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

Article abstract of DOI:10.1021/jm980684+

6H-Isoindolo[2,1-a]indoles (5, 7, 10, 13), 5,6-dihydroindolo[2,1- a]isoquinolines (20, 21), and 6,7-dihydro-5H-benzo[c]azepino[2,1-a]indoles (23, 25, 27, 30) have been prepared as melatonin analogues to investigate the nature of the binding site of the me

Full text of DOI:10.1021/jm980684+

1050
J . Med. Chem. 2000, 43, 1050-1061
Articles
Ma p p in g th e Mela ton in Recep tor . 6. Mela ton in Agon ists a n d An ta gon ists
Der ived fr om 6H-Isoin d olo[2,1-a ]in d oles, 5,6-Dih yd r oin d olo[2,1-a ]isoqu in olin es,
a n d 6,7-Dih yd r o-5H-ben zo[c]a zep in o[2,1-a ]in d oles
Ru¨diger Faust, Peter J . Garratt,* Rob J ones, and Li-Kuan Yeh
Department of Chemistry, University College London, 20 Gordon Street, London WC1H 0AJ , U.K.
Andrew Tsotinis and Maria Panoussopoulou
Department of Pharmacy, Division of Pharmaceutical Chemistry, University of Athens,
Panepistimiopolis-Zografou, Athens 157 71, Greece
Theodora Calogeropoulou
Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vas. Constantinou Ave.,
Athens 116 35, Greece
Muy-Teck Teh and David Sugden
Physiology Division, School of Biomedical Sciences, New Hunts House, King’s College London, Guy’s Campus,
London SE1 1UL, U.K.
Received December 7, 1998
6H-Isoindolo[2,1-a]indoles (5, 7, 10, 13), 5,6-dihydroindolo[2,1-a]isoquinolines (20, 21), and 6,7-
dihydro-5H-benzo[c]azepino[2,1-a]indoles (23, 25, 27, 30) have been prepared as melatonin
analogues to investigate the nature of the binding site of the melatonin receptor. The affinity
of analogues was determined in a radioligand binding assay using cloned human mt1 and MT2
receptor subtypes expressed in NIH 3T3 cells. Agonist and antagonist potency was measured
using the pigment aggregation response of a clonal line of Xenopus laevis melanophores. The
2-methoxyisoindolo[2,1-a]indoles (7a -d ) showed much higher binding affinities than the parent
isoindoles (5a -e), and whereas 7a -c were agonists in the functional assay, 7d and 5a -e were
antagonists. The 2-ethoxyisoindolo[2,1-a]indoles (10a -d ) showed reduced binding affinities
compared to their methoxy analogues, while the 5-chloro derivative 13 showed a considerable
reduction in binding affinity and potency compared to 7a . The 10-methoxy-5,6-dihydroindolo-
[2,1-a]isoquinolines (21a -c) had higher binding affinities than the corresponding parent
indoloisoquinolines (20a -c) in the human receptor subtypes, and the parent compounds were
antagonists whereas the 10-methoxy derivatives were agonists in the functional assay. The
N-cyclobutanecarbonyl derivatives of both the parent (20d ) and 10-methoxyl (21d ) series had
similar binding affinities and were both antagonists with similar potencies. The 11-methoxy-
6,7-5H-benzo[c]azepino[2,1-a]indoles (25a -d ) had higher binding affinities than the corre-
sponding parent compounds (23a -d ) at the MT2 receptor but similar affinities at the mt1 site;
all of the compounds were antagonists in the functional assay. Changing 11-methoxy for 11-
ethoxy decreased the binding affinity slightly, and this was more evident at the MT2 receptor.
All of the derivatives investigated had either the same or a greater affinity for the human MT2
receptor compared to the mt1 receptor (range 1:1-1:132). This suggests that the mt1 and MT2
receptor pockets differ in their ability to accommodate alkyl groups in the indole nitrogen region
of the melatonin molecule. Two compounds (7c and 25c) were tested in functional assays on
recombinant mt1 and MT2 melatonin receptors. Compound 7c is a potent agonist with some
selectivity (44-fold) for the MT2 receptor, while 25c is an MT2-preferring antagonist. Increasing
the carbon chain length between N-1 of indole and the 2-phenyl group from n ) 1 through n
) 3 leads to a fairly regular decrease in the binding affinity, but, remarkably, when n ) 3, it
converts the methoxy compounds from melatonin agonists to antagonists. The Xenopus
melatonin receptor thus cannot accommodate an N-n-alkyl chain attached to a 2-phenyl
substituent with n > 2 in the required orientation to induce or stabilize the active receptor
conformation.
In tr od u ction
Sleep problems are common, particularly among the
elderly, with up to 50% of people over the age of 65
reporting trouble sleeping some or part of the time.1,2
Night time insomnia is associated with increased day-
time sleepiness, reduced motor and cognitive perfor-
mance, and reduced productivity in the workplace, and
it is an important cause of industrial and road traffic
* To whom correspondence may be addressed. Tel: 44-2076794620.
Fax: 44-2076797463. E-mail: p.j.garratt@ucl.ac.uk.
10.1021/jm980684+ CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/23/2000

Mapping the Melatonin Receptor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1051
accidents. Current hypnotic drugs are recommended
only for short-term treatment of insomnia, but concerns
about “hangover” effects and problems upon withdrawal
persist. Many people with occasional sleep problems
resort to self-medication, and over-the-counter sales of
medicines for sleep problems are increasing rapidly.3
Melatonin (N-acetyl 5-methoxytryptamine, 1a ), a hor-
mone synthesized and released from the pineal gland
at night, has been shown to have a hypnotic action in
animals4 and humans,5-7 and there has been consider-
able recent interest in the therapeutic potential of
melatonin analogues as hypnotics and as agents for
restoring circadian rhythms disturbed by jet-lag, shift-
work, and aging.8-10
binding at the recombinant mt1 and MT2 subtypes and
receptor activation using the pigment aggregation assay
on a clonal Xenopus melanophore cell line33 and, for two
specific compounds, have investigated the agonist and
antagonist behavior in a functional assay on recombi-
nant mt1 and MT2 melatonin receptors expressed in the
NIH 3T3 cell line. These new analogues use a 2-phenyl
substituent annelated to the indole nitrogen by an alkyl
chain of one to three carbons to retain the 3-ethanamide
side chain in an optimum conformation.
Ch em istr y
The isoindolo[2,1-a]indoles and benzo[c]azepeno[2,1-
a] indoles were prepared by the method of Kozikowski
et al.34,35 The appropriate N-acetyl tryptamine was
alkylated on the indole nitrogen with 2-bromobenzyl
bromide (3a ) or 3-(2-bromophenyl)-1-bromopropane (3b),
and the derived N-alkyl indole was then cyclized with
Pd(PPh3)4 to give the desired product (Scheme 1).
Attempts to apply the same methodology to the
synthesis of the indolo[2,1-a]isoquinolines were unsuc-
cessful owing to a preferred elimination of the 2-(2-
bromophenyl)bromoethane to 2-bromostyrene. Substi-
tution of the tosylate 14 for the bromide led to the
desired alkylation of the indole nitrogen with 3-formyl-
indoles 15 to give the N-alkyl indoles 16.36 Cyclization
of the N-2-(2-bromophenyl)ethylindoles 16 with
Pd(PPh3)4 gave the tetracyclic indolo[2,1-a]isoquinoline
derivatives 17, and the sequence of the Henry reaction
followed by reduction gave the amines 19 which were
not purified but were immediately acylated with the
appropriate reagent to give the desired indolo[2,1-a]-
isoquinolines 20 and 21 (Scheme 2).
The physiological actions of melatonin in regulating
seasonal and circadian rhythms are thought to be
mediated through a family of specific, high affinity,
G-protein-coupled cell membrane receptors.11 Radio-
ligand binding studies using 2-[125I]iodomelatonin 1b
have revealed a widespread, heterogeneous distribution
of binding sites throughout the central nervous sys-
tem.12 Binding sites are particularly abundant in tissues
known to respond to melatonin, including the retina,
the suprachiasmatic nuclei of the hypothalamus (the
site of the biological clock in mammals), the pars
tuberalis of the pituitary, and cerebral and tail arter-
ies.13 Two receptor subtypes (mt1 and MT2) have been
cloned in mammals14,15 which, when expressed in host
cells, show the general pharmacological characteristics
of native melatonin receptors. A third subtype (termed
Mel1c), not detected in mammals, has been cloned from
chicken, Xenopus, and zebra fish.16
Notwithstanding the renewed interest in melatonin
and its actions in recent years, the pharmacology of
melatonin is in its infancy. In a series of studies we have
sought to understand how melatonin binds to and
activates its receptor17-21 and to use the knowledge
gained to design subtype specific receptor agonists and
antagonists which will be useful tools for defining the
full physiological and pathophysiological role of this
hormone. We and others22 have shown that the 5-meth-
oxyl group of melatonin is an important site for binding
to the receptor, but it is not an essential requirement
for agonist activity.17,21 The active conformation of the
3-ethanamide side chain has also been established from
studies with conformationally restricted indole18,21,23 and
non-indole24-28 analogues. Models for the binding of
melatonin to its receptor have been formulated on the
basis of structure-activity data, analogies with other
G-protein-coupled receptors for small molecular weight
ligands (such as the catecholamines and serotonin), and
comparative molecular field analysis.19,21,29-31 Recent
site-directed mutagenesis studies on the ovine mt1
subtype32 have given experimental support to aspects
of this model. We have now examined the role of the
indole nitrogen region of the molecule on radioligand
P h a r m a cology
The affinity of the analogues was determined in
competition radioligand binding assays using 2-[121I]-
iodomelatonin (specific activity 2200 Ci/mol, DuPont,
Stevenage, U.K.) as described previously37 on the re-
combinant human mt1 and MT2 subtypes expressed in
NIH 3T3 cells,14,15 kindly provided by Dr. S. M. Reppert
(Harvard Medical School, Boston, MA).The biological
activity of the analogues was assessed in a well-
established, specific model of melatonin action, the
pigment aggregation response of Xenopus laevis mel-
anophores.38,39 In these cells many thousands of black
pigment granules are normally distributed throughout
the cell, and addition of melatonin induces their rapid
movement toward the center of the cell. This response
is termed pigment aggregation and can be quantified
by measuring the change in light absorbance of the cells
as the pigment concentrates near the cell center. In the
present study, a clonal melanophore cell line, generously
provided by Dr. Michael Lerner (Department of Der-
matology, University of Texas), was used.
The agonist and antagonist potency of two analogues
(7c and 25c) was also determined on mt1 and MT2
receptors by measuring their ability to alter the con-
centration of intracellular cyclic AMP.
Resu lts a n d Discu ssion
We have previously examined the limiting structural
requirements, the optimal N-alkanoyl group, the pre-
ferred conformation of the 3-amidoethane side chain,

1052 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Faust et al.
Sch em e 1
and the range of C-5 substituents tolerated in melatonin
agonists. In continuing our attempts to define the
characteristics of the melatonin receptor, we have
turned our attention to the indole nitrogen region of the
molecule. It has previously been shown that a methyl
substituent is reasonably well tolerated on the indole
nitrogen but that longer alkyl groups or the benzyl
group markedly decreased binding affinity.18,19,40,41 Fol-
lowing the observation that 2-iodomelatonin was a more
potent agonist than melatonin itself, a number of groups

Mapping the Melatonin Receptor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1053
Sch em e 2
have prepared 2-substituted melatonin analogues and
have shown that these can be more potent than mela-
tonin.19,22,42,43 This increase in potency has been ascribed
to both the increased population of the active conforma-
tion for binding to the melatonin receptor and to the
presence of a C-2 binding site. To take advantage of this
increase in binding while examining the steric require-
ments at the indole nitrogen, we decided to synthesize
a series of indoles in which a 2-phenyl substituent was
attached at the ortho position to the indole nitrogen by
n methylene groups (n ) 1-3).
cells. Only partial sequences of Xenopus mt1 and MT2
receptors subtypes have been cloned.15 A correlation of
potency (IC50) in the pigment aggregation assay with
binding affinity at human mt1 or MT2 receptor subtypes
for the 28 melatonin antagonists in Tables 1, 2, and 3
was not significant (potency vs mt1 affinity r2 ) 0.002,
p ) 0.80; potency vs MT2 affinity r2 ) 0.063, p ) 0.20).
This lack of correlation suggests that melatonin-induced
pigment aggregation is not mediated by either mt1 or
MT2 receptor subtypes.
The 6H-isoindolo[2,1-a]indoles 5a -e with a proton at
R1 exhibit little change in binding affinity on going from
acetyl to butanoyl as the acylating group as is shown
by N-acyl 5-methoxytryptamines analogues 45 (Table
1), but cyclic N-acyl groups reduce affinity. The series
has higher binding affinities at the MT2 receptor and
the MT2/mt1 selectivities for the alkanoyl systems 5a -c
were all around 10-fold. All of these compounds were
weak antagonists in the Xenopus assay. Introduction
of a methoxyl group at R1 led to the expected increase
in binding affinity, the alkanoyl systems 7a -c being
comparable to melatonin but with a much greater
selectivity for MT2 over mt1. These compounds were
highly potent agonists in the Xenopus assay (Table 1,
Figure 1).
The cloning of the melatonin receptor14,44 and the
identification of high affinity melatonin subtypes11,15
have allowed the comparison of agonists at the two sites,
and for all compounds examined, the binding affinity
at the MT2 receptor is equal to or greater than that at
the mt1 receptor. As part of our study we have therefore
examined the binding affinities of a series of new
compounds at both the mt1 and MT2 receptor in order
to determine if there are marked differences in the
binding site of the receptors for this region of the
melatonin molecule.
The compounds prepared were characterized by stan-
dard methods as described in the Experimental Section.
The results of the binding and melanophore assays for
6H-isoindolo[2,1-a]indoles, 5,6-dihydroindolo[2,1-a]iso-
quinolines and 6,7-dihydro-5H-benzo[c]-azepino-[2,1-a]-
indoles are shown in Tables 1, 2, and 3, respectively.
The melatonin receptor subtype mediating pigment
aggregation has not been formally defined. Xenopus
leavis melanophores do express a Mel1c subtype, as these
cells were the source of the cDNA used for the first
melatonin receptor cloning,44 but it is not known if
Xenopus mt1 or MT2 subtypes are expressed in these
One analogue (7c), which was 89-fold selective for the
MT2 receptor (compared to the mt1 receptor, Table 1)
in radioligand binding assays, was also shown to have
potent agonist activity on the human mt1 and MT2
receptors expressed in NIH 3T3 cells (Figure 2).
Compound 7c, like melatonin, inhibited forskolin
stimulation of cyclic AMP (EC50, mean ( SEM, n ) 4:
melatonin, mt1 0.08 ( 0.04 nM, MT2 0.15 ( 0.02 nM;
7c, mt1 2.19 ( 0.8 nM, MT2 0.05 ( 0.01 nM). In this

1054 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Faust et al.
Ta ble 1
receptor binding (Ki, nM)
Xenopus melanophores
selectivity
MT2/mt1
compound
R1
R
human mt1
human MT2
agonist (EC50, nM)
antagonist (IC50, nM)
melatonin
0.66 ( 0.34
603 ( 37.9
0.33 ( 0.14
44.7 ( 21.8
2
13
0.08 ( 0.005
NA
NA
NA
2460 ( 460
3670
luzindolea
N-CBCPTb
5a
5b
5c
5d
5e
7a
7b
7c
H
H
H
H
Me
Et
Pr
c-C3H5
c-C4H7
Me
Et
Pr
c-C3H5
Me
Et
Pr
c-C3H5
Me
195 ( 70
17.0 ( 6.3
17.4 ( 4.8
12.0 ( 3.3
224 ( 6.7
1120 ( 229
0.06 ( 0.03
0.17 ( 0.06
0.05 ( 0.10
0.95 ( 0.42
0.21 ( 0.08
0.26 ( 0.05
0.16 ( 0.02
5.50 ( 0.98
33.9 ( 4.6
11
12
14
3
NA
NA
NA
NA
24600 ( 7600
4571 ( 1260
2340 ( 650
4170 ( 190
3160 ( 70
NA
NA
NA
7410 ( 490
NA
204 ( 49
174 ( 45
776 ( 228
1200 ( 406
1.82 ( 0.44
4.37 ( 0.72
4.47 ( 1.10
46.8 ( 9.7
18.2 ( 3.5
14.5 ( 3.1
8.91 ( 1.60
234 ( 60
H
1
NA
OMe
OMe
OMe
OMe
OEt
OEt
OEt
OEt
Cl
30
26
89
49
87
56
56
42
8
2.29 ( 1.0
21.9 ( 0.98
5.75 ( 0.38
NA
316 ( 70.8
178 ( 8.0
204 ( 4.65
NA
7d
10a
10b
10c
10d
13
NA
NA
28200 ( 6800
NA
282 ( 54
2090
a
b
See ref 58. N-(Cyclobutanecarbonyl)-2-phenyltryptamine, ref 19. NA ) no agonist or antagonist effect detected at 100 mM. mt1 and
MT2 data are the mean of quadruplicate determinations. Agonist and antagonist data on melanophores are the mean of triplicate
experiments.
Ta ble 2
receptor binding (Ki, nM)
Xenopus melanophores
agonist (EC50, nM) antagonist (IC50, nM)
selectivity
(MT2/mt1
compound
R1
R
human mt1
human MT2
melatonin
luzindolea
N-CBCPTb
20a
20b
20c
20d
21a
21b
21c
0.66 ( 0.34
603 ( 37.9
0.33 ( 0.14
44.7 ( 21.8
2
13
0.08 ( 0.005
NA
NA
NA
NA
NA
NA
12.6 ( 2.12
6.03 ( 0.66
9.33 ( 1.75
NA
NA
2460 ( 460
3670
H
H
H
H
OMe
OMe
OMe
OMe
Me
Et
Pr
c-C4H7
Me
Et
Pr
c-C4H7
389 ( 196
148 ( 130
70.8 ( 43.1
2040 ( 630
7.24 ( 0.43
5.89 ( 0.34
4.07 ( 0.24
437 ( 27
36.3 ( 21.2
9.33 ( 5.3
3.7 ( 1.2
355 ( 196
0.51 ( 0.26
0.12 ( 0.21
0.20 ( 0.018
324 ( 179
11
16
19
6
14
49
20
1
8910 ( 3290
2290 ( 250
447 ( 67
3720 ( 250
<100000
79400 ( 3600
42700 ( 8000
3720 ( 1020
21d
a
b
See ref 58. N-(Cyclobutanecarbonyl)-2-phenyltryptamine, ref 19. NA ) no agonist or antagonist effect detected at 100 mM. See
footnotes, Table 1, for methods.
biological assay 7c was considerably more potent (44-
fold) at the MT2 receptor subtype than the mt1 subtype
and is the most selective agonist yet described.
(Table 2). These derivatives were also antagonists in the
Xenopus assay, and 20c (R ) Pr) was reasonably potent.
Introducing the methoxyl group at R1 (21a -c) substan-
tially increased the binding affinity, and the compounds
were fairly potent agonists in the Xenopus assay. When
examined as antagonists, however, these compounds
also showed very weak antagonist potency. The N-
cyclobutanecarbonyl derivatives 20d (R1 ) H) and 21d
(R1 ) OMe) both exhibited similar low binding affinity
at the MT2 receptor and the same antagonist potency,
suggesting that the methoxyl group is not operating as
an important binding site in 21d at MT2 or melanophore
receptors.
The cyclopropanecarbonyl derivative 7d had a high
binding affinity at both subtypes but was a weak
antagonist in the Xenopus assay. Changing R1 from
methoxy to ethoxy led to some diminution in binding
affinity and melanophore potency, but otherwise the
compounds mirrored the methoxy derivatives. The
trends for the 6H-isoindolo[2,1-a]indoles series of com-
pounds are very similar to those found for the corre-
sponding 2-phenyltryptamine derivatives except that
the parent N-alkanoyl 2-phenyltryptamines were ago-
nists in the Xenopus assay.19
The
6,7-dihydro-5H-benzo[c]azepino[2,1-a]indoles
The 5,6-dihydroindolo[2,1-a]isoquinolines 20a -c had
similar binding affinities to the isoindoloindoles and had
relatively similar selectivities for the MT2 receptor
showed a profile of binding affinities similar to the other
two series. Introducing the methoxyl group at R1 in-
creased the binding affinity substantially at the MT2

Mapping the Melatonin Receptor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1055
Ta ble 3
receptor binding (Ki, nM)
Xenopus melanophores
selectivity
compound
R1
R
human mt1
human MT2
(MT2/mt1
agonist (EC50, nM)
antagonist (IC50, nM)
melatonin
luzindolea
N-CBCPTb
23a
23b
23c
23d
23e
25a
25b
25c
25d
25e
27a
27b
27c
27d
30
0.66 ( 0.34
603 ( 37.9
0.33 ( 0.14
44.7 ( 21.8
2
13
0.08 ( 0.005
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
2460 ( 460
3670
H
H
H
H
Me
Et
Pr
c-C3H5
c-C4H7
Me
Et
Pr
c-C3H5
c-C4H7
Me
Et
Pr
240 ( 76
115 ( 30
79.4 ( 23
117 ( 46
251 ( 111
275 ( 50
52.5 ( 16
66.1 ( 18
501 ( 123
759 ( 165
513 ( 152
269 ( 77
257 ( 70
2950 ( 845
288 ( 110
224 ( 55
1
2
1
2
1
44
37
132
42
24
4
12
40
35
57
3470 ( 520
1410 ( 1240
1860 ( 120
1180 ( 30
3470 ( 1330
2400 ( 620
1100 ( 360
525 ( 216
550 ( 103
3470 ( 1590
37100 ( 14200
3980 ( 590
380 ( 90
45.7 ( 15
63.1 ( 18
52.5 ( 37
309 ( 92
H
OMe
OMe
OMe
OMe
OMe
OEt
OEt
OEt
OEt
Cl
6.31 ( 1.94
1.41 ( 0.45
0.50 ( 0.14
12.0 ( 5.3
31.6 ( 10.7
117 ( 18.4
21.9 ( 3.4
6.46 ( 1.24
85.1 ( 14
5.01 ( 3.98
c-C3H5
Me
224 ( 33
282 ( 63
NA
a
b
See ref 58. N-(Cyclobutanecarbonyl)-2-phenyltryptamine, ref 19. NA ) no agonist or antagonist effect detected at 100 mM. See
footnotes, Table 1, for methods.
F igu r e 1. Changes in pigment distribution in Xenopus leavis
melanophores. (A) Aggregation of pigment in response to
F igu r e 2. Inhibition of cyclic AMP synthesis induced by
varying concentrations of melatonin, 7a (solid semicircle), 21a
forskolin by melatonin and 7c in NIH 3T3 cells expressing
(2), 20a ([), 5a (1), 23a (f), 25a (b). The change in pigment
human mt1 (A) or MT2 (B) melatonin receptor subtypes. Cells
distribution was determined by measuring cell absorbance
were treated with forskolin (10-5 M, 10 min) in the absence
before (Ai) and 60 min after (Af) addition of analogues. (B)
or presence of varying concentrations of agonists. For each cell
sample cyclic AMP was measured in duplicate by radioimmu-
noassay. Data are expressed as the percentage inhibition of
Antagonism of melatonin-induced (10-9 M) pigment aggrega-
tion by varying concentrations of 20a (]), 5a (3), 23a (f), and
25a (O). Each point is the mean ( SEM of quadruplicate wells.
the response to forskolin alone. Each point is the mean ( SEM
Error bars are omitted when the SEM was less than 0.02.
of quadruplicate cell samples. Error bars are omitted when
the SEM was less than the area covered by the symbol. *p <
receptor, and substitution of ethoxy for methoxy led to
a decrease in binding affinity. In the Xenopus assay,
however, no agonist activity was detected in any of these
compounds up to 10-4 M, and all the compounds,
including the methoxy and ethoxy derivatives, were
antagonists. The methoxy derivative 25c (R ) Pr)
showed the largest selectivity for MT2 over mt1 (132-
fold) of all the derivatives investigated in this study.
Furthermore, 25c was shown to act as a melatonin
receptor antagonist not only on Xenopus melanophores
but also on the mt1 and MT2 receptor subtypes ex-
pressed in NIH 3T3 cells (Figure 3). Compound 25c
0.05, ***p < 0.001 using Student’s t-test compared to forskolin-
stimulated cAMP level.
antagonized melatonin inhibition of forskolin stimula-
tion of cyclic AMP, but it did not have any agonist
activity at these receptor subtypes as no significant
inhibition of forskolin stimulation of cyclic AMP was
detected at up to 10 mM (data not shown). The antago-
nist potency of 25c (IC50, concentration producing half-
maximal antagonism of the melatonin response, mean
( SEM, n ) 3-4) was 7.7 ( 1.5 nM at the mt1 subtype

1056 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Faust et al.
F igu r e 3. Antagonism of melatonin inhibition of cyclic AMP
synthesis stimulated by forskolin in NIH 3T3 cells expressing
human mt1 (A) and MT2 (B) receptor subtypes. Cells were
pretreated (1 h) with vehicle or 25c (10-7 M for mt1 cells or
10-9 M for MT2 cells) before addition of forskolin (10-5 M) plus
melatonin (10-9 M) for 10 min. These concentrations were close
to the Ki values for 25c at each receptor subtype (Table 3).
Each bar represents the mean ( SEM of quadruplicate cell
samples. Significantly different from forskolin plus melatonin
alone, *p < 0.05, †p < 0.001 compared to forskolin-treated cells,
**p < 0.01 compared to melatonin-treated cells using Student’s
t-test.
F igu r e 4. PM3 minimized energy structures for compounds
7a , 21a , and 25a showing similar disposition of the methoxyl
with the ethanamide side chains.
and 0.14 ( 0.05 nM at the MT2 subtype). Thus 25c has
some selectivity (55-fold) for the MT2 receptor subtype.
An interesting difference can be noted for the substi-
tution of chlorine for methoxyl between the isoindoloin-
dole and benzazepinoindole series. Substitution of chlo-
rine in the isoindoloindole series (13) greatly reduces
the binding affinity for both the mt1 and MT2 receptors,
13 showing a lower binding affinity than the parent
compound 5a . It is, however, unlike 5a in being a weak
agonist rather than a weak antagonist (Table 1).
Substituting a chlorine in the benzazepinoindole series
gives a compound (30) with binding affinities very
similar to those of the methoxyl derivative 25a . The
binding affinities of 25a and 30 are similar to the parent
compound 23a at the mt1 receptor, but both methoxy
and chloro compounds have much higher binding af-
finities at the MT2 receptor. The chloro compound 30
is, however, 8-fold more potent as an antagonist than
25a , which is itself little more potent than 23a (Table
3).
In the present work, the three series of compounds
differ mainly in the number of atoms bridging the indole
to phenyl ring, although the changes in ring size that
this engenders results in a conformational change
between the phenyl ring and the indole portion of the
molecule. This is illustrated in Figure 4, which shows
energy minimized PM3 structures for 7a , 21a , and 25a .
In all three compounds, however, the methoxyl and
ethanamide side chain have a similar relationship to
each other. While introduction of a 2-phenyl group gave
agonists with improved binding affinity compared to
melatonin,19 a 2-phenyl ring bridged ortho to the indole
reduces affinity. A single carbon is more easily accom-
modated than two, which in turn is better tolerated than
three carbons. The requirements at the binding pocket
of the MT2 subtype appear less stringent than those at
the mt1 receptor, resulting in several analogues with
considerable MT2 subtype specificity. These included
compounds which mimicked the action of melatonin on
Xenopus melanophores (agonists) and others which
acted as competitive antagonists. Recent studies with
agonist analogues45 have also indicated that the MT2
subtype tolerates changes at the 5-position more readily
than the mt1 subtype, while changes to the N-acyl group
have a similar effect at both receptor subtypes.
The finding that many of the present series of
melatonin analogues acted as melatonin receptor ago-
nists or antagonists in the Xenopus melanophore model
system does not necessarily imply that they will have
the same activity at native or recombinant mt1 and MT2
receptor subtypes. However, two representative com-
pounds (7c and 25c) have been demonstrated to have
the same action (agonist and antagonist, respectively)
on human mt1 and MT2 receptor subtypes expressed in
NIH 3T3 cells (Figures 2 and 3). It will be interesting
to examine their activity at native melatonin receptors
in other mammalian model systems.
It was initially suggested that the mt1 subtype medi-
ated melatonin’s ability to entrain free-running circa-
dian rhythms because of the high level of expression of
its mRNA within the primary pacemaker of the brain,
the suprachiasmatic nuclei (SCN) of the hypothala-
mus.14 However, melatonin retains its phase-shifting
ability in mice in which the mt1 subtype has been
deleted,46 and receptor antagonists having MT2 subtype
selectivity have recently been shown to reduce melato-
nin-induced phase advances,47 indicating the impor-
tance of the MT2 receptor subtype in the actions of
melatonin on circadian rhythms. It follows that potent,
selective MT2 receptor subtype agonists might be useful
in treating various circadian rhythm disorders.8-10
Disturbances of circadian physiology are not simply an

Mapping the Melatonin Receptor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1057
using a curve-fitting program.53 For evaluation of antagonist
potency, cells were treated with vehicle (1% DMSO or metha-
nol) or varying concentrations (10-4-10-9 M) of the analogues
for 60 min before melatonin (10-9 M) was added. The concen-
tration of analogue reducing melatonin-induced pigment ag-
gregation by 50% (IC50) was determined.
inconvenience to transatlantic travellers; chronic rhythm
disturbance experienced by many shift workers is as-
sociated not only with sleep problems but also with
gastrointestinal disturbances48 and increased incidence
of cardiovascular disease49 and may contribute to in-
creased risk of breast cancer.50 It has been suggested
that melatonin’s sleep promoting action is related to its
ability to influence the circadian sleep timing mecha-
nisms by acting on the SCN,51 although acute sleep-
inducing effects of melatonin have been reported which
may be mediated by actions on other brain regions.4-7
Selective and potent MT2 agonists and antagonists such
as 7c and 25c, and compounds that can now be designed
with them as leads, will make possible experiments to
test the role of the receptor subtypes in sleep timing
mechanisms and in the acute hypnotic action of mela-
tonin. Continuing attempts to define the characteristics
of melatonin required for ligand binding, subtype se-
lectivity, and receptor activation are needed to provide
the tools necessary to define the full range of its
physiological actions and to realize the therapeutic
potential of this molecule.
NIH 3T3 cells expressing human mt1 or MT2 receptor
subtypes were seeded into 24-well plates and cultured for 48
h until they reached confluence (3-4 104 cells/well). After they
were washed twice with DMEM, 3-isobutyl-1-methylxanthine
(IBMX, 2.5 × 10-4 M) was added for 10 min at 37 °C before
the addition of the adenylate cyclase activator forskolin (10-5
M) in the absence or presence of melatonin or 7c to inhibit
stimulation of cyclic AMP. All drugs were diluted in DMEM
and contained IBMX as described above. After a 10 min
incubation at 37 °C, the medium in each well was aspirated
and 300 mL of ice-cold acetic acid (50 mM) was added, and
cells were scraped from the plate. The lysate was then collected
and boiled for 3 min. Antagonist action was determined by
treating cells with forskolin (10-5 M) and melatonin (10-9 M)
in the presence or absence of 25c. All samples were kept at
-20 °C until cyclic AMP was determined by RIA following
acetylation as described previously,54,55 using cyclic AMP 2′-
O-succinyl [125I]-tyrosine methyl ester (DuPont, Stevenage,
U.K.).
Physical data for one of each type of analogue is given, and
the data for the remaining analogues can be found in the
Supporting Information.
Exp er im en ta l Section
Melting points were determined on a Reichert melting point
apparatus or in glass capillary tubes on an Electrothermal
9100 apparatus and are uncorrected. EI mass spectra were
recorded on a VG ZAB-2F mass spectrometer, CI mass spectra
on a VG 12-250 mass spectrometer, and FAB mass spectra on
a M550 mass spectrometer. Only molecular ions (M+) or M+
+ 1 ions, base peaks, and the next two peaks due to ions of
maximum abundance are given. IR spectra were recorded on
Perkin-Elmer 883, PE-983, or 1650 FTIR spectrometers, using
KBr pellets unless otherwise stated. NMR spectra were taken
in CDCl3 unless otherwise stated. 1H NMR spectra were taken
on either a Varian VXR-400 or at 75 MHz on a Bruker AC300
MHz spectrometer, and the spectra are reported in δ. 13C NMR
spectra were recorded at 100 MHz on a Varian VXR-400
spectrometer and are reported in δ.
DC-Alufolien plates (Kieselgel 60 F254, Schichtdicke 0.2 mm,
Merck) were used for analytical TLC and were visualized with
ultraviolet light or developed with p-anisaldehyde, iodine, or
ninhydrin. Flash chromatography was performed using Sorbsil
c60-A silica as the stationary phase. Microanalyses were
carried out by either Service Central de Microanalyses of
CNRS in Vernaison, France, or Microanalytical Section, Dept.
of Chemistry, UCL.
NIH 3T3 cells expressing recombinant human mt1 or MT2
receptor subtypes were grown in complete Dulbecco’s Eagle
medium (DMEM) containing 100 i.u./mL penicillin, 0.1 mg/
mL streptomycin, 4 mM L-glutamine, 10% fetal bovine serum
(Imperial Laboratories, England, U.K.), and Geneticin (G418;
1 mg/mL; GIBCO/BRL) in humidified 5% CO2/95% air at 37
°C. The affinity constant (Kd) of the mt1 receptor subtype was
51 ( 7.8 pM, and the maximal binding density (Bmax) was
165.54.7 fmol/mg protein; the Kd of the MT2 receptor subtype
was 73.4 ( 9.9 pM, and the Bmax was 70.1 ( 0.6 fmol/mg
protein.
Melanophore cells were grown in 96-well tissue culture
plates, and growth medium33,52 was replaced with 0.7 × L-15
culture medium 18 h before analogues were tested. Initial
absorbance of cells (Ai, 630 nm) was measured in each well
using a Bio-Tek microtiter plate reader (model EL3115,
Anachem, U.K.), then cells were treated with the concentra-
tions of the analogues indicated. All experiments used qua-
druplicate wells at six concentrations of analogue. The final
absorbance (Af) was measured after 60 min, and the fractional
change in absorbance (1 - Af/Ai) was calculated. Vehicle did
not alter pigment granule distribution itself or inhibit re-
sponses to melatonin. The concentration of analogue producing
50% of the maximum agonist response (EC50) was determined
Gen er a l P r ep a r a t ion for t h e N-Alk yl Der iva t ives
4a -e, 6a -d , 9a -d , 12, 22a -e, 24a -e, 26a -d , a n d 29. NaH
(80%, 0.10 g, 2.3 mmol) was added to a stirred solution of the
required N-acylated 2-(indol-3-yl)ethylamine (0.9 mmol) in dry
THF (3 mL) at 0 °C, and a solution of the required 2-bro-
moalkylbromide (1.9 mmol) in dry THF (3 mL) was then added
slowly. The resulting mixture was stirred at room temperature
for 3 h, and saturated aqueous ammonium chloride was then
added. The mixture was extracted with ethyl acetate (2 × 50
mL), the organic layers were combined, washed with brine,
and dried (MgSO4). The N-acyl 2-(N-alkylindol-3-yl)ethyl-
amines were isolated by flash chromatography and, if solids,
recrystallized from ethyl acetate/petroleum ether 60-80.
Gen er a l P r ep a r a tion for th e Tetr a cyclic Der iva tives
5a -e, 7a -d , 10a -d , 13, 23a -d , 25a -e, 27a -d , a n d 30. The
required N-acyl 2-(N-alkylindol-3-yl)ethylamine (0.5 mmol),
prepared by the above method, was added to tetrakis(tri-
phenylphosphine)palladium(0) (ca. 30 mg) and potassium
acetate (ca. 50 mg) in dimethylamine (8 mL), and the mixture
was stirred at 160 °C under N2 for 16 h. The solvent was then
removed under reduced pressure and ethyl acetate (50 mL)
added to the residue. The mixture was washed with water (50
mL) and the aqueous phase extracted with ethyl acetate (3 ×
50 mL). The organic phases were combined, washed with water
(80 mL), brine, and dried (MgSO4). The tetracyclic ethana-
mides were isolated by flash chromatography and, if solids,
recrystallized from ethyl acetate/petroleum ether 60-80.
N-Acet yl 2-[1-(2-b r om ob en zyl)in d ol-3-yl]et h a n a m in e
(4a ): white solid, 60%, mp 110-115 °C; 1H NMR δ 1.9 (s, 3H),
2.97 (t, J ) 6.7 Hz, 2H), 3.57 (q, J ) 6.8 Hz, 2H), 5.32 (s, 2H),
5.68 (br s, 1H), 6.56 (dd, J ) 5.7, 3.5 Hz, 1H), 7.13 (m, 3H),
7.18 (m, 1H), 7.23 (d, J ) 8.4 Hz, 1H), 7.58 (dd, J ) 5.7, 3.7
Hz, 1H), 7.62 (d, J ) 7.5 Hz, 1H); 13C NMR δ 23.3, 25.2, 39.8,
50.0, 109.7, 112.5, 119.0, 119.4, 122.1, 122.3, 126.1, 127.8,
127.9, 128.1, 129.1, 132.8, 136.5, 136.7, 170.0; IR 3293, 2929,
1652, 1556, 1466, 1441, 1025, 740 cm-1; MS m/e 373, 372, 371,
370 (100), 314, 313, 312, 311, 300, 298, 171, 169. C19H19N2O-
79Br requires 370.2719; Found: 370.2735.
N-P r op a n oyl 2-[1-(2-b r om ob en zyl)in d ol-3-yl]et h a n -
a m in e (4b): yellow solid, 91%, mp 82-84 °C (lit.56 83-84 °C).
N-Bu tan oyl 2-[1-(2-br om oben zyl)in dol-3-yl]eth an am in e
(4c): yellow solid, 85%, mp 79-80 °C.
N-Cyclop r op a n eca r bon yl 2-[1-(2-br om oben zyl)in d ol-
3-yl]eth a n a m in e (4d ): yellow solid, 80%, mp 117.5-118.5 °C.
N-Cyclobu ta n eca r bon yl 2-[1-(2-br om oben zyl)in d ol-3-
yl]eth a n a m in e (4e): white solid, 67%, mp 107-108 °C.

1058 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
N-P r op a n oyl 2-[1-(2-br om oben zyl)-5-m eth oxyin d ol-3-
Faust et al.
N-Acetyl 2-(9-ch lor o-6H-isoin d ololo[2,1-a ]in d ol-11-yl)-
eth a n a m in e (13): yellow solid, 62%, mp 186-189 °C; 1H NMR
δ 1.85 (s, 3H), 3.21 (m, 2H), 3.60 (m, 2H), 5.05 (s, 2H), 5.6 (br
s, 1H), 7.16 (m, 1H), 7.25 (m, 1H), 7.33 (m, 1H), 7.44 (m, 1H),
7.48 (d, J ) 7.6 Hz, 1H), 7.08 (m, 1H), 7.82 (d, J ) 7.7 Hz,
1H); 13C NMR δ 23.6, 24.4, 40.3, 48.4, 102.5, 103.2, 110.0,
112.4, 120.9, 123.5, 126.6, 128.3, 129.3, 132.5, 133.3, 141.7,
153.3, 173.5; IR 3296, 2922, 1652, 1446, 1361, 1060, 788, 763
cm-1; MS m/e 327, 326, 325 (100), 324, 268, 267, 266, 265, 254,
252. C19H18N2O35Cl (M+ + H) requires 325.1108; Found:
325.1097. Anal. (C19H17ClN2O) C, H, N, Cl.
1
yl]eth a n a m in e (6a ): yellow oil, 91%; H NMR δ 1.08 (t, J )
7.6 Hz, 3H), 2.16 (q, J ) 7.6 Hz, 2H), 2.93 (t, J ) 6.7 Hz, 2H),
3.58 (q, J ) 6.5 Hz, 2H), 3.84 (s, 3H), 5.28 (s, 2H), 5.54 (br s,
1H), 6.55 (m, 1H), 6.83 (dd, J ) 8.8, 2.5 Hz, 1H), 7.04 (d, J )
2.3 Hz, 1H), 7.11 (m, 3H), 7.57 (m, 1H); 13C NMR δ 9.9, 25.4,
29.9, 39.6, 50.2, 56.0, 100.8, 110.6, 112.1, 112.4, 122.3, 126.7,
127.8, 128.2, 128.4, 129.2, 131.1, 132.8, 135.0, 154.2, 174.0;
IR 3298, 2929, 1641, 1454, 1228, 1043, 796 cm-1; MS m/e 417,
416, 415 (100), 414, 344, 343, 342, 341, 330, 328, 171, 169.
C21H24N2O279Br (M+ + H) requires 415.1010; Found: 415.1024.
N-Bu ta n oyl2-[1-(2-br om oben zyl)-5-m eth oxyin d ol-3-yl]-
eth a n a m in e (6b): yellow solid, 89%, mp 75-76 °C.
Tolu en e-4-su lfon ic Acid 2-(2-Br om op h en yl)eth yl Ester
(14). LiAlH4 (1.77 g, 46.64 mmol) was added in one portion at
0 °C to a stirred solution of 2-bromophenylacetic acid (2.50 g,
11.6 mmol) in tetrahydrofuran (150 mL). The resulting sus-
pension was allowed to reach ambient temperature and then
refluxed for 18 h. The mixture was then chilled (0 °C) and
quenched by the careful dropwise addition of H2O (12 mL).
The resulting white suspension was allowed to reach room
temperature and stirred for 15 min. The precipitated salts
were removed by filtration, and the filtrate was taken up in
ethyl acetate (150 mL). The organic layer was washed with
H2O (2 × 25 mL) and brine (25 mL), dried (Na2SO4), and
concentrated in vacuo to give 2-bromophenylethyl alcohol as
a colorless oil (2.00 g, 9.98 mmol, 86%) which was used without
any further purification in the next step. A solution of
2-bromophenylethyl alcohol (2.16 g, 10.75 mmol) in dichlo-
romethane (10 mL) was added dropwise at 0 °C to a stirred
solution of p-toluenesulfonyl chloride (5.74 g, 30.11 mmol) in
pyridine (10 mL) and dichloromethane (4 mL). After the
solution stood overnight at -18 °C, the TLC (dichloromethane/
ethyl acetate) (90:10) Rf ) 0.22, showed that the reaction was
complete. The solution was poured into ice-H2O (100 mL), the
mixture stirred for 30 min, the layers separated, and the
aqueous phase was extracted with dichloromethane (3 × 25
mL). The combined organic phase was washed with 2 N HCl
(5 mL), 5% NaHCO3 (5 mL), and brine (2 × 25 mL) and dried
(Na2SO4). The solvent was then removed under vacuum to give
14 as a colorless, viscous oil (3.32 g, 9.4 mmol, 87%): 1H NMR
δ 2.42 (s, 3H), 3.07 (t, 2H, J ) 6.8 Hz), 4.22 (t, 2H, J ) 6.8
Hz), 7.27-7.07 (m, 5H), 7.44 (d, 1H, J ) 7.9 Hz),7.67 (d, 2H,
J ) 8.2 Hz).
1-[2-(2-Br om op h en yl)et h yl]-1H -in d ole-3-ca r b oxa ld e-
h yd e (16a ). A solution of 14 (3.09 g, 8.70 mmol) in acetonitrile
(5 mL)was added dropwise to a reluxing, stirred suspension
of indole-3-carboxaldehyde (15a ) (1.26 g, 8.68 mmol) and
potassium carbonate (2.41 g, 17.41 mmol) in acetonitrile (30
mL). The mixture was refluxed for 4.5 h, cooled to room
temperature, and then poured into ice-H2O (100 mL). The
solution was stirred for 30 min, ethyl acetate (150 mL) was
added, and the mixture separated. The organic layer was
washed with H2O (2 × 50 mL) and brine (25 mL) and dried
(Na2SO4). The solvent was removed under vacuum, and the
residue was purified by flash chromatography (silica gel;
petroleum ether 40-60 °C/ethyl acetate) (70:30) to give 16a
as a pale yellow solid: 1.85 g, 5.66 mmol, 65%, mp 96-98 °C
(lit.36 98-100 °C).
N-Acetyl 2-(6H-isoin d olo[2,1-a ]in d ol-11-yl)eth a n a m in e
(5a ): pale yellow solid, 70%, mp 155-175 °C; 1H NMR δ 1.79
(s, 3H), 3.23 (t, J ) 6.5 Hz, 2H), 3.60 (m, 2H), 5.40 (s, 2H),
5.56 (br s, 1H), 7.11 (m, 1H), 7.20 (m, 1H), 7.29 (m, 1H), 7.33
(d, J ) 7.9 Hz, 1H), 7.40 (m, 1H), 7.46 (d, J ) 7.4 Hz, 1H),
7.59 (d, J ) 7.9 Hz, 1H), 7.78 (d, J ) 7.6 Hz, 1H); 13C NMR δ
23.3, 25.2, 39.8, 49.8, 103.5, 109.3, 119.3, 119.5, 121.1, 121.8,
123.7, 126.9, 128.3, 128.4, 132.3, 133.1, 133.8, 141.7, 175.0;
IR 3290, 3050, 2926, 2854, 1644, 1557, 1443, 1362, 1300, 1189,
734 cm-1; MS m/e 291, 290, 232, 231, 218, 204; C19H19N2O (M+
+ H) requires 291.1480: Found: 291.1497. Anal. (C19H18N2O)
C, H, N.
N-P r op a n oyl 2-(6H-isoin d olo[2,1-a ]in d ol-11-yl)eth a n -
a m in e (5b): yellow solid, 76%, mp 97-98 °C.
N-Bu t a n oyl 2-(6H -isoin d olo[2,1-a ]in d ol-11-yl)et h a n -
a m in e (5c): yellow crystals, 66%, mp 200-201 °C.
N-Cyclop r op a n eca r bon yl 2-(6H-isoin d olo[2,1-a ]in d ol-
11-yl)eth a n a m in e (5d ): white solid, 46%, mp 189-192 °C.
N-Cyclobu ta n eca r bon yl 2-(6H-isoin d olo[2,1-a ]in d ol-
11-yl)eth a n a m in e (5e): yellow solid, 37%, mp 190-191 °C.
N-Acetyl 2-(9-m eth oxy-6H-isoin d olo[2,1-a ]in d ol-11-yl)-
eth a n a m in e (7a ): white solid, 88%, mp 159-161 °C; 1H NMR
δ 1.75 (s, 3H), 3.08 (t, J ) 7.2 Hz, 2H), 3.29 (q, J ) 7.2 Hz,
2H), 3.79 (s, 3H), 5.11 (s, 2H), 6.78 (dd, J ) 8.7, 2.4 Hz, 1H),
7.09 (d, J ) 8.3 Hz, 1H), 7.34 (m, 1H), 7.43 (t, J ) 7.5 Hz,
1H), 7.56 (d, J ) 7.3 Hz, 1H), 7.84 (d, J ) 7.5 Hz, 1H), 8.00
(br t, 1H); 13C NMR δ 22.7, 24.3, 48.2, 55.4, 101.1, 103.2, 110.4,
111.2, 120.3, 123.9, 126.5, 127.9, 128.7, 132.7, 140.8, 142.0,
153.4, 169.2; IR 3308, 3074, 2928, 2860, 1643, 1559, 1486,
1440, 1372, 1300, 1238, 1225, 1164, 1127, 1106, 1042, 846, 762,
716 cm-1; MS m/e 321, 320, 262, 261, 248, 234, 43. Anal.
(C20H20N2O2) C, H, N
N-P r op a n oyl 2-(9-m eth oxy-6H-isoin d olo[2,1-a ]in d ol-
11-yl)eth a n a m in e (7b): pale yellow solid, 56%, mp 165-166
°C.
N-Bu ta n oyl 2-(9-m eth oxy-6H-isoin d olo[2,1-a ]in d ol-11-
yl)eth a n a m in e (7c): pale yellow solid, 68%, mp 154-155 °C.
N-Cyclop r op a n eca r bon yl 2-(9-m eth oxy-6H-isoin d olo-
[2,1-a ]in d ol-11-yl)eth a n a m in e (7d ): white solid, 81%, mp
154-156 °C.
N-Acetyl 2-(9-eth oxy-6H-isoin d olo[2,1-a ]in d ol-11-yl)-
eth a n a m in e (10a ): pale yellow solid, 37%, mp 166-170 °C;
1H NMR δ 1.46 (t, J ) 7.0 Hz, 3H), 1.83 (s, 3H), 3.21 (m, 2H),
3.62 (m, 2H), 4.11 (q, J ) 7.0 Hz, 2H), 5.03 (s, 2H), 5.6 (br s,
1H), 6.89 (dd, J ) 8.7, 2.3 Hz, 1H), 7.07 (d, J ) 2.3 Hz, 1H),
7.24 (d, J ) 8.7 Hz, 1H), 7.29 (m, 1H), 7.41 (t, J ) 7.6 Hz,
1H), 7.47 (d, J ) 7.4 Hz, 1H), 7.77 (d, J ) 7.6 Hz, 1H); 13C
NMR δ 15.1, 23.5, 24.6, 40.2, 48.4, 64.4, 102.5, 103.0, 110.2,
112.5, 120.7, 123.6, 126.7, 128.3, 129.2, 132.7, 133.3, 141.8,
153.3, 170.3; IR 2983-2865, 1650, 1559,1442, 1221, 765, 723
cm-1; MS m/e 335, 334, 276, 275, 262 (100). C21H23N2O2 (M+
+ H) requires 335.1750; Found: 335.1760. Anal. (C21H22N2O2)
C, H, N.
N-P r op a n oyl 2-(9-eth oxy-6H-isoin d olo[2,1-a ]in d ol-11-
yl)eth a n a m in e (10b): white solid, 50%, mp 160-164 °C.
N-Bu t a n oyl 2-(9-et h oxy-6H -isoin d olo[2,1-a ]in d ol-11-
yl)eth a n a m in e (10c): yellow solid, 66%, mp 153-155 °C.
N-Cyclop r op a n eca r b on yl 2-(9-et h oxy-6H -isoin d olo-
[2,1-a ]in d ol-11-yl)eth a n a m in e (10d ): yellow solid, 43%, mp
200-201 °C.
1-[2-(2-Br om oph en yl)eth yl]-5-m eth oxy-1H-in dole-3-car -
boxa ld eh yd e (16b). Compound 16b was obtained in the same
manner as a beige solid: 65%, mp 95-97 °C (lit.36 96-98 °C).
5,6-Dih yd r oin d olo[2,1-a ]isoq u in olin e-12-ca r b oxa ld e-
h yd e (17a ). Potassium acetate (0.69 g, 7.07 mmol) and
tetrakis(triphenylphosphine)palladium(0) (0.27 g, 0.24 mmol)
were added sequentially to a solution of 16a (1.47 g, 4.48
mmol) in dimethylformamide (65 mL). The resulting suspen-
sion was stirred at 110 °C overnight. The solvent was
evaporated under reduced pressure, and the resulting dark
suspension was chromatographed (flash column, petroleum
ether 40-60 °C/ethyl acetate, 90:10) to give 17a as a beige
powder: 0.97 g, 3.94 mmol, 88%, mp 123-125 °C (lit.36 124-
126 °C).
5,6-Dih yd r o-10-m eth oxyin d olo[2,1-a ]isoqu in olin e-12-
ca r boxa ld eh yd e (17b). Compound 17b was obtained in the

Mapping the Melatonin Receptor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1059
same manner as a beige solid: 77%, mp 136-138 °C (lit.36
135-137 °C).
1H, J ) 2.3, 8.9 Hz),7.39-7.07 (m, 5H), 7.89 (d, 1H, J ) 7.8
Hz); 13C NMR δ 23.3, 25.2, 30.0, 39.9, 40.1, 55.9, 100.2, 109.5,
112.6, 124.9, 127.0, 127.4, 128.5, 129.2, 129.6, 130.7, 131.8,
133.4, 154.1, 170.2. Anal. (C21H22N2O2) C, H, N.
N-P r op a n oyl 2-(10-m eth oxy-5,6-d ih yd r oin d olo[2,1-a ]-
isoqu in olin -12-yl)-eth a n a m in e (21b): white amorphous
solid, 64.5%, mp 159-160 °C.
N-Bu t a n oyl 2-(10-m et h oxy-5,6-d ih yd r oin d olo[2,1-a ]-
isoqu in olin -12-yl)eth a n a m in e (21c): 59%, mp 134-135 °C.
N-Cyclob u t a n eca r b on yl 2-(10-m et h oxy-5,6-d ih yd r o-
in d olo[2,1-a ]isoqu in olin -12-yl)eth a n a m in e (21d ): pale yel-
low oil, 21%.
2-(5,6-Dih yd r oin d olo[2,1-a ]isoqu in olin -12-yl)-1-n itr o-
eth en e (18a ). A solution of 17a (0.16 g, 0.65 mmol) and
ammonium acetate (0.032 g, 0.42 mmol) in nitromethane (2
mL) was refluxed for 2 h. After evaporation of the solvent
under reduced pressure, the residue was dissolved in dichlo-
romethane (3 mL) and H2O (3 mL) was added. The aqueous
layer was washed with dichloromethane (2 × 10 mL), and the
combined organic layers were washed with H2O (10 mL) and
brine (10 mL) and dried (Na2SO4). Removal of the solvent
under vacuo gave 18a as an orange powder: 0.183 g, 0.63
1
mmol, 97%, mp 109-111 °C; H NMR δ 3.17 (t, 2H, J ) 6.4
N-Acet yl 2-(5,6,7-t r ih yd r ob en zo[c]cycloh ep t [2,1-a ]-
in d ol-13-yl)eth a n a m in e (23a ): white foam, 68%; 1H NMR δ
1.73 (s, 3H), 2.23 (br s, 2H), 2.60 (t, J ) 6.9 Hz, 2H), 3.10 (t,
J ) 6.7 Hz, 2H), 3.52 (br d, 2H), 4.07 (br s, 2H), 5.4 (br s, 1H),
7.12 m, 1H), 7.24 (m, 1H), 7.30 (dd, J ) 7.2, 1.6 Hz, 1H), 7.30
(dd, J ) 7.2, 1.6 Hz, 1H), 7.34 (m, 2H), 7.37 (m, 1H), 7.49 (dd,
J ) 7.1, 1.6 Hz, 1H), 7.67 (d, J ) 7.9 Hz, 1H); 13C NMR δ
23.2, 24.4, 30.7, 31.1, 40.4, 108.6, 119.1, 121.9, 127.1, 127.7,
128.6, 129.2, 129.4, 132.2, 135.6, 137.7, 138.9, 169.8; IR 3294,
3074, 2937, 1693, 1560, 1489, 1437, 1364, 1239, 1222, 1169,
722, 696, 542 cm-1; MS m/e 319, 318, 260, 259, 246 (100).
Hz), 4.26 (t, 2H, J ) 6.4 Hz), 7.49-7.32 (m, 6H), 7.84-7.80
(m, 2H), 7.95 (d, 1H, J ) 13.3 Hz), 8.75 (d, 1H, J ) 13.3 Hz);
13C NMR δ 36.2, 48.8, 108.0, 110.6, 120.8, 122.5, 123.9, 125.7,
127.2, 128.6, 128.85, 132.1, 133.3, 135.9, 137.4, 137.5. Anal.
(C18H14N2O2) C, H, N.
10-Meth oxy-12-[(E)-2-n itr o-1-eth en yl]-5,6-dih ydr oin dolo-
[2,1-a ]isoqu in olin e (18b). Compound 18b was obtained in
the same manner as a bright yellow fluorescent solid: 44%,
mp 208-209 °C.
2-(5,6-Dih yd r oin d olo[2,1-a ]isoqu in olin -12-yl)-1-eth a n -
a m in e (19a ). A solution of 18a (0.19 g, 0.66 mmol) in
tetrahydrofuran (15 mL) was added dropwise at 0 °C to a
stirred suspension of LiAlH4 (0.25 g, 6.59 mmol) in THF (6
mL). After completion of addition, the mixture was refluxed
overnight and then allowed to reach ambient temperature.
After cooling to 0 °C, water (17 mL) was added. The mixture
was filtered, and the filtrate was taken up in ethyl acetate
(50 mL), washed with H2O (2 × 25 mL) and brine (25 mL),
and dried (Na2SO4). The solvent was removed under reduced
pressure to give crude amine 19a which was used without
further purification.
C
21H23N2O (M+ + H) requires 319.1800; Found: 319.1810.
N-P r op a n oyl 2-(5,6,7-tr ih yd r oben zo[c]cycloh ep t[2,1-a ]-
in d ol-13-yl)eth a n a m in e (23b): white foam, 95%.
N-Bu ta n oyl 2-(5,6,7-tr ih yd r oben zo[c]cycloh ep t[2,1-a ]-
in d ol-13-yl)eth a n a m in e (23c): white foam, 61%.
N-Cyclop r op a n eca r bon yl 2-(5,6,7-tr ih yd r oben zo[c]cy-
cloh ep t[2,1-a ]in d ol-13-yl)eth a n a m in e (23d ): brown solid,
50%, mp 142-144 °C.
N-Cyclobu ta n eca r bon yl 2-(5,6,7-tr ih yd r oben zo[c]cy-
cloh ep t[2,1-a ]in d ol-13-yl)eth a n a m in e (23e): white solid,
75%, mp 138-139 °C.
N-Acet yl 2-(5,6,7-t r ih yd r o-11-m et h oxyb en zo[c]cyclo-
h ep t[2,1-a ]in d ol-13-yl)eth a n a m in e (25a ): white foam, 97%;
1H NMR δ 1.74 (s, 3H), 2.23 (br s, 2H), 2.59 (m, 2H), 3.07 (t,
J ) 6.8 Hz, 2H), 3.51 (m, 2H), 3.89 (s, 3H), 4.10 (br s, 2H),
5.40 (br s, 1H), 6.90 (dd, J ) 8.8, 2.5 Hz, 1H), 7.12 (d, J ) 2.3
Hz, 1H), 7.23 (d, J ) 8.8 Hz, 1H), 7.31 (dd, J ) 6.9, 2.1 Hz,
1H), 7.34 (m, 1H), 7.37 (m, 1H), 7.50 (d, J ) 7.1 Hz, 1H); 13C
NMR δ 23.5, 24.4, 30.9, 31.1, 40.3, 40.6, 56.1, 100.7, 108.2,
109.4, 112.2, 127.1, 127.9, 128.5, 129.1, 129.4, 130.9, 132.3,
138.2, 140.3, 153.5, 174.0; IR 3275, 2935, 1652, 1558, 1486,
1438, 1222, 1168, 1119, 722 cm-1; MS m/e 349, 348, 290, 289,
276 (100). C22H25N2O2 (M+ + H) requires 349.1900; Found:
349.1916. Anal. (C22H24N2O2) C, H, N.
N-P r op a n oyl 2-(5,6,7-tr ih yd r o-11-m eth oxyben zo[c]cy-
cloh ep t[2,1-a ]-in d ol-13-yl)eth a n a m in e (25b): white foam,
82%.
N-Bu ta n oyl 2-(5,6,7-tr ih yd r o-11-m eth oxyben zo[c]cy-
cloh ep t[2,1-a ]in d ol-13-yl)eth a n a m in e (25c): white foam,
72%.
N-Cyclop r op a n eca r b on yl 2-(5,6,7-t r ih yd r o-11-m et h -
oxybenzo[c]cyclohept[2,1-a ]-indol-13-yl)ethanamine (25d):
white foam, 72%.
N-Cyclobu ta n eca r bon yl 2-(5,6,7-tr ih yd r o-11-m eth oxy-
b en zo[c]cycloh ep t [2,1-a ]in d ol-13-yl)et h a n a m in e (25e):
white foam, 76%.
2-(10-Meth oxy-5,6-d ih yd r oin d olo[2,1-a ]isoqu in olin -12-
yl)-1-eth a n a m in e (19b). Compound 19b was obtained in the
same manner as a clear, pale yellow oil, which was then used
without further purification.
Gen er a l P r oced u r e for th e P r ep a r a tion of Am id es 20,
21. To a cooled solution (0 °C) of 2-(5,6-dihydroindolo[2,1-a]-
isoquinolin-12-yl)-1-ethanamine (19a ) or 2-(10-methoxy-5,6-
dihydroindolo[2,1-a]isoquinolin-12-yl)-1-ethanamine (19b) (0.32
mmol) in tetrahydrofuran (3 mL) were added triethylamine
(0.15 mL) and the appropriate anhydride (0.10 mL). The ice
bath was removed and the solution stirred for 3 h. The solvent
was evaporated in vacuo, and the residue was taken up in
ethyl acetate (50 mL) and washed with H2O (50 mL), saturated
aqueous NaHCO3 (50 mL), and brine. The organic phase was
dried (Na2SO4) and concentrated in vacuo to give a brown oil.
Trituration of the oil with ethyl acetate (2 mL) afforded the
desired amide.
N-Acetyl 2-(5,6-d ih yd r oin d olo[2,1-a ]isoqu in olin -12-yl)-
eth a n a m in e (20a ): beige amorphous solid, 42%, mp 188-190
1
°C; H NMR δ 1.86 (s, 3H), 3.13 (t, 2H, J ) 6.0 Hz), 3.34 (t,
2H, J ) 6.7 Hz),3.66 (dt, 2H, J ) 6.5, 6.5 Hz), 4.24 (t, 2H, J )
5.9 Hz), 5.59 (bs, 1H), 7.38-7.12 (m, 6H), 7.63 (d, 1H, J ) 7.8
Hz), 7.92 (d, 1H, J ) 7.7 Hz); 13C NMR δ 23.3, 25.15, 30.1,
40.0, 40.1, 108.5, 108.8, 118.7, 119.5, 122.2, 125.2, 127.2, 127.5,
128.5, 129.0, 129.6, 131.4, 133.7, 135.4, 170.2. C20H20N2O
requires 305.1640; Found: 305.1654.
N-Acet yl
2-(5,6,7-t r ih yd r o-11-et h oxyb en zo[c]cyclo-
N-P r op a n oyl 2-(5,6-d ih yd r oin d olo[2,1-a ]isoqu in olin -
12-yl)eth a n a m in e (20b): white amorphous solid, 38%, mp
183-185 °C.
N-Bu ta n oyl 2-(5,6-d ih yd r oin d olo[2,1-a ]isoqu in olin -12-
yl)eth a n a m in e (20c): beige amorphous solid, 38%, mp 165-
167 °C.
N-Cyclob u t a n eca r b on yl 2-(5,6-d ih yd r oin d olo[2,1-a ]-
isoqu in olin -12-yl)eth a n a m in e (20d ): white powder, 34%,
mp 94-96 °C.
N-Acet yl 2-(10-m et h oxy-5,6-d ih yd r oin d olo[2,1-a ]iso-
qu in olin -12-yl)eth a n a m in e (21a ): beige amorphous solid,
68%, mp 153-154 °C; 1H NMR δ 1.87 (s, 3H), 3.11 (t, 2H, J )
6.30 Hz), 3.30 (t, 2H, J ) 6.95 Hz), 3.64 (q, 2H, J ) 6.6 Hz),
3.88 (s, 3H), 4.19 (t, 2H, J ) 6.3 Hz), 5.72 (bs, 1H), 6.89 (dd,
h ep t[2,1-a ]in d ol-13-yl)eth a n a m in e (27a ): white foam, 67%;
1H NMR δ 1.48 (t, J ) 7.0 Hz, 3H), 1.77 (s, 3H), 2.25 (br s,
2H), 2.63 (t, J ) 6.8 Hz, 2H), 3.09 (t, J ) 6.7 Hz, 2H), 3.52 (m,
2H), 4.02 (br s, 2H), 4.14 (m, 2H), 5.50 (br s, 1H), 6.93 (dd, J
) 8.9, 2.4 Hz, 1H), 7.16 (d, J ) 2.3 Hz, 1H),7.25 (d, J ) 8.8
Hz, 1H), 7.32 (m, 1H), 7.35 (m, 1H), 7.38 (m, 1H), 7.51 (dd, J
) 6.9, 1.6 Hz, 1H); 13C NMR δ 15.2, 23.2, 24.4, 31.1, 40.3, 40.6,
64.5,102.0, 108.1, 109,4, 112.7, 127.1, 127.9, 128.5, 129.1,
129.4, 131.0, 132.3, 138.2, 138.9, 153.1, 169.9; IR 3282, 2932,
1652, 1553, 1471, 1364, 1180, 789, 762 cm-1; MS m/e 363, 362,
304, 259, 290 (100). C23H27N2O2 (M+ + H) requires 363.2060;
Found: 363.2073. Anal. (C23H26N2O2) C, H, N.
N-P r op a n oyl 2-(5,6,7-tr ih yd r o-11-eth oxyben zo[c]cyclo-
h ep t[2,1-a ]in d ol-13-yl)eth a n a m in e (27b): white foam, 76%.

1060 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Faust et al.
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N-Bu ta n oyl 2-(5,6,7-tr ih yd r o-11-eth oxyben zo[c]cyclo-
h ep t[2,1-a ]in d ol-13-yl)eth a n a m in e (27c): white foam, 89%.
N-Cyclop r op a n eca r bon yl 2-(5,6,7-tr ih yd r o-11-eth oxy-
ben zo[c]cycloh ep t[2,1-a ]in d ol-13-yl)eth a n a m in e (27d ):
white foam, 77%.
Mod elin g. Molecular modeling on compounds 7a , 21a , and
25a was performed using the semiempirical PM3 parameter
set57 as implemented in MacSpartan Plus, Version 1.1.7,
available from Wave function Inc., 18401 Von Karman, Suite
370, Irvine, CA 92612, mounted on a Power Macintosh G3.
No geometry restrictions were applied, and no global minimum
search was performed.
Su p p or tin g In for m a tion Ava ila ble: Full physical data,
in the same format as for the representative examples, are
provided for all analogues. This material is available free of
charge via the Internet at http://pubs.acs.org.
Ack n ow led gm en t. We are grateful to Dr. Michael
R. Lerner, Department of Dermatology, University of
Texas Southwestern Medical Center at Dallas, Dallas,
TX 75235-9069, for supplying the Xenopus laevis mel-
anophore cell line, and Dr. Steven Reppert, Harvard
University, Boston, for providing us with NIH 3T3 cells
expressing human mt1 and MT2 melatonin receptor
subtypes. P.J .G. thanks the Leverhulme Trust for the
award of an Emeritus Fellowship.
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