1,6-dihydro-2H-indeno[5,4-b]furan derivatives: Design, synthesis, and pharmacological characterization of a novel class of highly potent MT 2-selective agonists

Article abstract of DOI:10.1021/jm200221q

Figure Presented. A novel series of 1,6-dihydro-2H-indeno[5,4-b]furan derivatives were designed and synthesized as MT₂-selective ligands. This scaffold was identified as a potent mimic of the 5-methoxy indole core of melatonin, and introduction of a cyclohexylmethyl group at the 7-position of this scaffold afforded an MT₂-selective ligand 15 (K_i = 0.012 nM) with high MT₁/MT₂ selectivity (799). Compound 15 was identified as a potent full agonist for the MT₂ subtype and exhibited reentrainment effects to a new light/dark cycle in ICR mice at 3-30 mg/kg. This result demonstrated the involvement of the MT₂ receptors in chronobiotic activity.

Full text of DOI:10.1021/jm200221q

ARTICLE
pubs.acs.org/jmc
1,6-Dihydro-2H-indeno[5,4-b]furan Derivatives: Design, Synthesis,
and Pharmacological Characterization of a Novel Class of Highly
Potent MT2-Selective Agonists
Tatsuki Koike,* Yasutaka Hoashi, Takafumi Takai, Masaharu Nakayama, Nobuhito Yukuhiro,
Takashi Ishikawa, Keisuke Hirai, and Osamu Uchikawa
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 17-85 Jusohonmachi, 2-Chome, Yodogawa-ku,
Osaka 532-8686, Japan
S Supporting Information
b
ABSTRACT: A novel series of 1,6-dihydro-2H-indeno[5,4-b]furan derivatives were designed and
synthesized as MT2-selective ligands. This scaffold was identified as a potent mimic of the 5-methoxy
indole core of melatonin, and introduction of a cyclohexylmethyl group at the 7-position of this scaffold
afforded an MT2-selective ligand 15 (Ki = 0.012 nM) with high MT1/MT2 selectivity (799). Compound
15 was identified as a potent full agonist for the MT2 subtype and exhibited reentrainment effects to a new
light/dark cycle in ICR mice at 3ꢀ30 mg/kg. This result demonstrated the involvement of the MT2
receptors in chronobiotic activity.
’ INTRODUCTION
which melatonin causes phase-shifting effects, also support the
involvement of this receptor on chronobiotic activity. However,
most of these MT2-selective ligands behave as antagonists, and
only a few MT2-selective agonists23 have been reported to date.
To clarify the distinct functions of MT2 subtype, MT2 full agonist
with high potency and sufficient selectivity is still required.
In our previous studies aimed at identifying MT1/MT2
agonists, we reported potent 1,6,7,8-tetrahydro-2H-indeno[5,4-
b]furan derivatives,14 including ramelteon. A key element con-
tributing to the high binding affinity of ramelteon (MT1, Ki =
0.014 nM; MT2, Ki = 0.112 nM)15 is its angular furan-fused
tricyclic ring. We also designed and synthesized indene derivative
1,24 a potent mimic of the indole core of melatonin (Figure 1).
For the exploration of potent MT2-selective ligands, we designed
a novel 1,6-dihydro-2H-indeno[5,4-b]furan core, an indene-type
of tricyclic indeno[5,4-b]furans (Figure 2). Substitution at the
7-position of this scaffold may enhance selectivity toward MT2
subtype in preference to the MT1 because the common structural
feature in the most of the reported MT2-selective ligands17 bear
bulky substituent at the 2-position of the indole core of melato-
nin, like luzindole. In this paper, we report the synthesis and
pharmacological characterization of MT2-selective 1,6-dihydro-
2H-indeno[5,4-b]furan derivatives.
Melatonin (5-methoxy-N-acetyltryptamine, Figure 1) is a
neurohormone synthesized and secreted from the pineal gland
in a circadian manner, and it reaches its peak concentration at
night.1 Melatonin plays an important role in the circadian aspects
of human physiology2 such as regulation of body temperature,
control of seasonal cycles, and regulation of the sleep/wake cycle.
In humans, it has been suggested that melatonin might have a
variety of clinical applications such as the delayed sleep phase
syndrome,3 jet lag,4 and seasonal affective disorders5 and as a
hypnotic agent.6 Moreover, melatonin is reported to have
neuroprotective,7 cardiovascular,8 antioxidant,9 and antitumor
properties.10 In mammals, melatonin activates two high-affinity
G-protein-coupled receptors (MT1 and MT2).11 MT1 receptors
are expressed in several areas of the brain, especially in the
suprachiasmatic nuclei (SCN) and the pars tuberalis of the
pituitary, whereas MT2 receptors are essentially localized in the
SCN and retina. Another melatonin receptor binding site, MT3,
has lower affinity than do MT1 and MT2 and has been char-
acterized as the hamster homologue of quinone reductase 2.12
Over the last two decades, many MT1/MT2 agonists have
been reported, and some have been evaluated in clinical trials.13
Ramelteon,14,15 designed and synthesized in the course of our
investigation on melatonin-mimic MT1/MT2 agonists, reduces
sleep latency and increases the total sleep time and has been
marketed for the treatment of insomnia (in the USA in 2005, and
in Japan in 2010).16 Several MT2-selective ligands have also been
reported recently.17 The representative ligands luzindole18 and
4-phenyl-2-propionamidotetralin (4P-PDOT)19 act as antago-
nists for the MT2 receptor and block the melatonin-mediated
phase advances of circadian rhythms in mice.20 This result
suggests the involvement of the MT2 receptor in the entrainment
of circadian rhythms.21 MT1 knockout studies on mice,22 in
’ CHEMISTRY
7-Alkyl derivatives 8ꢀ16 were synthesized from the pre-
viously reported 1,2,6,7-tetrahydro-8H-indeno[5,4-b]furan-8-
one 214 (Scheme 1). Aldol reaction of 2 with various aldehydes
or ketones under basic conditions, followed by hydrogenation in
the presence of 10% Pd/C, gave the R-alkylated tricyclic ketones
Received: February 26, 2011
Published: April 07, 2011
r
2011 American Chemical Society
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|

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Figure 1. Chemical structures of melatonin, MT1/MT2 agonists (ramelteon and indene derivative 1) and representative MT2-selective ligands
(luzindole and 4P-PDOT).
4.6-fold decrease in the binding affinity for the MT2 subtype. Because
sufficient MT1/MT2 selectivity was not achieved by replacement of
the amide side chain, the effect of various substituents at the
7-position of the scaffold was investigated, keeping the amido moiety
as acetamido group.
Introduction of a benzyl group (compound 14) resulted in a
drastic decrease in the binding affinity for MT1 (Ki = 2.68 nM)
Figure 2. Design of 1,6-dihydro-2H-indeno[5,4-b]furan derivatives as
but no notable change in the affinity for MT2 (Ki = 0.0085 nM), and
MT2-selective ligands.
this led to enhanced MT1/MT2 selectivity (316). This result
suggested that the MT2 receptor has a larger hydrophobic pocket
and wider structural diversity than the MT1 receptor at the 7-position
4bꢀe. Compounds 2 and 4bꢀe were subjected to the Hornerꢀ
Emmons reaction in the presence of diethyl cyanomethylpho-
sphonate to give unsaturated nitriles 5aꢀe, which were converted to
amines 6aꢀe by hydrogenation over Raney cobalt (ODHT-60).
Isomerization of exo olefins 6aꢀe with hydrogen chloride in ethyl
acetate gave the 1,6-dihydro-2H-indeno[5,4-b]furan derivatives
7aꢀe. N-acylated derivatives 8ꢀ12 and 14ꢀ16 were prepared by
treatment with the appropriate acyl chlorides, and the N-ureide
derivative 13 was prepared by treatment with ethyl isocyanate.
Derivatives 18ꢀ27, which have an aromatic ring group at the
7-position, were synthesized as described in Scheme 2. Regiose-
lective bromination of 8 with 1.0 equiv of Br2 in dichloromethane
provided the desired compound 17 in 41% yield. Aromatic ring
groups were introduced by Suzuki coupling in the presence of
Pd(PPh3)4, Na2CO3, and the corresponding boronic acids.
of this scaffold. Furthermore, this result agreed with the result that
luzindole,18 which has a benzyl group at the 2-position of the indole
core, shows MT2 selectivity. The high MT2 binding affinity of 14 was
maintained when the benzyl group of 14 was converted to a
cyclohexylmethyl (compound 15) or 3-thienylmethyl (compound
16) group. 15 showed an MT2 binding affinity 18 times higher (Ki =
0.012 nM) than that of melatonin and exhibited enhanced MT1/
MT2 selectivity (799).
Introduction of aromatic ring groups was also explored.
Phenyl and furyl derivatives (18 and 19) showed enhanced
binding affinities for both MT1 and MT2, while the pyridyl
derivative 20 maintained its affinity. This result suggested that
the substituents whose sizes were similar to those of phenyl and
furyl were tolerated for the hydrophobic pockets around the
7-position for both MT1 and MT2 receptors. Then, bulky
aromatic ring groups were investigated. As in the case of benzyl
derivative 14, the MT1 receptor was less tolerant to structural
diversity at the 7-position of the scaffold, and introduction of a
methoxy group at the ortho, meta, or para positions of the
7-phenyl ring of 18 (derivatives 21, 22, and 23) led to enhanced
MT1/MT2 selectivity ratios of 55, 6.3, and 81, respectively.
Bulkier substituents such as ortho-O-i-propylphenyl and meta-i-
propyl-phenyl (compounds 24 and 25) helped enhance the MT1/
MT2 selectivity ratios (>292 and 1200, respectively), whereas the
para-t-butyl-phenyl substituent (compound 26) resulted in the poor
MT1/MT2 ratio (1.8). The presence of bulky heteroaromatic groups
such as 3-benzothienyl (compound 27) also resulted in enhanced
MT1/MT2 selectivity (220). 25 was the most selective; it showed
high MT1/MT2 selectivity (1200), and its MT2 binding affinity (Ki =
0.011 nM) was 19 times that of melatonin itself.
The structureꢀactivity relationship at the 7-position of this
1,6-dihydro-2H-indeno[5,4-b]furan core was consistent with
that of the reported melatonin-mimic ligands17 which bear bulky
substituents at the topologically equivalent position to the
2-position of the indole core of melatonin. The formation of
angular furan-fused tricyclic ring system might contribute to the
high binding affinity of this series as observed in the derivatives of
ramelteon,14 thus the introduction of bulky substituents at the
7-position of this tricyclic core provided the MT2-selective
ligands with high binding affinity.
’ RESULTS AND DISCUSSION
1. Binding Affinity for MT1 and MT2. The binding affinities of
8ꢀ27 for human MT1 and MT2 melatonin receptors were evaluated
by using 2-[125I]iodomelatonin as a radioligand in Chinese hamster
ovary (CHO) cells expressing these receptors.15 The chemical
structures of 8ꢀ27 and their binding affinities for MT1 and MT2
are shown in Table 1.
Compound 8, which has no substituent at the 7-position of the
1,6-dihydro-2H-indeno[5,4-b]furan core, showed strong binding
affinity both for MT1 (Ki = 0.045 nM) and MT2 (Ki = 0.022 nM).
The affinities of 8 were 5ꢀ10 times those of melatonin, which
indicated that the 1,6-dihydro-2H-indeno[5,4-b]furan core is a
potent mimic of the 5-methoxy indole core of melatonin. Introduc-
tion of an i-propyl group at the 7-position of the core (R1) was
tolerated, and the binding affinity of i-propyl derivative 9 for MT1
and MT2 was comparable to that of 8. Before further exploration of
the 7-position of the scaffold, the acetamido group (R2) of com-
pound 9 was investigated. Replacement of the acetamido group by a
propionylamido, cyclopropylcarboxamido, or trifluoroacetamido
group (corresponding derivatives: 10, 11, and 12, respectively)
cause only slight change in the binding affinity and MT1/MT2
selectivity. On the other hand, replacement of the acetamido group
by an ethylureido group (compound 13) resulted in moderate MT1/
MT2 selectivity (13), however, this replacement also resulted in a
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ARTICLE
Scheme 1a
a Reagents and conditions: (a) R3COR4, NaOH, H2O, THF, 60 °C; (b) R3COR4, A12O3, CH2Cl2; (c) H2, Pd/C, MeOH or EtOH; (d) NaH,
(EtO)2POCH2CN, THF, 40 °C; (e) H2, Raney Co, NH3, EtOH; (f) HCl, EtOAc, 60 °C; (g) R2COCl, Et3N, CH2Cl2; (h) EtNCO, pyridine.
Scheme 2a
a Reagents and conditions: (a) Br2, CH2Cl2; (b) R1B(OH)2, Pd(PPh3)4, Na2CO3, EtOH, toluene, H2O, 80 °C.
2. Functional Activity of 14, 15, and 25. The functional
activities of the most selective compounds (14, 15, and 25) were
evaluated for the forskolin-induced cAMP accumulation in CHO
cells expressing human MT1 or MT2 receptors, based on the
method of Kato et al.,15 with some modifications. The agonist
potency was expressed as EC50 (nM), while the maximal efficacy
(Emax) was expressed as a percentage of the efficacy observed
with 1 μM melatonin.
compounds exhibited potent agonistic activity for the MT2 subtype.
Interestingly, their maximal efficacy (Emax) wasobservedinthediffer-
ent manner. While the benzyl (14, Emax = 62) and meta-i-propyl-
phenyl (25, Emax = 73) derivatives behaved as partial agonists for
MT2 receptors, the cyclohexylmethyl derivative (15, Emax = 99)
behaved as a full agonist. This result indicated that the hydrophobic
substituents at the 7-position of the scaffold can contribute not only
to the selectivity for MT2 receptors, but also to the intrinsic activity.
Because the aromaticity and electronic density of the cyclohexyl-
methyl substituent of 15 differ greatly from those of the benzyl and
meta-i-propyl-phenyl substituents (derivatives 14 and 25), these
Compounds 14, 15, and 25 exhibited moderate agonistic
activity for the MT1 subtype, which corresponds to their low binding
affinities compared to melatonin (Figure 3). On the other hand, these
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Table 1. Binding Affinities of 8ꢀ27 for Human MT1 and MT2 Obtained in Competition Radioligand Binding Assays Using
2-[125I]Iodomelatonin
Kia (nM)
compd
R1
R2
MT1
MT2
MT1/MT2
melatonin
8
0.24 ( 0.06
0.045 ( 0.005
0.024 ( 0.003
0.013 ( 0.001
0.11 ( 0.03
0.032 ( 0.003
1.1 ( 0.2
0.21 ( 0.03
0.022 ( 0.002
0.018 ( 0.002
0.011 ( 0.002
0.035 ( 0.005
0.010 ( 0.002
0.084 ( 0.016
0.0085 ( 0.0018
0.012 ( 0.002
0.0067 ( 0.0014
0.014 ( 0.004
0.0065 ( 0.0015
0.0096 ( 0.0021
0.034 ( 0.005
0.020 ( 0.005
0.0090 ( 0.0023
0.037 ( 0.011
0.34 ( 0.16
1.1
2.1
ꢀH
Me
9
ꢀi-Pr
ꢀi-Pr
ꢀi-Pr
ꢀi-Pr
ꢀi-Pr
Me
Et
1.3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1.3
c-Pr
CF3
-NH-Et
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
3.2
3.1
13
ꢀCH2ꢀPh
2.7 ( 1.4
316
799
132
0.61
1.3
ꢀCH2-c-Hex
ꢀCH2-3-thienyl
ꢀBr
9.2 ( 1.6
0.88 ( 0.12
0.0087 ( 0.0013
0.0082 ( 0.0020
0.0065 ( 0.0014
0.069 ( 0.009
1.1 ( 0.3
ꢀPh
ꢀ3-furyl
0.68
2.1
ꢀ3-pyridyl
ꢀPh(OMe) (o)
ꢀPh(OMe) (m)
ꢀPh(OMe) (p)
ꢀPh(O-i-Pr) (o)
ꢀPh(i-Pr) (m)
ꢀPh(t-Bu) (p)
ꢀ3-benzothienyl
53
0.057 ( 0.007
3.0 ( 0.5
6.3
81
>100
>292
1200
1.8
13 ( 4
0.011 ( 0.003
0.77 ( 0.18
1.4 ( 0.2
1.3 ( 0.4
0.0060 ( 0.0014
220
a Ki values are the mean of at least three measurements.
parameters may strongly influence the intrinsic activity. To the best of
our knowledge, the cyclohexylmethyl derivative 15 is the most potent
MT2-selective ligand (Ki = 0.012 nM), with high MT1/MT2
selectivity (799) and full agonistic activity.
’ CONCLUSION
The results of this studyconfirm that the tricyclic 1,6-dihydro-2H-
indeno[5,4-b]furan skeleton is a potent mimic of the 5-methoxy
indole core of melatonin. Introduction of a cyclohexylmethyl group
at the 7-position of the core afforded a potent MT2-selective agonist
(Ki = 0.012 nM, Emax = 99) with high MT1/MT2 selectivity (799).
Treatment with 3ꢀ30 mg/kg of 15 accelerated reentrainment to a
new light/dark cycle in ICR mice, thus indicating the involvement of
the MT2 receptor in chronobiotic activity.
3. Chronobiotic Activity of 15. The selective MT2 agonist 15
was assessed in terms of its effect on reentrainment to a new
light/dark cycle in ICR mice, based on the method of Hirai
et al.,25 with some modifications (Figure 4). The mice were
maintained in a 12 h light/dark cycle for at least 2 weeks and then
subjected to 8 h advances of the cycle. Individual actograms
showed that daily treatment of the mice with 30 mg/kg of 15
accelerated reentrainment to the new light/dark cycle. Daily
treatment with 3 mg/kg (mean ( SEM; 5.80 ( 0.95, n = 10) and
30 mg/kg (4.20 ( 0.57, n = 10) of 15 accelerated days to
reentrainment, while it was slow in the case of treatment with the
vehicle (6.67 ( 1.07, n = 9). This result demonstrates that MT2
receptors might mediate the chronobiotic activity and is in
agreement with the studies using MT2-selective antagonists20
or MT1 knockout mice.22 Ramelteon, the MT1/MT2 agonists as
a hypnotic agent, also showed chronobiotic activity in rats25 as
one of the feature of its pharmacological effects. This result
suggests that the chronobiotic activity of ramelteon might be
mediated through MT2 receptors.
’ EXPERIMENTAL SECTION
Melting points were determined on a Buchi melting point apparatus
and were not corrected. Proton nuclear magnetic resonance (1H NMR)
spectra were recorded on Varian Gemini-200 (200 MHz), Varian Gemini-
300 (300 MHz), or Bruker DPX300 (300 MHz) instruments. Chemical
shifts are reported as δ values (ppm) downfield from internal tetramethylsi-
lane of the indicated organic solution. Peak multiplicities are expressed as
follows. Abbreviations are used as follows: s, singlet; d, doublet; t, triplet;
q, quartet; dd, doublet of doublet; ddd, doublet of doublet of doublets;
dt, doublet of triplet; brs, broad singlet; m, multiplet. Coupling constants
(J values) are given in hertz (Hz). LC/MS (ESIþ) was performed on a ZQ-
2000 apparatus with acetonitrile/water mobile phase. Preparative HPLC
were performed an automated Gilson HPLC system using a YMC C-18
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column (S-5 μM 50 mm ꢁ 20 mm I.D.) with 10ꢀ100% gradient
waterꢀacetonitrile containing 0.1% TFA. Reaction progress was determined
by thin layer chromatography (TLC) analysis on silica gel 60 F254 plate
(Merck) or NH TLC plates (Fuji Silysia Chemical Ltd.). The purities
(>95%) of all compounds tested in biological systems were established by
elemental analyses. Element analyses were carried out by Takeda Analytical
Laboratories, and the results were within 0.4% of theoretical values.
7-(1-Methylethylidene)-1,2,6,7-tetrahydro-8H-indeno[5,4-b]-
furan-8-one (3b). To a solution of 2 (10.0 g, 57.4 mmol) and acetone
(42.1 mL, 574 mmol) in THF (100 mL), a solution of sodium hydroxide
(2.52 g, 63.0 mmol) in water (100 mL) was added and stirred at 60 °C
for 3 days. After being cooled to room temperature, saturated aqueous
sodium hydrogen carbonate was added, and the mixture was extracted
with EtOAc. The extract was washed with brine and dried over
anhydrous magnesium sulfate. The filtrate was concentrated and then
washed with diethyl ether to give 3b (5.69 g, 46%) as a white solid. 1H
NMR (CDCl3) δ 1.99 (3H, s), 2.42 (3H, s), 3,54 (2H, t, J = 8.9 Hz), 3.58
(2H, s), 4.66 (2H, t, J = 8.9 Hz), 6.98 (1H, d, J = 8.1 Hz), 7.19 (1H, d, J =
8.1 Hz). MS (ESI) m/z 215 (M þ H)þ.
7-Benzylidene-1,2,6,7-tetrahydro-8H-indeno[5,4-b]furan-
8-one (3c). Benzaldehyde (7.00 mL, 68.9 mmol) and activated basic
aluminum oxide (150 g) were added to a vigorously stirred solution of 2
(10.0 g, 57.4 mmol) in methylene chloride (300 mL). After 1 h of stirring
at room temperature, aluminum oxide was filtered and washed abun-
dantly with methylene chloride. The filtrate was concentrated and then
recrystallized from a mixture of EtOAc and hexane to give 3c (9.65 g,
64%, single isomer) as a white solid. 1H NMR (CDCl3) δ 3.58 (2H, t, J =
8.9 Hz), 4.00 (2H, d, J = 1.7 Hz), 4.69 (2H, t, J = 8.9 Hz), 7.04 (1H, d, J =
8.1 Hz), 7.28 (1H, d, J = 8.1 Hz), 7.37ꢀ7.53 (3H, m), 7.60ꢀ7.71 (3H,
m). MS (ESI) m/z 263 (M þ H)þ.
7-(Cyclohexylmethylene)-1,2,6,7-tetrahydro-8H-indeno[5,4-
b]furan-8-one (3d). By a similar procedure that described for 3b, 3g
(71%) was obtained as a white solid (single isomer). 1H NMR (CDCl3) δ
1.18ꢀ1.43 (5H, m), 1.65ꢀ1.86 (5H, m), 2.25ꢀ2.43 (1H, m), 3.54 (2H, t,
J = 8.9 Hz), 3.63 (2H, s), 4.67 (2H, t, J = 8.9 Hz), 6.68 (1H, dt, J = 9.8, 2.1
Hz), 7.01 (1H, d, J = 7.9 Hz), 7.22 (1H, d, J = 8.3 Hz). MS (ESI) m/z 269
(M þ H)þ.
7-(3-Thienylmethylene)-1,2,6,7-tetrahydro-8H-indeno[5,4-
b]furan-8-one (3e). By a similar procedure that described for 3c, 3e
(52%) was obtained as a colorless solid (single isomer). 1H NMR
(CDCl3) δ 3.56 (2H, t, J = 8.9 Hz), 3.91 (2H, d, J = 1.5 Hz), 4.68 (2H,
t, J =8.9Hz), 7.03(1H, d, J= 8.1Hz), 7.27 (1H, d, J = 8.1 Hz), 7.41 (2H, d,
J = 2.1 Hz), 7.58ꢀ7.71 (2H, m). MS (ESI) m/z 269 (M þ H)þ.
7-Isopropyl-1,2,6,7-tetrahydro-8H-indeno[5,4-b]furan-8-
one (4b). A solution of 3b (5.69 g, 26.6 mmol) in EtOH (1000 mL)
was hydrogenated in the presence of 10% palladium on carbon (3 g,
containing 50% water) at room temperature for 30 min. The catalyst was
removed by filtration, and the filtrate was concentrated. The residue was
purified by silica gel column chromatography (EtOAc/hexane) to give 4b
(4.84 g, 84%) as a colorless oil. 1HNMR(CDCl3) δ1.20 (6H, d, J=6.7Hz),
2.63ꢀ2.91 (2H, m), 3.06ꢀ3.25 (1H, m), 3.45 (2H, t, J = 8.8 Hz), 4.65 (2H,
t, J = 8.8 Hz), 6.99 (1H, d, J = 8.0 Hz), 7.16 (1H, d, J = 8.0 Hz). MS (ESI)
m/z 217 (M þ H)þ.
Figure 3. Functional analysis of melatonin, 14, 15, and 25 on forskolin-
stimulated cAMP formation in CHO cells expressing human MT1 or human
MT2 receptor. Each data point represents the mean ( standard error of two
dependent experiments performed in triplicate. Curves are fit using nonlinear
regression analysis with a variable slope (four parameters) to obtain potency
(EC50) values; GraphPad Prism software is used for this purpose. The
LogEC50 values for MT1 are ꢀ10.59 (0.03 (melatonin), ꢀ9.29 (0.09 (14),
ꢀ8.35 (0.09 (15),andꢀ8.52 (0.06 (25).TheLogEC50 values for MT2 are
ꢀ9.07 (0.07(melatonin), andꢀ8.88 (0.08 (15). Emax (%) values for MT2
are 61.7 ( 3.1 (14), 99.0 ( 2.9 (15), and 72.8 ( 2.7 (25).
Figure 4. Effect of 15 on running-wheel activity in ICR mice after an 8 h advance of the light/dark cycle. Representative double-plotted actograms are
shown for the vehicle, as well as for 3 and 30 mg/kg of 15. The arrows indicate the day during the shifting phase, and the vertical bar represents the time of
injection. The upper horizontal bar indicates the light (white) and dark (black) periods during preshifting, and the lower bar shows those for postshifting.
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7-Benzyl-1,2,6,7-tetrahydro-8H-indeno[5,4-b]furan-8-one
(4c). By a similar procedure that described for 4b, 4c (84%) was
obtained as a white solid. 1H NMR (CDCl3) δ 2.64 (1H, dd, J = 14.0,
10.3 Hz), 2.79 (1H, dd, J = 16.1, 3.4 Hz), 2.90ꢀ3.17 (2H, m), 3.38 (1H,
dd, J = 14.0, 4.1 Hz), 3.50 (2H, t, J = 8.9 Hz), 4.66 (2H, t, J = 8.9 Hz), 6.99
(1H, d, J = 8.1 Hz), 7.13 (1H, d, J = 8.0 Hz), 7.18ꢀ7.38 (5H, m). MS
(ESI) m/z 265 (M þ H)þ.
(2H, d, J = 7.2 Hz), 2.82ꢀ3.09 (4H, m), 3.27 (2H, s), 3.40 (2H, t, J = 8.5
Hz), 4.56 (2H, t, J = 8.5 Hz), 6.52 (1H, d, J = 7.9 Hz), 7.08 (1H, d, J = 7.9
Hz). MS (ESI) m/z 298 (M þ H)þ.
2-[7-(3-Thienylmethyl)-1,6-dihydro-2H-indeno[5,4-b]furan-
8-yl]ethanamine hydrochloride (7e). By a similar procedure that
1
described for 7a, 7e (63%) was obtained as a white solid. H NMR
(methanol-d4) δ 2.97ꢀ3.09 (4H, m), 3.24 (2H, s), 3.43 (2H, t, J = 8.6 Hz),
3.87 (2H, s), 4.57 (2H, t, J = 8.6 Hz), 6.52 (1H, d, J = 7.9 Hz), 6.97 (1H, dd,
J = 5.0, 1.2 Hz), 7.01ꢀ7.13 (2H, m), 7.35 (1H, dd, J = 5.0, 2.8 Hz). MS
(ESI) m/z 298 (M þ H)þ.
7-(Cyclohexylmethyl)-1,2,6,7-tetrahydro-8H-indeno[5,4-b]-
furan-8-one (4d). A solution of 3d (8.00 g, 29.8 mmol) in MeOH
(200 mL) was hydrogenated in the presence of 10% palladium on carbon
(500 mg) at room temperature for 3 h. THF (200 mL) was added to the
mixture, the catalyst was removed by filtration, and the filtrate was
concentrated. The residue was washed with MeOH to give 4d (6.23 g,
77%) as a white solid. 1H NMR (CDCl3) δ 0.85ꢀ1.13 (2H, m), 1.15ꢀ
1.39 (4H, m), 1.39ꢀ1.58 (1H, m), 1.62ꢀ1.94 (6H, m), 2.66ꢀ2.81 (2H,
m), 3.28 (1H, dd, J = 17.6, 8.6 Hz), 3.48 (2H, t, J = 8.9 Hz), 4.66 (2H, t, J =
8.9 Hz), 7.01 (1H, d, J = 8.2 Hz), 7.18 (1H, d, J = 8.0 Hz). MS (ESI) m/z
271 (M þ H)þ.
N-[2-(1,6-Dihydro-2H-indeno[5,4-b]furan-8-yl)ethyl]acet-
amide (8). To a solution of 7a (1.20 g, 5.05 mmol) in methylene chloride
(50 mL) was added triethylamine (1.41 mL, 10.1 mmol) and acetyl chloride
(431 μL, 6.06 mmol). After the mixture was stirred at room temperature for
10 min, water and EtOAc were added to the mixture. The organic layer was
washed with saturated aqueous sodium hydrogen carbonate and brine and
dried over anhydrous magnesium sulfate. The filtrate was concentrated, and
the residue was purified by silica gel column chromatography (EtOAc/
hexane) and then recrystallized from a mixture of EtOAc and hexane to give 8
(0.95 g, 77%) as a white solid; mp 157ꢀ158 °C (EtOAc/hexane). 1H NMR
(CDCl3) δ 1.98 (3H, s), 2.68ꢀ2.87 (2H, m), 3.31 (2H, d, J = 1.5 Hz), 3.43
(2H, t, J = 8.7 Hz), 3.57 (2H, q, J = 6.8 Hz), 4.60 (2H, t, J = 8.7 Hz), 5.63
(1H, s), 6.29 (1H, s), 6.67 (1H, d, J = 7.9 Hz), 7.19 (1H, d, J = 7.9 Hz). MS
(ESI) m/z 244 (M þ H)þ. Anal. (C15H17NO2) C, H, N.
7-(3-Thienylmethyl)-1,2,6,7-tetrahydro-8H-indeno[5,4-b]-
furan-8-one (4e). By a similar procedure that described for 4b, 4e
(78%) was obtained as a white solid. 1H NMR (CDCl3) δ 2.70ꢀ2.87
(2H, m), 2.89ꢀ3.04 (1H, m), 3.09ꢀ3.24 (1H, m), 3.30 (1H, dd, J = 14.3,
4.0 Hz), 3.48 (2H, t, J=8.9Hz), 4.66(2H, t,J= 8.9 Hz), 6.85ꢀ7.07(3H, m),
7.14 (1H, d, J=8.1Hz), 7.20ꢀ7.30 (1H, m). MS (ESI) m/z271 (M þ H)þ.
2-(1,6-Dihydro-2H-indeno[5,4-b]furan-8-yl)ethanamine hy-
drochloride (7a). A 65% suspension of sodium hydride in mineral oil
(3.18 g, 86.1 mmol) was added to a solution of diethyl cyanomethylpho-
sphonate (16.8 g, 94.7 mmol) in THF (100 mL). After stirred at room
temperature for 30 min, the mixture was added dropwise to a stirred solution
of 2(5.00g, 28.7mmol) inTHF(100mL) at40°Candthenstirredat40°C
for 1 h. Water was added, and the mixture was extracted with EtOAc. The
extract was washed with brine and dried over anhydrous magnesium sulfate.
The filtrate was concentrated, and the residue was purified by silica gel
column chromatography (EtOAc/hexane) to give 5a (5.03 g, 89%) as a
mixture of cisꢀtrans isomers. A solution of 5a (19.4 g, 98.4 mmol) in EtOH
(saturated with NH3, 500 mL) and EtOH (500 mL) was hydrogenated in
the presence of Raney cobalt ODHT-60 (50 g, washed with EtOH two times
before use) at room temperature for 3 days. The catalyst was removed by
filtration. The filtrate was concentrated to afford 6a as a crude oil. To a
solution of crude 6a in MeOH (300 mL), 4 M hydrogen chloride in EtOAc
(300 mL) was added and stirred at 60 °C for 3 h. After being cooled to room
temperature, the resulting solid was collected by filtration and washed with
EtOAc to give 7a (16.2 g, 69%) as a white solid; mp 246ꢀ248 °C
(recrystallized from MeOH). 1H NMR (methanol-d4) δ 2.92ꢀ3.06 (2H,
m), 3.16ꢀ3.34 (4H, m), 3.42 (2H, t, J = 8.6 Hz), 4.56 (2H, t, J = 8.6 Hz),
6.40 (1H, d, J=1.5Hz),6.59(1H, d,J= 8.1 Hz), 7.15 (1H, d, J=8.1Hz).MS
(ESI) m/z 202 (M þ H)þ. Anal. (C13H16NOCl) C, H, N, Cl.
N-[2-(7-Isopropyl-1,6-dihydro-2H-indeno[5,4-b]furan-8-yl)-
ethyl]acetamide (9). By a similar procedure that described for 8, 9
(57%) was obtained as a white solid; mp 132ꢀ134 °C (EtOAc/hexane).
1H NMR (CDCl3) δ 1.16 (6H, d, J = 6.9 Hz), 1.96 (3H, s), 2.76 (2H, d, J =
7.2 Hz), 2.97ꢀ3.10 (1H, m), 3.28 (2H, s), 3.35ꢀ3.48 (4H, m), 4.58 (2H, t,
J = 8.5 Hz), 5.62 (1H, brs), 6.60 (1H, d, J = 8.0 Hz), 7.13 (1H, d, J = 8.0
Hz). MS (ESI) m/z 286 (M þ H)þ. Anal. (C18H23NO2) C, H, N.
N-[2-(7-Isopropyl-1,6-dihydro-2H-indeno[5,4-b]furan-8-yl)-
ethyl]propanamide (10). By a similar procedure that described for 8,
10 (69%) was obtained as a white solid; mp 144ꢀ146 °C (EtOAc/
hexane). 1H NMR (CDCl3) δ 1.15 (3H,t, J = 7.7 Hz), 1.16 (6H, d, J = 6.6
Hz), 2.18 (2H, q, J = 7.7 Hz), 2.76 (2H, t, J = 7.3 Hz), 2.97ꢀ3.08 (1H, m),
3.27 (2H, s), 3.37ꢀ3.47 (4H, m), 4.58 (2H, t, J = 8.5 Hz), 5.62 (1H, brs),
6.60 (1H, d, J = 7.7 Hz), 7.13 (1H, d, J = 7.7 Hz). MS (ESI) m/z 300 (M þ
H)þ. Anal. (C19H25NO2) C, H, N.
N-[2-(7-Isopropyl-1,6-dihydro-2H-indeno[5,4-b]furan-8-yl)-
ethyl]cyclopropanecarboxamide (11). By a similar procedure that
described for 8, 11 (75%) was obtained as a white solid; mp 197ꢀ200 °C
(EtOAc/hexane). 1H NMR (CDCl3) δ 0.70ꢀ0.79 (2H, m), 0.93ꢀ1.03
(2H, m), 1.17 (6H, d, J = 6.9 Hz), 1.17ꢀ1.33 (1H, m), 2.77 (2H, t, J = 7.1
Hz), 2.97ꢀ3.10 (1H, m), 3.28 (2H, s), 3.37ꢀ3.50 (4H, m), 4.58 (2H, t, J =
8.5 Hz), 5.78 (1H, brs), 6.60 (1H, d, J = 8.0 Hz), 7.13 (1H, d, J = 8.0 Hz).
MS (ESI) m/z 312 (M þ H)þ. Anal. (C20H25NO2) C, H, N.
2-(7-Isopropyl-1,6-dihydro-2H-indeno[5,4-b]furan-8-yl)etha-
namine hydrochloride (7b). By a similar procedure that described for
7a, 7b (69%) was obtained as a white solid; mp 212ꢀ215 °C (EtOH). 1H
NMR (DMSO-d6) δ 1.14 (6H, d, J = 6.6 Hz), 2.85 (4H, brs), 3.00ꢀ3.13
(1H, m), 3.23 (2H, s), 3.40 (2H, t, J = 8.6 Hz), 4.54 (2H, t, J = 8.6 Hz), 6.51
(1H, d, J = 8.0 Hz), 7.09 (1H, d, J = 8.0 Hz), 8.15 (2H, brs). Anal.
(C16H22NOCl) C, H, N, Cl.
2,2,2-Trifluoro-N-[2-(7-isopropyl-1,6-dihydro-2H-indeno-
[5,4-b]furan-8-yl)ethyl]acetamide (12). By a similar procedure
that described for 8, 12 (38%) was obtained as a white solid; mp
162ꢀ164 °C (EtOAc/hexane). 1H NMR (CDCl3) δ 1.16 (6H, d, J = 6.9
Hz), 2.84 (2H, t, J = 7.3 Hz), 2.93ꢀ3.06 (1H, m), 3.30 (2H, s), 3.41 (2H,
t, J = 8.5 Hz), 3.53 (2H, q, J = 7.3 Hz), 4.59 (2H, t, J = 8.5 Hz), 6.48 (1H,
brs), 6.62 (1H, d, J = 7.8 Hz), 7.15 (1H, d, J = 7.8 Hz). MS (ESI) m/z 340
(M þ H)þ. Anal. (C18H20F3NO2) C, H, N.
2-(7-Benzyl-1,6-dihydro-2H-indeno[5,4-b]furan-8-yl)etha-
namine hydrochloride (7c). By a similar procedure that described
for 7a, 7c (48%) was obtained as a white solid. 1H NMR (methanol-d4) δ
3.05 (4H, s), 3.19(2H, s), 3.43 (2H, t, J = 8.5 Hz), 3.86 (2H, s), 4.57 (2H,
t, J = 8.5 Hz), 6.50 (1H, d, J = 8.0 Hz), 7.02 (1H, d, J = 8.0 Hz), 7.10ꢀ7.40
(5H, m). MS (ESI) m/z 292 (M þ H)þ.
N-Ethyl-N0-[2-(7-isopropyl-1,6-dihydro-2H-indeno[5,4-b]-
furan-8-yl)ethyl]urea (13). Ethyl isocyanate (165 mg, 2.32 mmol)
was added to a solution of 7b (500 mg, 1.79 mmol) in pyridine (5 mL) at
room temperature, and the reaction mixture was stirred at 60 °C for 2 h.
The solvent was evaporated, and aqueous hydrogen chloride was added
and extracted with methylene chloride. The filtrate was concentrated,
and the residue was purified by silica gel column chromatography
(MeOH/chloroform) to give 13 (499 mg, 89%) as a white solid; mp
167ꢀ169 °C (EtOAc). 1H NMR (CDCl3) δ 1.10 (3H, t, J = 7.3 Hz),
2-[7-(Cyclohexylmethyl)-1,6-dihydro-2H-indeno[5,4-b]furan-
8-yl]ethanamine hydrochloride (7d). By a similar procedure that
described for 7a, 7d (55%) was obtained as a white solid. 1H NMR (metha-
nol-d4) δ0.90ꢀ1.10 (2H, m), 1.12ꢀ1.40 (3H, m), 1.44ꢀ1.83(6H, m),2.39
3441
dx.doi.org/10.1021/jm200221q |J. Med. Chem. 2011, 54, 3436–3444

Journal of Medicinal Chemistry
ARTICLE
1.15 (6H, d, J = 7.0 Hz), 2.76 (2H, t, J = 7.1 Hz), 2.97ꢀ3.23 (3H, m),
3.26 (2H, s), 3.33 (2H, q, J = 7.0 Hz), 3.43 (2H, t, J = 8.5 Hz), 4.17 (1H,
brs), 4.41 (1H, brs), 4.57 (2H, t, J = 8.5 Hz), 6.58 (1H, d, J = 7.7 Hz),
7.11 (1H, d, J = 7.7 Hz). MS (ESI) m/z 315 (M þ H)þ. Anal.
(C19H26N2O2) C, H, N.
N-[2-(7-Pyridin-3-yl-1,6-dihydro-2H-indeno[5,4-b]furan-8-yl)-
ethyl]acetamide (20). By a similar procedure that described for 18, 20
(26%) was obtained as a yellow solid; mp 168ꢀ170 °C (EtOAc/hexane). 1H
NMR (CDCl3) δ 1.87 (3H, s), 2.95 (2H, t, J = 7.8 Hz), 3.36ꢀ3.50 (2H, m),
3.58 (2H, t, J = 8.6 Hz), 3.71 (2H, s), 4.65 (2H, t, J = 8.6 Hz), 5.59 (1H, s),
6.73 (1H, t, J= 4.0 Hz), 7.23 (1H, d, J=4.9Hz),7.38(1H,dd,J=7.7,4.9Hz),
7.72ꢀ7.82 (1H, m), 8.55 (1H, dd, J = 4.9, 1.6 Hz), 8.70 (1H, d, J = 1.6 Hz).
MS (ESI) m/z 321 (M þ H)þ. Anal. (C20H20N2O2) C, H, N.
N-[2-(7-Benzyl-1,6-dihydro-2H-indeno[5,4-b]furan-8-yl)-
ethyl]acetamide (14). By a similar procedure that described for 8,
14 (38%) was obtained as a white solid; mp 132ꢀ133 °C (EtOAc/hexane).
1H NMR (CDCl3) δ 1.89 (3H, s), 2.86 (2H, t, J = 7.3 Hz), 3.23 (2H, s),
3.35ꢀ3.56 (4H, m), 3.81 (2H, s), 4.60 (2H, t, J = 8.6 Hz), 5.58 (1H, s), 6.60
(1H, d, J = 7.7 Hz), 7.07 (1H, d, J = 7.7 Hz), 7.13ꢀ7.35 (5H, m). MS (ESI)
m/z 334 (M þ H)þ. Anal. (C22H23NO2) C, H, N.
N-{2-[7-(2-Methoxyphenyl)-1,6-dihydro-2H-indeno[5,4-b]-
furan-8-yl]ethyl}acetamide (21). By a similar procedure that de-
scribed for 18, 21 (49%) was obtained as a white solid; mp 137ꢀ138 °C
(EtOAc/hexane). 1H NMR (CDCl3) δ 1.74 (3H, s), 2.75 (2H, t, J = 6.5
Hz), 3.36 (2H, q, J = 6.5 Hz), 3.47 (2H, t, J = 8.6 Hz), 3.65 (2H, s), 3.83
(2H, s), 4.62 (2H, t, J = 8.6 Hz), 5.57 (1H, s), 6.69 (1H, d, J = 7.9 Hz),
6.94ꢀ7.07 (2H, m), 7.12ꢀ7.22 (2H, m), 7.28ꢀ7.40 (1H, m). MS (ESI)
m/z 350 (M þ H)þ. Anal. (C22H23NO3) C, H, N.
N-{2-[7-(Cyclohexylmethyl)-1,6-dihydro-2H-indeno[5,4-b]-
furan-8-yl]ethyl}acetamide (15). By a similar procedure that de-
scribed for 8, 15 (79%) was obtained as a white solid; mp 133ꢀ134 °C
(EtOAc/hexane). 1H NMR (CDCl3) δ 0.82ꢀ1.03 (2H, m), 1.06ꢀ1.32
(3H, m), 1.42ꢀ1.78 (6H, m), 1.96 (3H, s), 2.32 (2H, d, J = 7.2 Hz), 2.74
(2H, t, J = 7.2 Hz), 3.26 (2H, s), 3.32ꢀ3.52 (4H, m), 4.59 (2H, t, J = 8.6
Hz), 5.60 (1H, s), 6.59 (1H, d, J = 7.9 Hz), 7.11 (1H, d, J = 7.9 Hz). MS
(ESI) m/z 340 (M þ H)þ. Anal. (C22H29NO2) C, H, N.
N-{2-[7-(3-Methoxyphenyl)-1,6-dihydro-2H-indeno[5,4-b]-
furan-8-yl]ethyl}acetamide (22). By a similar procedure that de-
scribed for 18, 22 (40%) was obtained as a white solid; mp 132ꢀ134 °C
(EtOAc/hexane). 1H NMR (CDCl3) δ 1.80 (3H, s), 2.96 (2H, t, J = 7.3
Hz), 3.35ꢀ3.47 (2H, m), 3.53 (2H, t, J = 8.6 Hz), 3.68 (2H, s), 3.85 (3H,
s), 4.63 (2H, t, J = 8.6 Hz), 5.42 (1H, s), 6.70 (1H, d, J = 7.9 Hz),
6.83ꢀ7.04 (3H, m), 7.21 (1H, d, J = 7.9 Hz), 7.35 (1H, t, J = 7.9 Hz). MS
(ESI) m/z 350 (M þ H)þ. Anal. (C22H23NO3) C, H, N.
N-{2-[7-(3-Thienylmethyl)-1,6-dihydro-2H-indeno[5,4-b]-
furan-8-yl]ethyl}acetamide (16). By a similar procedure that
described for 8, 16 (34%) was obtained as a pale-yellow solid; mp
1
125ꢀ127 °C (EtOAc/hexane). H NMR (CDCl3) δ 1.87 (3H, s), 2.84
(2H, t, J =7.3 Hz), 3.29 (2H, s), 3.34ꢀ3.55 (4H, m), 3.81 (2H, s), 4.60 (2H,
t, J=8.6Hz), 5.58(1H, s), 6.61(1H, d, J=7.7Hz), 6.88ꢀ6.98(2H, m), 7.09
(1H, d, J = 7.7 Hz), 7.21ꢀ7.31 (1H, m). MS (ESI) m/z 340 (M þ H)þ.
Anal. (C20H21NO2S) C, H, N.
N-{2-[7-(4-Methoxyphenyl)-1,6-dihydro-2H-indeno[5,4-b]-
furan-8-yl]ethyl}acetamide (23). By a similar procedure that de-
scribed for 18, 23 (28%) was obtained as a white solid; mp 204ꢀ205 °C
(EtOAc/hexane). 1H NMR (CDCl3) δ 1.81 (3H, s), 2.95 (2H, t, J = 7.3
Hz), 3.34ꢀ3.58 (4H, m), 3.66 (2H, s), 3.85 (3H, s), 4.63 (2H, t, J = 8.6
Hz), 5.45 (1H, s), 6.68 (1H, d, J = 7.9 Hz), 6.92ꢀ7.00 (2H, m), 7.20 (1H,
d, J = 7.9 Hz), 7.32ꢀ7.43 (2H, m). MS (ESI) m/z 350 (M þ H)þ. Anal.
(C22H23NO3) C, H, N.
N-[2-(7-Bromo-1,6-dihydro-2H-indeno[5,4-b]furan-8-yl)-
ethyl]acetamide (17). To a solution of 8 (8.48 g, 34.9 mmol) in
methylene chloride (50 mL), bromine (1.79 mL, 34.9 mmol) was added
dropwise and stirred at room temperature for 30 min. Then 1 M aqueous
Na2S2O3 solution and EtOAc were added to the mixture. The organic layer
was separated, washed with saturated aqueous sodium hydrogen carbonate
and brine, and dried over anhydrous magnesium sulfate. The filtrate was
concentrated, and the residue was purified by silica gel column chromatog-
raphy (MeOH/EtOAc) and preparative HPLC to give 17 (4.56 g, 41%) as a
white solid; mp 145ꢀ146 °C (EtOAc). 1H NMR (CDCl3) δ 1.98 (3H, s),
2.83 (2H, t, J = 7.2 Hz), 3.34ꢀ3.50 (4H, m), 3.56 (2H, s), 4.61 (2H, t, J =8.7
Hz), 5.72 (1H, s), 6.65 (1H, d, J = 7.9 Hz), 7.10 (1H, d, J = 7.9 Hz). MS
(ESI) m/z 323 (M þ H)þ. Anal. (C15H16BrNO2) C, H, N, Br.
N-{2-[7-(2-Isopropoxyphenyl)-1,6-dihydro-2H-indeno-
[5,4-b]furan-8-yl]ethyl}acetamide (24). By a similar procedure that
described for 18, 24 (47%) was obtained as a white solid; mp 135ꢀ136 °C
(EtOAc/hexane). 1H NMR (CDCl3) δ 1.30 (6H, d, J = 6.0 Hz), 1.74 (3H,
s), 2.76 (2H, t, J = 6.5 Hz), 3.36 (2H, q, J = 6.5 Hz), 3.48 (2H, t, J = 8.6 Hz),
3.66 (2H, s), 4.53ꢀ4.69 (3H, m), 5.57 (1H, s), 6.70 (1H, d, J = 7.9 Hz),
6.92ꢀ7.04 (2H, m), 7.11ꢀ7.36 (3H, m). MS (ESI) m/z 378 (M þ H)þ.
Anal. (C24H27NO3) C, H, N.
N-{2-[7-(3-Isopropylphenyl)-1,6-dihydro-2H-indeno[5,4-b]-
furan-8-yl]ethyl}acetamide (25). By a similar procedure that de-
scribed for 18, 25 (53%) was obtained as a white solid; mp 176ꢀ178 °C
(EtOAc/hexane). 1H NMR (CDCl3) δ 1.29 (6H, d, J = 7.0 Hz), 1.76 (3H,
s), 2.89ꢀ3.03 (3H, m), 3.43 (2H, q, J = 6.7 Hz), 3.52 (2H, t, J = 8.6 Hz),
3.71 (2H, s), 4.63 (2H, t, J = 8.6 Hz), 5.37 (1H, s), 6.70 (1H, d, J = 7.9 Hz),
7.15ꢀ7.28 (4H, m), 7.36 (1H, t, J = 7.5 Hz). MS (ESI) m/z 362 (M þ
H)þ. Anal. (C24H27NO2) C, H, N.
N-{2-[7-(4-tert-Butylphenyl)-1,6-dihydro-2H-indeno[5,4-b]-
furan-8-yl]ethyl}acetamide (26). By a similar procedure that de-
scribed for 18, 26 (47%) was obtained as a white solid; mp 212ꢀ213 °C
(EtOAc/hexane). 1H NMR (CDCl3) δ 1.36 (9H, s), 1.77 (3H, s), 2.98
(2H, t, J = 7.2 Hz), 3.29ꢀ3.58 (4H, m), 3.69 (2H, s), 4.63 (2H, t, J = 8.7
Hz), 5.43 (1H, s), 6.69 (1H, d, J = 7.9 Hz), 7.20 (1H, d, J = 7.9 Hz),
7.35ꢀ7.49 (4H, m). MS (ESI) m/z 376 (M þ H)þ. Anal. (C25H29NO2)
C, H, N.
N-[2-(7-Phenyl-1,6-dihydro-2H-indeno[5,4-b]furan-8-yl)-
ethyl]acetamide (18). A mixture of 17 (200 mg, 0.621 mmol), phenyl
boronic acid (189 mg, 1.55 mmol), Pd(PPh3)4 (72 mg, 0.062 mmol), and
2 M aqueous Na2CO3 (4 mL) in EtOH (3 mL) and toluene (3 mL) was
stirred at 80 °C for 30 min. Water and EtOAc were added to the mixture. The
organic layer was washed with saturated aqueous sodium hydrogen carbonate
and brine and dried over anhydrous magnesium sulfate. The filtrate was
concentrated, and the residue was purified by silica gel column chromatog-
raphy (EtOAc/hexane) and preparative HPLC and then recrystallized from a
mixture of EtOAc and hexane to give 18 (124 mg, 63%) as a white solid; mp
1
149ꢀ151 °C (EtOAc/hexane). H NMR (CDCl3) δ 1.78 (3H, s), 2.96
(2H, t, J= 7.3 Hz), 3.35ꢀ3.47 (2H, m), 3.53 (2H, t, J=8.6Hz), 3.70(2H, s),
4.63 (2H, t, J=8.6Hz), 5.43(1H, s), 6.70(1H, d,J= 7.9 Hz), 7.21 (1H, d, J=
7.9 Hz), 7.28ꢀ7.37 (1H, m), 7.38ꢀ7.48 (4H, m). MS (ESI) m/z 320 (M þ
H)þ. Anal. (C21H21NO2) C, H, N.
N-{2-[7-(3-Furyl)-1,6-dihydro-2H-indeno[5,4-b]furan-8-yl]-
ethyl}acetamide (19). Bya similar procedure that described for 18, 19
(33%) was obtained as a pale-yellow solid; mp 156ꢀ157 °C (EtOAc/
N-{2-[7-(1-Benzothien-3-yl)-1,6-dihydro-2H-indeno[5,4-b]-
furan-8-yl]ethyl}acetamide (27). By a similar procedure that described
for 18, 27 (11%) was obtained as a pale-brown solid; mp 208ꢀ210 °C
(EtOAc/hexane). 1H NMR (CDCl3) δ 1.60 (3H, s), 2.81 (2H, t, J = 7.1
Hz), 3.25ꢀ3.36 (2H, m), 3.55 (2H, t, J = 8.6 Hz), 3.73 (2H, s), 4.66 (2H, t,
J = 8.6 Hz), 5.26 (1H, s), 6.74 (1H, d, J = 7.9 Hz), 7.24 (1H, d, J = 7.9 Hz),
7.32 (1H, s), 7.35ꢀ7.45 (2H, m), 7.58ꢀ7.73 (1H, m), 7.83ꢀ8.00 (1H, m).
MS (ESI) m/z 376 (M þ H)þ. Anal. (C23H21NO2S) C, H, N.
1
hexane). H NMR (CDCl3) δ 1.90 (3H, s), 3.00 (2H, t, J = 7.4 Hz),
3.40ꢀ3.55 (4H, m), 3.61 (2H, s), 4.62 (2H, t, J = 8.6 Hz), 5.67 (1H, s),
6.65 (1H, d, J = 7.9 Hz), 6.75 (1H, dd, J = 1.9, 0.9 Hz), 7.16 (1H, d, J = 7.9
Hz), 7.49 (1H, t, J = 1.9 Hz), 7.76 (1H, s). MS (ESI) m/z 310 (M þ H)þ.
Anal. (C19H19NO3) C, H, N.
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Journal of Medicinal Chemistry
ARTICLE
Preparation of CHO Membrane for Melatonin Receptor
(MT1 or MT2) Binding Assays. Cell lines stably expressing human
MT1 receptor (hMT1-CHO/binding, cell line: TPCCB0117) or human
MT2 receptor (hMT2-CHO, cell line: TPCCB0098) were used for the
experiments. hMT1-CHO/binding cells and hMT2-CHO cells were cultured
in F-12 nutrient mixture (Ham) supplemented with 10% fetal bovine serum
(FBS) and 500 μg/mL of Geneticin, 100 U/mL penicillin, and 100 μg/mL
streptomycin in a 5% CO2/95% air atmosphere. Cells were harvested at
confluence in Ca2þ-Mg2þ-free phosphate buffered saline (PBS) containing
0.5 mM ethylenediaminetetraacetic acid (EDTA) and collected by centrifu-
gation. Cells were washed with PBS, pelleted, and homogenized in ice-cold
10 mM NaHCO3 buffer (pH 7.4 at 25 °C) containing 5 mM EDTA and 1ꢁ
Complete proteinase inhibitor (Roche). Cell homogenates were centrifuged
(1000g, 10min, 4°C). Supernatant was collected by centrifugation (140000g,
60 min, 4 °C). Pellets were resuspended in ice-cold Tris-HCl buffer (pH 7.4
at 25 °C) containing 1 mM EDTA and 1ꢁ Complete proteinase inhibitor
and stored at ꢀ80 °C until binding assays.
the cells were incubated for 30 min at room temperature. The reaction
was terminated by the addition of 10 μL of anti-cAMP acceptor beads
solution and 10 μL of lysis/detection buffer (the assay buffer containing
0.3% Tween-20, biotinylated cAMP, and donor bead) of AlphaScreen
cAMP Assay Kit (PerkinElmer). The plates were mixed for 30 s on an
orbital shaker, covered, and incubated in the dark for 12 h at room
temperature and then read on EnVision Multilabel plate reader
(PerkinElmer). The 50% effective concentration (EC50) and maximal
effect for agonists (Emax) were calculated using nonlinear regression
analysis (a three-parameter doseꢀresponse curve) in GraphPad Prism
software (GraphPad Software). Emax was expressed as a percentage of
that observed with 1 μM melatonin (= 100%).
Reentrainment for 8 h Advance of the Dark Phase in Mice
Running Wheel Activity. Male ICR mice (6 weeks old) were
purchased from CLEA Japan, Inc. (Tokyo, Japan). Mice were housed
individually in cages equipped with 20 cm diameter running wheels
under 12/12 light/dark cycle (lights-on at 6 a.m., lights-off at 6 p.m.)
with food and water ad libitum. A data acquisition system (ClockLab
software, Actimetrics, Wilmette, IL, USA) was used to record the
number of wheel rotations in 1 min bins. After the data were down-
loaded, the ClockLab software package (MATLAB 6.5, MathWorks,
Natick, MA, USA) was used to calculate the activity onset and display the
double-plotted actograms. Animals were habituated in the running
wheel cages until a reliable activity pattern was established (at least for
2 weeks). Following habituation period, the dark cycle was advanced by
8 h and animals were kept in a new 12/12 light/dark cycle for 14 days.
Compound 15 (3 and 30 mg/kg) was suspended in 0.5% methylcellu-
lose saline and administered in a volume of 10 mL/kg intraperitoneally
at 1 h before dark period once daily from the day of phase shifting. Days
to reentrainment was defined as the day which activity onset was
advanced more than 7 h from the day before phase shifting and
maintained for at least 3 consecutive days. In case animals did not meet
this criterion, days to reentrainment of these animals were defined as 14
days. All animal handling and procedures were carried out according to
the guidelines of the Takeda Experimental Animal Care and Use
Committee (Takeda Pharmaceutical Company Ltd., Osaka, Japan).
Affinity for the Human MT1/MT2 Receptors. The frozen
homogenate was thawed, suspended in ice-cold 50 mM Tris-HCl buffer
(pH 7.7 at 25 °C), and used for the binding assay. For the assay using
CHO cell membrane homogenate, 2 μL of DMSO solution of a test
compound was mixed with homogenate and 40 (MT1) or 80 pM (MT2)
2-[125I]iodomelatonin in a total volume of 200 μLandincubatedat25°Cfor
150 min. The binding reaction was terminated by rapid filtration using a cell
harvestor (PerkinElmer), followed by four 300 μL washes with ice-cold
incubation buffer. Nonspecific binding was defined in the presence of 1 μM
melatonin. GF/C filter plates were dried and radioactivity determined after
addition of 25 μL of Microscint-0 using a TopCount (PerkinElmer). The
50% inhibitory concentration (IC50) was calculated using nonlinear regres-
sion analysis (a three-parameter doseꢀresponse curve) in GraphPad Prism
software (GraphPad Software). The dissociation constant of the compound
for the receptor (Ki) was calculated using the following equation:
Ki ¼ IC50=ð1 þ L=KdÞ
where L and Kd represent the concentration and the affinity constant of
2-[125I]iodomelatonin in the binding assay, respectively. The specific binding
of 2-[125I]iodomelatonin for human MT1 receptors in hMT1-CHO/binding
was saturable. The one site specific binding plot in GraphPad Prism software
(GraphPad Software) of saturation isotherm using ligand concentrations
ranging from 5 to 160 pM revealed affinity binding sites with a Kd value of
33.9 ( 4.2 pM and a Bmax of 33.8 ( 1.6 fmol/mg protein (one experiment).
Human MT2 receptors expressed in hMT2-CHO showed lower affinities
than those of human MT1 receptors, and its Kd value and Bmax were 72.6 (
9.4 pM and 72.1 ( 4.4 fmol/mg protein (one experiment), respectively,
determined with ligand concentrations ranging from 5 to 160 pM.
’ ASSOCIATED CONTENT
S
Supporting Information. Elemental analyses for com-
b
pounds 8ꢀ27. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ81-6-6300-6546. Fax: þ81-6-6300-6306; E-mail:
Inhibition of Forskolin-Induced cAMP Formation in CHO
Cells Expressing Human Melatonin Receptor (MT1 or MT2).
CHO cell lines stably expressing human MT1 receptor (hMT1-CHO/
cAMP, cell line: TPCCB229) or human MT2 receptor (hMT2-CHO,
cell line: TPCCB0098) were used for cAMP assays. Cells were cultured
in F-12 nutrient mixture (Ham) supplemented with 10% fetal bovine
serum (FBS) and 500 μg/mL of Geneticin, 100 U/mL penicillin, and
100 μg/mL streptomycin in a 5% CO2/95% air atmosphere. hMT1-
CHO/cAMP cells or hMT2-CHO cells were suspended with assay
buffer (modified Hanks’ balanced salt solution (HBSS) containing
5 mM HEPES-buffer [pH 7.4], 0.1% BSA, 100 μM 3-isobutyl-1-
methylxanthine (IBMX), and 100 μM 4-(3-butoxy-4-methoxyphenyl)
methyl-2-imidazolidone (Ro20-1724) after cells were harvested at
confluence in Ca2þ-Mg2þ-free PBS containing 0.5 mM EDTA and
collected by centrifugation. Ten μL of hMT1-CHO/cAMP cells and
hMT2-CHO cells were plated at a density of 2 ꢁ 104 and 6 ꢁ 104 cells/
well on 384-well plates, respectively. After 10 μL of a test compound or
melatonin solution in the assay buffer were added, 10 μL of forskolin
solution in the assay buffer was added (final concentration 2 μM), and
Koike_Tatsuki@takeda.co.jp.
’ ACKNOWLEDGMENT
We thank Dr. Shigenori Ohkawa and Dr. Yuji Ishihara for their
helpful discussions.
’ ABBREVIATIONS USED
MLT, melatonin; SCN, suprachiasmatic nuclei; 4P-PDOT,
4-phenyl-2-propionamidotetralin; CHO, Chinese hamster ovary; PBS,
phosphate buffered saline
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