Mapping the melatonin receptor. 7. Subtype selective ligands based on β-substituted N-acyl-5-methoxytryptamines and β-Substituted N-acyl-5-methoxy-1-methyltryptamines

Article abstract of DOI:10.1021/jm0512544

A series of β-substituted and β,β-disubstituted N-acyl 5-methoxy-1-methyltryptamines and 5-methoxytryptamines 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 of all analogues was measured using the pigment aggregation response of a clonal line of Xenopus laevis melanophores, β-Methylmelatonin (17a) and β,β-dimethylmelatonin (17b), though showing a slight decrease in binding at human receptors, show an increase in potency on Xenopus, N-Butanoyl 5-methoxy-1-methyl-β,β-trimethylenetryptamine (12c) is an antagonist at human MT₁ receptors but an agonist at MT₂, while N-butanoyl 5-methoxy-1-methyl-β,β-tetramethylenetryptamine (13c) is an antagonist at MT₁ but had no action at MT₂ and is one of the first examples of an MT₁ selective antagonist.

Full text of DOI:10.1021/jm0512544

J. Med. Chem. 2006, 49, 3509-3519
3509
Mapping the Melatonin Receptor. 7. Subtype Selective Ligands Based on â-Substituted
N-Acyl-5-methoxytryptamines and â-Substituted N-Acyl-5-methoxy-1-methyltryptamines
Andrew Tsotinis,*,† Margarita Vlachou,† Demetris P. Papahatjis,‡ Theodora Calogeropoulou,‡ Spyros P. Nikas,‡ Peter J. Garratt,§
Vincent Piccio,§ Stefan Vonhoff,§ Kathryn Davidson, Muy-Teck Teh,∧ and David Sugden
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, UniVersity of Athens, Panepistimioupoli-Zografou, Athens 157 71, Greece,
Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vas. Constantinou AVenue, 116 35 Athens,
Greece, Department of Chemistry, UniVersity College London, 20 Gordon Street, London WC1H 0AJ, U.K., and DiVision of ReproductiVe
Health, Endocrinology & DeVelopment, School of Biomedical Sciences, Hodgkin Building, Kings College London, London SE1 1UL, U.K.
ReceiVed December 16, 2005
A series of â-substituted and â,â-disubstituted N-acyl 5-methoxy-1-methyltryptamines and 5-methoxy-
tryptamines 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 of all
analogues was measured using the pigment aggregation response of a clonal line of Xenopus laeVis
melanophores. â-Methylmelatonin (17a) and â,â-dimethylmelatonin (17b), though showing a slight decrease
in binding at human receptors, show an increase in potency on Xenopus. N-Butanoyl 5-methoxy-1-methyl-
â,â-trimethylenetryptamine (12c) is an antagonist at human MT1 receptors but an agonist at MT2, while
N-butanoyl 5-methoxy-1-methyl-â,â-tetramethylenetryptamine (13c) is an antagonist at MT1 but had no
action at MT2 and is one of the first examples of an MT1 selective antagonist.
Introduction
The 5-methoxy group of melatonin has been shown to be
important for binding to the receptor,9,21 but it is not an essential
requirement for agonist activity.10 The active conformation of
the 3-ethanamide side chain has been established from studies
with conformationally restricted indole10,13,22 and non-indole
analogues.15-18 Models for the binding of melatonin with its
receptor have been formulated on the basis of structure-activity
data, analogies with other G-protein-coupled receptors for
ligands of low molecular mass, comparative molecular field
analysis, and site-directed and chimeric receptor mutagenesis
studies.12-14,17,23-29 The conformational preferences of jet-cooled
melatonin have recently been explored, and the active confor-
mation is one of the four lowest minima.30 There has been
considerable interest in developing receptor subtype selective
compounds. MT2 subtype selective agonists and antagonists have
been reported,14,25-28,30-37 and MT1 selective agonists and
antagonists have recently been obtained.34,38,39
The pineal hormone melatonin (N-acetyl 5-methoxytryptamine,
1a)1,2 is an important component in the regulation of seasonal
and circadian rhythms. Its action is believed to be mediated
through a family of specific, high-affinity, G-protein-coupled
cell membrane receptors,3 and radioligand binding studies using
2-[125I]-iodomelatonin (1b) have revealed a widespread, het-
erogeneous distribution of binding sites throughout the central
nervous system.4 Two receptor subtypes (MT1 and MT2) have
been cloned in mammals5,6 which, when expressed in host cells,
show the general pharmacological characteristics of native
melatonin receptors. A third subtype, Mel1c, has been cloned
from chicken, Xenopus, and zebrafish7 but has not been detected
in mammals. A fourth melatonin binding site, MT3, has been
identified as the enzyme quinone reductase.8
We have now examined a series of â-substituted N-acyl
5-methoxytryptamines and â-substituted N-acyl 5-methoxy-1-
methyltryptamines on binding at the recombinant MT1 and MT2
subtypes and receptor activation using the pigment aggregation
assay on a clonal Xenopus melanophore line.40 For compounds
that proved of interest in these assays, we have investigated
the agonist and antagonist behavior in a functional assay on
recombinant MT1 and MT2 melatonin receptors expressed in
the NIH 3T3 cell line.
A large number of studies have been directed toward
understanding how melatonin binds to and activates these
receptors using both indole9-15 and non-indole16-20 derivatives.
Chemistry
1,â-Dimethylmelatonin (6a), 1,â,â-trimethylmelatonin (6d),
1-methyl-â,â-tetramethylenemelatonin (13h), and their ana-
logues were prepared through the Bischler reaction by the route
shown in Scheme 1. N-Methyl-p-anisidine (2) was treated with
the appropriate R-bromoketone 3a-c to give the corresponding
3-indolylethanoic ester, which was hydrolyzed to the acid 4a-c
and then converted to the amide 5a-c. Reduction of the amide
and acylation of the resulting amine gave 6a-g and 13a-e.
This route proved unsuccessful in attempts to prepare the
* To whom correspondence may be addressed. Tel: 30 210 7274812.
Fax: 30 210 7274747. E-mail: tsotinis@pharm.uoa.gr.
† University of Athens.
‡ National Hellenic Research Foundation.
§ University College London.
Kings College London.
∧ Current address: Dr. Teck Teh, Centre for Clinical & Diagnostic Oral
Sciences, Barts & the London School of Medicine & Dentistry, Queen Mary
University of London, The Blizard Building, 4 Newark Street, London E1
2AT, U.K.
10.1021/jm0512544 CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/09/2006

3510 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Tsotinis et al.
Scheme 1
Scheme 2
analogues 11a-12e, and the alternative route shown in Scheme
2 was used to obtain these and related systems. Indole (7a) or
5-methoxyindole (7b) was converted to the corresponding nitrile
by the method of Pe´rez-Alvarez et al.,41 and the nitrile was then
N-methylated to 9a,b. Treatment of 9a,b with potassium bis-
(trimethylsilyl)amide followed by the appropriate R,ω-dihaloal-
kane gave 10a-c which were reduced to the corresponding
amine and acylated to give 11a-12e and 14a-e. To determine
the effect of the N-1 methyl group, the cyanide 7b was N1-
Boc-protected and then methylated with MeI and NaH to give
either 16a or 16b depending on the amount of MeI used. The
nitriles 16a,b were deprotected with (n-Bu)4NF, reduced with
LAH, and acetylated to give 17a and 17b. The N1-Boc-protected
cyanide 15 was also treated with sodium hydride followed by
1,3-dibromopropane and MeOH to give 18 (Scheme 3). Reduc-
tion of 18 with LAH and acylation of the resulting amine gave
19a-e.
Pharmacology
The affinity of the analogues was determined in competition
radioligand binding assays using 2-[125I]-iodomelatonin (specific
activity 2200 Ci/mol, DuPont, Stevenage, U.K.), as described
previously,42 on the recombinant human MT1 and MT2 subtypes
expressed in NIH 3T3 cells. The biological activity of the
analogues was assessed in a well-established, specific model
of melatonin action, the pigment aggregation response of

Mapping the Melatonin Receptor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3511
Scheme 3
Xenopus laeVis melanophores. In these cells, melatonin triggers
a translocation of pigment, normally distributed throughout the
cell, toward the cell center. This response is termed pigment
aggregation and is quantified by measuring the change in light
(630 nm) absorbance of the cells as the pigment concentrates
near the cell center. In the present study, a clonal melanophore
line, generously provided by Dr. Michael Lerner (Department
of Dermatology, University of Texas) was used.
For specific analogues whose properties in the affinity and
potency tests were of interest, the agonist and antagonist
potencies were determined on human MT1 and MT2 receptors
expressed in NIH 3T3 cells by measuring their ability to inhibit
forskolin stimulation of intracellular cyclic AMP.
at MT2 than at the MT1 receptor, indicating that the MT2
receptor cavity is larger and/or more flexible than that of the
MT1 receptor. This suggested to us that a combination of
suitably placed substituents might provide compounds that were
agonists at the MT2 receptor but inactive or antagonists at the
MT1 receptor. In solution, the 3-N-acetyl ethylamine side chain
of melatonin is probably fairly evenly distributed between the
staggered conformations at C-3-âC, with those in which the
side chain is away from the ring predominating. Substituents
on the 3-side-chain, particularly at the â position, should initially
increase the preference for the active conformation but, as the
size of the substituents is increased, the population of the
preferred conformation may decline. With sufficiently large
substituents, for example the cyclopentane derivatives 13, the
preferred conformation may be with the ring and 3-side-chain
orthogonal to the plane of the indole ring. In the active site,
rotation of the side chain into the active conformation will
orientate the â-substituents away from the indole ring. Introduc-
tion of a substituent on the indole nitrogen could further affect
the binding space required around the N-1 to C-3 regions of
the molecule, which again might be more readily tolerated by
the MT2 receptor. We had previously examined â- and
R-substituted compounds having a phenylethyl group at N-1
which had a promising profile,43 and we therefore prepared a
series of 5-methoxy-1-methyltryptamines substituted at the
â-position by one or two methyl groups or by a three-, four-,
or five-membered ring. We also prepared 5-methoxytryptamines
substituted at the â-position by a four-membered ring in order
to determine the effect of the methyl substituent at N-1. The
compounds were characterized by standard methods as described
in the Experimental Section.
Results and Discussion
The generally accepted active conformation of melatonin at
its receptor site has the methyl group of the 5-methoxy moiety
and the 3-ethylamino side chain orientated above the indole ring
(Figure 1).26 Substituents at C-2 of the indole ring increase the
binding affinity of the compounds probably by a combination
of an increase in the population of the active conformation and
from binding to a specific pocket available to C-2 substitu-
ents.9,10 For many C-2 substituted analogues, affinity is greater
The cloning of the melatonin receptor4 and the identification
of high-affinity melatonin subtypes2,5 have allowed the com-
parison of agonists and antagonists at the two sites. As we have
previously reported,14 the potency results obtained on pigment
aggregation in X. laeVis melanophores do not correlate well with
either the MT1 or MT2 receptor subtypes. The results of the
binding and melanophore assays for the three sets of compounds
are shown in Tables 1, 2, and 3, respectively.
From Table 1 it can be seen that the introduction of methyl
groups at the â-position of melatonin (17a,b) reduces the binding
affinity to both the MT1 and MT2 receptors, but that a small
Figure 1. Probable active conformation of melatonin at its receptor.
Displacements of the C-3 side chain require little energy.30

3512 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Tsotinis et al.
Table 1a
Xenopus melanophores
receptor binding (Ki, nM)
human MT1 human MT2
MT2/
MT1
agonist
antagonist
(IC50, nM)
compd
R1
R2
R3
R
(EC50, nM)
melato nin
17a
17b
6a
(+)6a
(-)6a
6b
6c
6d
6e
6f
H
H
H
H
H
H
Me
H
H
H
H
H
Me
Me
Me
Me
Me
0.39 ( 0.08
1.67 ( 0.17
1.12 ( 0.38
5.48 ( 1.70
1.22 ( 0.36
7.71 ( 0.71
4.90 ( 0.83
4.27 ( 0.29
11.5 ( 2.2
0.35 ( 0.03
2.94 ( 0.88
2.75 ( 0.33
0.41 ( 0.12
0.50 ( 0.05
0.27 ( 0.07
1.02 ( 0.07
0.47 ( 0.04
1.29 ( 0.24
1.15 ( 0.13
0.74 ( 0.08
33.1 ( 2.2
1.1
0.6
0.4
13
2
28.5
5
9
9
5
11.5
5
0.063 ( 0.004
0.049 ( 0.001
0.0072 ( 0.0001
1.72 ( 0.08
0.98 ( 0.01
10.8 ( 0.08
3.72 ( 0.17
1.00 ( 0.24
6.31 ( 0.94
1.26 ( 0.11
1.00 ( 0.37
57.5 ( 5.1
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
Pr
Me
Et
Me
Me
Me
Me
Me
Me
Me
Me
Me
6.03 ( 0.66
8.51 ( 0.92
170 ( 11
Pr
c-C4H7
6g
a MT1 and MT2 data are the mean of quadruplicate determinations. Agonist and antagonist data on melanophores are the mean of triplicate experiments.
NA ) no antagonist effect detected at 10 µM.
Table 2a
Xenopus melanophore s
receptor binding (Ki nM)
MT2/
MT1
agonist
(EC50, nM)
antagonist
(IC50, nM)
compd
n
R
human MT1
human MT2
melatonin
11a
11b
11c
12a
12b
12c
12d
12e
13a
13b
13c
13d
13e
0.39 ( 0.08
176 ( 26
86 ( 20
0.35 ( 0.03
90 ( 11
1.1
2
2
28
7
8
12
2
0.2
2
3
7
0.063 ( 0.004
10.5 ( 3.0
6.8 ( 2.7
1.7 ( 0.3
1010 ( 210b
980 ( 160b
2.00 ( 0.8
NA
NA
NA
NA
NA
NA
NA
NA
NA
1
1
1
2
2
2
2
2
3
3
3
3
3
Me
Et
Pr
Me
Et
Pr
c-C3H5
c-C4H7
Me
Et
Pr
c-C3H5
c-C4H7
39 ( 9
212 ( 39
483 ( 16
34.0 ( 2.0
10.6 ( 1.8
307 ( 53
714 ( 14
148 ( 33
302 ( 27
589 ( 76
468 ( 21
479 ( 124
7.5 ( 2.8
72 ( 3
637 ( 17
826 ( 6
NA
2066 ( 126
>10000
628 ( 34
811 ( 60
1635 ( 13
1175 ( 16
1766 ( 165
4.40 ( 0.15
0.86 ( 0.13
138 ( 12
3767 ( 569
66.1 ( 4.4
102 ( 46
85.1 ( 33.8
257 ( 28
407 ( 105
2
1
NA
NA
a See footnote to Table 1 for methods. NA ) no agonist or antagonist effect detected at 10 µM. b Partial agonist with very low efficacy (see Figure 2).
preference for the MT1 site occurs. â-Methylmelatonin (17a)
is chiral and, from studies on the corresponding 1-methyl
derivative described below, it is likely that the enantiomers
exhibit different binding affinities. Introducing methyl groups
at positions 1 and â (6a-c) also causes a drop in binding affinity
with the effect being greater on the MT1 site. Increasing the
size of the alkyl chain on the amide group from methyl (6a) to
propyl (6c) leads to only a small increase in binding. Introduc-
tion of a second methyl group at the â-position (6d-g) reduces
the binding affinity a little further with the effect most
pronounced at the MT1 receptor to give compounds showing a
small preference for MT2. As the alkyl chain is increased (6e-
f), there is a small increase in MT2 affinity, with the butanoyl
derivative 6f having the maximal affinity for MT2 and the
propanoyl derivative 6e for MT1. For both receptors, cyclobu-
tanecarbonyl as the N-acylating group dramatically reduces
affinity. Most of theses compounds show a small selectivity
(up to 13-fold) for the MT2 receptor.
All the compounds are agonists in the melanophore assay;
17b (EC50 ) 7.2 pM) was 9-fold more potent than melatonin.
Compounds 6a-c are chiral, and since it is known that
enantiomers can have different affinities and potencies at the
receptor, the enantiomers of 6a were separated by chiral
chromatography and tested. Enantiomer (+)-6a has a 10-fold
higher potency for pigment aggregation than (-)-6a. In previous
studies,14,17 it was found that in compounds that were chiral at
the equivalent â-position, the (-)-enantiomers had higher
potency on melanophores; however, in these compounds the
chiral atom was in a six-membered ring and the compounds
had much greater magnitudes of rotation. The (+)-enantiomer
shows a similar binding affinity at the two receptors, whereas
the (-)-enantiomer binds more strongly to the MT2 receptor,
giving (-)-6a 28-fold selectivity for MT2. Interestingly, (+)-
6a has a higher affinity than (-)-6a at MT1 receptors (and a
higher potency on melanophores), but at the MT2 subtype it is
the (-)-enantiomer which has the higher affinity (∼2-fold). The
absolute configuration of the enantiomers of 6a is not known,
and in order to gain more insight into the receptor stereochem-
istry we are now examining the affinities of a range of chiral
compounds.
Table 2 shows the binding and potency results for melatonin
analogues with a ring attached at the â-position of the side chain
and a methyl group at N-1. The compounds 11a-c with a
cyclopropane ring are all agonists in the melanophore assay but
show a reduced potency compared to melatonin. Potency
increases on changing the amine acylating group from methyl
through propyl, with 11c the most potent. Increasing the length
of the acylating group also improves binding affinity at MT2
but not MT1, 11c having a 28-fold selectivity. The cyclobutane
derivatives 12a-c showed a remarkable (45-84-fold) increase
in affinity at both melatonin receptors on changing the acyl
group from methyl through propyl. On melanophores, the methyl
(12a) and ethyl (12b) compounds were partial agonists giving
only modest aggregation (8% and 20%, respectively, of the
maximum response to melatonin) at 10 µM. However, the propyl
derivative (12c) was essentially a full agonist (92% of the
response of melatonin) with an EC50 of 2.00 nM. Changing the
acyl group to cyclopropanecarbonyl (12d) or cyclobutanecar-

Mapping the Melatonin Receptor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3513
Xenopus melanophores
Table 3a
receptor binding (Ki, nM)
MT2/
MT1
agonist
(EC50, nM)
antagonist
(IC50, nM)
compd
R1, R2
R
human MT1
human MT2
melatonin
19a
19b
19c
19d
H
Me
Me
Et
Pr
c-C3H5
c-C4H7
0.39 ( 0.08
8.20 ( 1.93
65.7 ( 10.7
116 ( 10
57.8 ( 10.0
493 ( 37
0.35 ( 0.03
3.30 ( 0.17
32 ( 2
57.4 ( 24.0
28.9 ( 1.7
62.2 ( 13.5
1.1
2.5
2
2
2
0.063 ( 0.004
1.67 ( 0.12
0.99 ( 0.05
2.56 ( 0.20
NA
NA
NA
NA
NA
2498 ( 116
>10000
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)3-
19e
8
NA
a See footnote to Table 1 for methods. NA ) no agonist or antagonist effect detected at 10 µM.
Table 4a
Xenopus melanophores
receptor binding (Ki nM)
MT2/
MT1
agonist
antagonist
(IC50, nM)
compd
R
human MT1
human MT2
(EC50, nM)
14a
14b
14c
14d
14e
Me
Et
Pr
c-C3H5
c-C4H7
2188 ( 1276
1514 ( 790
724 ( 79
1259 ( 378
1047 ( 24
1738 ( 40
1445 ( 215
2344 ( 899
2
1
NA
NA
NA
NA
NA
1248 ( 153
1149 ( 56
3031 ( 432
3443 ( 321
2006 ( 293
0.4
0.4
>4
617 ( 13
>10000
a See footnote to Table 1 for methods. NA ) no agonist effect detected.
Figure 2. Concentration-response curves for pigment aggregation by melatonin analogues in melanophores. Cells were grown in 96-well plates,
and growth medium was replaced by 0.7 × L-15 culture medium 18 h before testing. Initial absorbance (Ai, 630 nm) of the cells was measured in
each well, and cells were then treated with the concentrations of the analogue indicated. The final absorbance (Af) was measured after 60 min, and
the fractional change [1 - (Af/Ai)] was calculated. This figure shows the effect on agonist potency of changing the R group of the N-(2-[5-methoxy-
1-methyl-1H-indol-3-yl]-2,2-trimethylene-ethyl)alkanamides (12) and N-acyl 5-methoxy-â,â-trimethylenetryptamines (19) shown in Tables 2 and
3. Each point represents the mean response of duplicate wells of melanophores at each concentration in a single experiment. Results for a single
experiment are shown, but similar results were obtained in a second experiment.
bonyl (12e) dramatically reduced affinity at both receptor
subtypes and gave weak antagonists on the melanophore
response. Compound 13a with a cyclopentane ring had a similar
affinity to the corresponding cyclopropyl analogue 11a, but it
was a weak antagonist at the melanophore receptor rather than
an agonist. In contrast to the cyclopropyl (11a-c) and cyclobutyl
(12a-c) series, lengthening the acyl group (13a-c) tended to
have little effect (MT2) or reduced (MT1) affinity. In this series,
cyclopropanecarbonyl (13d) and cyclobutanecarbonyl (13e)
acylating groups had little influence on affinity. All compounds
with cyclopentane rings (13a-e) were weak antagonists on
melanophores.
The compounds in Table 3 illustrate the effect of replacing
the N-methyl group by hydrogen for the series of compounds
with a four-membered ring. The derivatives 19a,b are agonists
in the melanophore assay, unlike their N-methyl analogues
12a,b. Binding affinity at MT1 and MT2 receptors declines 15-
17-fold as the acyl group lengthens from methyl through propyl
(19a-c) rather than increasing dramatically as is seen for the
N-methyl analogues (12a-c). Again, all show a small preference
for the MT2 receptor. The N-cyclopropanecarbonyl 19d and
N-cyclobutanecarbonyl 19e derivatives are both antagonists in
the melanophore assay, and both have a somewhat higher
affinity at MT1 and MT2 receptors than their N-methyl ana-
logues.
Table 4 shows the binding potency data for a series of
cyclobutane derivatives with H rather than OMe at position 5
of the indole ring. All of the compounds are antagonists in the
melanophore assay, and they all bind weakly at the human MT1
and MT2 receptors.
The dramatic change in activity on melanophores, illustrated
in Figure 2, between 19a-c (all agonists) and 12a-c (12a,b
very weak, low-efficacy partial agonists, 12c agonist) by
substituting a methyl group for hydrogen at N-1 in the
cyclobutane series, led us to investigate their functional response
at the MT1 and MT2 human receptors. While all of the
compounds bind reasonably strongly to the human receptors,
12a-c show an increase in binding with increasing length of
side chain (Me, Et, Pr) whereas the NH series 19a-c show a
decrease with increasing side chain length. Compound 12c is
of particular interest in that increasing the side chain length from
Me to Et to Pr (12a to b to c) has converted the cyclobutane

3514 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Tsotinis et al.
Figure 3. Inhibition of forskolin-induced cyclic AMP synthesis by melatonin, 12c, and 13c in NIH 3T3 cells expressing human MT1 or MT2
receptors. Cells were treated with 3-isobutyl-1-methylxanthine (IBMX, 25 µM, 10 min) then forskolin (1 µM, 10 min)14 in the absence or presence
of the compounds indicated: melatonin (1 nM), 12c (1 µM), or 13c (10 µM). For each cell sample, cyclic AMP was measured in duplicate by
radioimmunoassay. Data are expressed as mean ( SEM of quadruplicate cell samples. *p < 0.05 compared to forskolin alone. **p < 0.01 compared
to forskolin alone. †p < 0.02 compared to forskolin + melatonin. ‡p < 0.0001 compared to forskolin + melatonin.
derivatives from weak (EC50 ∼ 1000 nM) partial agonists
(12a,b) with very low efficacy (8-20%) to a potent (EC50 ) 2
nM) agonist with virtually full efficacy. In two independent
experiments, maximal aggregation with 12c was 88% and 96%
of that achieved with melatonin (1 nM). This may indicate that
in the cyclobutane series (12) the methoxy group is not well
accommodated in its binding site, perhaps because of the indole
N-methyl group, and so cannot readily induce the receptor
conformation needed for agonist action. Extra binding affinity
obtained at the N-acyl bonding site by increasing the length of
the alkyl group may allow full receptor activation.
We have examined the potency of 12c and, initially for
comparison, the cyclopentane analogue 13c at the human MT1
and MT2 human receptors using the inhibition of forskolin-
induced formation of cyclic AMP as a marker of receptor
activation. The results are shown in Figure 3.
In NIH 3T3 cells expressing MT1 receptors, melatonin (1 nM)
inhibited forskolin-stimulated cyclic AMP synthesis but 12c (1
µM) did not, indicating that 12c does not have an agonist activity
at MT1 receptors. However, 12c did block melatonin-induced
suppression of cyclic AMP (Figure 3), indicating that it acts as
an MT1 antagonist. At the MT2 receptor, 12c (1 µM) acted as
an agonist inhibiting cyclic AMP synthesis as effectively as
melatonin. Compound 13c (10 µM) also had no agonist action
at MT1 receptors even at this higher concentration, but it did
act as an MT1 antagonist. Unlike 12c, 13c had no significant
MT2 agonist action, nor did it block melatonin inhibition of
cyclic AMP. Compounds 6f, 11c, 17b, and 19c also acted as
MT1 and MT2 receptor agonists inhibiting forskolin stimulation
of cyclic AMP synthesis (data not shown).
affinity for both human receptors, particularly at MT1. This
effect can largely be attributed to the (-)-enantiomer and
illustrates the much greater steric requirement of the MT1
receptor in this region. The introduction of a second â-methyl
group (6d) further decreases the affinity, but mainly at the MT1
receptor. The changes in potency at the Xenopus receptor reflect
the change in affinity at the MT1 receptor. The introduction of
a three-membered (11a) ring drastically reduces the affinity at
both human receptors and also the potency on Xenopus
melanophores. Affinity is also decreased with four- and five-
membered rings (12a, 13a), but in these cases the compounds
are antagonists on melanophores.
Comparison of the N-methyl derivative 6a with its NH
analogue 17a shows that the methyl group increases affinity
∼5-fold at MT2 while decreasing it ∼5-fold at MT1. The same
pattern of effects is observed on comparing 6d and 17b. In the
case of the cyclobutyl derivatives, introducing the methyl group
on the indole nitrogen (13a) greatly reduces the affinity to both
human receptors from that found in the NH analogue 19a and
converts a melanophore agonist into an antagonist. The related
analogue 13c also acted as an antagonist on human MT1
receptors (Figure 3) though not at MT2 receptors despite its ∼10-
fold higher affinity at the MT2 site. Compound 13c is one of
the first examples of a MT1 selective antagonist and warrants
further investigation. Further, whereas extending the N-acyl side
chain (Me < Et < Pr) decreases the affinity of the indole NH
compounds 19a-c, extending the side chain with the NMe
compounds (12a-c) dramatically increases affinity. In the
melanophore assay, the indole NH compounds (19a-c) are all
agonists while 12a and b show only very weak partial agonist
activity with only 12c having near full efficacy as an agonist.
Indeed, 12c has different effects on the two mammalian
Taking N-methylmelatonin as the basic structure, these results
illustrate the effect of adding methyl and cycloalkyl groups at
the â-position. A single â-methyl group (6a) decreases the

Mapping the Melatonin Receptor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3515
with CH2Cl2 (2 × 50 mL). The alkaline mixture was then poured
into an excess of ice-cold HCl (10%) when the product precipitated.
It was removed by filtration under reduced pressure, washed with
water, and then dried at 25 °C/1 mmHg.
receptors, acting as an antagonist at MT1 but as an agonist at
the MT2 site.
The four compounds, 11c, 19c, 12c, and 13c, nicely illustrate
the subtle differences between the three receptors, with the small
incremental changes in size and possibly shape affecting first
the human MT1 receptor, then the melanophore receptor, while
the human MT2 receptor remains much more tolerant to change.
Thus, while all four compounds show a higher binding for MT2
over MT1, the cyclopentane derivative 13c is an antagonist at
melanophores while the cyclopropane derivative 11c and the
cyclobutane NH derivative 19c are agonists.
Some of these compounds, particularly 12c and 13c, should
be valuable tools for determining whether specific melatonin
effects are mediated by the MT1 or MT2 receptors. We are
currently examining the effects of 12c and 13c in a study on
the sleep behavior of rats, hoping to establish the role of
melatonin in sleep onset and to discover whether it acts through
a melatonin receptor.
General Procedure for the Conversion of Acids to Amides.
The carboxylic acid (1 equiv) was dissolved in CH2Cl2 (10 mL/g),
Et3N (1.1 equiv) was added, and the mixture was cooled to 0 °C
and stirred. After 10 min methyl chloroformate (1.1 equiv) was
added dropwise, and stirring was then continued for 30 min at room
temperature. Ammonia was then bubbled through the solution for
2 min when a white precipitate formed. The mixture was stirred
for 1 h and was then washed with water (20 mL), 2 M HCl (2 ×
20 mL), and 2 M NaOH (2 × 20 mL) and dried (MgSO4). The
solvent was removed to leave the crude product which was of
sufficient purity for subsequent reactions.
General Procedure for the Preparation of Amides. A solution
of amine in dry CH2Cl2 (1 mL) was treated with triethylamine at
0 °C. The appropriate anhydride or acid chloride was then added
dropwise, and the resulting mixture was left stirring at room
temperature for 30-60 min (the reaction was monitored by TLC).
The reaction mixture was then poured into water, and CH2Cl2 was
added. The organic layer was washed with H2O (15 mL) and brine
and dried (Na2SO4). The solvent was evaporated under reduced
pressure to give the crude product, which was purified either by
trituration with ethyl acetate or by SPC.
General Procedure for Reduction of Nitriles. A solution of
the nitrile in benzene (3 mL) was added dropwise to a well-stirred
suspension of LAH in dry ether (15 mL) at 0 °C. After the addition
was completed, the mixture was allowed to thaw and then stirred
at room temperature for 1 h. The resulting suspension was cooled
to 0 °C, H2O (2 mL) was added cautiously, and stirring continued
for 30 min. The reaction mixture was filtered, and the filter cake
was washed with ethyl acetate (3 × 30 mL). The filtrate collected
was washed with H2O (15 mL) and brine and dried (Na2SO4). The
solvent was removed under reduced pressure and the resulting amine
then used without further purification.
General Procedure for Reduction of Amides. The amide (1
equiv) was dissolved in anhydrous THF (10 mL/g) and added
dropwise to a suspension of LAH (10 equiv) in anhydrous THF
(20 mL/g). After completion of addition the mixture was refluxed
for 2 h and allowed to cool. Water (2 mL) was then added dropwise,
the mixture was filtered, and the residue was washed with EtOAc.
The filtrate was washed with water (20 mL) and extracted with 2
M HCl (2 × 20 mL). The aqueous solution was then washed with
EtOAc (20 mL), and 2 M NaOH was then added until the reaction
mixture was basic. The mixture was extracted with EtOAc (2 ×
20 mL) and the organic extracts dried (MgSO4). Removal of the
solvent gave the amine as a colorless oil in sufficient purity for
subsequent reactions.
Experimental 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 and base
peaks are reported. 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
1
otherwise stated. H NMR spectra were taken on either a Varian
VXR-400 or a Bruker AC 300 spectrometer, and the spectra are
reported in δ. 13C NMR spectra were recorded on either a Varian
VXR-400 or Bruker AC200 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-anisidine, iodine, or ninhydrin.
Flash chromatography was performed using Sorbsil c60-A silica
as the stationary phase. Spinning plate chromatography (SPC) was
performed in a Chromatotron apparatus (Model 7924), using plates
of 4 mm thickness coated with Merck Kieselgel GF254 silica gel.
Chiral HPLC was performed on a Varian PrepStar 218 instrument
using Chiracel AD analytical and semipreparative columns with
hexane/ethyl acetate as eluant. Optical rotations were recorded on
a Polaar 2000 instrument as solutions in EtOAc. Circular dichroism
spectra were determined at the Department of Pharmacy, King’s
College London, on a Jasco J720 spectropolarimeter as solutions
in methanol. Recombinant human MT1 and MT2 subtypes expressed
in NIH 3T3 cells were kindly provided by Dr. S. M. Reppert
(Harvard Medical School, Boston, MA). Microanalyses were carried
out by either the Microanalytical Section of the Institute of Organic
and Pharmaceutical Chemistry, NHRF, or the Microanalytical
Section, Department of Chemistry, UCL.
General Procedure for the Bischler Reaction. A mixture of
N-methyl-p-anisidine (2 equiv) and the R-bromoketone (1 equiv)
was stirred under nitrogen at 50 °C for 3 h. The resulting dark
mixture was dissolved in propan-2-ol (100 mL/0.1 mol aniline),
and ZnCl2 (3 equiv, dried at 25 °C/1 mmHg for 2 d) was added.
The mixture was refluxed under N2 for 16 h. The solvent was
removed by evaporation and the residue extracted with a mixture
of 2 M HCl and EtOAc (3 × 100 mL HCl/150 mL EtOAc). The
red organic layer was washed with water (2 × 100 mL) and
saturated Na2CO3 solution (2 × 100 mL) and dried (MgSO4).
Evaporation of the solvent gave the crude product of sufficient
purity for use in subsequent reactions.
2-(5-Methoxy-1-methyl-1H-indol-3-yl)propanoic Acid (4a).
N-Methyl-p-anisidine (2) (15.50 g, 0.11 mol) was treated with ethyl
4-bromo-2-methyl-3-oxobutyrate (3a) (12.10 g, 0.055 mol) and
ZnCl2 (22.5 g) according to the general procedure to give a brown
oil. This was dissolved in MeOH (50 mL) and saponified with 10%
NaOH according to the general procedure to give 4a (5.20 g, 20
mmol, 36%) as a yellow solid, mp 118-120 °C (lit.44 123-124
°C).
2-(5-Methoxy-1-methyl-1H-indol-3-yl)-2-methylpropanoic Acid
(4b). N-Methyl-p-anisidine (2) (21.90 g, 0.16 mol) was treated with
ethyl 4-bromo-2,2-dimethyl-3-oxobutyrate (3b) (19.20 g, 0.08 mol)
and ZnCl2 (25 g) according to the general procedure to give a brown
oil. This was dissolved in MeOH (50 mL) and saponified with 10%
NaOH according to the general procedure to give 4b (5.90 g, 24
mmol, 30%) as a pale brown solid, mp 78-81 °C (lit.44 80.5-82
°C).
1-(5-Methoxy-1-methyl-1H-indol-3-yl)cyclopentanecarboxyl-
ic Acid (4c). N-Methyl-p-anisidine (2) (13.70 g, 0.10 mol) was
treated with ethyl 1-bromoacetyl cyclopentane-1-carboxylate (3c)
(13.10 g, 0.05 mol) and ZnCl2 (15 g) according to the general
procedure to give a brown oil. This was dissolved in MeOH (100
mL) and saponified with 10% NaOH according to the general
General Procedure for Saponification of Esters. The crude
product from the Bischler reaction was dissolved in hot 90%
aqueous MeOH or EtOH, NaOH (10 equiv) was added, and the
mixture refluxed for 6 h. The alcohol was removed by evaporation
under reduced pressure and the remaining alkaline solution washed

3516 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Tsotinis et al.
procedure to give 4c (7.10 g, 26 mmol, 54%) as a pale brown solid,
mp 60-70 °C. 1H NMR δ 1.75 (m, 4H), 2.06-2.09 (m, 2H), 2.63-
2.67 (m, 2H), 3.70 (s, 3H), 3.80 (s, 3H), 6.86-6.89 (m, 1H), 6.93
(s, 1H), 7.15-7.17 (m, 2H). 13C NMR δ 23.9, 32.9, 35.8, 53.3,
55.8, 102.7, 110.0, 111.8, 116.0, 126.7, 126.7, 132.8, 153.5, 182.4.
IR 3430, 2929, 1715, 1218 cm-1. MS m/e 273, 228 (100). Anal.
(C16H19NO3) C, H, N.
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]-2,2-tetramethylene-
ethyl)cyclobutanecarboxamide (13e). From 5c, 66%, mp 121-
123 °C.
Preparation of 5-Methoxy-1H-indole-3-acetonitrile (8b).41,43
A mixture of diethylamine (0.70 mL, 6.8 mmol), acetic acid (0.90
mL, 16 mmol), aqueous formaldehyde (37%, 0.70 mL, 24.5 mmol),
and water (2 mL) was added in one portion to either indole or
5-methoxyindole (6.80 mmol). The mixture was stirred at room
temperature for 3 h and was then made alkaline (pH 8) by addition
of 10% aqueous NaOH. The mixture was extracted with EtOAc
(3 × 5 mL), and the combined organic extracts were washed with
brine and dried (Na2SO4). The solvent was removed under reduced
pressure and the residue used directly without further purification.
The residue (1.42 g) was dissolved in MeOH (23 mL) and stirred,
and a solution of KCN (1.12 g, 17.3 mmol) in water (2 mL) and
then iodomethane (1.30 mL, 20.7 mmol) were added at such a rate
that the temperature did not rise above 35 °C. After completion of
addition the mixture was stirred at room temperature for 10 h when
TLC indicated that the reaction was complete. The solvent was
removed under reduced pressure, and the residue was extracted with
EtOAc. The extracts were washed with water then brine and dried
(Na2SO4). The solvent was removed under reduced pressure to give
2-(5-Methoxy-1-methyl-1H-indol-3-yl)propanamide (5a). Com-
pound 4a (5.10 g, 22 mmol) was treated with Et3N (2.20 g, 22
mmol) and methyl chloroformate (2.10 g, 22 mmol) in CH2Cl2 by
the general procedure to give 5a (3.50 g, 15 mmol, 68%) as an
1
amorphous solid, mp 80-89 °C. H NMR δ 1.63 (d, J ) 7.3 Hz,
3H), 3.74 (s, 3H), 3.82 (m, 1H), 3,84 (s, 3H), 5.62-5.68 (br s,
2H), 6.91 (dd, J ) 2.4, 8.9 Hz, 1H), 6.98 (s, 1H), 7.02 (d, J ) 2.3
Hz, 1H), 7.20 (d, J ) 8.8 Hz, 1H). 13C NMR δ 17.7, 32.9, 38.1,
55.9, 100.7, 110.3, 112.4, 113.9, 126.9, 127.0, 132.5, 154.0, 177.8.
IR 3410, 2939, 1650, 1492, 1228 cm-1. MS m/e 232, 188 (100),
97. Anal. (C13H16N2O2) C, H, N.
2-(5-Methoxy-1-methyl-1H-indol-3-yl)-2-methylpropana-
mide (5b). From 4b, mp 43-51 °C.
1-(5-Methoxy-1-methyl-1H-indol-3-yl)cyclopentanecarboxa-
mide (5c). From 4c, mp 63-74 °C.
1
the products as yellow oils. 8b: 1.08 g, 95%. H NMR δ 3.64 (s,
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]propyl)acetamide (6a).
Compound 5a (2.50 g, 11 mmol) was reduced with LAH (3.0 g)
as described in the general procedure to give the amine (1.00 g,
4.6 mmol, 42%) as a yellow oil. The oil (0.40 g, 1.8 mmol) was
treated with acetic anhydride (0.20 g) by the general procedure to
give 6a (0.25 g, 1.0 mmol, 54%), mp 45-47 °C. 1H NMR δ 1.31
(d, J ) 7.0 Hz, 3H), 1.86 (s, 3H), 3.18-3.22 (m, 1H), 3.41-3.48
(m, 2H), 3.62 (s, 3H), 3.80 (s, 3H), 6.39 br t, 1H), 6.78 (s, 1H),
6.83 (dd, J ) 2.2, 9.0 Hz, 1H), 7.09 (d, J ) 2.3 Hz, 1H), 7.10 (d,
J ) 8.7 Hz, 1H). 13C NMR δ 18.4, 22.6, 30,5, 32.3, 45.5, 55.4,
100.7, 109.7, 111.3, 116.4, 125.5, 127.0, 132.1, 153.2, 170.3. IR
3305, 2943, 1639, 1498, 1218 cm-1. MS m/e 260, 201 (100). Anal.
(C15H20N2O2) C, H, N
2H), 3.78 (s, 3H), 6.84 (dd, J ) 2.2, 8.8 Hz, 1H), 6.95 (m, 2H),
7.16 (d, J ) 8.8 Hz, 1H), 8.53 (bs, 1H). 13C NMR δ 13.8, 55.5,
99.4, 103.3, 112.2, 112.5, 118.4, 123.5, 126.0, 131.1, 153.9.
Methylation of 8a,b. Potassium hydroxide 85% (5.45 mmol)
was added at 0 °C to a stirred solution of nitrile 8a or 8b (5.45
mmol) in DMF (30 mL). Iodomethane (5.98 mmol) was then added
dropwise at the same temperature, and the mixture was left stirring
for 30 min at ambient temperature. The resulting suspension was
then poured into water and extracted with EtOAc. The organic layer
was washed with water and brine and dried (Na2SO4), and the
solvent was removed under reduced pressure to give the crude
product, which was triturated with EtOAc.
The racemic mixture (40 mg of 6a) was dissolved in hexane:2-
propanol (9:1, 2 mg/mL) and injected in 0.5 mL aliquots onto a 25
cm × 1 cm Chiral AD semipreparative HPLC column, eluting with
hexane:2-propanol (9:1) (6a, 20.5 min; 6b, 22.0 min). Removal of
the solvent gave the separate solid enantiomers, each of which was
then examined by analytical HPLC for purity.
(1-Methyl-1H-indol-3-yl)acetonitrile (9a): 0.86 g, 93%, mp
58-60 °C (lit.45 59-60 °C).
(5-Methoxy-1-methyl-1H-indol-3-yl)acetonitrile (9b): 0.53 g,
88%, mp 105-106 °C (lit.45 104-106 °C).
General Method for the Synthesis of 10a-c. Potassium bis-
(trimethylsilyl)amide (3 equiv for 10a,c, 5 equiv for 10b) was added
to a solution of the nitrile 9a,b (2.94 mmol) in dry tetrahydrofuran
(23 mL) at 0 °C, under an argon atmosphere. The reaction mixture
was then stirred for 3 min at 0 °C, and a solution of either 1,3-
dibromopropane (1.2 equiv) or 1,2-dichloroethane (3 equiv) in dry
THF (6 mL) was added dropwise. The reaction was stirred until
completion (TLC) and then poured into a saturated solution of NH4-
Cl. The aqueous phase was extracted with EtOAc, and the combined
organic layers were washed with water and brine and dried (Na2-
SO4). The solvent was removed under reduced pressure and the
product purified by flash column chromatography (diethyl ether/
petroleum ether 40-60 °C) (25:75) to give the desired nitriles as
off-white solids.
(+)-6a: 17.2 mg; 100%. [R]D +9.0° (c ) 0.0086). (-)-6a: 14.0
mg, 97%. [R]D -8.3° (c ) 0.0070).
UV and CD spectra of the enantiomers were acquired on a Jasco
J720 spectropolarimeter in the range 400-185 nm as solutions (0.40
mg/mL) in methanol. A 0.02 cm cell path length was used, and all
spectra were corrected for solvent baseline and normalized for ꢀ
and ∆ꢀ. The UV spectra are virtually identical, and the CD spectra
are mirror images
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]propyl)propana-
mide (6b). From 5a, 60%, yellow oil.
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]propyl)butana-
mide (6c). From 5a, 70%, yellow oil.
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]-2-methylpropyl)ac-
etamide (6d). From 5b, 83%, mp 32-35 °C.
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]-2-methylpropyl)-
propanamide (6e). From 5b, 55%, yellow oil.
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]-2-methylpropyl)bu-
tanamide (6f). From 5b, 40%, yellow oil.
1-Cyano-1-(1-methyl-1H-indol-3-yl)]cyclobutane (10a): 58%,
mp 80-81 °C. H NMR δ 2.19 (m, 1H), 2.40 (m, 1H), 2.75 (m,
2H), 2.90 (m, 2H), 3.77 (s, 3H), 7.02 (s, 1H), 7.39 (m, 3H), 7.72
(d, J ) 9.0 Hz, 1H). 13C NMR δ 17.6, 32.7, 33.9, 64.9, 109.6,
113.3, 119.3, 119.6, 121.0, 122.3, 124.2, 125.3, 125.8, 137.6. Anal.
(C14H14N2) C, H, N.
1
N-(2-(5-Methoxy-1-methyl-1H-indol-3-yl)-2-methylpropyl)cy-
clobutanecarboxamide (6g). From 5b, 62%, mp 135-137 °C.
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]-2,2-tetramethylene-
ethyl)acetamide (13a). From 5c, 53%, mp 97-98 °C.
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]-2,2-tetramethylene-
ethyl)propanamide (13b). From 4c, 72%, mp 118-119 °C.
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]-2,2-tetramethylene-
ethyl)butanamide (13c). From 5c, 68%, mp 108-108.5 °C.
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]-2,2-tetramethylene-
ethyl)cyclopropanecarboxamide (13d). From 5c, 84%, mp 134-
135 °C.
1-Cyano-1-(5-methoxy-1-methyl-1H-indol-3-yl)cyclopro-
pane (10b): 42%, mp 105-107 °C. H NMR δ 1.26-1.36 (m,
2H), 1.60-1.68 (m, 2H), 3.55 (s, 3H), 3.91 (s, 3H), 6.91-6.97
(m, 2H), 7.17-7.25 (m, 2H). 13C NMR δ 18.0, 32.8, 38.8, 55.2,
102.0, 110.3, 112.4, 112.6, 124.1, 125.6, 126.4, 133.2, 154.3. Anal.
(C14H14N2O) C, H, N.
1
1-Cyano-1-(5-methoxy-1-methyl-1H-indol-3-yl)cyclobutane
1
(10c): 56%, mp 114-116 °C. H NMR δ 2.18 (m, 1H), 2.40 (m,
1H), 2.70 (m, 2H), 2.90 (m, 2H), 3.72 (s, 3H), 3.87 (s, 3H), 6.95
(dd, J ) 2.3, 9.0 Hz, 1H) 6.98 (s, 1H), 7.11 (d, J ) 2.3 Hz, 1H),
7.21 (d, J ) 9.0 Hz, 1H). 13C NMR δ 17.6, 32.9, 33.6, 33.7, 56.0,

Mapping the Melatonin Receptor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3517
101.3, 110.5, 112.4, 112.6, 124.1, 125.6, 126.4, 133.0, 154.1. Anal.
(C15H16N2O) C, H, N.
give the methylated Boc-derivative as a brown oil (1.02 g, 76%).
A solution of (n-Bu)4NF in THF (7.4 mL, 7.4 mmol) was added to
a stirred portion of the oil (0.47 g, 1.5 mmol) in THF (10 mL).
The mixture was stirred and heated to 65 °C for 8 h and the resulting
solution poured onto water (20 mL) and extracted with EtOAc.
The organic layer was washed with water and brine and then dried
(Na2SO4). The solvent was removed under reduced pressure and
the residue purified by flash chromatography to give 16b as an oil
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]-2,2-dimethylene-
ethyl)acetamide (11a). The cyanide 10b46 was reduced to the
corresponding amine by the general method for the reduction of
nitriles, and the amine was then acetylated by the general method
for the acylation of amines to give 11a: 48%, mp 114-116 °C.
1H NMR δ 0.77-0.94 (m, 4H), 1.90 (s, 3H), 3.43 (d, J ) 5.8 Hz,
2H), 3.72 (s, 3H), 3.88, (s, 3H), 5.22 (bs, 1H), 6.86-6.96 (m, 2H),
7.13-7.28 (m, 2H). 13C NMR δ 12.4, 22.9, 32.5, 46.5, 46.8, 55.5,
102.5, 110.0, 111.2, 118.7, 126.1, 126.6, 133.0, 153.1, 172.8. Anal.
(C16H20N2O2) C, H, N.
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]-2,2-dimethylene-
ethyl)propanamide (11b): 52%, mp 104-105 °C.
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]-2,2-dimethylene-
ethyl)butanamide (11c): 62%, mp 110-112 °C.
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]-2,2-trimethylene-
ethyl)acetamide (12a). From 10c,46 48%, mp 88-89 °C. 1H NMR
δ 1.86 (s, 3H), 1.90-2.46 (m, 6H), 3.70-3.74 (m, 5H), 3.81 (s,
3H), 5.45 (bs, 1H), 6.78-6.95 (m, 2H), 7.18-7.28 (m, 2H). 13C
NMR δ 15.7, 23.0, 30.0, 32.5, 46.6, 46.8, 55.5, 102.5, 109.9, 111.5,
118.6, 126.0, 126.6, 132.9, 153.0, 170.0. Anal. (C17H22N2O2) C,
H, N.
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]-2,2-trimethylene-
ethyl)propanamide (12b): 46%, mp 93-95 °C.
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]-2,2-trimethylene-
ethyl)butanamide (12c): 52%, mp 106-107 °C.
1
(0.31 g, 1.45 mmol). H NMR δ 1.78 (s, 6H), 3.84 (s, 3H), 6.88
(dd, J ) 2.2, 8.8 Hz, 1H), 7.22 (d, J ) 8.8 Hz, 1H), 7.28 (s, 1H),
8.55 (bs, 1H).
2-(5-Methoxy-1H-indol-3-yl)propanonitrile (16a). Prepared as
for 16b except that only a slight molar excess of iodomethane was
added. The yield was substantially less (10%) than that for 16b.
1H NMR δ 1.72 (d, J ) 6 0.9 Hz, 3H), 3.86 (3H), 3.99 (q, J ) 6.9
Hz, 1H), 6.89 (dd, J ) 2.2, 8.8 Hz, 1H), 7.09 (d, J ) 8.8 Hz, 1H),
7.27 (s, 1H), 8.37 (bs, 1H).
â,â-Dimethylmelatonin (17b). A solution of the nitrile 16b (0.33
g, 1.55 mmol) in benzene (1.0 mL) was added dropwise to a stirred
suspension of LAH (0.18 g, 4.7 mmol) in dry ether (8.0 mL) at 0
°C. The mixture was allowed to warm to room temperature, and
stirring continued for a further 45 min. The resulting mixture was
cooled to 0 °C and stirred, and water (1.0 mL) was added dropwise
followed by EtOAc (10 mL). Stirring was continued for 30 min,
and the mixture was then filtered. The residue was washed with
EtOAc (3 × 30 mL), and the combined filtrates were washed with
water and brine and dried (Na2SO4). The solvent was removed under
reduced pressure to give the amine as a pale yellow oil that was
used without further purification. The amine (0.29 g, 1.33 mmol)
was dissolved in dry CH2Cl2 (4.0 mL), and the solution was treated
with Et3N (0.30 mL, 2.2 mmol) at 0 °C and stirred. Ac2O (0.17
mL, 1.2 mmol) was then added dropwise, the mixture allowed to
come to room temperature, and stirring continued for 1 h. The
reaction mixture was poured into water (20 mL), and CH2Cl2 (20
mL) was added. The organic layer was separated, washed with water
and brine, and dried (Na2SO4), The solvent was removed under
reduced pressure and the residue purified by column chromatog-
raphy, eluting with EtOAc:cyclohexane (95:5) to give 17b as a buff
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]-2,2-trimethylene-
ethyl)cyclopropanecarboxamide (12d): 46%, mp 120-122 °C.
N-(2-[5-Methoxy-1-methyl-1H-indol-3-yl]-2,2-trimethylene-
ethyl)cyclobutanecarboxamide (12e): 42%, mp 121-123 °C.
N-(2-[1-Methyl-1H-indol-3-yl]-2,2-trimethylene-ethyl)aceta-
mide (14a). Compound 10a46 was reduced by LAH according to
the general procedure and then acetylated according to the general
1
procedure to give 14a: 52%, mp 89-90 °C. H NMR δ 1.89 (s,
3H), 1.96-2.08 (m, 1H), 2.14-2.46 (m, 5H.), 3.79 (d, J ) 5.8 Hz,
2H), 3.81 (s, 3H), 5.20 (bs, 1H), 6.85 (s, 1H), 7.06-7.12 (m, 1H),
7.22-7.35 (m, 2H) 7.48-7.54 (m, 1H). 13C NMR δ 15.8, 22.3,
29.8, 33.5, 46.8, 47.8, 101.5, 110.1, 111.4, 118.9, 125.8, 126.4,
133.0, 140.9, 170.9. Anal. (C16H20N2O2) C, H, N.
1
solid (0.22 g, 62%), mp 120-122 °C. H NMR δ 1.40 (s, 6H),
1.84 (s, 3H), 3.63 (d, J ) 5.8 Hz, 2H), 3.83 (s, 3H), 5.32 (br t, 5.5
Hz, 1H), 6.85 (dd, J ) 2.4, 8.8 Hz, 1H), 6.97 (d, J ) 2.4 Hz, 1H),
N-(2-[1-Methyl-1H-indol-3-yl]-2,2-trimethylene-ethyl)propan-
amide (14b): 46%, mp 96-97 °C.
N-(2-[1-Methyl-1H-indol-3-yl]-2,2-trimethylene-ethyl)butana-
mide (14c): 62%, mp 109-110 °C.
N-(2-[1-Methyl-1H-indol-3-yl]-2,2-trimethylene-ethyl)cyclo-
propanecarboxamide (14d): 42%, mp 123-124 °C.
7.18 (d, J ) 2.4 Hz), 7.27 (d, J ) 8.8 Hz, 1H), 8.54 (bs, 1H). 13
C
NMR δ 23.1, 26.1, 35.4, 48.6, 55.7, 103.1, 111.6, 112.2, 120.7,
122.5, 125.6, 132.5, 153.4, 170.4. Anal. (C15H20N2O2) C, H, N.
â-Methylmelatonin (17a). Prepared from 16a as for 17b, pale
1
yellow oil (78%). H NMR δ 1.35 (d, J ) 7.1 Hz, 3H), 1.87 (s,
3H), 3.23 (m, 1H), 3.42 (m, 1H), 3.61 (m, 1H), 3.84 (s, 3H), 5.44
(bs, 1H), 6.86 (dd, J ) 2.4, 8.8 Hz, 1H), 6.99 (d, J ) 2.4 Hz, 1H),
7.07 (d, J ) 2.4 Hz, 1H), 7.25 (d, J ) 8.8 Hz, 1H), 8.05 (bs, 1H).
13C NMR δ 18.7, 23.3, 30.9, 45.6, 55.9, 100.8, 112.1, 112.2, 118.2,
121.5. 126.9, 131.7, 153.8, 170.3. Anal. (C14H18N2O2) C, H, N
1-(5-Methoxy-1H-indol-3-yl)cyclobutanecarbonitrile (18). A
solution of 1,3-dibromopropane (0.1 mL, 0.95 mmol) and 15 (0.27
g, 0.95 mmol) in a mixture of DMSO (3.5 mL) and ether (3.5 mL)
was added dropwise to a slurry of NaH (2.8 mmol) in DMSO (2.5
mL) at ambient temperature). The resulting mixture was stirred at
ambient temperature for 2 h, cooled to 0 °C, and treated with MeOH
(3 mL). The cooling bath was removed, and the reaction was stirred
at ambient temperature for 20 min. The mixture was extracted with
EtOAc, and the organic layer was washed with water and brine
and dried (Na2SO4). The solvent was removed under reduced
pressure, and the residue was purified by flash column chroma-
tography (petroleum ether 40-60 °C/ethyl acetate) (75:25) to give
N-(2-[1-Methyl-1H-indol-3-yl]-2,2-trimethylene-ethyl)cyclobu-
tanecarboxamide (14e): 48%, mp 125-127 °C.
(5-Methoxy-1-t-butoxycarbonyl-1H-indol-3-yl)acetonitrile (15).
A solution of 8b (0.37 g, 2.0 mmol) in CH2Cl2 (5 mL) was treated
with 4-(dimethylamino)pyridine (0.24 g, 2 mmol) and di-tert-butyl
dicarbonate (0.46 mL, 2 mmol). The mixture was stirred at ambient
temperature for 3 h, and the resulting suspension was then poured
into water and extracted with EtOAc. The organic layer was washed
with water and brine and dried (Na2SO4). The solvent was
evaporated under reduced pressure, and the residue was purified
by flash column chromatography to give 15 as a beige solid (0.59
1
g, 95%), mp 118-120 °C. H NMR δ 1.66 (s, 9H), 3.74 (s, 2H),
3.87 (s, 3H,), 6.93 (d, J ) 2.4 Hz, 1H), 6.97 (dd, J ) 2.4, 9.2 Hz,
1H), 7.60 (s, 1H), 8.05 (d, J ) 8.6 Hz, 1H). Anal. (C16H18N2O3)
C, H, N.
2-(5-Methoxy-1H-indol-3-yl)-2-methylpropanonitrile (16b). A
mixture of the acetonitrile 15 (1.22 g, 4.50 mmol) and iodomethane
(0.53 mL, 11 mmol) in DMF (9.2 mL) was added dropwise to a
stirred slurry of NaH (0.420 g, 10.5 mmol) in DMF (9.2 mL) at 0
°C. The mixture was then allowed to warm to room temperature
and stirred for 4 h. The mixture was then treated with saturated
aqueous NH4Cl and extracted with EtOAc, and the organic extract
was washed with water and brine and dried (Na2SO4). The solvent
was removed under reduced pressure and the residue purified by
flash chromatography, eluting with cyclohexane/EtOAc (96:4) to
1
18 (0.15 g, 70%) as a brown viscous oil; H NMR δ 2.12-2.18
(m, 1H), 2.28-2.44 (m, 1H), 2.62-2.73 (m, 2H), 2.81-2.95 (m,
2H), 3.86 (s, 3H), 6.89 (dd, J ) 2.4, 9.2 Hz, 1H), 7.08-7.12 (m,
2H), 7.26 (d, J ) 9.0 Hz, 1H), 8.15 (br s, 1H). Anal. (C14H14N2O)
C, H, N.
N-(2-[5-Methoxy-1H-indol-3-yl]-2,2-trimethyleneethyl)aceta-
mide (19a). Compound 18 was reduced with LAH following the
general procedure and acetylated by the general procedure to give

3518 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Tsotinis et al.
19a, 65%, light yellow viscous oil. 1H NMR δ 1.86 (s, 3H), 1.96-
2.44 (m, 6H), 3.75 (d, J ) 5.5 Hz, 2H), 3.81 (s, 3H), 5.29 (br s,
1H), 6.86 (dd, J ) 2.3, 8.7 Hz, 1H), 6.93 (d, J ) 2.2 Hz, 1H), 6.97
(13) Garratt, P. J.; Travard, S.; Vonhoff, S.; Tsotinis, A.; Sugden, D.
Mapping the melatonin receptor. 4. Comparison of the binding
affinities of a series of substituted phenylalkylamides. J. Med. Chem.
1996, 39, 1797-1805.
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et al. Mapping the melatonin receptor. 5. Melatonin agonists and
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carbazoles, and hexahydrocyclohept[b]indoles. J. Med. Chem. 1998,
41, 451-467.
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Mapping the melatonin receptor. 6. Melatonin agonists and antago-
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(d, J ) 2.3 Hz, 1H), 7.23 (d, J ) 8.7 Hz, 1H), 8.53 (br s, 1H). 13
C
NMR δ 16.5, 23.3, 30.8, 41.5, 47.5, 55.9, 101.9, 112.0, 112.2, 120.9,
122.1, 125.7, 132.3, 153.6, 170.5. Anal. (C16H20N2O2) C, H, N.
N-(2-[5-Methoxy-1H-indol-3-yl]-2,2-trimethylene-ethyl)pro-
panamide (19b): 55%, light brown viscous oil.
N-(2-[5-Methoxy-1H-indol-3-yl]-2,2-trimethylene-ethyl)bu-
tanamide (19c): 57%, viscous oil.
N-(2-[5-Methoxy-1H-indol-3-yl]-2,2-trimethylene-ethyl)cyclo-
propanecarboxamide (19d): 40%, light brown viscous oil.
N-(2-[5-Methoxy-1H-indol-3-yl]-2,2-trimethylene-ethyl)cy-
clobutanecarboxamide (19e): 44%, light brown viscous oil.
Acknowledgment. The University of Athens group thanks
EPEAEK II Program Pythagoras II - Support of UniVersities
Research Groups (KA: 70/3/7993) for financial support. The
groups at King’s College London and University College
London thank the Wellcome Trust for funding this project (Grant
GR065816). We are grateful to Dr. Michael R. Lerner, Depart-
ment of Dermatology, University of Texas Southwestern Medi-
cal Center, Dallas, TX 75235-9069, for supplying the clonal
Xenopus laeVis melanophore cell line used in this work. We
thank Drs. Alex Drake and Tam Bui of the Department of
Pharmacy, King’s College London, for obtaining the CD spectra.
(17) Jansen, J. M.; Copinga, S.; Gruppen, G.; Molinari, E. J.; Dubocovich,
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Supporting Information Available: Experimental details and
1H NMR and MS spectral data for listed compounds, elemental
analysis results, and CD spectra. This material is available free of
charge via the Internet at http://pubs.acs.org.
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