Synthesis of substituted N-[3-(3-methoxyphenyl)propyl] amides as highly potent MT2-selective melatonin ligands

Article abstract of DOI:10.1016/j.bmcl.2010.02.084

A series of substituted N-[3-(3-methoxyphenyl)propyl] amides were synthesized and their binding affinities towards human melatonin MT₁and MT₂receptors were evaluated. It was discovered that a benzyloxyl substituent incorporated at C6 position of the 3-methoxyphenyl ring dramatically enhanced the MT₂binding affinity and at the same time decreased MT₁binding affinity.

Full text of DOI:10.1016/j.bmcl.2010.02.084

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2582–2585
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of substituted N-[3-(3-methoxyphenyl)propyl] amides as highly
potent MT2-selective melatonin ligands
*
Yueqing Hu, Maurice K. C. Ho, King H. Chan, David C. New, Yung H. Wong
Department of Biochemistry, the Biotechnology Research Institute, and the Molecular Neuroscience Centre, Hong Kong University of Science and Technology,
Clear Water Bay, Kowloon, Hong Kong
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of substituted N-[3-(3-methoxyphenyl)propyl] amides were synthesized and their binding affin-
ities towards human melatonin MT1 and MT2 receptors were evaluated. It was discovered that a benzyl-
oxyl substituent incorporated at C6 position of the 3-methoxyphenyl ring dramatically enhanced the MT2
binding affinity and at the same time decreased MT1 binding affinity.
Received 30 October 2009
Revised 27 January 2010
Accepted 22 February 2010
Available online 25 February 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Melatonin
Ligand
SAR
Melatonin (N-acetyl-5-methoxytryptamine) is a vertebrate neu-
rohormone secreted by the pineal gland during darkness.1 It regu-
lates the circadian rhythm and can be used to treat diseases
associated with the desynchronization of biological rhythms, such
as jet-lag, disturbed sleep-wake cycles and seasonal disorders.2
Melatonin is also involved in a number of other physiological ef-
fects and has a variety of therapeutic potentials for the treatment
of depression, cancer and neurodegenerative pathologies.3a–c
Melatonin exerts its physiological effects through the activation
nists.11a–c It is hoped that the new analogues would not only be
more metabolically stable, but also be more subtype selective. High
affinity and subtype-selective analogues represent potential candi-
dates for drug development as well as valuable experimental tools
for the delineation of melatonin receptor pharmacology.12a,b
Our research goals were to design and synthesize novel com-
pounds that exhibit potent binding affinity and good subtype
selectivity at MT1 and/or MT2 receptors. A simplified form of mel-
atonin, N-[3-(3-methoxyphenyl)propyl] propionamide was chosen
as our starting compound. Despite its simple structure, it has good
binding affinity (5.6 nM) towards melatonin receptors.13 Surpris-
ingly, there are only a few studies on substituted phenylalkyl
amides analogues.14a–c Here, we report a series of benzyloxyl
substituted phenylpropyl amides which showed high binding
affinities toward MT2 receptors.
of specific receptors, including two Gi-coupled receptor subtypes
4a,b
MT1 and MT2
that are widely expressed in different tissues.5
Both receptors are expressed in the suprachiasmatic nucleus
(SCN) which is the master control center of circadian rhythm. Stud-
ies in mice suggest that the firing of SCN neurons is suppressed by
MT1 thereby implicating MT1 in sleep promotion.6a–c However,
opposing results in meta-analyses of human studies have been re-
ported for the efficacy of melatonin to alleviate sleep distur-
bance.7a,b A more recent study showed that MT2 may promote
sleep in rats.8 While it has been shown that the phase advance-
ment of rat SCN neuron firing is mediated by MT2, discrepancies
have been observed between different strains of mice.9a,b Sub-
type-selective ligands might help to clarify these inconsistencies.
Although melatonin is involved in a number of biological and
physiological processes, its use in clinical applications is quite lim-
ited because of its short half-life (15–30 min)10 and lack of subtype
selectivity. Considerable interests have been devoted in the design
and synthesis of novel melatonin receptor agonists and antago-
O
O
HN
HN
R2
R1
O
O
O
N
H
O
2
Melatonin
O
O
HN
HN
R1
O
O
R2
3
1
N-[3-(3-methoxyphenyl)propyl]
Designed Compounds
acetamide
* Corresponding author. Tel.: +852 2358 7328; fax: +852 2358 1552.
E-mail address: boyung@ust.hk (Y.H. Wong).
Figure 1. Structures of melatonin, N-[3-(3-methoxyphenyl)propyl] acetamide and
the designed compounds.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.084

Y. Hu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2582–2585
2583
Table 1
In terms of structure, N-[3-(3-methoxyphenyl)propyl] acetam-
ide 1 is a truncated form of melatonin without the nitrogen-con-
taining five member ring (Fig. 1). It has all the key components
(aromatic ring, methoxy group, amido trimethylene chain), which
are essential for potent binding to melatonin receptors. We
decided to use N-[3-(3-methoxyphenyl)propyl] acetamide as a
template for further optimization in order to discover novel, potent
and subtype selective melatonin receptor ligands. A variety of dif-
ferent substituents were incorporated at the C6 (compound 2) or
C4 (compound 3) positions of the 3-methoxyphenyl ring in order
to modulate the binding affinity and selectivity. It was expected
that an aromatic ring attached to the 3-methoxylphenyl ring
through a linker might provide selectivity towards MT2 subtype,
as an extra phenyl group attached to the adjacent position of the
amido ethyl chain was a common structure motif in a number of
MT2-selective ligands discovered so far.12a
Binding affinity of the compounds 1a–1b, 2a–2d towards human MT1 and MT2
receptors expressed in CHO cells
O
O
O
O
R1
N
H
R1
N
H
O
1a-b
2a-d
R1
Ki (nM)
MT1/MT2
MT1
MT2
MT
1a
0.296
23.3
85.4
879
263
1172
—
0.429
0.751
7.07
0.04705
0.00055
0.00103
2300
0.69
31
12
Me
Et
Me
Et
1b
2aa
2ba
2ca
2d
1.87 Â 104
4.73 Â 105
1.14 Â 106
—
The synthetic pathway towards the designed compounds of
interest is shown in Scheme 1. The process began with commer-
cially available 2-hydroxy-5-methoxybenzaldehyde or vanillin as
starting materials. Alkylation of the hydroxyl group with benzyl
bromide, followed by Horner–Emmons olefination with diethylcy-
n-Pr
Ph
a
Spectral data for these compounds are given as Supplementary data.
anomethylphosphate of the aldehydes afforded the
a,b-unsatu-
Table 2
rated nitriles as a mixture of cis- and trans-isomers in excellent
yield. Reaction of the unsaturated nitriles with lithium aluminum
hydride in refluxing ether reduced both the double bond and nitrile
triple bond in one step to give the amines with modest yield (65%),
which were converted to the desired amides by treatment with dif-
ferent acyl chlorides. To modify the C6 or C4 positions of the 3-
methoxyphenyl ring, the benzyl group was removed under cata-
lytic hydrogenation. The resulting free phenols were then alkylated
with different halides to afford the desired products.15
Binding affinity of the compounds 2a, 2e–2q
O
O
O
O
O
N
H
O
N
H
O
R2
2e-p
2q
R2
Ki (nM)
MT1/MT2
Competitive binding characteristics of the compounds towards
human MT1 and MT2 melatonin receptor subtypes stably ex-
pressed in Chinese hamster ovary (CHO) cells were determined
by whole cell binding assays using 1 nM [3H]melatonin as the
probe.16 The Kd of melatonin for MT1 and MT2 receptors was
0.296 nM and 0.429 nM, respectively, as determined by saturation
binding assays (Supplementary Fig. 1). The Ki values of the com-
pounds for MT1 and MT2 as well as their MT1/MT2 selectivity ratio
are reported in Tables 1–3 and representative displacement curves
are shown in Figure 2.
MT1
MT2
2a
2e
2f
2g
2i
2j
2k
2l
Bn
H
Me
Et
879
379
1280
183
400
2930
—
2700
441
—
0.047
46.2
26.1
1.59
2.12
13.4
72.2
27.9
0.0238
102
1.87 Â 104
8.22
49.1
116
189
n-Pr
Ph–(CH2)2–
Ph–(CH2)3–
4-MeO–Bn
3-MeO–Bn
4-Br-2-F–Bn
3,5-Di-MeO-Bn
2-Py–CH2–
3-MeO–Bn
219
—
97
2ma
2n
2o
2p
2qa
1.86 Â 104
—
—
—
1840
705
0.0326
1.61
0.00069
Carboxamides with different R1 groups were evaluated first.
1150
1.03 Â 106
Table
1 shows the melatonin receptor binding affinities for
N-[3-(3-methoxyphenyl)propyl] amides with a benzyloxyl group
substituted at the phenyl ring C6 position. For comparison, the
binding data for melatonin and two analogues (1a and 1b) that
are unsubstituted at C6 position were also determined. Melatonin
showed potent affinity towards both MT1 and MT2 receptors with
little selectivity. As compared to melatonin, N-[3-(3-methoxy-
a
Spectral data for these compounds are given as Supplementary data.
phenyl)propyl] acetamide 1a and propionamide 1b showed lower
affinities toward both receptors with minor changes in selectivity.
It was evident that an additional benzyloxy group at C6 position of
the 3-methoxyphenyl ring dramatically enhanced the MT2 affinity
to sub-nM and sub-pM range while it decreased MT1 affinity to
over 200 nM. The different amides (with a different R1 group) also
modulated the MT2 affinity. For compounds 2a, 2b, 2c, and 2d, the
MT2 binding affinity increased according to the following order of
R1 group: Ph << Me < n-Pr ꢀ Et, with R1 equals ethyl group as the
best. The Ki of compounds 2a–c towards MT2 were all in the low
or sub-pM range, while their Ki towards MT1 were over 200 nM
(Fig. 2).
NH2
3
1
O
O
CN
c
a,b
O
O
OH
OBn
4-OH-
or
OBn
6-OH-
d
O
O
O
HN
R1
HN
R1
HN
R1
Next, the effects of a variety of R2 groups were evaluated, while
R1 was kept as methyl group (Table 2). Compared to compound 2a
(Ki for MT2 = 47 pM), compound 2e without the benzyl group
showed only modest binding affinity (Ki = 46.2 nM) towards MT2.
Considering compound 1a without the extra hydroxyl group was
reasonably active towards MT1 (Ki = 23.3 nM) and MT2 (Ki =
0.751 nM) binding, the hydroxyl group at C6 position of the
e
f
O
O
O
OR2
OH
OBn
Scheme 1. Reagent and conditions: (a) BnBr, NaH, THF, rt, 100%; (b) NaH,
(EtO)2PO(CH2CN), THF, 95%; (c) LiAlH4, Et2O, reflux, ꢀ65%; (d) R1COCl, Et3N, CH2Cl2,
rt, 86–95%); (e) Pd/C, H2 balloon, rt, 96%; (f) R2Br or R2Cl, K2CO3, DMF, rt, 100%.

2584
Y. Hu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2582–2585
After the optimal groups for R1 and R2 groups were identified,
Table 3
compound 2q incorporating the optimal R1 at the terminal end of
the amide chain and R2 groups at the C6 position of the 3-
methoxyphenyl core (R1 = Et, R2 = 3-methoxybenzyl) was synthe-
sized. As predicted, 2q was extremely potent toward MT2 receptor
with sub-pM binding affinity, while its binding affinity toward MT1
receptor was 705 nM.
Melatonin receptor binding of compounds 3a–3d
O
O
N
H
R1
O
R2
3a-3d
Finally, a couple of alkyloxyl substitutions at C4 position were
also evaluated. As shown in Table 3, it was obvious that the benzyl-
oxyl group at C4 position was not as good as at C6 position for
enhancing the binding affinities towards both MT1 and MT2
receptors.
R1
R2
Ki (nM)
MT1/MT2
MT1
MT2
69.3
121
12.6
105
3a
3b
3c
3d
Me
Et
Me
Me
Bn
Bn
H
8980
2010
257
130
16.7
20.5
13.9
In summary, we have identified a novel series of C6-benzyloxyl
substituted N-[3-(3-methoxyphenyl)propyl] amides which showed
potent binding affinities toward MT2 receptor with high selectivity.
In particular, several compounds (2b, 2c, 2q) exhibited pM to sub-
pM affinity towards MT2 and over 200 nM affinity towards MT1.
Preliminary results from FLIPR assays suggest that these com-
pounds are potent MT2-selective agonists as they stimulated intra-
cellular Ca2+ release in CHO cells expressing the MT2 receptor. The
experimentally determined log P values of the test compounds are
generally in the range of 2.66–3.34, which is comparable to mela-
tonin (log P = 2.16) and that they are compatible with the Lipinski’s
Rule of Five. These easily synthesized compounds represent useful
pharmacological tools to further investigate the biological func-
tions of the MT2 receptor. It remains to be determined if these
compounds have better pharmacokinetic properties than melato-
nin to warrant their evaluation as drug candidates.
(3-MeO)Bn
1460
Acknowledgements
This study was supported in part by grants from the Hong Kong
Jockey Club, the Innovation and Technology Fund (ITS/113/03),
RGC (660108) and UGC (AoE/B-15/01) of Hong Kong.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.02.084.
Figure 2. Competitive binding of selected compounds towards MT1- or MT2-
expressing CHO cells. Data are mean SEM of four individual experiments done in
duplicates. Correlation coefficients of the fitted dose response curve are in the range
of 0.70–0.99 for MT1, and 0.80–0.94 for MT2.
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with modifications for intact cells. Briefly, 1.5 Â 105 cells were suspended in
binding buffer (50 mM Tris, 2 mM MgCl2, 1 mM EGTA, pH7.4) containing 1 nM
[3H]melatonin and increasing concentrations of a test compound. Assays were
carried out at 4 °C for 60 min with occasional agitation and then terminated by
rapid filtration through GF/C filters pre-soaked in 10 mM Tris, pH 7.4. Bound
radioactivity was counted in Wallac 1450 Microbeta Jet scintillation counter.
15. All of the compounds submitted for bioassays were characterized by 1H NMR,
13C NMR, ESI-MS, etc.
16. CHO cells stably expressing human MT1 or MT2 receptor have been described
and characterized previously (New, D. C.; Wong, Y. H. Assay Drug Dev. Technol.
2004, 2, 269–280). Dissolution of test compounds and dilution methods were
as described in the Supplementary data. Competitive binding assays were
performed as described (Ho, M. K.; New, D. C.; Wong, Y. H. Neurosignals 2002,
11, 115–122.; Tian, Y.; New, D. C.; Yung, L. Y.; Allen, R. A.; Slocombe, P. M.;
Competitive curves were fitted using a one-site competition nonlinear
regression (GraphPad Prism 3.03). Data were means of 2–3 independent
experiments performed in duplicates. Standard errors were typically within
10% of the mean value. Melatonin was employed as standard reference in every
assay with reproducible Ki. Ki values were calculated using the Cheng–Prusoff
equation.