Conformationally restrained melatonin analogues: Synthesis, binding affinity for the melatonin receptor, evaluation of the biological activity, and molecular modeling study

Article abstract of DOI:10.1021/jm960651z

The design, synthesis, and biological profile of several indole melatonin analogues with a conformationally restricted C3 amidoethane side chain are presented. Examination of the accessible conformations of the melatonin side chain led us to explore some of its fully or partially restricted analogues, 2-12, the binding affinity values of which were utilized to gain further insight on the melatonin binding site. Two pharmacophoric models have been devised for melatonin and the active compounds by conformational analysis and superimposition performed using the DISCO program. In these models, the melatonin side chain can adopt a gauche/anti conformation out of the indole plane. Another contribution of this study regards the observation of a possible binding point interaction around the C2 position of the indole, as suggested by the remarkably increased binding affinity observed in the C2-substituted analogues 6 and 9 and especially in the more rigid analogue 5. The biological activity and the efficacy of the new compounds were tested by measuring the inhibition of the forskolin-stimulated cAMP accumulation and the GTPγS index. Both analyses demonstrated that all of the compounds were full agonists with the exception of 4 and 9, which showed a slight reduction in efficacy and would seem to be partial agonists.

Full text of DOI:10.1021/jm960651z

1990
J . Med. Chem. 1997, 40, 1990-2002
Con for m a tion a lly Restr a in ed Mela ton in An a logu es: Syn th esis, Bin d in g Affin ity
for th e Mela ton in Recep tor , Eva lu a tion of th e Biologica l Activity, a n d
Molecu la r Mod elin g Stu d y
Gilberto Spadoni,* Cesarino Balsamini, Giuseppe Diamantini, Barbara Di Giacomo, and Giorgio Tarzia
Istituto di Chimica Farmaceutica e Tossicologica, Universita` degli Studi di Urbino, Piazza Rinascimento 6,
I-61029 Urbino, Italy
Marco Mor, Pier Vincenzo Plazzi, and Silvia Rivara
Dipartimento Farmaceutico, Universita` degli Studi di Parma, Viale delle Scienze, I-43100 Parma, Italy
Valeria Lucini, Romolo Nonno, Marilou Pannacci, Franco Fraschini, and Bojidar Michaylov Stankov
Cattedra di Chemioterapia, Dipartimento di Farmacologia, Universita` degli Studi di Milano, Via Vanvitelli, 32,
I-20129 Milano, Italy
Received September 17, 1996X
The design, synthesis, and biological profile of several indole melatonin analogues with a
conformationally restricted C3 amidoethane side chain are presented. Examination of the
accessible conformations of the melatonin side chain led us to explore some of its fully or
partially restricted analogues, 2-12, the binding affinity values of which were utilized to gain
further insight on the melatonin binding site. Two pharmacophoric models have been devised
for melatonin and the active compounds by conformational analysis and superimposition
performed using the DISCO program. In these models, the melatonin side chain can adopt a
gauche/ anti conformation out of the indole plane. Another contribution of this study regards
the observation of a possible binding point interaction around the C2 position of the indole, as
suggested by the remarkably increased binding affinity observed in the C2-substituted
analogues 6 and 9 and especially in the more rigid analogue 5. The biological activity and the
efficacy of the new compounds were tested by measuring the inhibition of the forskolin-
stimulated cAMP accumulation and the GTPγS index. Both analyses demonstrated that all
of the compounds were full agonists with the exception of 4 and 9, which showed a slight
reduction in efficacy and would seem to be partial agonists.
In tr od u ction
physiology shows few seasonal changes. Melatonin
receptors have recently been cloned from Xenopus
dermal melanophores10 and from hamsters, sheep, and
human brains.11
The pineal hormone melatonin (N-acetyl-5-methoxy-
tryptamine, aMT, 1) is now recognized to be the regula-
tor of circadian rhythmicity in humans and of seasonal
breeding in different animal species.1 Melatonin also
appears to be involved in a number of physio/pathologi-
cal conditions, and there is strong evidence for its use
in pathologies associated with circadian rhythm disor-
ders. The administration of aMT in humans was shown
to alleviate jet lag,2 to induce sleep,3 and to advance the
sleep rhythm of subjects with delayed sleep phase
syndrome.4 Numerous studies on elderly people and on
depressed patients have also reported a decrease in
overnight melatonin biosynthesis, thus suggesting a role
for this hormone in the aging process5 and in seasonal
depression.6 Recently aMT was reported to be eventu-
ally useful as an immunostimulant7 and as coadjuvant
in some antitumoral therapies.8 Melatonin receptors
are found in the CNS of mammals, and evidence for the
presence of different subtypes of melatonin receptors
has been reported.9 These findings suggest that aMT
should have more complex regulatory activities than
those recognized to date, especially in humans, whose
Despite major advances in our understanding of the
role of aMT, the knowledge of the physiological functions
of aMT receptors has been hampered by a lack of potent
and selective aMT-receptor antagonists and the poor
selectivity of action of the known aMT agonists.9b
The 3-ethylamido chain of aMT is flexible and can
adopt several energetically equivalent conformations by
rotation around the τ1 (C3a-C3-Câ-CR), τ2 (C3-Câ-
CR-N), and τ3 (Câ-CR-N-CO) bonds;12 this confor-
mational flexibility is probably responsible for the broad
spectrum of biological activities of aMT. Assuming a
correlation between the lack of selectivity of aMT and
the conformational freedom of the ethylamido side chain
and given the fact derived in other fields that confor-
mationally restricted compounds often lead to increased
binding affinity and selectivity over receptor subtypes,13
other authors recently reported a number of rigid aMT
analogues with a conformationally restricted ethylamido
side chain.14 Structure-activity studies on aMT ana-
logues showed that both the N-acyl and 5-methoxy
groups are necessary for high binding affinity15 and that
the relative spatial distance between these groups is
also an important factor.14a Thus, a knowledge of the
active conformation(s) of aMT, and therefore of the
spatial relationship of its presumed pharmacophoric
* Corresponding author: Gilberto Spadoni, Istituto di Chimica
Farmaceutica, Universita` di Urbino, Piazza Rinascimento 6, 61029
Urbino, Italy. (Tel) +39-722-2545. (Fax) +39-722-2737. (E-mail)
chimfarm@chim.uniurb.it.
X Abstract published in Advance ACS Abstracts, May 15, 1997.
S0022-2623(96)00651-6 CCC: $14.00 © 1997 American Chemical Society

Conformationally Restrained Melatonin Analogues
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 1991
F igu r e 1.
Ta ble 1. Binding Affinitya and Biological Activity of Compounds 2-16
relative
affinityb
GTPγS
cAMP
indexd
compd
aMT (1)
2
3
4
5
6
7
8
9
10
11
12
13
14
15
IC50
2.2
33000 ( 3160
4300 ( 387
1200 ( 80
73 ( 8
Ki
indexc
activitye
0.61
9100
1200
330
20
1
15000
1950
550
33
0.77
4.5
13.2
0.64
nt
1360
100
590
nt
nt
nt
81
1.64
131
3230
0.059
0.028
1.6
1
nt
nt
0.62
1
0.85
1.05
1.25
0.6
nt
0.88
0.98
0.97
nt
1
nt
nt
A
nt
nt
PA
A
A
A
0.75
0.98
0.9
1
1.18
0.78
nt
0.9
1.02
1
1.7 ( 0.2
9.9 ( 1.2
29 ( 2
0.48
2.7
8.0
0.39
nt
820
63
360
nt
nt
nt
A
1.4 ( 0.1
>10000
PA
nt
A
A
A
nt
nt
nt
3000 ( 370
230 ( 20
1300 ( 145
>10000
>10000
>10000
nt
nt
nt
nt
nt
16
(S)-29f
30g
31h
32
7100 ( 1050
1900
0.08
1.02
0.82
0.91
0
AN
A
A-PA
A
2-Br-melatonin
2-Ph-melatonin
6-Cl-melatonin
0.13 ( 0.008
0.036
0.017
0.99
1.15
0.78
1.04
0.062 ( 0.01
3.6 ( 0.28
a
IC50 and Ki values are expressed in nM and are the means of 3-20 independent determinations, derived from nonlinear fitting strategies.
b
The SEM values were below 15% of the mean. Relative affinity ) (IC50 compd/IC50 aMT) determined in parallel, in the same experiment.
c GTPγS index ) [(IC50 with GTPγS)/(IC50 without GTPγS)] compd/[(IC50 with GTPγS)/(IC50 without GTPγS)] aMT. cAMP index ) (percent
d
inhibition compd)/(percent inhibition aMT) determined in parallel in the same experiment. e nt, not tested; A, agonist; PA, partial agonist;
AN, antagonist. f Values from J ansen et al.14e Values from Garrat et al.14a Values from Gruppen et al.17
g
h
groups (i.e. the aromatic moiety, the ethylamido, and
the methoxy substituent) is of fundamental importance
both for a rational design of potent and selective new
molecules and for the clarification of the topography of
the aMT receptor. Therefore, along this line of thought
and expanding on what had been already reported, we
undertook a project aimed at exploring the possibility
of limiting the conformational freedom of the 3-ethyl-
amido side-chain in ways thus far unexplored. To this
end, a variety of restricted or rigid aMT analogues with
the 3-ethylamido side chain included in different struc-
tures (2-12) (Figure 1) were prepared and tested in
binding assays. The biological results obtained for the
most interesting compounds, i.e. 4, 7, 8, 12 (Table 1),
were submitted to a computer molecular modeling study
(DISCO),16 together with some of the previously de-
scribed conformationally restricted ligands (i.e. (S)-
29,14e,f 30,14a 3117) in order to devise possible pharma-
cophore models. The results of this analysis will be
presented and discussed in comparison to other recently
proposed models.14e,f,15,18
It had been reported by several groups, including
ours, that appropriate substituents, either at the C2
position of the indole nucleus15,19,20 or ortho to the
ethylamido side chain in the naphthalenic melatonin
analogues,21 modulate the binding affinity to the mela-
tonin receptor. We therefore decided to further inves-
tigate the consequences of suitable C2 indole substitu-
ents on the binding affinity of these new rigid analogues,
in order to better understand the role of these groups
in the binding to the aMT receptor. With regard to the
goal of determining the bioactive conformation(s), the

1992 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13
Spadoni et al.
182-3 °C; 84% yield), was prepared according to a
previously reported procedure for the ethyl ester deriva-
tive.26 Key intermediates in the synthesis of new
compounds 6 and 10 were the corresponding keto
derivatives 24 and 25, which were prepared as outlined
in Scheme 1. Briefly, the palladium-catalyzed coupling
(Heck reaction) of 4-bromo-5-methoxyindole derivatives
1727 and 18 with methyl or benzyl acrylate gave the
derivatives 19 and 20, to which the (E)-stereochemistry
was assigned on the basis of the J value (16.5 and 16.2
Hz, respectively) reported for the ethenyl protons, in
accordance with that reported for similar compounds.28
Compounds 19 and 20 were converted to the acids 22
and 23 by catalytic hydrogenation and hydrolysis; these
acids were then cyclized in polyphosphoric acid (80 °C,
80 min) to the ketones 24 and 25. The reactions carried
out on the 2-carbomethoxy derivatives 18 and 23 gave
a better yield than those obtained on the unsubstituted
compounds 17 and 22. The ketone 24 was transformed
in two steps (condensation with benzylamine, catalytic
hydrogenation and debenzylation) to the crude amine
intermediate 26, which was acylated with acetic anhy-
dride/TEA, obtaining the desired compound 10 in 15%
overall yield (Scheme 2). As shown in Scheme 3, the
ketone 25 was converted to the corresponding 2,4,6-
triisopropyl hydrazone by reaction with 2,4,6-triisopro-
pylbenzenesulfonyl hydrazide (TPSH), which was then
transformed, without any previous purification, into the
cyano ethyl ester derivative 27 (29% yield) by heating
in ethanol with an excess of potassium cyanide using a
procedure previously described for a related com-
pound.29 By reduction of 27 with H2, Raney nickel in
the presence of Ac2O, we obtained the desired compound
6. Derivative 7 was prepared by ester hydrolysis of 6
followed by decarboxylation of the corresponding acid
in boiling quinoline in the presence of Cu powder. The
(E)-3-ethenyl alkylamido derivatives 8 and 9 were
synthesized in poor yields (see the Experimental Sec-
tion) by reduction of the nitro group (H2, Raney nickel,
room temperature) of the corresponding (E)-5-methoxy-
3-(2-nitroethenyl)indoles in the presence of acetic an-
hydride. High amounts of melatonin or 2-phenylmela-
tonin were isolated from this reaction. The (E)-
stereochemistry was assigned on the basis of the
coupling constant values of the ethenyl protons (J )
14.62 Hz), which were consistent with the values
expected for the (E)-stereoisomers.30 By catalytic hy-
drogenation over 10% Pd-C of 3-nitro-6-methoxy-9H-
carbazole31 and concomitant acylation with acetic an-
hydride we obtained a mixture of compounds 11 (30%
yield), and 12 (30% yield) which were then separated
by flash chromatography. The new compound 13 (37%
yield) was prepared by reducing 4-(2-nitroethenyl)-5-
methoxyindole26 with LiAlH4 and acylating the resulting
crude amine with Ac2O/TEA. The palladium-catalyzed
coupling of 18 with acrylonitrile gave the derivative 28,
which was converted to the propylacetamido derivative
14 by hydrogenation over Raney nickel and N-acylation
with Ac2O (Scheme 4). By reduction of 4-cyano-5-
methoxyindole26 or 3-cyano-5-methoxyindole32 with H2,
Raney nickel in the presence of a suitable anhydride,
we obtained the corresponding alkylamidomethyl de-
rivatives 15 and 16 (Figure 2). Compound 32, N-[(2-
phenyl-1H-indol-3-yl)ethyl]cyclobutanecarboxamide, used
F igu r e 2.
accessible conformations of compounds 2-12 (Figure 1)
were superimposed to those of aMT, looking for common
relative spatial distances among the putative pharma-
cophoric groups.
Compounds 6 and 7, in which the C3-Câ portion of
the side chain is incorporated into a cyclic structure,
and 8 and 9, which are characterized by the presence
of the CR-Câ double bond, represent aMT analogues
with a partially constrained 3-ethylamido side chain.
The carbazole derivatives 11 and 12 and compounds 4
and 5 represent two different ways in which the con-
formational flexibility is limited around both the τ1 and
the τ2 bonds. In derivatives 422-7, which have a ring
that bridges the R- or â-carbon of the ethylamido chain
and 4-position of the indole ring, thus limiting the
conformational flexibility of the side chain, the alkyl-
amido group is conformationally free. On the contrary,
in the aza tricyclic compounds 2 and 3, formally
obtained by cyclization of the acyl moiety to the 4- and
2-positions of the indole, the conformational freedom is
restricted not only around the τ1 and τ2 bonds but also
around the τ3 one, so that they are more rigid analogues
of aMT, with the CONH group fixed in a cis conforma-
tion. Finally, we synthesized four compounds (Figure
2) with the mobile side chain attached at position 4 of
the indole (13-15), or with a chain shorter than that of
aMT (16), in order to test the hypothesis that the
relative distances between the pharmacophoric points
could be sufficient to determine the affinity for the aMT
receptor, regardless of the attachment point of the side
chain.
Although a clear stereoselectivity of the binding of
conformationally restricted compounds to the melatonin
receptor had recently been demonstrated,14e,23 no at-
tempt was made at this stage to resolve the enantio-
meric mixtures. When a chiral center was present,
compounds were tested as racemates.
Ch em istr y
The tricyclic compounds 224 and 325 were synthesized
according to previously reported procedures. Catalytic
hydrogenation (H2, 10% Pd-C, 50 psi) of the known
6-methoxy-4-nitro-1,3,4,5-tetrahydrobenz[cd]indole26 and
acetylation of the resulting crude amine provided the
desired product (6-methoxy-4-acetamido-1,3,4,5-tetrahy-
drobenz[cd]indole) 4 (65% yield). Bromination of 4 with
N-bromosuccinimide (NBS) in dioxane/AcOH (2:1) at 0
°C yielded the 2-bromo derivative 5 (52% yield). The
4-bromo-2-(carboxymethyl)-5-methoxyindole, 18 (mp

Conformationally Restrained Melatonin Analogues
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 1993
Sch em e 1a
a
Reagents: (a) PdCl2(PPh3)2, TEA, AcOH, CH2dCHCOOR1, 120 °C, 7 h; (b) H2, 3 atm, Pd/C (10%), THF, room temperature, 6 h; (c)
3 N KOH, MeOH, THF, room temperature, 16 h; (d) PPA, 80 °C, 80 min.
Sch em e 2a
Sch em e 4a
a
Reagents: (a) benzylamine, p-TsOH‚H2O, reflux, 20 h; (b) H2,
a
Reagents: (a) PdCl2(PPh3)2, TEA, AcOH, CH2dCHCN, 120 °C,
3 atm, Pd/C (10%), THF, 50 °C, 2 h; (c) Ac2O, THF, TEA, room
temperature (overall yield: 15%).
7 h; (b) H2, 4 atm, Ra-Ni, THF, Ac2O, 50 °C, 6 h.
Sch em e 3a
Assuming a good resemblance between quail and chick
brains,35 we presume that our binding studies involved
a receptor population in which the Mel1a receptor
subtype prevails. The IC50 values were determined and
Ki values calculated by using nonlinear fitting strate-
gies; compounds having low affinity (IC50 > 10-5 M)
were excluded from further analysis.
Biologica l Activity Deter m in a tion . Biological ac-
tivity was evaluated using two methods: (i) effects on
the forskolin-stimulated cAMP accumulation in quail
optic tecta explants and (ii) effects of coincubation with
GTPγS (10-4 M) on the IC50 values (GTPγS index).
(i) Effects on F or sk olin -Stim u la ted cAMP Ac-
cu m u la tion . It is well-known that aMT inhibits for-
skolin-stimulated cAMP accumulation;36 therefore, the
agonist/antagonist profile of new aMT analogues can be
evaluated by measuring their influence on the cAMP
synthesis.
a
Reagents: (a) TPSH, THF, room temperature, 3 h; (b) KCN,
EtOH, reflux, 24 h; (c) H2, 4 atm, Ra-Ni, THF, Ac2O, 50 °C, 6 h;
(d) 3 N KOH, MeOH, THF, room temperature, 16 h; (e) quinoline,
Cu, reflux, 4 hr.
(ii) Effects of Coin cu ba tion w ith GTP γS on th e
IC50 Va lu es (GTP γS In d ex). The choice of introducing
the GTPγS index was based on the knowledge that the
aMT receptor is coupled to a regulatory G-protein in its
signal-transduction pathway.35,37 It is well-known that
guanine nucleotides shift a significant part of the
available receptors from the state of high-affinity con-
formation (Rh) to a state of lower affinity conformation
(Rl).38 Agonists possess higher affinity for the Rh form,
while antagonists cannot distinguish between Rh and
Rl or have a higher affinity for Rl.38,39 This is also the
mechanism by which the difference between the efficacy
of agonists and antagonists is explained,40 and this
method was previously used to evaluate the agonist
as reference antagonist, was synthesized as previously
described.33
P h a r m a cology
Bin d in g Stu d ies a n d Deter m in a tion of Affin ity.
The affinity of the analogues at the melatonin binding
site in the quail optic tecta was determined in competi-
tion binding analyses using 2-[125I]iodomelatonin as a
labeled ligand (100 pM). There are no data in the
literature indicating the type of receptor subtypes
present in the quail optic tecta; however, data available
for the chick34 indicate that Mel1a is expressed more
abundantly than Mel1c, whereas Mel1b is not present.

1994 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13
Spadoni et al.
profile of a naphthalenic aMT ligand.21 Assuming that
under basal, unstimulated conditions there is an exist-
ing ratio of Rh/Rl < 1, the system is prevalently inactive.
In that case the agonists, because of their higher affinity
for the Rh, would create a shift in the equilibrium
toward the Rh state, with a consequent activation of the
signal-transduction pathway, while the antagonists
would keep the basal conditions unaltered or at best
change the ratio of Rh/Rl toward values less than basal
values.41 This type of interaction would allow prediction
of the efficacy of a compound after having measured its
affinity for both Rh and Rl.42 Our prediction was that,
under conditions of competition analysis (2-[125I]iodo-
melatonin, 200 pM, see the Experimental Section),
coincubation with GTPγS would result in increased IC50
values for an agonist (the curve would shift to the right),
while the IC50 values for an antagonist would remain
unchanged or decrease (the curve would shift to the left).
The series of pilot studies using melatonin (agonist) and
compound 3233 (antagonist) completely confirmed the
prediction of this model. In the presence of GTPγS, the
aMT IC50 values increased by 3-6 times while the IC50
values for compound 32 showed a 2-3 times decrease.
These results completely justified the application of this
new approach to assess the agonist/antagonist nature
of all the compounds reported in this study.
In order to be able to compare these results with those
of the cAMP analysis, a numerical index was introduced
(GTPγS index) ) [(IC50 + GTPγS)/(IC50 - GTPγS)]-
compound/[(IC50 + GTPγS)/(IC50 - GTPγS)]aMT.
As seen in Table 1, the GTPγS index evaluation
thoroughly corresponded to that obtained by using the
cAMP data. The final evaluation of the compounds
(agonist, partial agonist, antagonist) was made taking
into consideration both indices (GTPγS index and
cAMP). Compounds with indices having numerical
values > 0.8 were considered full agonists; values
ranging from 0.2 to 0.8 were indicative of partial
agonists, and values < 0.2 were considered character-
istic for the antagonists. In the few cases in which
values of both indices were close to, but not within, the
theoretically determined limits of the range 0.2-0.8, a
double denomination was adopted, i.e. if a compound
had a GTPγS index ) 0.16 and cAMP index ) 0.25, it
was considered to be an antagonist-partial agonist.
conformation of the amide bond is not appropriate for
receptor binding, or due to an inappropriate conforma-
tion of the ethyl chain.
The semirigid analogues 8 and 9 provide fewer
possible conformations for recognition by the receptor
than aMT itself. Although in these compounds there
is a rotatable bond between the indole 3-carbon and the
ethenyl moiety, this chain is somewhat constrained to
near-planar conformations (s-cis, s-trans) because of
conjugation between the indole ring and the double
bond. These compounds showed good but not optimal
binding affinity. In order to determine the preferred
s-cis or s-trans conformation of 8 and 9 we investigated
the carbazole derivatives 11 and 12 in which the
ethylamido side chain is fixed in the coplanar extended
s-cis conformation. This transformation led to a reduc-
tion in binding affinity (i.e. 8 nM for 8, compared to 63
nM for 12). This result may indicate that the aMT
receptor links preferentially to melatonin in its folded
s-trans conformation, although it is impossible to ex-
clude a priori a negative sterical interaction between
the C ring of carbazole and the receptor site. Constraint
of the C3-Câ bond, which led to the derivatives 6 and
7 with the side chain partially folded toward the C4
indole position ring system, gave markedly better
results than the compounds which led to the more rigid
back-folded compounds 4 and 5.
In order to build a topographical model of the binding
site, the conformational space of the compounds with
higher affinity was investigated, looking for a common
set of distances among the hypothetical pharmacophoric
points by DISCO module of SYBYL 6.3.16 Compounds
endowed with at least one hundredth of the affinity of
aMT were chosen for this task, i.e. compounds 7, 8, 12,
and aMT itself. Compound 4 was also included, as its
2-Br derivative 5 is only 33 times less potent than aMT
in receptor binding. Other conformationally constrained
compounds with known affinity for the aMT receptor
were included in the search, i.e. compounds (S)-29,14e
30,14a and 3117 (Figure 5).
With regard to the choice of the hypothetical phar-
macophoric groups, the possible anchor points were the
methoxy group, the phenyl ring, and the carboxamido
group. The indole NH was not considered because it is
not present in compounds 29, 30, and 31 and in other
known aMt receptor ligands, such as naphthalene
derivatives.43
Resu lts a n d Discu ssion
Keeping aMT as the reference compound, the search
for common pharmacophoric distances was performed
using DISCO on conformer databases of molecular
mechanics minimized conformations. The amide and
the methoxy groups were considered in fixed positions,
as described in the Experimental Section. A recent
paper14e presented a pharmacophoric model in which
the methoxy groups in aMT and in 29 had opposite
relative orientations. This choice was based on the
observation that 6-Cl-aMT has higher receptor binding
affinity than aMT itself. As we observed an opposite
rank of affinity (Table 1), we decided to keep the
methoxy group in the same orientation for all of the
compounds.
The results of the biological activity determinations
and binding studies are summarized in Table 1. The
biological activity and the efficacy of the new compounds
were tested by measuring the inhibition of the forskolin-
stimulated cAMP accumulation and the GTPγS index.
Both analyses demonstrated that all of the compounds
were full agonists with the exception of 4 and 9 (Figures
3 and 4), which showed a slight reduction in efficacy
behaving like partial agonists. The compounds synthe-
sized exhibited a broad range of binding affinities: from
as high as 0.39 nM to inactive. The affinities of
compounds 6 and 7, and 8 and 9, with the 3-ethylamido
side chain partially constrained, were the highest.
Attempts to explore full rigidity of the side chain by
linking the alkylamido moiety to the 4- or 2-position of
the indole ring implemented with the structures 2 or 3
led to compounds that exhibited very poor binding
affinity. This could be due to the fact that the cis
Eight features were assigned to the pharmacophoric
groups, including the aromatic centroid, the three
heteroatoms, and four points in the directions of the
possible hydrogen bonds. One of the two lone pair

Conformationally Restrained Melatonin Analogues
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 1995
F igu r e 3. Examples of the evaluation of the biological activity of the compounds by using the GTPγS index: competition
experiments performed with a fixed concentration of the labelled ligand (200 pM 2-[125I]iodomelatonin) and varying concentrations
of the competing drug, in absence or presence of GTPγS (10-4 M). Ratio ) (IC50 + GTPγS)/IC50. GTPγS index ) ratio compound/
ratio aMT. IC50 values are expressed in nM. Activity: when GTPγS index < 0.2 ) AN (antagonist); between 0.2 and 0.8 ) PA
(partial agonist); >0.8 ) A (agonist).
F igu r e 5.
Among the models belonging to the group with the
lowest distance tolerance (3.0 Å), the two with the
smallest union volumes were selected and named A and
B. The resulting superimpositions are represented in
Figure 6.
In describing the relative dispositions of the three
putative pharmacophoric groups, for sake of clarity only
the distances among the four points indicated in Figure
7 are reported in Table 2. In fact, these are the most
informative features because the positions of amide N
and O are dependent on those of their hydrogen bond
direction prolongations, and the lone pair prolongations
on the methoxy group are less important because its
orientation is somewhat arbitrary (see the Experimental
Section). The four points can be seen as the vertices of
a tetrahedron, and the interpoint distances remain the
same upon space reflection. Therefore, for each super-
F igu r e 4. Examples of the evaluation of the biological activity
of the compounds by measuring the effect on forskolin (10-5
M)-stimulated cAMP accumulation in explants from quail optic
tecta. C, control; F, forskolin. Agonist: ratio of percent
inhibition compound/percent inhibition aMT > 0.8. Partial
agonist: ratio of percent inhibition compound/percent inhibi-
tion aMT is between 0.8 and 0.2. Antagonist: ratio of percent
inhibition compound/percent inhibition aMT < 0.2.
prolongations of the carbonyl oxygen was not considered
as it was found to be close to the rest of the molecule
and therefore discarded as unlikely to represent a
putative hydrogen bond donor site of the receptor.

1996 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13
Spadoni et al.
F igu r e 6. Superimposition of compounds with good affinity for aMT receptor, resulting from DISCO analysis, according to models
A and B. Melatonin (reference compound) is highlighted in capped sticks. Lone pairs are represented in red; the prolongations of
the lone pairs, the prolongations of the N-H bond, and the hydrophobic centroid are represented in purple.
Ta ble 2. Range of Distances (Å) among the Four
Pharmacophoric Points represented in Figure 7 for the
Conformers Superimposed by DISCO, and Corresponding
Distances for aMT (Reference Compound)
distance melatonina all compounds other aMt conformers
Model A
d1
d2
d3
d4
6.70b
9.71b
7.64b
8.37b
6.70-8.48
7.47-10.72
7.64- 8.67
6.34- 8.92
Model B
d1
d2
d3
d4
9.88c
8.03c
9.40c
7.32c
7.51-10.07
7.08-10.72
7.96-9.46
7.01-8.91
9.74,d 9.67,e 7.81f
8.24,d 9.21,e 9.21f
8.97,d 8.91,e 8.83f
7.72,d 8.16,e 7.81f
F igu r e 7. Pharmacophoric points employed for a synthetic
description of models A and B.
a
b
Conformation used as reference. τ1 ) 79.1°, τ2 ) 178.6°, τ3
) 78.4°. c τ1 ) 81.6°, τ2 ) -179.8°, τ3 ) -174.7°. τ1 ) -4.2°, τ2
d
imposition model, a specular one exists, which for
flexible compounds corresponds to specular conforma-
tions with inversion of the torsional angles, and which
for rigid chiral compounds can correspond to the op-
posite enantiomer. For compound 29 only the (S)-
isomer was considered, in accordance with recent
findings,14e but it is not clearly able to discriminate
between specular models as it can fit both in different
conformations. In both models A and B aMT adopted
a gauche/ anti side chain conformation (A, τ1 ) 79.1°,
τ2 ) 178.4°; B, τ1 ) 81.6°, τ2 ) -179.8°), whereas the
τ3 torsional angle was 78.3° and -174.7°, respectively
(energy: B - A ) 0.24 kcal/mol). Three other aMT
conformers had distances d1-d4 within the ranges of
model B: one of which (τ1 ) -4.2°, τ2 ) -175.5°; τ3 )
-78.4°) fit the aMT conformer of model B well, while
the other two had poorer fits (see Table 2). Two
pharmacophore models (afterward named G-A and
G-B) were recently reported14e,f which, although based
on a partially different set of compounds and different
) -175.5°, τ3 ) -78.4°. e τ1 ) -175.8°, τ2 ) 178.3°, τ3 ) 80.2°.
f τ1 ) 0.0°, τ2 ) 180.0°, τ3 ) -179.6°.
assumptions (methoxy group orientation, see above), are
can be compared to our models. The first model, G-A
(with a aMT conformation referred to as a gauche/ -
gauche/ -90°), is not consistent with ours. In particu-
lar, the very active newly synthesized compound 7 gave
a good fit to the aMT conformers of our models A and B
(maximum interfeature distances difference of 0.90 and
0.68 Å, respectively) and a much poorer fit to the aMT
conformer of G-A model (2.05 Å); moreover, the car-
bazole 12 did not fit the G-A model at all. The second
model, G-B, of aMT conformer (gauche/ anti/ -90°) had
the same chain orientation as the conformers in our
models A and B, differing only in τ3, which assumes
the third possible minimum energy value. With respect
to our superimposition protocol, the alignment of our
compounds on the G-B conformer of aMT gave a union
volume larger than that obtained with our models A and

Conformationally Restrained Melatonin Analogues
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 1997
B; the G-B model therefore seems less likely than A or
B. Summarizing the results of the pharmacophore
search, we proposed two possible pharmacophore models
(A and B) on the basis of the interfeature distances and
union volume of all the set compounds; the orientation
of the CR-N bond among the three alternative mini-
mum energy values for τ3 proposed by models A, B, and
G-B, remains undetermined.
a further binding point interaction may be the cause of
the increased affinity.
The implication of the results presented in this study
is that the orientation of the C3 amido side chain and
the C2 indole substituent are critical determinants for
receptor binding affinity. The present work extends
what had been presented so far, offering additional
information on the binding of melatonin for its receptor
site. In particular, we demonstrate that in addition to
the three pharmacophoric elements (the 5-methoxy
group, 3-ethanamido side chain, and the aromatic-
aromatic interaction of the indole ring), the C2 indole
substituent contributes significantly to the receptor
binding of this series, thus establishing a putative four-
point pharmacophore. Furthermore, our results suggest
that one of the preferred binding conformations of the
C3-ethylamido side chain is out of the plane of the
indole, gauche/ anti orientated as depicted in Figure 6,
and this conclusion is in agreement with recently
literature data on the high-affinity compound 29.14a
The only constrained compound which did not reveal
affinity for the aMT receptor, compound 10, did not have
the four pharmacophoric points within the predefined
ranges. More flexible compounds, such as 13-16
(Figure 2), are less informative in this kind of study.
Compound 13 can be easily superimposed to compounds
4 and 5, given their topological similarity; its low affinity
compared to that of 5 is probably due to the lack of a
positive effect of a substituent in the 2-position of the
indole. The compound with the longest chain (14) can
accommodate its points at the distances required by
models A and B, but was inactive; this seems to be due
to its excessive flexibility, rather than a negative effect
of the different attachment point of the chain (in C4).
In fact, the other C4-substituted compound 13 retained
residual affinity. The compound with the shortest chain
(15) had no minimum-energy conformation able to fit
the pharmacophore requirements and was inactive.
Compound 16 is a borderline case: it can accommodate
the pharmacophoric groups as required for model B, but
not for model A; its low affinity does not however allow
definitive statements based on this observation. In the
above-mentioned compounds 2 and 3, the amide group
is located at distances from the methoxy group and from
the aromatic ring which are different than those re-
quired by the two models, apart from their cis confor-
mation; it is therefore impossible to assume an active
trans conformation of aMT based only on these com-
pounds.
Exp er im en ta l Section
(a ) Ch em ica l Meth od s. Melting points were determined
on a Bu¨chi SMP-510 capillary melting point apparatus and
are uncorrected. 1H-NMR spectra were recorded on a Bruker
AC 200 spectrometer; chemical shifts are reported in ppm and
given in δ units. EI-MS spectra (70 eV) were taken on a Fisons
Trio 1000. Only molecular ions (M+) and base peaks are given.
Infrared spectra were obtained on a Bruker FT-48 spectrom-
eter; absorbances are reported in ν (cm-1). Elemental analyses
were performed on a Carlo Erba analyzer.
6-Met h oxy-1-oxo-1,2,3,4-t et r a h yd r o-â-ca r b olin e (2).
Compound 2 was prepared according to the literature method.24
8-Met h oxy-1,4,5,7-t et r a h yd r oa zocin o[4,5,6-cd ]in d ol-
6(3H)-on e (3). Compound 3 was prepared according to the
literature method:25 1H NMR (DMSO-d6) δ 3.13 (t, 2H), 3.59
(q, 2H), 3.77 (s, 3H), 3.86 (s, 2H), 6.85 (d, 1H, J ) 8.8 Hz),
7.13 (br s, 1H), 7.17 (d, 1H, J ) 8.8 Hz), 7.35 (br t, 1H), 10.84
(br s, 1H); IR (Nujol) 3291, 3195, 1651 cm-1; MS (EI) m/ z 230
(M+, 100).
(()-4-Acetam ido-6-m eth oxy-1,3,4,5-tetr ah ydr oben z[cd]-
in d ole (4). A solution of 6-methoxy-4-nitro-1,3,4,5-tetrahy-
drobenz[cd]indole26 (0.1 g, 0.43 mmol) in MeOH (6 mL) was
hydrogenated over 10% Pd-C (0.03 g) at 50 psi of H2 until
hydrogen uptake ceased (∼2 h). The catalyst was filtered on
Celite, and the filtrate was evaporated at reduced pressure,
giving a crude residue which was dissolved in THF (4 mL) and
then acylated with Ac2O (0.05 mL, 0.68 mmol) and Et3N (0.05
mL, 0.69 mmol) at room temperature for 3 h. The solvent was
evaporated in vacuo, and the residue was dissolved in EtOAc,
washed with a saturated NaHCO3 solution and brine, dried
(Na2SO4), and concentrated under reduced pressure. The
crude product was purified by flash chromatography over silica
gel using EtOAc as eluent to give 4 (0.063 g, 57%) as a white
amorphous solid: 1H NMR (CDCl3) δ 1.94 (s, 3H), 2.85-3.10
(m, 4H), 3.86 (s, 3H), 4.76 (m, 1H), 5.52 (br d, 1H), 6.89 (d,
1H, J ) 8.6 Hz), 6.93 (br s, 1H), 7.17 (d, 1H, J ) 8.6 Hz), 7.79
(br s, 1H); IR (Nujol) 3380, 3280, 1639 cm-1; MS (EI) m/ z 244
(M+), 185 (100). Anal. (C14H16N2O2‚2/3H2O) C, H, N.
(()-4-Acet a m id o-2-b r om o-6-m et h oxy-1,3,4,5-t et r a h y-
d r oben z[cd ]in d ole (5). NBS (1.05 equiv) was added portion
wise to an ice-cooled solution of 4 (0.24 g, 1 mmol) in AcOH
(2.8 mL) and dioxane (1.4 mL). The mixture was stirred at 0
°C for 0.5 h and for 1.5 h at room temperature. The mixture
was then poured onto an ice-cooled 30% NaOH solution and
extracted (3×) with EtOAc, and the combined extracts were
washed with brine and dried (Na2SO4). Evaporation of the
solvent gave a crude residue which was purified by chroma-
tography (silica gel, cyclohexane/EtOAc, 3:7, as eluent) and
crystallization from EtOAc to give 5 as a beige solid (0.17 g,
53%): mp 200 °C; 1H NMR (CDCl3 and MeOD) δ 1.80 (s, 3H),
2.54-3.02 (m, 4H), 3.74 (s, 3H), 4.49 (m, 1H), 6.72 (d, 1H, J )
We also investigated the role of appropriate C2 indole
substituents in the binding affinity for the aMT recep-
tor. In fact, we had previously reported that substitu-
tion with bromine19 or phenyl20 at C2 leads to a large
increase in binding affinity. The favorable role of the
methoxy group in the ortho position of the side chain
in a naphthalenic aMT analogue had also been re-
ported.21 Garrat et al.14a justified the higher activity of
2-halo or 2-phenyl analogues of aMT, suggesting that
the C-2 substituent plays a role in orienting the side
chain toward a favorable folded conformation. In order
to test this hypothesis, the molecular mechanics energy
profile upon rotation of torsional angle C3a-C3-Câ-
CR was calculated for compounds 8 and 9 by the Grid
search routine of SYBYL (see the Experimental Section).
The two compounds gave very similar energy vs tor-
sional angle plots, with minimum energy at 180° for 8
and at 150° for 9, and a second minimum (which fits
the pharmacophoric distances) at 40° for both com-
pounds, 1.3-1.4 kcal/mol above the global minimum.
The rotational barrier was also the same in the two
cases, with maximum energy around 4.5 kcal/mol above
the global minimum, at 90°.
On the contrary, the remarkable increase in binding
affinity observed in the C2-substituted analogues 6 and
9 and especially in the more rigid analogue 5 in
comparison to the unsubstituted analogues indicate that

1998 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13
Spadoni et al.
8.75 Hz), 6.98 (d, 1H, J ) 8.75 Hz); IR (Nujol) 3349, 3226,
1657 cm-1; MS (EI) m/ z 324-322 (M+), 263-265 (100). Anal.
(C14H15BrN2O2) C, H, N.
(silica gel, THF/cyclohexane, 1:1) to give 24 as a white solid
(0.14 g, 35%): mp 220-230 °C dec; 1H NMR (acetone-d6) δ
2.69 (t, 2H), 3.23 (t, 2H), 3.87 (s, 3H), 6.96 (d, 1H, J ) 8.8 Hz),
7.30 (d, 1H, J ) 8.8 Hz), 7.77 (d, 1H, J ) 1.46 Hz), 10.92 (br
s, 1H); IR (Nujol) 3110, 1646, 1605 cm-1; MS (EI) m/ z 201
(M+).
Meth yl (E)-3-(5-Meth oxy-1H-in dol-4-yl)pr open oate (19).
A mixture of 4-bromo-5-methoxyindole27 (2.26 g, 10 mmol),
methyl acrylate (2.58 g, 30 mmol), and bis(triphenylphos-
phine)palladium(II) chloride (0.7 g, 1 mmol) in AcOH (17 mL)
and TEA (17 mL) was stirred for 6 h at 120 °C in a closed
vessel. The mixture was cooled to room temperature and then
poured onto water and CH2Cl2. The biphasic mixture was
filtered through a pad of Celite, and the aqueous layer of the
filtrate was extracted twice with CH2Cl2. The combined
organic layers were washed with a saturated NaHCO3 aqueous
solution and then with brine and dried over Na2SO4. After
removal of the solvent the desired product was chromato-
graphed over silica gel (cyclohexane/ethyl acetate, 7:3 as
eluent) to give pure 19 (0.92 g, 40%). Crystallization from
2-Ca r bom eth oxy-6-m eth oxy-4,5-d ih yd r oben z[cd ]in d ol-
3(1H)-on e (25). Polyphosphoric acid (PPA) (9 g) was pre-
heated with mechanical stirring at 80-85 °C under nitrogen
atmosphere. The acid 23 (0.554 g, 2 mmol) was then added
over a 5 min period, the mixture was stirred at 80 °C for 60
min under N2, and ice was added and left under stirring for 1
h.The mixture was extracted three times with CHCl3, and the
combined extracts were washed with 10% aqueous Na2SO4
solution and 5% aqueous NaHCO3 solution, dried (Na2SO4),
and concentrated under reduced pressure. The solid was
triturated with EtOAc to obtain a yellow solid (0.43 g, 84%):
1
CH2Cl2/hexane: mp 135-138 °C; H NMR (CDCl3) δ 3.84 (s,
1H NMR (CDCl3) δ 2.92 (t, 2H), 3.32 (t,
mp 220-221 °C dec;
3H), 3.93 (s, 3H), 6.81 (m, 1H), 6.83 (d, 1H, J ) 16.3 Hz), 6.91
(d, 1H, J ) 8.9 Hz), 7.31 (t, 1H), 7.39 (dd, 1H, J ) 8.91 Hz, J
) 0.95 Hz), 8.32 (d, 1H J ) 16.3 Hz), 8.49 (br s, 1H); IR (Nujol)
3323, 1685 cm-1; MS (EI) m/ z 231 (M+, 100).
2H), 3.92 (s, 3H), 4.04 (s, 3H), 7.10 (d, 1H, J ) 8.8 Hz), 7.29
(d, 1H, J ) 8.8 Hz), 9.33 (br s, 1H); IR (Nujol) 3300, 1688,
1616 cm-1; MS (EI) m/ z 259 (M+), 227 (100).
(()-3-Acetam ido-6-m eth oxy-1,3,4,5-tetr ah ydr oben z[cd]-
in d ole (10). Under a nitrogen atmosphere a mixture of the
ketone 24 (0.2 g, 1 mmol), benzylamine (0.14 g, 1.3 mmol),
and p-toluenesulfonic acid monohydrate (0.0025 g, 0.013 mmol)
in dry benzene (25 mL) was heated at reflux with a Dean-
Stark water separator for 20 h. The benzene and the excess
benzylamine were removed in vacuo, and the crude residue
was dissolved in absolute EtOH (10 mL). This solution was
hydrogenated at 3 atm of H2 for 2 h at 50 °C in the presence
of 10% Pd-C catalyst (0.02 g). The catalyst was filtered off,
and the solvent was evaporated under reduced pressure to give
the crude oily amine 26 which was then dissolved in THF (5
mL) and stirred at room temperature for 6 h with Ac2O (0.07
mL, 0.75 mmol) in the presence of TEA (0.1 mL, 0.75 mmol).
The solvent was evaporated in vacuo, and the residue was
dissolved in ethyl acetate, washed with NaHCO3 solution
followed by brine, dried (Na2SO4), and evaporated again.
Purification by flash chromatography (silica gel; EtOAc as
eluent) and crystallization from CH2Cl2/hexane yielded the
desired product 10 as a white solid (0.037 g, 15%): mp 205-
208 °C dec; 1H NMR (CDCl3) δ 2.01 (s, 3H), 1.95-2.27 (m, 4H),
3.88 (s, 3H), 5.34 (m, 1H), 5.67 (br d, 1H), 6.89 (d, 1H, J ) 8.9
Hz), 7.05 (d, 1H, J ) 1.6 Hz), 7.15 (d, 1H, J ) 8.89 Hz), 7.94
(br s, 1H); IR (Nujol) 3296, 1616 cm-1; MS (EI) m/ z 244 (M+),
185 (100). Anal. (C14H16N2O2) C, H, N.
Ben zyl (E)-3-(5-Meth oxy-2-ca r bom eth oxy-1H-in d ol-4-
yl)p r op en oa te (20). The compound 20 was prepared by the
same procedure described above, using benzyl acrylate instead
of methyl acrylate (yield: 3.06 g, 84%). Crystallization from
MeOH: mp 181-183 °C; 1H NMR (CDCl3) δ 3.94 (s, 3H), 3.96
(s, 3H), 5.30 (s, 2H), 6.86 (d, 1H, J ) 16.16 Hz), 7.08 (d,1H, J
) 8.9 Hz), 7.40 (m, 7H), 8.31 (d, 1H, J ) 16.16 Hz), 8.90 (br s,
1H); IR (Nujol) 3327, 1689 cm-1; MS (EI) m/ z 365 (M+, 100%).
Meth yl 3-(5-Meth oxy-1H-in d ol-4-yl)p r op a n oa te (21). A
solution of 19 (2.31 g, 10 mmol) in THF (150 mL) was
hydrogenated over 10% Pd-C (0.23 g) at 3 atm of H2 for 6 h
at room temperature. The catalyst was filtered on Celite, and
the filtrate was evaporated at reduced pressure, giving a
nearly quantitative yield of the desired compound 21 as yellow
solid, which was directly used without further purification: 1H
NMR (CDCl3) δ 2.69 (t, 2H), 3.26 (t, 2H), 3.77 (s, 3H), 3.87 (s,
3H), 6.54 (m, 1H), 6.91 (d, 1H, J ) 8.9 Hz), 7.19-7.25 (m, 2H),
8.17 (br s, 1H); IR (Nujol) 3320, 1700 cm-1; MS (EI) m/ z 233
(M+), 160 (100).
5-Meth oxy-1H-in d ole-4-p r op a n oic Acid (22). A solution
of 21 (2.33 g, 10 mmol) in THF (20 mL), MeOH (24 mL), and
3 N KOH (10 mL) was stirred at room temperature for 16 h.
The solvents were removed in vacuo, and the residue was
dissolved in water and ethyl acetate and then acidified with 6
N HCl. The layers were separated, and the organic phase was
washed with brine, dried (Na2SO4), and evaporated in vacuo
to obtain the desired compound 22 which was purified by
crystallization from CHCl3 (1.86 g, 85%): mp 152-153 °C; 1H
NMR (CDCl3) δ 2.73 (t, 2H), 3.28 (t, 2H), 3.88 (s, 3H), 6.54 (m,
1H), 6.91 (d, 1H, J ) 8.8 Hz), 7.23 (m, 2H), 8.07 (br s, 1H); IR
(Nujol) 3430, 1712, 1682 cm-1; MS (EI) m/ z 219 (M+), 160
(100).
(()-2-(Car boxyeth yl)-3-cyan o-6-m eth oxy-1,3,4,5-tetr ah y-
d r oben z[cd ]in d ole (27). A suspension of 25 (0.26 g, 1 mmol)
and TPSH (0.37 g, 1.25 mmol) in dry THF (10 mL) was stirred
at room temperature under N2 atmosphere for 3 h. The solvent
was removed under vacuum, and the crude residue was
dissolved in EtOH (8 mL). KCN (1.4 g; 21 mmol) was added,
and the solution was heated, under reflux, for 24 h under N2
.
The solvent was removed at reduced pressure, and the residue
was taken up with CHCl3, and H2O. The aqueous phase was
extracted three times with CHCl3 and the collected organic
layers were washed sequentially with a saturated solution of
NaHCO3, 2N HCl, H2O, and brine, then dried (Na2SO4), and
concentrated under vacuum. The crude product was purified
by flash chromatography (silica gel, CHCl3/EtOAC, 98:2) and
then by crystallization from EtOAc/hexane, to obtain the
desired compound 27 as a yellowish solid (0.085 g, 29%): mp
160-161 °C dec; 1H NMR (CDCl3) δ 1.47 (t, 3H), 2.15 (m, 1H),
2.54 (m, 1H), 2.91 (m, 1H), 3.22 (m, 1H), 3.88 (s, 3H), 4.47 (m,
3H), 7.05 (d, 1H, J ) 8.9 Hz), 7.20 (d, 1H, J ) 8.9 Hz), 8.93
(br s, 1H); IR (Nujol) 3295, 2234, 1698 cm-1; MS (EI) m/ z 284
(M+), 238 (100).
(()-3-(Aceta m id om eth yl)-2-(ca r boxyeth yl)-6-m eth oxy-
1,3,4,5-tetr a h yd r oben z[cd ]in d ole (6). A solution of 27
(0.284 g, 1 mmol) in THF (10 mL) and acetic anhydride (2 mL)
was hydrogenated over Raney nickel at 4 atm of H2 for 6 h at
50 °C. The catalyst was filtered on Celite, and the filtrate was
concentrated in vacuo and partitioned between CH2Cl2 and 2
N NaOH. The organic layer was washed with brine, then dried
(Na2SO4), and evaporated under reduced pressure to give a
crude residue which was purified by flash chromatography
5-Met h oxy-2-ca r b oxym et h yl-1H -in d ole-4-p r op a n oic
Acid (23). A solution of 20 (3.65 g, 10 mmol) in THF (150
mL) was hydrogenated over 10% Pd-C (0.4 g) at 3 atm of H2
for 16 h at room temperature. The catalyst was filtered on
Celite, and the filtrate was evaporated at reduced pressure to
give the title compound 23 as yellow solid, which was purified
by heating in refluxing EtOAc (2.38 g, 86%): mp 209-215 °C;
1H NMR (CDCl3) δ 2.71 (t, 2H), 3.25 (t, 2H), 3.88 (s, 3H), 3.95
(s, 3H), 7.06 (d, 1H, J ) 9.2 Hz), 7.23 (d, 1H, J ) 1.59 Hz),
7.25 (d, 1H, J ) 9.2 Hz), 8.88 (br s, 1H); MS (EI) m/ z 277
(M+), 186 (100); IR (Nujol) 3300, 1688, 1631 cm-1
.
6-Met h oxy-4,5-d ih yd r ob en z[cd ]in d ol-3(1H )-on e (24).
Polyphosphoric acid (PPA) (9 g) was preheated with mechan-
ical stirring at 80-85 °C under nitrogen atmosphere. The acid
22 (0.438 g, 2 mmol) was then added over a 5 min period, and
the mixture was stirred at 80 °C for 80 min under N2 and ice
was added and left under stirring for 1 h. The crude brown
solid precipitate was filtered, dissolved in hot THF (100 mL),
diluted with CH2Cl2 (50 mL), and washed with saturated
aqueous solution of NaHCO3 and brine. After drying (Na2-
SO4), the solvents were evaporated under vacuum, and the
crude black solid residue was purified by flash chromatography

Conformationally Restrained Melatonin Analogues
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 1999
(silica gel; ethyl acetate as eluent) and crystallization from
(s, 1H), 9.91 (br s, 1H), 11.16 (br s, 1H); IR (cm-1, Nujol) 3415,
3276, 1641; MS (EI) m/ z 224 (M+), 182 (100). Anal.
(C14H12N2O‚1.2H2O) C, H, N.
CH2Cl2/hexane to obtain 6 as a white solid (0.261 g, 79%): mp
1
123 °C; H NMR (CDCl3) δ 1.45 (t, 3H), 1.92 (s, 3H), 2.01 (m,
1H), 2.18 (m, 1H), 2.79 (m, 1H), 3.06 (m, 1H), 3.48 (m, 2H),
3.65 (m, 1H), 3.87 (s, 3H), 4.44 (m, 2H), 6.96 (br s, 1H), 7.05
(d, 1H, J ) 8.9 Hz), 7.15 (d, 1H, J ) 8.9 Hz), 8.43 (br s, 1H);
IR (Nujol) 3351, 3305, 1685, 1647 cm-1; MS (EI) m/ z 330 (M+),
271 (100). Anal. (C18H22N2O4) C, H, N.
12: white solid, 0.081 g, 30%; mp 206 °C (EtOAc/hexane);
1H NMR (DMSO-d6) δ 2.06 (s, 3H), 3.83 (s, 3H), 6.99 (dd, 1H,
J ) 8.97 Hz, J ) 2.56 Hz), 7.35 (m, 3H), 7.52 (d, 1H, J ) 2.56
Hz), 8.35 (s, 1H), 9.88 (s, 1H), 10.94 (br s, 1H); IR (Nujol) 3378,
3182, 1668 cm-1; MS (EI) m/ z 254 (M+, 100%). Anal.
(C15H14N2O2‚H2O) C, H, N.
(()-3-(Acet a m id om et h yl)-6-m et h oxy-1,3,4,5-t et r a h y-
d r oben z[cd ]in d ole (7). A solution of 6 (0.075 g, 0.23 mmol)
in THF (0.5 mL), EtOH (0.5 mL), and 3 N KOH (0.2 mL) was
stirrred at room temperature for 24 h. The solution was
concentrated in vacuo, diluted with water, and acidified with
6 N HCl. The solid precipitate was filtered, washed with
water, and dried at 30 °C under vacuum to obtain 0.065 g of
the crude acid intermediate. This acid was dissolved in
quinoline (2 mL) and heated under reflux for 4 h in the
presence of 0.01 g of Cu powder in N2 atmosphere. The
mixture was acidified with 2 N HCl and extracted with CH2-
Cl2 (5 × 10 mL). The collected organic extracts were washed
with 2 N HCl, NaHCO3, H2O, and brine, dried (Na2SO4), and
evaporated in vacuo to obtain a crude product which was
purified by flash chromatography on silica gel (ethyl acetate
as eluent) to obtain the desired pure compound as white solid
(0.025 g, 42%). An analytical sample was recrystallized from
EtOAc/cyclohexane: mp 151-152 °C; 1H NMR (CDCl3) δ 1.83
(m, 1H), 1.99 (s, 3H), 2.12 (m, 1H), 3.01 (m, 2H), 3.21 (m, 1H),
3.58 (m, 2H), 3.87 (s, 3H), 5.62 (br s, 1H), 6.88 (d, 1H, J ) 8.7
Hz), 6.95 (br d, 1H), 7.14 (d, 1H, J ) 8.6 Hz), 7.79 (br s, 1H);
IR (Nujol) 3490, 3390, 1643 cm-1; MS (EI) m/ z 258 (M+), 199
(100). Anal. (C15H18N2O2‚1.5H2O) C, H, N.
(E )-N -[2-(5-M e t h o x y -1H -i n d o l-3-y l)e t h e n y l)a c e t -
a m id e (8). A solution of (E)-3-(2-nitroethenyl)-5-methoxyin-
dole44 (0.218 g, 1 mmol) in THF (10 mL) and acetic anhydride
(2 mL) was hydrogenated over Raney nickel at 4 atm of H2 for
10 h at room temperature. The catalyst was filtered on Celite,
and the filtrate was concentrated in vacuo and partitioned
between ethyl acetate and 2 N NaOH. The organic layer was
washed with brine, dried (Na2SO4), and evaporated under
reduced pressure to give a crude residue which was chromato-
graphed (silica gel; ethyl acetate as eluent) to give 0.04 g of a
mixture of the desired product 8 and its N1-acetyl derivative
(considerable amount of melatonin was also isolated). This
mixture was dissolved in MeOH (8 mL) and stirred at room
temperature for 4 h in the presence of 2 N NaOH (1.5 mL).
The solvent was evaporated in vacuo, and the residue was
taken up in ethyl acetate, washed with brine, and dried (Na2-
SO4). After removal of the solvent at reduced pressure, pure
8 was obtained by crystallization from EtOAc/hexane (0.023
g, 10%): mp 170-173 °C dec; 1H NMR (CDCl3) δ 2.13 (s, 3H),
3.89 (s, 3H), 6.28 (d, 1H, J ) 14.62 Hz), 6.87, 6.91 (dd, 1H, J
) 8.58 Hz and J ) 2.23 Hz), 7.20 (d, 1H, J ) 2.23 Hz), 7.21
(br s, 1H), 7.27 (d, 1H J ) 8.58 Hz), 7.41, 7.46 (dd, 1H J )
14.62 Hz), 8.04 (br s, 1H); IR (cm-1, Nujol) 3292, 1653; MS
(EI) m/ z 230 (M+, 100). Anal. (C13H14N2O2) C, H; N: calcd,
12.17; found, 11.67.
N-[2-(5-Met h oxy-1H -in d ol-4-yl)et h yl]a cet a m id e (13).
4-(2-Nitroethenyl)-5-methoxyindole26 (0.22 g, 1 mmol) was
added portion wise to a stirred ice-cooled suspension of LiAlH4
(0.23 g, 6 mmol) in dry THF (15 mL) under nitrogen and the
mixture was stirred at room temperature for 5 h. After cooling
at 0 °C, water was added dropwise to destroy the excess
hydride, the mixture was filtered on Celite, and the filtrate
was concentrated in vacuo and partitioned between water and
ethyl acetate. The organic layer was washed with brine, dried
(Na2SO4), and concentrated under reduced pressure to give a
crude oily amine which was then used without further
purification. This crude amine was dissolved in THF (5 mL)
and stirred at room temperature for 6 h with Ac2O (0.093 mL,
1 mmol) in the presence of TEA (0.13 mL, 1 mmol). The
solvent was evaporated in vacuo, and the residue was dissolved
in ethyl acetate, washed with NaHCO3 solution followed by
brine, dried (Na2SO4), and evaporated again. Purification by
flash chromatography (silica gel; EtOAc as eluent) gave the
desired product 13 (0.085 g, 37%) as yellow oil: 1H NMR
(CDCl3) δ 1.87 (s, 3H), 3.13 (t, 2H), 3.56 (q, 2H), 3.87 (s, 3H),
6.09 (br s, 1H), 6.47 (m, 1H), 6.90 (d, 1H J ) 8.8 Hz), 7.17,
7.18 (dd, 1H, J ) 3.1 Hz, J ) 2.6 Hz), 7.24 (d, 1H, J ) 8.8 Hz),
8.56 (br s, 1H); IR (neat) 3400, 3290, 1652 cm-1; MS (EI) m/ z
232 (M+), 173 (100). Anal. (C13H16N2O2) C, H; N: calcd, 12.06;
found, 11.61.
3-[2-(Ca r b oxym et h yl)-5-m et h oxy-1H -in d ol-4-yl]a cr y-
lon itr ile (28). A mixture of methyl 4-bromo-5-methoxyindole-
2-carboxylate (0.284 g, 1 mmol), acrylonitrile (0.16 g, 3 mmol),
and bis(triphenylphosphine)palladium(II) chloride (0.07 g, 0.1
mmol) in AcOH (2 mL) and TEA (2 mL) was stirred for 6 h at
120 °C in a closed vessel. The mixture was cooled to room
temperature and then poured onto water and CH2Cl2. The
biphasic mixture was filtered through a pad of Celite and the
aqueous layer of the filtrate was extracted twice with CH2Cl2.
The combined organic layers were washed with a saturated
NaHCO3 aqueous solution and with brine and dried over Na2-
SO4. After removal of the solvent the desired product was
chromatographed over silica gel (cyclohexane/ethyl acetate, 1:1,
as eluent) to give the title compound (0.12 g, 45%) as a cis-
trans mixture. It could be crystallized from MeOH: mp 225-
232 °C; 1H NMR (acetone-d6) δ 3.90 (s, 3H), 3.97 (s, 3H), 6.50
(d, 1H, J ) 16.8 Hz), 7.19 (d, 1H, J ) 8.96 Hz), 7.44 (m, 1H),
7.62 (dd, 1H, J ) 8.96 Hz, J ) 0.86 Hz), 7.95 (d, 1H, J ) 16.8
Hz), 11.01 (br s, 1H); IR (Nujol) 3336, 2212, 1711 cm-1; MS
(EI) m/ z 256 (M+, 100).
N-[3-(2-Ca r boxym et h yl-5-m et h oxy-1H-in d ol-4-yl)p r o-
p yl]a ceta m id e (14). A solution of 28 (0.256 g, 1 mmol) in
THF (10 mL) and acetic anhydride (2 mL) was hydrogenated
over Raney nickel at 4 atm of H2 for 6 h at 50 °C. The catalyst
was filtered on Celite, and the filtrate was concentrated under
vacuum and partitioned between ethyl acetate and 2 N NaOH.
The organic layer was washed with brine, dried (Na2SO4), and
evaporated under reduced pressure to give a crude residue
which was purified by flash chromatography (silica gel; ethyl
acetate as eluent) and crystallization from EtOAc/hexane to
obtain 14 as a white solid (0.192 g, 63%): mp 139-140 °C dec;
1H NMR (CDCl3) δ 1.74 (t, 2H), 2.83 (t, 2H), 3.09 (q, 2H), 1.90
(s, 3H), 3.75 (s, 3H), 3.79 (s, 3H), 6.20 (br t, 1H), 6.92 (d, 1H,
J ) 8.9 Hz), 7.04 (m, 1H), 7.17 (d, 1H, J ) 8.8 Hz), 9.60 (br s,
1H); IR (cm-1, Nujol) 3303, 1712, 1653; MS (EI) m/ z 304 (M+),
43 (100). Anal. (C16H20N2O4) C, H, N.
(E)-N-[2-(5-Met h oxy-2-p h en yl-1H -in d ol-3-yl)et h en yl]-
a ceta m id e (9). This compound was prepared starting from
(E)-3-(2-nitroethenyl)-5-methoxy-2-phenylindole20 (0.294 g, 1
mmol) according to the procedure described above (0.046 g,
1
15%): mp 202-204 °C; H NMR (CDCl3) δ 2.12 (s, 3H), 3.92
(s, 3H), 6.32 (d, 1H, J ) 14.62 Hz), 6.89, 6.94 (dd, 1H, J )
8.58 Hz and J ) 2.22 Hz), 7.19 (br s, 1H), 7.31-7.64 (m, 8H),
8.07 (br s, 1H); IR (Nujol) 3280, 1653 cm-1; MS (EI) m/ z 306
(M+), 232 (100). Anal. (C19H18N2O2) C, H, N.
3-Aceta m id o-9H-ca r ba zole (11) a n d 3-Aceta m id o-6-
m eth oxy-9H-ca r ba zole (12). A solution of 3-nitro-6-meth-
oxy-9H-carbazole31 (0.24 g, 1 mmol) in THF (15 mL) was
hydrogenated over 10% Pd-C (0.05 g) at 4 atm of H2 for 16 h
at room temperature, then for 2 h at 50 °C. The catalyst was
filtered on Celite, and the filtrate was evaporated at reduced
pressure giving a crude mixture of 11 and 12. These com-
pounds were separated by flash chromatography (silica gel,
cyclohexane/EtOAc, 3:7).
N-[(5-Meth oxy-1H-in d ol-4-yl)m eth yl]a ceta m id e (15). A
solution of 4-cyano-5-methoxyindole26 (0.17 g, 1 mmol) in THF
(10 mL) and acetic anhydride (2 mL) was hydrogenated over
Raney nickel at 4 atm of H2 for 6 h at 50 °C. The catalyst
was filtered on Celite, and the filtrate was concentrated in
vacuo and partitioned between ethyl acetate and 2N NaOH.
11: white solid, 0.075 g, 30%; mp 205 °C (EtOAc/hexane);
1H NMR (DMSO-d6) δ 2.06 (s, 3H), 7.11-8.02 (m, 6H), 8.33

2000 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13
Spadoni et al.
The organic layer was washed with brine, dried (Na2SO4), and
evaporated under reduced pressure to give a crude residue
which was purified by flash chromatography (silica gel; ethyl
acetate as eluent) and crystallization from CHCl3/hexane to
Gr id sea r ch . The SYBYL 6.3 Grid search routine45 was
used to calculate energy profiles upon rotation of single bonds.
Grid search performs rotations of the selected bond followed
by energy minimization of the rest of the molecule. The
standard Tripos force field with Gasteiger-Hu¨ckel charge
calculation was employed, with energy minimization per-
formed by the method of Powell and termination set at an
energy gradient of 0.02 kcal/mol‚Å.
(c) Mela ton in Bin d in g Assa ys. The source of the ani-
mals, the characterization of the melatonin receptor and the
isolation of the crude membrane preparations used in the
present study have been described in detail elsewhere.35,46
(d ) Deter m in a tion of th e Biologica l Activity. Biological
activity was evaluated using two methods:
1
obtain 15 as a white solid (0.12 g, 55%): mp 151-152 °C; H
NMR (CDCl3) δ 1.96 (s, 3H), 3.89 (s, 3H), 4.75 (d, 2H), 5.90
(br s, 1H), 6.65 (m, 1H), 6.92 (d, 1H, J ) 8.9 Hz), 7.25 (m, 1H),
7.32 (d, 1H, J ) 8.9 Hz), 8.30 (br s, 1H); IR (Nujol) 3325, 3268,
1616 cm-1; MS (EI) m/ z 218 (M+), 175 (100). Anal.
(C12H14N2O2) C, H, N.
N-[(5-Meth oxy-1H-in dol-3-yl)m eth yl]pr opan am ide (16).
A solution of 3-cyano-5-methoxyindole32 (0.17 g, 1 mmol) in
THF (10 mL) and acetic anhydride (2 mL) was hydrogenated
over Raney nickel at 4 atm of H2 for 6 h at 50 °C. The catalyst
was filtered on Celite, and the filtrate was concentrated in
vacuo and partitioned between ethyl acetate and 2 N NaOH.
The organic layer was washed with brine, dried (Na2SO4), and
evaporated under reduced pressure to give a crude residue
which was purified by flash chromatography (silica gel; ethyl
acetate as eluent) and crystallization from EtOAc/hexane to
(i) Effects on F or sk olin -Stim u la ted cAMP Accu m u la -
tion . The punching technique, the handling of tissue explants,
and cAMP determinations have been described in detail
elsewhere.47
The concentrations of the compounds to be used
were calculated on the basis of their affinity, as follows: the
experimentally derived maximum effective dose of melatonin
was around 10-7 M. Therefore, the relative affinity for each
compound was determined as IC50 compound/IC50 aMT and
the dose of the compound equivalent to that of melatonin
(MED, melatonin equivalent dose) in terms of receptor oc-
cupancy was calculated: [MED compound ) (relative affinity
compound) 10-7]. Afterward, two doses of each compound were
assayed: one that was equal (or closest) to the calculated MED
and another, greater by one order of magnitude. For example,
a compound with a relative affinity of 0.025 has a MED value
of 2.5 × 10-9. Therefore the doses utilized were 10-9 and 10-8
M. This avoided the use of inadequately high or low doses in
the analyses, as has been common practice to date. In all cases
the two concentrations employed gave similar results, thus
confirming the validity of the theoretical assumption regarding
the receptor occupancy. In only a few cases, the higher dose
could not be used, due to problems related to the solubility of
the compounds. This was the case with compound 32. On
the basis of the data obtained, a cAMP index, assigning a rank
of potency for the compounds, was calculated as follows:
percent inhibition compound/percent inhibition aMT.
1
obtain 16 as a white solid (0.11 g, 47%): mp 157-158 °C; H
NMR (CDCl3) δ 1.18 (t, 3H), 2.23 (q, 2H), 3.86 (s, 3H), 4.60 (d,
2H), 5.59 (br s, 1H), 6.87, 6.92 (dd, 1H, J ) 8.8 Hz and J )
2.54 Hz), 7.08 (d, 1H, J ) 2.54 Hz), 7.15 (d, 1H, J ) 2.54 Hz),
7.20 (d, 1H, J ) 8.8 Hz), 8.09 (br s, 1H); IR (Nujol) 3378, 3232,
1652 cm-1; MS (EI) m/ z 232 (M+), 159, 160 (53.75, 53.75).
Anal. (C13H16N2O2) C, H, N.
N-[(2-P h en yl-1H-in d ol-3-yl)eth yl]cyclobu ta n eca r box-
a m id e (32). Compound 32, used as reference antagonist, was
synthesized as previously described.33
(b) Molecu la r Mod elin g. Molecular modeling was per-
formed on a Silicon Graphics R4400 200 MHz 64 Mb RAM
Indigo2 workstation, using SYBYL 6.3 software from Tripos
Inc., St. Louis, MO. DISCO is a module of SYBYL.
Con for m a tion a l An a lyses a n d DISCO Mod el Develop -
m en t. Three-dimensional models of all molecules were con-
structed using the sketch option of the building component of
Sybyl 6.3, and their geometry was subsequently optimized
using the Tripos standard force field after Gasteiger-Hu¨ckel
charge calculation.
(ii) Effects of Coin cu ba tion w ith GTP γS on th e IC50
Va lu es (GTP γS In d ex). Briefly, in each experiment aMT
was assayed as a reference standard, GTPγS was used in a
constant concentration of 10-4 M and the labeled ligand was
always 200 pM. The rest of the conditions of the experiment
Nine features were selected among those automatically
assigned by DISCO in order to define the hypothetical phar-
macophore; in particular, an aromatic centroid, 2 hydrogen-
bond (H-B) acceptor atoms (methoxy and carbonyl oxygens),
1 H-B donor atom (amide NH), 4 H-B donor sites (dummy
points in the direction of the lone pairs, 3 Å apart form the
oxygen atoms), and 1 H-B acceptor site (in the direction of
amide NH) were defined before subsequent calculations.
Conformational databases for the compounds included in the
analysis were prepared using the Multisearch option of the
DISCO routine, which generates unique energy minimum
conformations with different internal geometry for those atoms
that determine the features. Each database was then in-
spected in order to verify that the conformational space was
adequately represented. The carboxamido group was consid-
ered in trans configuration; as no constrained amide group was
present in the set of selected active compounds, the cis
configuration is also possible. Therefore, the superimposition
results cannot discriminate between cis and trans configura-
tion. The same applies for the orientation of the methoxy
group which was fixed in a conformation coplanar with the
indole nucleus of aMT, with the methyl group opposite the
nearest bridge position. In fact, the other coplanar conforma-
tion is sterically hindered in some tricyclic compounds, as
resulted from Grid searches on the O-C5 bond. This orienta-
tion is probable but not mandatory, given the unavailability
of crucial information about it. A master database containing
one conformer for each molecule was created and submitted
to the DISCO routine; the natural agonist melatonin was
selected as the reference compound. The choice of the super-
imposition models was based on the inclusion of at least eight
features and on the lowest level of distance tolerance, in steps
of 0.5 Å. Distance tolerance is the maximum allowed differ-
ence between an interfeatures distance for any conformation
of each compound and the same distance for a fixed conforma-
tion of melatonin.
were as described elsewhere.35,46 GTPγS index ) [(IC50
GTPγS)/(IC50 - GTPγS)]compound/[(IC50 + GTPγS)/(IC50
GTPγS)]aMT.
+
-
Ack n ow led gm en t. This work was supported by
grants from Ministero della Ricerca Scientifica e Tec-
nologica (MURST) and Consiglio Nazionale delle Ricerche
(CNR)
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