHz, CDCl3): δ 162.0, 150.7, 147.8, 132.9, 130.4, 118.9, 109.4, 106.9, 106.0, 55.7, 36.9.

N-(2-Benzenesulfinyl-ethyl)-3-hydroxy-4-methoxy-N-methylbenzamide (9).

Diisopropyl carbodiimide (7.9 ml, 50.3 mmol) was added dropwise to a solution of compound 2 (8.38 g, 45.7 mmol), compound 8 (8.46 g, 50.3 mmol) and hydroxybenzotriazole (6.84 g, 50.3 mmol) in a mixture of CH2Cl2 (150 ml) and DMF (40 ml) under N2. After stirring at room temperature for 2 days, the reaction was stopped and concentrated under reduced pressure. CH2Cl2 was added to the residue and the white urea salt was filtered. The filtrate was treated with saturated NH4Cl, extracted with CH2Cl2, dried and concentrated. The crude product was purified by flash column chromatography on silica gel (MeOH∶CH2Cl2 = 1∶20) yielding 9 (18.83 g, 84.3%) as a foam solid, which was used in the next step without further purification.

N-(2-Benzenesulfinyl-ethyl)-3-benzyloxy-4-methoxy-N-methylbenzamide (10).

Benzyl bromide (8.1 ml, 68.6 mmol) and K2CO3 (9.5 g, 68.6 mmol) were added to the crude product 9 (18.83 g, 56.5 mmol) in 100 ml DMF. The mixture was stirred at room temperature for 17 hours. The solvent was removed under reduced pressure. Water was added to the residue and then extracted with ethyl acetate for 3 times. The combined ethyl acetate extract was dried, filtered and concentrated. The crude product was purified by flash column chromatography with silica gel (ethyl acetate∶hexane, from 3∶2 to 4∶1, then ethyl acetate) yielding 10 (17.7 g, 41.8 mmol, 91% for two steps) as a pale yellow oil.

General procedure for the preparation of compounds 12 and 13.

2,4,6-Collidine (16.6 ml, 125.4 mmol) was added to a solution of the product 10 (17.7 g, 41.8 mmol) in 210 ml CH2Cl2 under N2 at 0°C, followed by adding TFAA (29.5 ml, 208.95 mmol) dropwise. After stirring for 30 min, the reaction was quenched by slow addition of 180 ml 10% K2CO3. The mixture was then warmed to room temperature. The layers were separated and the aqueous layer was extracted twice with CH2Cl2. The combined CH2Cl2 was washed twice with 10% HCl, dried, filtered, concentrated and then dissolved in 210 ml toluene. p-Toluenesulfonic acid monohydrate (39.75 g, 209.0 mmol) was then added and the mixture was refluxed for 40 min. The reaction was cooled to room temperature, and saturated NaHCO3 was added until pH was 8. The aqueous layer was separated and extracted with CH2Cl2 several times until TLC of the aqueous layer did not show the desired products. The combined organic layers were dried, filtered and concentrated. The crude products were purified by flash column chromatography on silica gel (ethyl acetate∶hexane∶ammonia = 60∶40∶1) yielding 13 and (ethyl acetate∶hexane = 4∶1) 12 as white solids.

7-Hydroxy-6-methoxy-2-methylisoquinolin-1(2H)-one (12).

The crude products were purified by flash column chromatography on silica gel (ethyl acetate∶hexane = 4∶1) yielding 12 (3.2 g, 15.6 mmol, 37% from 10) as a white solid. 1H-NMR (400 MHz, CDCl3) δ 7.78 (1H, s), 6.98 (1H, d, J = 7.4 Hz), 6.87 (1H, s), 6.46 (1H, d, J = 7.4 Hz), 3.98 (3H, s), 3.60 (3H, s).

5-Hydroxy-6-methoxy-2-methylisoquinolin-1(2H)-one (13).

The crude products were purified by flash column chromatography on silica gel (ethyl acetate∶hexane∶ammonia = 60∶40∶1) yielding 13 (2.5 g, 10 mmol, 24% from 10) as a white solid. 1H-NMR (400 MHz, CDCl3) δ 7.96 (1H, d, J = 8.8 Hz), 7.03 (1H, d, J = 8.8 Hz), 6.97 (1H, d, J = 7.6 Hz), 6.83 (1H, d, J = 7.6 Hz), 3.86 (3H, s), 3.55 (3H, s).

Common alkylation procedure for the preparation of compounds 7a–g, 14a–d and15a–d.

Desired alkyl bromide (∼1.3 equiv.) and K2CO3 (2 equiv.) was added to a solution of 1 equiv. of either 6, 12 or 13 in DMF (0.2 M), and the mixture was stirred overnight at room temperature. Water was added after the reaction was completed as shown by TLC, and the mixture was extracted with ethyl acetate, dried, concentrated and finally purified by column chromatography to give the desired products.

6-Benzyloxy-7-methoxy-2-methylisoquinolin-1(2H)-one (7a).

The crude product was purified by flash column chromatography on silica gel (ethyl acetate∶petroleum ether = 17∶3) yielding 7a (98%) as a white solid. mp: 102–104°C. ESI-MS: 296.23 (M+1). HR-TOF-MS: calculated for C18H17NO3, 296.1287 (M+1); found 296.1296 (M+1). 1H-NMR (300 MHz, CDCl3): δ 7.82 (1H, s), 7.45–7.48 (2H, m), 7.32–7.41(3H, m), 6.96 (1H, d, J = 7.4 Hz), 6.88 (1H, s), 6.34 (1H, d, J = 7.4 Hz), 5.23 (2H, s), 4.00 (3H, s), 3.58 (3H, s). 13C-NMR (75 MHz, CDCl3): δ161.7, 152.3, 149.6, 136.1, 132.3, 130.9, 128.6, 128.1, 127.2, 107.7, 105.5, 70.7, 56.1, 37.0.

7-Methoxy-6-(3-methoxy-benzyloxy)-2-methylisoquinolin-1(2H)-one (7b).

The crude product was purified by flash column chromatography on silica gel (ethyl acetate∶petroleum ether, from 3∶2 to 9∶1) yielding 7b (100%) as a pale yellow solid. mp: 85–87°C. ESI-MS: 326.33 (M+1), 348.23 (M+Na). HR-TOF-MS: calculated for C19H19NO4, 326.1392 (M+1); found 326.1404 (M+1). 1H-NMR (300 MHz, CDCl3): δ 7.80 (1H, s), 7.28 (1H, t, J = 7.8 Hz), 7.01–7.04 (2H, m), 6.92 (1H, d, J = 6.3 Hz), 6.86–6.83 (1H, d, J = 8.8 Hz), 6.31 (1H, d, J = 7.2 Hz), 5.19 (2H, s), 3.98 (3H, s), 3.78 (3H, s), 3.55 (3H, s). 13C-NMR (100 MHz, CDCl3): δ 161.7, 159.7, 152.1, 149.4, 137.6, 132.2, 130.8, 129.6, 120.1, 119.2, 113.4, 112.6, 107.7, 107.6, 105.3, 70.4, 56.0, 55.0, 36.9.

6-(3,5-Dimethoxy-benzyloxy)-7-methoxy-2-methylisoquinolin-1(2H)-one (7c).

ESI-MS: 356.27 (M+1), 378.13 (M+Na). HR-TOF-MS: calculated for C20H21NO5, 356.1498 (M+1); found 356.1514 (M+1). 1H-NMR (400 MHz, CDCl3): δ 7.82 (1H, s), 6.97 (1H, d, J = 7.2 Hz), 6.87 (1H, s), 6.61 (2H, s), 6.40 (1H, s), 6.34 (1H, d, J = 7.2 Hz), 5.19 (2H, s), 4.01 (3H, s), 3.79 (6H, s), 3.59 (3H, s). 13C-NMR (100 MHz, CDCl3): δ 160.9, 152.1, 149.5, 138.5, 132.2, 130.8, 120.3, 107.8, 107.7, 104.5, 104.9 (2), 99.8, 70.7, 56.2, 55.4 (2), 37.1.

7-Methoxy-6-(2-methoxy-benzyloxy)-2-methylisoquinolin-1(2H)-one (7d).

The crude product was purified by flash column chromatography on silica gel (ethyl acetate∶hexane = 7∶3) yielding 7d (100%) as a white solid. mp: 150–152°C. ESI-MS: 326.27 (M+1). HR-TOF-MS: calculated for C19H19NO4, 326.1392 (M+1); found 326.1398 (M+1). 1H-NMR (400 MHz, CDCl3): δ 7.82 (1H, s), 7.48 (1H, d, J = 6.4 Hz), 7.30 (1H, m), 6.91–7.00 (4H, m), 6.35 (1H, d, J = 7.1 Hz), 5.30 (2H, s), 4.01 (3H, s), 3.89 (3H, s), 3.59 (3H, s). 13C-NMR (100 MHz, CDCl3): δ 161.8, 156.5, 152.4, 149.6, 132.4, 130.8, 128.9, 128.2, 124.4, 120.7(2), 120.1, 110.2, 107.7, 105.6, 65.7, 56.2, 55.4, 37.1.

7-Methoxy-6-(4-methoxy-benzyloxy)-2-methylisoquinolin-1(2H)-one (7e).

The crude product was purified by flash column chromatography on silica gel (ethyl acetate∶hexane, from 3∶2 to 4∶1) yielding 7e (98%) as a white solid. mp: 170–172°C. ESI-MS: 326.27 (M+1). HR-TOF-MS: calculated for C19H19NO4, 326.1392 (M+1); found 326.1404 (M+1). 1H-NMR (300 MHz, CDCl3): δ 7.81 (1H, s), 7.39 (1H, d, J = 6.9 Hz), 6.89–6.96 (m, 4H), 6.34 (1H, d, J = 7.2 Hz), 5.15 (2H, s), 3.98 (3H, s), 3.80 (3H, s), 3.57 (3H, s). 13C-NMR (75 MHz, CDCl3): δ 161.8, 159.4, 152.3, 149.5, 132.3, 130.8, 129.0, 128.1, 120.1, 113.0, 107.7, 107.6, 105.4, 70.5, 56.0, 55.2, 37.0.

7-Methoxy-2-methyl-6-(4-methyl-benzyloxy)-isoquinolin-1(2H)-one (7f).

The crude product was purified by flash column chromatography on silica gel (ethyl acetate∶petroleum ether = 4∶1) yielding 7f (100%) as a white solid. mp: 120–122°C. ESI-MS: 310.27 (M+1), 332.20 (M+Na). HR-TOF-MS: calculated for C19H19NO3, 310.1443 (M+1); found 310.1448 (M+1). 1H-NMR (400 MHz, CDCl3): δ 7.81 (1H, s), 7.35 (2H, d, J = 7.6 Hz), 7.19 (2H, d, J = 7.6 Hz), 6.96 (1H, d, J = 7.2 Hz), 6.34 (1H, d, J = 7.2 Hz), 5.20 (2H, s), 3.99 (3H, s), 3.59 (3H, s), 2.35 (3H, s). 13C-NMR (100 MHz, CDCl3): δ 161.8, 152.3, 149.5, 137.8, 133.0, 132.3, 130.8, 129.3, 127.3, 120.1, 107.7(2), 105.5, 70.7, 56.2, 37.1, 21.3.

6-(3,5-Dimethyl-benzyloxy)-7-methoxy-2-methylisoquinolin-1(2H)-one (7g).

ESI-MS: 324.26 (M+1). HR-TOF-MS: calculated for C20H21NO3, 324.1600 (M+1); found 310.1448 (M+1). 1H-NMR (400 MHz, CDCl3): δ 7.82 (1H, s), 7.07 (2H, s), 6.96 (2H, d, J = 7.2 Hz), 6.90 (1H, s), 6.35 (1H, d, J = 7.6 Hz), 5.16 (2H, s), 4.01 (3H, s), 3.59 (3H, s), 2.32 (6H, s). 13CNMR (75 MHz, CDCl3): δ 161.8, 152.4, 149.5, 138.2, 135.9, 132.3, 130.8, 129.7, 125.0 (2), 120.2, 107.7, 105.5, 70.9, 56.2, 37.1, 21.3.

7-Benzyloxy-6-methoxy-2-methylisoquinolin-1(2H)-one (14a).

The crude product was purified by flash column chromatography on silica gel (ethyl acetate∶petroleum ether = 4∶1) yielding 14a (100%) as a white solid. White solid. 100% yield. mp: 132–134°C. ESI-MS: 296.26 (M+1), 318.16 (M+Na). HR-TOF-MS: calculated for C18H17NO3, 296.1287 (M+1); found 296.1285 (M+1). 1H-NMR (300 MHz, CDCl3): δ 7.87 (1H, s), 7.49 (2H, m), 7.27–7.40 (3H, m), 6.95 (1H, d, J = 7.4 Hz), 6.85 (1H, s), 6.37 (1H, d, J = 7.3 Hz), 5.25 (2H, s), 3.95 (3 H, s), 3.56 (3 H, s). 13C-NMR (75 MHz, CDCl3): δ 161.8, 153.5, 148.2, 136.3, 132.6, 131.0, 128.5, 127.9, 127.5, 119.9, 109.2, 106.0, 105.3, 70.7, 55.9, 36.9.

6-Methoxy-7-(3-methoxy-benzyloxy)-2-methylisoquinolin-1(2H)-one (14b).

The crude product was purified by flash column chromatography on silica gel (MeOH∶CH2Cl2 = 3∶200) yielding 14b (78%) as a white solid. White solid. 78% yield. ESI-MS: 326.23 (M+1). HR-TOF-MS: calculated for C19H19NO4, 326.1392 (M+1); found 326.1391 (M+1). 1H-NMR (400 MHz, CDCl3): δ 7.86 (1H, s), 7.26–7.30 (1H, s), 7.05–7.08 (2H, m), 6.94 (1H, d, J = 7.2 Hz), 6.83–6.85 (2H, m), 6.36 (1H, d, J = 7.2 Hz), 3.94 (3H, s), 3.79 (3H, s), 3.54 (3H, s). 13C-NMR (75 MHz, CDCl3): δ 161.6, 159.6, 153.4, 148.1, 137.8, 132.6, 131.0, 129.5, 119.8, 119.7, 113.5, 112.8, 109.2, 106.0, 105.3, 70.6, 56.0, 55.2, 37.1.

7-(3,5-Dimethoxy-benzyloxy)-6-methoxy-2-methylisoquinolin-1(2H)-one (14c).

The crude product was purified by flash column chromatography on silica gel yielding 14c (75%) as a white solid. ESI-MS: 356.02 (M+1). HR-TOF-MS: calculated for C20H21NO5, 356.1498 (M+1); found 356.1502 (M+1). 1H-NMR (400 MHz, CDCl3): δ 7.86 (1H, s), 6.98 (1H, d, J = 7.6 Hz), 6.88 (1H, s), 6.65 (2H, s), 6.40 (1H, s), 6.39 (1H, d, J = 7.2 Hz), 5.20 (2H, s), 3.97 (3H, s), 3.79 (6H, s), 3.57 (3H, s). 13C-NMR (75 MHz, CDCl3): δ 160.8, 153.3, 148.1, 147.9, 138.6, 132.6, 131.1, 119.9, 109.3, 106.1, 105.4, 105.2, 100.0, 70.2, 56.0, 55.4 (2), 37.1.

6-Methoxy-2-methyl-7-(4-methyl-benzyloxy)-isoquinolin-1(2H)-one (14d).

The crude product was purified by flash column chromatography on silica gel (MeOH∶CH2Cl2 = 3∶200) yielding 14d (92%) as a white solid. mp: 150–152°C. ESI-MS: 310.12 (M+1). HR-TOF-MS: calculated for C19H19NO3, 310.1443 (M+1); found 310.1437 (M+1). 1H-NMR (400 MHz, CDCl3): δ 7.87 (1H, s), 7.38 (2H, d, J = 8.0 Hz), 7.18 (2H, d, J = 8.0 Hz), 6.97 (1H, d, J = 7.2 Hz), 6.86 (1H, s), 6.39 (1 H, d, J = 7.2 Hz), 5.22 (2H, s), 3.95 (3 H, s), 3.57 (3H, s), 3.17 (3H, s). 13C-NMR (100 MHz, CDCl3): δ 161.7, 153.5, 148.2, 137.6, 133.2, 132.5, 130.9, 129.1, 127.6, 119.9, 109.2, 106.0, 105.4, 70.7, 56.0, 37.1, 21.2.

5-Benzyloxyl-6-methoxy-2-methylisoquinolin-1(2H)-one (15a).

The crude product was purified by flash column chromatography on silica gel yielding 15a (100%) as a white solid. mp: 125–127°C. ESI-MS: 296.23 (M+1), 318.13 (M+Na). HR-TOF-MS: calculated for C18H17NO3, 296.1287 (M+1); found 296.1294 (M+1). 1H-NMR (400 MHz, CDCl3): δ 8.20 (1H, d, J = 8.8 Hz), 7.35–7.47 (5H, m) 7.14 (1H, d, J = 8.8 Hz), 6.94 (1H, d, J = 7.0 Hz), 6.67 (1H, d, J = 7.0 Hz), 5.07 (2H, s), 3.97 (3H, s), 3.53 (3H, s). 13C-NMR (75 MHz, CDCl3): δ 162.2, 154.4, 140.7, 137.3, 132.7, 132.5, 128.4, 128.3, 128.1, 124.6, 120.4, 112.2, 100.4, 75.3, 56.0, 36.8.

6-Methoxy-5-(3-methoxy-benzyloxy)-2-methyl-2H-isoquinolin-1-one (15b).

The crude product was purified by flash column chromatography on silica gel (ethyl acetate∶hexane = 7∶3) yielding 15b (100%) as a white solid. mp: 124–125°C. ESI-MS: 326.11 (M+1). HR-TOF-MS: calculated for C19H19NO4, 326.1392 (M+1); found 326.1408 (M+1). 1H-NMR (300 MHz, CDCl3): δ 8.20 (1H, d, J = 8.9 Hz), 7.29 (1H, t, J = 8.1 Hz), 7.14 (1H, d, J = 8.9 Hz), 7.02–7.05 (2H, m), 6.96 (1H, d, J = 7.5 Hz), 6.86–6.89 (1H, m), 6.69 (1H, d, J = 7.6 Hz), 5.05 (2H, s), 3.98 (3H, s), 3.82 (3H, s), 3.55 (3H, s). 13C-NMR (75 MHz, CDCl3): δ 162.2, 159.6, 154.4, 140.8, 138.9, 132.7, 132.5, 129.4, 124.6, 120.5, 120.4, 113.6, 112.2, 100.4, 75.1, 56.0, 55.2, 36.8.

5-(3,5-Dimethoxy-benzyloxy)-6-methoxy-2-methylisoquinolin-1(2H)-one (15c).

The crude product was purified by flash column chromatography on silica gel (MeOH∶CH2Cl2 = 1∶99) yielding 15c (100%) mp: 85–86°C. ESI-MS: 355.98 (M+1). HR-TOF-MS: calculated for C20H21NO5, 356.1498 (M+1); found 356.1499 (M+1). 1H-NMR (300 MHz, CDCl3): δ 8.20 (1H, d, J = 9.1 Hz), 7.14 (1H, d, J = 8.8 Hz), 6.96 (1H, d, J = 7.6 Hz), 6.70 (1H, d, J = 7.0 Hz), 6.64 (2H, s), 6.43 (1H, d, J = 2.1 Hz), 5.01 (2H, s), 3.99 (3H, s), 3.80 (6H, s), 3.55 (3H, s). 13C-NMR (75 MHz, CDCl3): δ 162.0, 160.7, 154.2, 140.7, 139.6, 132.6, 132.5, 124.6, 120.5, 112.2, 105.9, 100.4, 100.0, 75.2, 56.1, 55.4, 36.9.

6-Methoxy-2-methyl-5-(4-methyl-benzyloxy)-isoquinolin-1(2H)-one (15d).

The crude product was purified by flash column chromatography on silica gel yielding 15d (95%). ESI-MS: 310.11 (M+1). HR-TOF-MS: calculated for C19H19NO3, 310.1443 (M+1); found 310.1453 (M+1). 1H-NMR (400 MHz, CDCl3): δ 89.19 (1H, d, J = 8.8 Hz), 7.34 (1H, d, J = 7.6 Hz), 7.17 (2H, d, J = 7.6 Hz), 7.13 (1H, d, J = 9.2 Hz), 6.93 (1H, d, J = 7.6 Hz), 6.67 (1H, d, J = 7.6 Hz), 2.02 (2H, s), 3.97 (3H, s), 3.53 (3H, s), 2.35 (3H, s). 13C-NMR (100 MHz, CDCl3): δ 162.0, 154.2, 140.7, 137.7, 134.2, 132.6, 132.3, 128.9, 128.3, 124.3, 120.3, 112.2, 100.5, 75.2, 56.0, 36.8, 21.3.

Biology

Stable cell lines.

Individual melatonin receptor subtype (either MT1 or MT2) was stably co-expressed with a G protein chimera 16z25 in Chinese hamster ovary (CHO) cells, hereafter denoted as CHO-hMT1 and CHO-hMT2, respectively. The generation and characterization of the two stable cell lines have been described previously [39]. Single cell-derived colonies of the stable lines with sufficiently high receptor density and robust responses in the fluorometric assay (as described below) were isolated from the pool of transfected cell. Usage of these clonal lines was limited to 15 passages to prevent degradation of signal detection.

Competitive binding assay.

Competitive binding assays were performed as described [26]. Cells were suspended in binding buffer (50 mM Tris, 2 mM MgCl2, 1 mM EGTA, pH 7.4) in the density of 1.5×105 cell/ml. 1 nM [3H]melatonin and increasing concentrations of a tested compound was included. Cells were incubated at 4°C for 60 min with intermittent agitation and the reaction was stopped by rapid filtration through GF/C filters pre-soaked in 10 mM Tris, pH 7.4 and washed with 1 ml of prechilled binding buffer. Retained radioactivity was measured with Wallac 1450 Microbeta Jet scintillation counter. Competition curves were drawn as one-site competition nonlinear regression model using GraphPad Prism 3.03 to the data which were means ± SEM of 3–4 independent experiments performed in duplicates. In every assay, melatonin was included as standard reference with highly reproducible Ki values, which were calculated using the Cheng-Prusoff equation.

Fluorescence-based assay of intracellular Ca2+ mobilization using FLIPRTETRA.

4×104 Cells were seeded and grown for overnight in Costar #3603 96-well plates before assay. The growth medium was replaced by 200 µl of 2 µl Fluo-4 AM (Invitrogen) in the assay buffer (Ca2+-containing HEPES-buffered HBSS with 2.5 mM probenecid) to label the cells for 1 h at 37°C. Tested compounds in different concentrations were diluted in assay buffer in 5× of the final desired concentrations in a V-bottomed microplate, and both the cell and compound plates were put into the fluorometric imaging plate reader FLIPRTETRA (Molecular Devices, Sunnyvale, CA). Upon the addition of tested compounds or melatonin, the fluorescent signal was monitored real-time for 3 min with an excitation wavelength of 488 nm as described previously [25]. The peak of the time course of fluorescence changes was expressed in relative fluorescence units (RFU). Dose response curves were constructed by non-linear regression using GraphPad Prism 3.

cAMP accumulation assay.

2×105 cells were seeded in each well of 12-well plates and grown for overnight. Each well was labeled with 1 ml of [3H]adenine (1 µCi/ml) in F-12K medium containing 1% FBS (vol/vol) for overnight. The labeling media were then replaced by 1 ml of 20 mM HEPES-buffered F-12K containing 1 mM isobutylmethylxanthine (IBMX) and the tested compounds at desired concentrations, and incubated at 37°C for 30 min. The reaction was stopped by aspiration and adding 1 ml prechilled 5% trichloroacetic acid (w/v) with 1 mM ATP, and stored at 4°C for 30 min. [3H]cAMP was extracted from the pool of labeled adenosine nucleotides by sequential ion-exchange chromatography as previously described [40]. The radioactivity of labeled cAMP and total labeled nucleotide fractions separated by chromatography were estimated by scintillation counting. Inhibition curves were constructed by non-linear regression using GraphPad Prism 3.

Immunodetection of ERK phosphorylation.

2×105 cells were seeded into each well of 12-well plates for overnight before serum withdrawal for 4 h to reduce the basal ERK phosphorylation. Tested compounds were diluted in serum-free culture medium at desired concentrations and treated the cells for 5 min. Reactions was stopped by aspiration and then adding 150 µl of lysis buffer with protease inhibitors (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA, 40 mM NaP2O7, 1% Triton X-100, 1 mM dithiothreitol, 200 µM Na3VO4, 100 µM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 4 µg/ml aprotinin, and 0.7 µg/ml pepstain). Cells were allowed to lyse for 30 min at 4°C with agitation, and the collected total cell lysates were cleared by centrifugation. Protein samples were resolved in SDS-PAGE and transferred to nitrocellulose membranes using iBlot system (Invitrogen). Phosphorylated ERK was detected using specific antibodies as described previously [41]. Chemiluminescence signals on the blots were detected on X-ray films.

Supporting Information

Figure S1.

Phosphorylation of ERK induced by compounds 6 and 12. Experimental details were as to the legend of Figure 5.

https://doi.org/10.1371/journal.pone.0113638.s001

(TIF)

Figure S2.

Regulation of intracellular Ca2+ mobilization in CHO cells expressing MT1 or MT2 by 7e or luzindole. Experimental details were as to the legend of Figure 2. Estimation of maximal responses and EC50 were tabulated in Table 2.

https://doi.org/10.1371/journal.pone.0113638.s002

(TIF)

Figure S3.

Comparison of ERK phosphorylation inhibition by para-substituted benzyloxyl derivatives. Experimental details were as to the legend of Figure 7.

https://doi.org/10.1371/journal.pone.0113638.s003

(TIF)

Acknowledgments

We thank Dr. David New for his contribution to analysis of FLIPR data.

Author Contributions

Conceived and designed the experiments: YH YHW. Performed the experiments: KHC. Analyzed the data: KHC MKCH YHW. Contributed reagents/materials/analysis tools: YH XH. Wrote the paper: YH KHC MKCH YHW.

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