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

Article abstract of DOI:10.1371/journal.pone.0113638

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 receptormediated 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. Copyright:

Full text of DOI:10.1371/journal.pone.0113638

RESEARCH ARTICLE
Synthesis and Functional Characterization
of Substituted Isoquinolinones as MT2-
Selective Melatoninergic Ligands
Yueqing Hu1, King H. Chan1, Xixin He1, Maurice K. C. Ho1, Yung H. Wong1,2
*
1. Division of Life Science and the Biotechnology Research Institute, Hong Kong University of Science and
Technology, Clear Water Bay, Kowloon, Hong Kong, 2. State Key Laboratory of Molecular Neuroscience, and
the Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay,
Kowloon, Hong Kong
*boyung@hkust.hk
Abstract
OPEN ACCESS
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.
Citation: Hu Y, Chan KH, He X, Ho MKC, Wong
YH (2014) Synthesis and Functional
Characterization of Substituted Isoquinolinones as
MT2-Selective Melatoninergic Ligands. PLoS
ONE 9(12): e113638. doi:10.1371/journal.pone.
0113638
Editor: Alessio Lodola, University of Parma, Italy
Received: July 3, 2014
Accepted: October 27, 2014
Published: December 5, 2014
Copyright: ß 2014 Hu et al. This is an open-
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and repro-
duction in any medium, provided the original author
and source are credited.
Data Availability: The authors confirm that all data
underlying the findings are fully available without
restriction. All relevant data are within the paper
and its Supporting Information files.
Funding: This work was supported in part by the
National Key Basic Research Program of China
(2013CB530900), Research Grant Council
Introduction
(HKUST 660108), University Grant Council of
Hong Kong (T13-607/12R), and the Hong Kong
Jockey Club Charities Trust. The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the manu-
script. All the authors confirm that this does not
alter the authors’ adherence to all the PLOS ONE
policies on sharing data and materials.
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
Competing Interests: The authors have declared
that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
1 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
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 melatoni-
nergic 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+
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
2 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
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.
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 mM) or basically no significant binding towards MT1 (Table 1). Contrarily,
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
3 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
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%.
doi:10.1371/journal.pone.0113638.g001
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, K CO , DMF, r.t., ,95%.
˚
2
3
doi:10.1371/journal.pone.0113638.g002
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
4 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
Table 1. Binding affinities of the tested compounds towards human MT1 and MT2 expressed in CHO cells.
MT1
MT2
Compound
MT
R
pIC50¡SEM
9.53¡0.14
NSBc
Ki (nM) %Respa¡SEM
pIC50¡SEM
9.37¡0.21
5.55¡0.18
7.05¡0.16
5.93¡0.54
5.65¡0.17
5.95¡0.34
5.96¡0.40
5.46¡0.27
4.83¡0.24
6.00¡0.24
4.74¡0.60
4.86¡0.15
5.92¡0.37
8.26¡0.06
6.85¡0.16
5.02¡0.38
Ki (nM) %Resp¡SEM
Selectivityb
Bn
0.296
95¡2
0.429
852
91¡6
78¡7
92¡4
73¡9
79¡5
47¡5
70¡6
86¡2
78¡8
92¡3
94¡11
73¡10
85¡8
85¡8
94¡13
74¡11
0.69
7a
Bn
-
-
-
7b
(3-MeO)Bn
(3,5-MeO)Bn
(2-MeO)Bn
(4-MeO)Bn
(4-Me)Bn
(3,5-Me)Bn
Bn
4.79¡0.14
4.24¡0.24
NSB
3700
90¡8
26.6
350
139
7c
13100
41¡9
37.6
7d
-
-
674
-
7e
NSB
-
-
334
-
7f
5.05¡0.06
NSB
2040
38¡10
333
6.13
7g
-
-
1050
4460
302
-
14a
14b
14c
14d
15a
15b
15c
15d
NSB
-
-
-
(3-MeO)Bn
(3,5-MeO)Bn
(4-Me)Bn
Bn
NSB
-
-
-
NSB
-
-
5490
4150
362
-
NSB
-
-
-
5.04¡0.42
4.88¡0.15
5.03¡0.12
5.47¡0.35
2070
2980
2130
770
84¡10
71¡13
63¡4
34¡5
5.72
1800
50.7
0.27
(3-MeO)Bn
(3,5-MeO)Bn
(4-Me)Bn
1.66
42.0
2890
a%Resp – Percentage responses of the melatonin (MT)-mediated displacement of radioligand binding.
bSelectivity was defined as the ratio Ki(MT1)/Ki(MT2).
cNSB – no specific binding for up to 30 mM of tested compounds in the binding buffer.
The pIC50 and %Resp were mean ¡ SEM of 3–5 trials done in duplicates. The corresponding Ki values were calculated using the mean pIC50 values.
doi:10.1371/journal.pone.0113638.t001
most of the tested compounds showed detectable binding at MT2. Among
compounds 7a–7g 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.
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 7a–7g. 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 (Ki51.7 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
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
5 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
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).
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 a 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
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.
doi:10.1371/journal.pone.0113638.g003
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
6 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
Table 2. Isoquinolinone-induced intracellular Ca2+ mobilization in MT1-CHO and MT2-CHO cells.
MT1
MT2
Compound
MT
pEC50 ¡SEM
EC50 (nM)
0.16
NSRb
%Respa ¡SEM
pEC50 ¡SEM
9.23¡0.07
6.79¡0.30
9.54¡0.56
7.68¡0.59
7.59¡0.62
-
EC50 (nM)
0.6
%Resp ¡SEM
100¡30
84¡20
92¡11
90¡16
85¡22
-
9.85¡0.21
100¡26
7a
-
-
163
7b
7.51¡0.17
31.1
63¡7
0.30
21.0
25.7
NSR
NSR
110
7c
6.58¡0.29
266
65¡10
7d
-
-
-
-
-
-
-
-
-
-
-
-
NSR
NSR
NSR
NSR
NSR
NSR
NSR
NSR
NSR
NSR
NSR
NSR
-
-
-
-
-
-
-
-
-
-
-
-
7e
7f
-
-
7g
6.96¡0.41
-
83¡6
-
14a
14b
14c
14d
15a
15b
15c
15d
NSR
48.9
228
7.31¡0.19
6.64¡0.40
-
96¡16
71¡17
-
NSR
215
6.67¡0.05
9.84¡0.28
8.39¡0.55
6.89¡0.48
70¡9
89¡20
85¡19
40¡17
0.14
4.08
128
a%Resp – Percentage responses of the melatonin (MT)-mediated stimulation of Ca2+ signal.
bNSR – no significant response for up to 30 mM of tested compounds in the binding buffer.
The pEC50 and %Resp were mean ¡ SEM of 3–5 trials done in duplicates. The corresponding EC50 values were calculated using the mean pEC50 values.
doi:10.1371/journal.pone.0113638.t002
melatonin induced a slightly more potent response in MT1-expressing cells as
expected. Compound 12 partially activated both MT1 and MT2 receptors at
10 mM 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 mM; 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.
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
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
7 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
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 7a–7c 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 mM, whereas the concentration of 7c to induce exclusive MT2 activation was
even lower (,10 nM).
Very different responses were observed for another two mono-methoxyben-
zyloxyl 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
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.
doi:10.1371/journal.pone.0113638.g004
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
8 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
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-methoxybenzy-
loxyl 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 mM, and all the
other tested compounds were ineffective. In contrast, most of the tested
compounds activated ERK phosphorylation in MT2-CHO cells in a concentra-
tion-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
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
9 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
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.
doi:10.1371/journal.pone.0113638.g005
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.
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
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
10 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
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-mM
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.
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
Table 3. Isoquinolinone-induced inhibition of cAMP production in MT1-CHO and MT2-CHO cells.
MT1
MT2
a
Compound
MT
pIC50 ¡SEM
IC50 (nM)
0.090
1201
NSRb
396
%Inhmax ¡SEM
pIC50 ¡SEM
9.36¡0.09
7.20¡0.12
5.61¡0.24
6.61¡0.18
7.16¡0.24
6.49¡0.11
9.09¡0.25
7.39¡0.16
IC50 (nM)
0.44
%Inhmax ¡SEM
69¡2
10.05¡0.13
59¡2
7b
5.92¡0.21
69¡8
92.9
70¡3
72¡3c
7c
-
-
2460
247
7d
6.40¡1.11
33¡13
59¡5
14b
14c
-
-
-
-
NSR
-
-
-
-
69.4
71¡6
NSR
326
58¡3
15b
15c
NSR
0.81
61¡4
NSR
40.6
64¡3
The pIC50 and %Inhmax were mean ¡ SEM of 2 trials done in duplicates. The corresponding IC50 values were calculated using the mean pIC50 values.
a%Inhmax – Maximal percentage inhibition of the forskolin-elevated cAMP level.
bNSR – no significant response for up to 10 mM of tested compounds in the assay buffer.
cExtrapolated %Inhmax from non-linear regression.
doi:10.1371/journal.pone.0113638.t003
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
11 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
12 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
Figure 6. Isoquinolinone derivative-induced inhibition of forskolin-stimulated cAMP production. CHO
cells expressing MT1 or MT2 were treated with 50 mM 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.
doi:10.1371/journal.pone.0113638.g006
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 mM 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 mM; 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 mM (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 (Ki5102 nM) and
MT2 (Ki52.9 nM) under our experimental conditions. In MT1-CHO cells,
luzindole completely eliminated basal ERK phosphorylation at 1 mM. 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.
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 out-
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
13 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
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 rM – 1 mM) of melatonin in the absence or presence of 10 mM or 1 mM of 7e or
7f. Other experimental details were as to the legend of Figure 4.
doi:10.1371/journal.pone.0113638.g007
performed 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.
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
14 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
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-
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
15 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
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
1
Varian Mercury 300 spectrometer. Chemical shifts of the H-NMR were
referenced to residual solvent (chloroform at 7.26 ppm) or TMS (0.00 ppm).
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
16 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
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 com-
pounds 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:NH3510:90:1) 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
1
(MeOH/CH2Cl251:25) yielding 4 (4.8 g, 14.4 mmol, 84%) as a white solid. H-
NMR (400 MHz, CDCl3) d 7.64 (1H, dd, J58.4, 1.2 Hz), 7.56 (1H, d, J51.2 Hz),
7.30 (5H, m), 6.93 (1H, d, J58.4 Hz), 4.49 (2H, t, J55.6 Hz), 3.92 (3H, s), 3.31
(2H, t, J55.6 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:hexane51:1) yielding
5 (6.1 g, 100%) as a brown oil, which was used in the next step without further
1
purification. H-NMR (400 MHz, CDCl3): d 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
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
17 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
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:CH2Cl251:25) yielding 6 (2.1 g, 10.2 mmol, 71%) as a white
1
solid. H-NMR (400 MHz, CDCl3): d 7.82 (1H, s), 6.99 (1H, s), 6.98 (1H, d,
J57.2 Hz), 6.38 (1H, d, J57.2 Hz), 3.99 (3H, s), 3.60 (3H, s). 13C-NMR
(100 MHz, CDCl3): d 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:CH2Cl251:20) 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 CH Cl under N at 0 C, followed by adding TFAA
˚
2
2
2
(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
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
18 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
chromatography on silica gel (ethyl acetate:hexane:ammonia560:40:1) yielding 13
and (ethyl acetate:hexane54:1) 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:hexane54:1) yielding 12 (3.2 g, 15.6 mmol, 37% from 10) as a white
1
solid. H-NMR (400 MHz, CDCl3) d 7.78 (1H, s), 6.98 (1H, d, J57.4 Hz), 6.87
(1H, s), 6.46 (1H, d, J57.4 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:ammonia560:40:1) yielding 13 (2.5 g, 10 mmol, 24% from
1
10) as a white solid. H-NMR (400 MHz, CDCl3) d 7.96 (1H, d, J58.8 Hz), 7.03
(1H, d, J58.8 Hz), 6.97 (1H, d, J57.6 Hz), 6.83 (1H, d, J57.6 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 ether517:3) yielding 7a (98%) as a white solid. mp: 102–
104 C. ESI-MS: 296.23 (M+1). HR-TOF-MS: calculated for C H NO , 296.1287
˚
18 17
3
1
(M+1); found 296.1296 (M+1). H-NMR (300 MHz, CDCl3): d 7.82 (1H, s),
7.45–7.48 (2H, m), 7.32–7.41(3H, m), 6.96 (1H, d, J57.4 Hz), 6.88 (1H, s), 6.34
(1H, d, J57.4 Hz), 5.23 (2H, s), 4.00 (3H, s), 3.58 (3H, s). 13C-NMR (75 MHz,
CDCl3): d161.7, 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:
˚
1
calculated for C19H19NO4, 326.1392 (M+1); found 326.1404 (M+1). H-NMR
(300 MHz, CDCl3): d 7.80 (1H, s), 7.28 (1H, t, J57.8 Hz), 7.01–7.04 (2H, m),
6.92 (1H, d, J56.3 Hz), 6.86–6.83 (1H, d, J58.8 Hz), 6.31 (1H, d, J57.2 Hz),
5.19 (2H, s), 3.98 (3H, s), 3.78 (3H, s), 3.55 (3H, s). 13C-NMR (100 MHz,
CDCl3): d 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.
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
19 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
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,
1
356.1498 (M+1); found 356.1514 (M+1). H-NMR (400 MHz, CDCl3): d 7.82
(1H, s), 6.97 (1H, d, J57.2 Hz), 6.87 (1H, s), 6.61 (2H, s), 6.40 (1H, s), 6.34 (1H,
d, J57.2 Hz), 5.19 (2H, s), 4.01 (3H, s), 3.79 (6H, s), 3.59 (3H, s). 13C-NMR
(100 MHz, CDCl3): d 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:hexane57:3) 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): d 7.82 (1H, s), 7.48 (1H, d,
J56.4 Hz), 7.30 (1H, m), 6.91–7.00 (4H, m), 6.35 (1H, d, J57.1 Hz), 5.30 (2H, s),
4.01 (3H, s), 3.89 (3H, s), 3.59 (3H, s). 13C-NMR (100 MHz, CDCl3): d 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 C H NO ,
˚
19 19
4
1
326.1392 (M+1); found 326.1404 (M+1). H-NMR (300 MHz, CDCl3): d 7.81
(1H, s), 7.39 (1H, d, J56.9 Hz), 6.89–6.96 (m, 4H), 6.34 (1H, d, J57.2 Hz), 5.15
(2H, s), 3.98 (3H, s), 3.80 (3H, s), 3.57 (3H, s). 13C-NMR (75 MHz, CDCl3): d
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 ether54:1) 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
˚
1
C19H19NO3, 310.1443 (M+1); found 310.1448 (M+1). H-NMR (400 MHz,
CDCl3): d 7.81 (1H, s), 7.35 (2H, d, J57.6 Hz), 7.19 (2H, d, J57.6 Hz), 6.96 (1H,
d, J57.2 Hz), 6.34 (1H, d, J57.2 Hz), 5.20 (2H, s), 3.99 (3H, s), 3.59 (3H, s), 2.35
(3H, s). 13C-NMR (100 MHz, CDCl3): d 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): d 7.82 (1H, s), 7.07 (2H, s),
6.96 (2H, d, J57.2 Hz), 6.90 (1H, s), 6.35 (1H, d, J57.6 Hz), 5.16 (2H, s), 4.01
(3H, s), 3.59 (3H, s), 2.32 (6H, s). 13CNMR (75 MHz, CDCl3): d 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.
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
20 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
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 ether54:1) 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): d 7.87 (1H, s), 7.49 (2H, m), 7.27–7.40 (3H, m),
6.95 (1H, d, J57.4 Hz), 6.85 (1H, s), 6.37 (1H, d, J57.3 Hz), 5.25 (2H, s), 3.95 (3
H, s), 3.56 (3 H, s). 13C-NMR (75 MHz, CDCl3): d 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:CH2Cl253:200) 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
1
(M+1); found 326.1391 (M+1). H-NMR (400 MHz, CDCl3): d 7.86 (1H, s),
7.26–7.30 (1H, s), 7.05–7.08 (2H, m), 6.94 (1H, d, J57.2 Hz), 6.83–6.85 (2H, m),
6.36 (1H, d, J57.2 Hz), 3.94 (3H, s), 3.79 (3H, s), 3.54 (3H, s). 13C-NMR
(75 MHz, CDCl3): d 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:
1
calculated for C20H21NO5, 356.1498 (M+1); found 356.1502 (M+1). H-NMR
(400 MHz, CDCl3): d 7.86 (1H, s), 6.98 (1H, d, J57.6 Hz), 6.88 (1H, s), 6.65 (2H,
s), 6.40 (1H, s), 6.39 (1H, d, J57.2 Hz), 5.20 (2H, s), 3.97 (3H, s), 3.79 (6H, s),
3.57 (3H, s). 13C-NMR (75 MHz, CDCl3): d 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:CH Cl 53:200) yielding 14d (92%) as a white solid. mp: 150–152 C. ESI-
˚
2
2
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): d 7.87 (1H, s), 7.38 (2H, d,
J58.0 Hz), 7.18 (2H, d, J58.0 Hz), 6.97 (1H, d, J57.2 Hz), 6.86 (1H, s), 6.39 (1
H, d, J57.2 Hz), 5.22 (2H, s), 3.95 (3 H, s), 3.57 (3H, s), 3.17 (3H, s). 13C-NMR
(100 MHz, CDCl3): d 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
1
296.1294 (M+1). H-NMR (400 MHz, CDCl3): d 8.20 (1H, d, J58.8 Hz), 7.35–
7.47 (5H, m) 7.14 (1H, d, J58.8 Hz), 6.94 (1H, d, J57.0 Hz), 6.67 (1H, d,
J57.0 Hz), 5.07 (2H, s), 3.97 (3H, s), 3.53 (3H, s). 13C-NMR (75 MHz, CDCl3): d
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
21 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
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:hexane57:3) 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);
1
found 326.1408 (M+1). H-NMR (300 MHz, CDCl3): d 8.20 (1H, d, J58.9 Hz),
7.29 (1H, t, J58.1 Hz), 7.14 (1H, d, J58.9 Hz), 7.02–7.05 (2H, m), 6.96 (1H, d,
J57.5 Hz), 6.86–6.89 (1H, m), 6.69 (1H, d, J57.6 Hz), 5.05 (2H, s), 3.98 (3H, s),
3.82 (3H, s), 3.55 (3H, s). 13C-NMR (75 MHz, CDCl3): d 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:CH Cl 51:99) yielding 15c (100%) mp: 85–86 C. ESI-MS: 355.98 (M+1).
˚
2
2
HR-TOF-MS: calculated for C20H21NO5, 356.1498 (M+1); found 356.1499
1
(M+1). H-NMR (300 MHz, CDCl3): d 8.20 (1H, d, J59.1 Hz), 7.14 (1H, d,
J58.8 Hz), 6.96 (1H, d, J57.6 Hz), 6.70 (1H, d, J57.0 Hz), 6.64 (2H, s), 6.43
(1H, d, J52.1 Hz), 5.01 (2H, s), 3.99 (3H, s), 3.80 (6H, s), 3.55 (3H, s). 13C-NMR
(75 MHz, CDCl3): d 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
1
C19H19NO3, 310.1443 (M+1); found 310.1453 (M+1). H-NMR (400 MHz,
CDCl3): d 89.19 (1H, d, J58.8 Hz), 7.34 (1H, d, J57.6 Hz), 7.17 (2H, d,
J57.6 Hz), 7.13 (1H, d, J59.2 Hz), 6.93 (1H, d, J57.6 Hz), 6.67 (1H, d,
J57.6 Hz), 2.02 (2H, s), 3.97 (3H, s), 3.53 (3H, s), 2.35 (3H, s). 13C-NMR
(100 MHz, CDCl3): d 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.
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
22 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
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 1.56105 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
46104 Cells were seeded and grown for overnight in Costar #3603 96-well plates
before assay. The growth medium was replaced by 200 ml of 2 ml 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 56 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
26105 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 mCi/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 isobutylmethyl-
xanthine (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.
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
23 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
Immunodetection of ERK phosphorylation
26105 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 ml 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 mM Na3VO4, 100 mM phenylmethylsulfonyl fluoride, 2 mg/ml
leupeptin, 4 mg/ml aprotinin, and 0.7 mg/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.
doi: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.
doi: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.
doi: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.
References
1. Dubocovich ML (2007) Melatonin receptors: role on sleep and circadian rhythm regulation. Sleep Med 8
Suppl 3: 34–42.
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
24 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
2. Slominski RM, Reiter RJ, Schlabritz-Loutsevitch N, Ostrom RS, Slominski AT (2012) Melatonin
membrane receptors in peripheral tissues: Distribution and functions. Mol Cell Endocrinol.
3. Arendt J (2006) Melatonin and human rhythms. Chronobiol Int 23: 21–37.
4. Ebisawa T, Karne S, Lerner M, Reppert S (1994) Expression cloning of a high-affinity melatonin
receptor from Xenopus dermal melanophores. Proc Natl Acad Sci U S A 91: 6133–6137.
5. Reppert S, Weaver D, Ebisawa T (1994) Cloning and characterization of a mammalian melatonin
receptor that mediates reproductive and circadian responses. Neuron 13: 1177–1185.
6. Reppert S, Godson C, Mahle C, Weaver D, Slaugenhaupt S, et al. (1995) Molecular characterization
of a second melatonin receptor expressed in human retina and brain: the Mel1b melatonin receptor. Proc
Natl Acad Sci U S A 92: 8734–8738.
7. von Gall C, Stehle JH, Weaver DR (2002) Mammalian melatonin receptors: molecular biology and
signal transduction. Cell Tissue Res 309: 151–162.
8. Liu C, Weaver D, Jin X, Shearman L, Pieschl R, et al. (1997) Molecular dissection of two distinct
actions of melatonin on the suprachiasmatic circadian clock. Neuron 19: 91–102.
9. Jin X, von Gall C, Pieschl R, Gribkoff V, Stehle J, et al. (2003) Targeted disruption of the mouse
Mel(1b) melatonin receptor. Mol Cell Biol 23: 1054–1060.
10. Ram P, Dai J, Yuan L, Dong C, Kiefer T, et al. (2002) Involvement of the mt1 melatonin receptor in
human breast cancer. Cancer Lett 179: 141–150.
11. Frungieri M, Mayerhofer A, Zitta K, Pignataro O, Calandra R, et al. (2005) Direct effect of melatonin
on Syrian hamster testes: melatonin subtype 1a receptors, inhibition of androgen production, and
interaction with the local corticotropin-releasing hormone system. Endocrinology 146: 1541–1552.
12. Peschke E, Mu¨hlbauer E, Musshoff U, Csernus VJ, Chankiewitz E, et al. (2002) Receptor (MT(1))
mediated influence of melatonin on cAMP concentration and insulin secretion of rat insulinoma cells INS-
1. J Pineal Res 33: 63–71.
13. Drazen D, Nelson R (2001) Melatonin receptor subtype MT2 (Mel 1b) and not mt1 (Mel 1a) is associated
with melatonin-induced enhancement of cell-mediated and humoral immunity. Neuroendocrinology 74:
178–184.
14. Dubocovich M, Masana M, Iacob S, Sauri D (1997) Melatonin receptor antagonists that differentiate
between the human Mel1a and Mel1b recombinant subtypes are used to assess the pharmacological
profile of the rabbit retina ML1 presynaptic heteroreceptor. Naunyn Schmiedebergs Arch Pharmacol 355:
365–375.
15. Doolen S, Krause DN, Dubocovich ML, Duckles SP (1998) Melatonin mediates two distinct responses
in vascular smooth muscle. Eur J Pharmacol 345: 67–69.
16. Waldhauser F, Waldhauser M, Lieberman HR, Deng MH, Lynch HJ, et al. (1984) Bioavailability of oral
melatonin in humans. Neuroendocrinology 39: 307–313.
17. Tetsuo M, Markey SP, Kopin IJ (1980) Measurement of 6-hydroxymelatonin in human urine and its
diurnal variations. Life Sci 27: 105–109.
18. Nonno R, Pannacci M, Lucini V, Angeloni D, Fraschini F, et al. (1999) Ligand efficacy and potency at
recombinant human MT2 melatonin receptors: evidence for agonist activity of some mt1-antagonists.
Br J Pharmacol 127: 1288–1294.
19. Faust R, Garratt PJ, Jones R, Yeh LK, Tsotinis A, et al. (2000) Mapping the melatonin receptor. 6.
Melatonin agonists and antagonists derived from 6H-isoindolo[2,1-a]indoles, 5,6-dihydroindolo[2,1-
a]isoquinolines, and 6,7-dihydro-5H-benzo[c]azepino[2,1-a]indoles. J Med Chem 43: 1050–1061.
20. Lee AWM, Chan WH, Chan ETT (1992) A facile total synthesis of isoquinolone alkaloids. Journal of the
Chemical Society, Perkin Transactions 1: 309–310.
¨
21. Pummerer R (1909) Uber Phenyl-sulfoxyessigsa¨ure.: Ber. Dtsch. Chem. Ges. 42. 2282–2291.
¨
22. Pummerer R (1910) Uber Phenylsulfoxy-essigsa¨ure. (II.). Ber. Dtsch. Chem. Ges. 43. 1401–1412.
23. Zhu J, Hu Y, Ho MK, Wong YH (2012) Pharmacokinetics, oral bioavailability and metabolism of a novel
isoquinolinone-based melatonin receptor agonist in rats. Xenobiotica 42: 1138–1150.
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
25 / 26

Synthesis and Characterization of MT2-Selective Isoquinolinones
24. Mody S, Ho M, Joshi S, Wong Y (2000) Incorporation of Galpha(z)-specific sequence at the carboxyl
terminus increases the promiscuity of galpha(16) toward G(i)-coupled receptors. Mol Pharmacol 57: 13–
23.
25. Liu A, Ho M, Wong C, Chan J, Pau A, et al. (2003) Galpha(16/z) chimeras efficiently link a wide range
of G protein-coupled receptors to calcium mobilization. J Biomol Screen 8: 39–49.
26. Hu Y, Ho M, Chan K, New D, Wong Y (2010) Synthesis of substituted N-[3-(3-methoxyphenyl)propyl]
amides as highly potent MT(2)-selective melatonin ligands. Bioorg Med Chem Lett 20: 2582–2585.
27. Zlotos DP (2012) Recent progress in the development of agonists and antagonists for melatonin
receptors. Curr Med Chem 19: 3532–3549.
28. Rivara S, Mor M, Bedini A, Spadoni G, Tarzia G (2008) Melatonin receptor agonists: SAR and
applications to the treatment of sleep-wake disorders. Curr Top Med Chem 8: 954–968.
29. Farce A, Chugunov AO, Loge´ C, Sabaouni A, Yous S, et al. (2008) Homology modeling of MT1 and
MT2 receptors. Eur J Med Chem 43: 1926–1944.
30. Karageorge GN, Bertenshaw S, Iben L, Xu C, Sarbin N, et al. (2004) Tetrahydroisoquinoline
derivatives as melatonin MT2 receptor antagonists. Bioorganic and Medicinal Chemistry Letters 14:
5881–5884.
31. Gerdin M, Mseeh F, Dubocovich M (2003) Mutagenesis studies of the human MT2 melatonin receptor.
Biochem Pharmacol 66: 315–320.
32. Conway S, Mowat E, Drew J, Barrett P, Delagrange P, et al. (2001) Serine residues 110 and 114 are
required for agonist binding but not antagonist binding to the melatonin MT(1) receptor. Biochem Biophys
Res Commun 282: 1229–1236.
33. Spadoni G, Stankov B, Duranti A, Biella G, Lucini V, et al. (1993) 2-Substituted 5-methoxy-N-
acyltryptamines: synthesis, binding affinity for the melatonin receptor, and evaluation of the biological
activity. J Med Chem 36: 4069–4074.
34. Mor M, Spadoni G, Di Giacomo B, Diamantini G, Bedini A, et al. (2001) Synthesis, pharmacological
characterization and QSAR studies on 2-substituted indole melatonin receptor ligands. Bioorg Med
Chem 9: 1045–1057.
35. Rivara S, Lorenzi S, Mor M, Plazzi PV, Spadoni G, et al. (2005) Analysis of structure-activity
relationships for MT2 selective antagonists by melatonin MT1 and MT2 receptor models. J Med Chem
48: 4049–4060.
36. Hogan MV, El-Sherif Y, Wieraszko A (2001) The modulation of neuronal activity by melatonin: in vitro
studies on mouse hippocampal slices. J Pineal Res 30: 87–96.
37. Podda MV, Deriu F, Giaconi E, Milia M, Tolu E (2003) Melatonin inhibits rat medial vestibular nucleus
neuron activity in vitro. Neurosci Lett 341: 209–212.
38. Wallez V, Durieux-Poissonnier S, Chavatte P, Boutin J, Audinot V, et al. (2002) Synthesis and
structure-affinity-activity relationships of novel benzofuran derivatives as MT(2) melatonin receptor
selective ligands. J Med Chem 45: 2788–2800.
39. New D, Wong Y (2004) Characterization of CHO cells stably expressing a G alpha 16/z chimera for high
throughput screening of GPCRs. Assay Drug Dev Technol 2: 269–280.
40. Wong Y (1994) Gi assays in transfected cells. Methods Enzymol 238: 81–94.
41. Liu A, Wong Y (2004) G16-mediated activation of nuclear factor kappaB by the adenosine A1 receptor
involves c-Src, protein kinase C, and ERK signaling. J Biol Chem 279: 53196–53204.
PLOS ONE | DOI:10.1371/journal.pone.0113638 December 5, 2014
26 / 26