Tricyclic alkylamides as melatonin receptor ligands with antagonist or inverse agonist activity

Article abstract of DOI:10.1021/jm040768k

This work reports the design and synthesis of novel alkylamides, characterized by a dibenzo[a,d] cycloheptene nucleus, as melatonin (MLT) receptor ligands. The tricyclic scaffold was chosen on the basis of previous quantitative structure-activity studies on MT₁ and MT₂ antagonists, relating selective MT₂ antagonism to the presence of an aromatic substituent out of the plane of the MLT indole ring. Some dibenzo seven-membered structures were thus selected because of the noncoplanar arrangement of their benzene rings, and an alkylamide chain was introduced to fit the requirements for MLT receptor binding, namely, dibenzocycloheptenes with an acylaminoalkyl side chain at position 10 and dibenzoazepines with this side chain originating from the nitrogen atom bridging the two phenyl rings. Binding affinity at human cloned MT₁ and MT₂ receptors was measured by 2[¹²⁵I] iodomelatonin displacement assay and intrinsic activity by the GTPγS test. The majority of the compounds were characterized by higher affinity at the MT₂ than at the MT ₁ receptor and by very low intrinsic activity values, thus confirming the importance of the noncoplanar arrangement of the two aromatic rings for selective MT₂ antagonism. Dibenzocycloheptenes generally displayed higher MT₁ and MT₂ affinity than dibenzoazepines. N-(8-Methoxy-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-10-ylmethyl)propionamide (4c) and -butyramide (4d) were the most selective MT₂ receptor antagonists of the series, with MT₂ receptor affinity comparable to that of melatonin and as such among the highest reported in the literature for MLT receptor antagonists. The acetamide derivative 4b produced a noticeable reduction of GTPγS binding at MT₂ receptor, thus being among the few inverse agonists described.

Full text of DOI:10.1021/jm040768k

4202
J . Med. Chem. 2004, 47, 4202-4212
Tr icyclic Alk yla m id es a s Mela ton in Recep tor Liga n d s w ith An ta gon ist or
In ver se Agon ist Activity
Valeria Lucini,† Marilou Pannacci,† Francesco Scaglione,† Franco Fraschini,† Sivia Rivara,*,‡ Marco Mor,‡
Fabrizio Bordi,‡ Pier Vincenzo Plazzi,‡ Gilberto Spadoni,§ Annalida Bedini,§ Giovanni Piersanti,§
Giuseppe Diamantini,§ and Giorgio Tarzia§
Dipartimento di Farmacologia, Chemioterapia e Tossicologia Medica, Universita` degli Studi di Milano, Via Vanvitelli 32,
I-20129 Milano, Italy, Dipartimento Farmaceutico, Universita` degli Studi di Parma, Parco Area delle Scienze 27/ A,
I-43100 Parma, Italy, and Istituto di Chimica Farmaceutica e Tossicologica, Universita` degli Studi di Urbino “Carlo Bo”,
Piazza Rinascimento 6, I-61029 Urbino, Italy
Received J anuary 12, 2004
This work reports the design and synthesis of novel alkylamides, characterized by a dibenzo-
[a,d]cycloheptene nucleus, as melatonin (MLT) receptor ligands. The tricyclic scaffold was chosen
on the basis of previous quantitative structure-activity studies on MT1 and MT2 antagonists,
relating selective MT2 antagonism to the presence of an aromatic substituent out of the plane
of the MLT indole ring. Some dibenzo seven-membered structures were thus selected because
of the noncoplanar arrangement of their benzene rings, and an alkylamide chain was introduced
to fit the requirements for MLT receptor binding, namely, dibenzocycloheptenes with an
acylaminoalkyl side chain at position 10 and dibenzoazepines with this side chain originating
from the nitrogen atom bridging the two phenyl rings. Binding affinity at human cloned MT1
and MT2 receptors was measured by 2-[125I]iodomelatonin displacement assay and intrinsic
activity by the GTPγS test. The majority of the compounds were characterized by higher affinity
at the MT2 than at the MT1 receptor and by very low intrinsic activity values, thus confirming
the importance of the noncoplanar arrangement of the two aromatic rings for selective MT2
antagonism. Dibenzocycloheptenes generally displayed higher MT1 and MT2 affinity than
dibenzoazepines. N-(8-Methoxy-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-10-ylmethyl)propi-
onamide (4c) and -butyramide (4d ) were the most selective MT2 receptor antagonists of the
series, with MT2 receptor affinity comparable to that of melatonin and as such among the
highest reported in the literature for MLT receptor antagonists. The acetamide derivative 4b
produced a noticeable reduction of GTPγS binding at MT2 receptor, thus being among the few
inverse agonists described.
In tr od u ction
(MT3), has recently been characterized as the hamster
homologue of the human enzyme quinone reductase 2.14
Melatonin (N-acetyl-5-methoxytryptamine, MLT), a
neurohormone primarily produced in the pineal gland
at night, is known to have a central role in the
regulation of daily and seasonal rhythms in vertebrates.
In mammals, the exact role of MLT in the coordination
of circadian rhythms remains to be clarified.1 Ac-
cumulating evidence indicates that exogenously admin-
istered MLT restores some circadian rhythms disturbed
by jet lag, shift work, blindness, and aging.2-5 MLT, in
addition to its chronobiologic effects, has important
immune regulatory,6 antioxidant,7 and oncostatic8 prop-
erties. It has been suggested that MLT might be useful
in some degenerative pathologies, like Alzheimer’s
disease,9 and in reducing edema formation in ischemic
animals.10
The physiological role of these receptors has yet to
be clarified. Early data suggested that these receptor
subtypes have distinct biological roles in mediating the
circadian15 and vascular16 functions of MLT, as well as
its neuroprotective17 and antitumor18 effects and the
enhancement of humoral and cellular immunity.19 While
these findings have provided some understanding of the
roles of MT1 and MT2 receptors, the availability of high-
affinity subtype-selective ligands would greatly assist
in probing the pharmacology associated with the two
receptor subtypes. Therefore, medicinal chemistry re-
search in the melatonin area is being directed toward
the discovery of subtype-selective melatonin ligands,
both as pharmacological tools and as potential thera-
peutic agents.20
Most physiological MLT effects are mediated through
the activation of at least two high-affinity G-protein-
coupled receptors (named MT1 and MT2),11-13 localized
both in the central nervous system and in peripheral
tissues. In addition to these high-affinity MLT receptors,
another low-affinity melatonin binding site, termed
Numerous studies have focused on the discovery of
high-affinity ligands for melatonin receptors, and sev-
eral have been described.21-24 However, only a few
selective melatonin antagonists/partial agonists have
been reported to date (Figure 1),25-33 and very few
agonists, displaying some selectivity for the MT2 recep-
tor, have appeared in the literature.34,35
* To whom correspondence should be addressed. Phone: ++39 0521
905062. Fax: ++39 0521 905006. E-mail: silvia.rivara@unipr.it.
† Universita` degli Studi di Milano.
In the course of our studies, various modifications of
the melatonin structure have been examined36-39 in
‡ Universita` degli Studi di Parma.
§ Universita` degli Studi di Urbino “Carlo Bo”.
10.1021/jm040768k CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/17/2004

Tricyclic Alkylamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17 4203
F igu r e 1. Chemical structures of selective MT2 receptor antagonists or partial agonists.
order to determine which structural features are re-
quired for receptor affinity, intrinsic activity, and/or
subtype selectivity. We have discovered, for example, a
new series of melatonin receptor antagonists by trans-
location of the MLT side chain from the C3 to the C2
position of the indole nucleus.40 Further modifications
aimed at developing more potent and selective MT2
ligands have revealed the necessity of shortening the
length of the side chain by one C unit and of introducing
a benzyl substituent into the N1-indole29 (UCM 454 in
Figure 1).
A comparison of the molecular models of the potent
and selective UCM 454 and of other MT2 selective
antagonists reported in the literature (e.g., 4P-PDOT,25,41
K185,28 luzindole25 in Figure 1) has allowed us to
formulate a hypothesis for the structural requirements
for receptor subtype selectivity.29 This hypothesis has
been tested by performing three-dimensional quantita-
tive structure-affinity and structure-intrinsic activity
relationship (3D-QSAR) studies by means of the CoMFA
approach.42 3D-QSAR models for MLT antagonists have
shown the presence of a bulky group in the region
corresponding to positions 1 and 2 of MLT, located out
of the plane of the melatonin indole ring, to be the key
feature conferring MT2 selective antagonism. The oc-
cupation of this region by a substituent was tolerated
at the MT2 receptor only and it was correlated with a
limited intrinsic activity of the compounds.
analogues, bearing an acylaminoalkyl side chain of
different length and in different positions, to reproduce
the spatial disposition of the pharmacophore elements
of known MT2 selective antagonists. In this work the
synthesis of these compounds, their binding affinity at
human MT1 and MT2 receptors, and their intrinsic
activity are reported.
Ch em istr y
The synthetic strategies (Schemes 1-5) for the target
compounds are different according to the seven-mem-
bered central ring, the length of the acylaminoalkyl side
chain, and the nature of the substituent on the phenyl
ring.
Useful intermediates for the synthesis of the new
tricarbocyclic compounds were dibenzocycloheptenes
bearing functional groups suitable for further conver-
sion.
We synthesized (Scheme 1) the 10-bromoketone 143
by dehydrobromination of trans-10,11-dibromodibenzo-
suberone by potassium tert-butoxide and converted it
by classical methods (LiAlH4/AlCl3 reduction of the keto
group, bromo substitution with cuprous cyanide) into
the dibenzocycloheptencarbonitrile 3.44 Raney nickel
hydrogenation of the nitrile and concomitant N-acyla-
tion with acetic anhydride gave the saturated derivative
4a ; reduction with lithium aluminum hydride/AlCl3,
followed by N-acylation with acetic anhydride gave
compound 5.
A more complex procedure was required for the
preparation of derivatives 4b-d bearing a methoxy
substituent on the phenyl ring (Scheme 2). In accord-
ance with a previously reported procedure for similar
compounds,45 condensation of 3-methoxyphenylaceto-
nitrile and 2-carboxybenzaldehyde gave exclusively (Z)-
2-[2-cyano-2-m-methoxyphenyl)vinyl]benzoic acid, which
was converted into the dibenzocycloheptanone deriva-
On the basis of these findings, we looked for scaffolds
fulfilling the requirements for selective antagonism and
other than the tetralin, indole, naphthalene, and iso-
steric structures reported in Figure 1. We focused on
compounds having a short N-acylaminoalkyl chain and
a tricyclic scaffold; in particular, we observed that
dibenzo seven-membered ring systems held the two
benzene rings in a skewed out-of-plane conformation.
We thus designed a small series of dibenzocycloheptene
and dibenzoazepine derivatives, their dihydro or oxo

4204 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17
Lucini et al.
Sch em e 1a
genation of the N-cyanomethyldibenzoazepine 20 in the
presence of Raney nickel and propionic anhydride gave
the saturated derivative 21 (Scheme 4), whereas reduc-
tion of dibenzoazepines 24a ,b with lithium aluminum
hydride followed by N-acylation with acetic or propionic
anhydride gave the unsaturated compounds 25a ,b
(Scheme 5). Synthesis of the starting unsymmetrical
3-methoxydibenzoazepine 23b was obtained by PPA
rearrangement of 6-methoxy-1-phenylindole29 to diben-
zoazepine, in accordance with a previously described
procedure for similar compounds.49 The N-acetylami-
noacetyl derivative 28 was synthesized by acylation of
23a 50 with bromoacetyl bromide, treatment with potas-
sium phthalimide, N-deprotection, and N-acylation with
acetic anhydride (Scheme 5).
Reagents: (i) t-BuO-K+, t-BuOH; (ii) LiAlH4, AlCl3, THF; (iii)
a
CuCN, N-methylpyrrolidone; (iv) H2, Raney-Ni, 4 atm, Ac2O, THF;
(v) LiAlH4, AlCl3, Et2O; (vi) Ac2O, TEA, THF.
P h a r m a cology
Compounds were tested by measuring their binding
affinity for human MT1 and MT2 receptors and their in
vitro functional activity.
Binding affinity was assessed in competition experi-
ments, using 2-[125I]iodomelatonin as the labeled ligand,
on cloned human MT1 and MT2 receptors expressed in
NIH3T3 rat fibroblast cells.
The relative intrinsic activity (IAr) was measured by
means of the GTPγS test by measuring the direct
activation of the G protein after binding of the tested
compound to the cloned human MT1 or MT2 receptor.
Full agonists increase the binding of [35S]GTPγS in a
concentration-dependent manner, similar to the natural
ligand MLT, partial agonists increase the binding to a
lesser extent than MLT, and antagonists are without
effect, whereas inverse agonists reduce the basal
[35S]GTPγS binding. The IAr values were obtained by
dividing the maximum ligand-induced stimulation of
[35S]GTPγS binding by that of MLT as measured in
the same experiment. The interaction between ligands
and MLT was investigated by competition experiments
in which increasing concentrations of the antagonist
inhibit the maximum MLT-induced stimulation of
[35S]GTPγS binding.
tive 6 by double bond reduction with NaBH4 and
Friedel-Crafts cyclization. Reduction of the cyanoke-
tone 6 by successive treatment with NaBH4 and then
Me2SiCl2/NaI46 led to a mixture of dibenzocyclohep-
tencarbonitrile 7 and dibenzocycloheptencarboxamide
8. Reduction of 8 with LiAlH4/AlCl3 or hydrogenation
of 7 over Raney nickel in the presence of NH3/EtOH
gave crude (8-methoxy-10,11-dihydro-5H-dibenzo[a,d]-
cyclohepten-10-yl)methylamine, which was subsequently
acylated with the suitable anhydride in the pre-
sence of triethylamine (TEA) to yield the desired tri-
cyclic derivatives 4b-d . The 5-oxo analogue 9 was syn-
thesized by hydrogenation of the cyanoketone 6 over
Raney nickel in the presence of NH3/EtOH and succes-
sive N-acylation of the intermediate amine with acetic
anhydride.
Key intermediates in the synthesis of the new tricyclic
compounds 18a and 18b were the dibenzocyclohep-
tanones 16a 47 and 16b,48 which were prepared as
outlined in Scheme 3 in accordance with previously
reported procedures. Briefly, reaction of phthalic anhy-
dride with the Grignard reagent from p-bromoanisole
gave the 2-benzoylbenzoic acid derivative 10b. The
ketones 10a ,b were reduced to the corresponding hy-
drocarbons by heating an alkaline solution of the
ketoacid with excess Zn dust. The carboxylic acid
moieties were transformed by ester formation and
LiAlH4 reduction to the benzyl alcohol derivatives 12a ,b.
These were converted to the corresponding bromides
13a ,b by treatment with hydrogen bromide; displace-
ment of bromine with potassium cyanide yielded the
cyanomethyl derivatives 14a ,b, which were then hy-
drolyzed to the carboxylic acids 15a ,b by refluxing in a
mixture of sulfuric and acetic acid. The dibenzocyclo-
heptanones 16a 47 and 16b48 were formed by polyphos-
phoric acid (PPA) internal Friedel-Crafts acylation of
the acids 15a ,b. The cycloheptanone underwent a two-
carbon homologation by the Wittig-Horner reaction
with diethyl (cyanomethyl)phosphonate to give the
nitriles 17a ,b; they were directly transformed into the
saturated acetamidoethyl derivatives 18a ,b by hydro-
genation over Raney nickel in the presence of acetic
anhydride.
Resu lts a n d Discu ssion
Table 1 reports the pharmacological results obtained
with compounds synthesized to investigate the effect of
a dibenzo seven-membered cyclic scaffold on human
melatonin receptor binding and intrinsic activity. These
derivatives are also characterized by the presence of an
acylaminoalkyl side chain.51
The nature of the tricyclic ring and the position and
the length of the side chain were varied by selecting
those combinations that gave good superposition to
known MT2 selective antagonists (e.g., UCM 454 and
4P-PDOT in Figure 1) in the conformations chosen from
a previous 3D-QSAR model.42 Both dibenzocycloheptene
(4a -d , 5, 9, 18a ,b in Table 1) and dibenzoazepine (21,
25a ,b, 28) derivatives were prepared. The acylami-
noalkyl chain originated from position 10 in the first
series and from the nitrogen atom in the second one.
Mono- or dimethylene spacers connected the amide
function to the tricyclic scaffold directly or through a
carbonyl (28). The effect of different geometries of the
tricyclic nucleus was evaluated by employing the aro-
matic scaffolds, their 10,11-dihydro analogues, or the
The dibenzo[b,f]azepine derivatives 21 and 25a ,b
were synthesized by alkylation of the suitable diben-
zoazepine 19 or 23a ,b with bromoacetonitrile. Hydro-

Tricyclic Alkylamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17 4205
Sch em e 2a
a
Reagents: (i) NaOMe, MeOH; (ii) NaBH4, i-PrOH; (iii) SOCl2, ClCH2CH2Cl; (iv) SnCl4, ClCH2CH2Cl; (v) NaBH4, dry MeOH; (vi) NaI,
(CH3)2SiCl2, CH3CN; (vii) H2, Raney-Ni, NH3/EtOH, THF, 4 atm, 60 °C; (viii) (R1CO)2O, TEA, THF; (ix) LiAlH4, AlCl3, THF, reflux 4 h.
Sch em e 3a
a
Reagents: (i) Mg, Et2O, I2, toluene; (ii) Zn, NaOH; (iii) CH2N2, Et2O; (iv) LiAlH4, Et2O; (v) HBr 48%; (vi) KCN, EtOH; (vii) H2SO4,
CH3COOH; (viii) PPA, 90 °C; (ix) (EtO)2P(O)CH2CN, NaH, THF; (x) H2, Raney-Ni, 4 atm, Ac2O, THF, 60 °C.
Sch em e 4a
Our design strategy yielded compounds with remark-
able affinity, particularly at MT2 receptors. Almost all
compounds showed higher affinity at the MT2 receptor,
with compounds 4a -c characterized by the highest
selectivity. Dibenzocycloheptenes were more potent than
dibenzoazepines, and, within the first series, the monom-
ethylene spacer conferred higher affinity at both recep-
tor subtypes (4a versus 18a ; 4b versus 18b). A methoxy
group at position 8 led to an increase in binding affinity
at both MT1 and MT2 receptors with a more relevant
effect for the monomethylene derivatives, enhancing
their potency by about 30 times (4a versus 4b). On the
other hand, it did not influence subtype selectivity. The
N-propionyl and N-butyryl derivatives showed higher
affinity at both receptor subtypes compared to the
a
Reagents: (i) BrCH2CN, ∆; (ii) H2, Raney-Ni, 4 atm, (EtCO)2O,
THF.
oxo derivative (9); the role of the methoxy group, placed
in a position topographically corresponding to position
5 of the indole ring of MLT, was also considered.
Different acylating groups were selected for the amino
function to investigate the importance of steric effects
on biological activity.

4206 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17
Lucini et al.
Sch em e 5a
a
Reagents: (i) PPA, 80 °C; (ii) Na2CO3, BrCH2CN, CH2Cl2; (iii) LiAlH4, THF; (iv) (R1CO)2O, TEA, THF; (vi) potassium phthalimide,
DMF; (vii) NH2-NH2, ∆.
Ta ble 1. Binding Affinitya and Intrinsic Activity (IAr)b of New
Tricyclic Derivatives for the Human MT1 and MT2 Melatonin
Receptors Stably Expressed in NIH3T3 Cells
pounds showed lower affinity and subtype selectivity.
Within this series, hydrogenation of the 10-11 double
bond was not favorable, and the positive effect of the
ethyl substituent in the amide side chain and of the
methoxy group was less marked than in the dibenzo-
cycloheptenes; the presence of a carbonyl function in the
side chain of 28 was not tolerated and produced a huge
drop in affinity at both receptors.
Concerning intrinsic activity, dibenzoazepine deriva-
tives 21 and 25a ,b did not significantly influence GTPγS
binding, showing IAr values around zero. The same
was observed for the majority of dibenzocycloheptenes,
with some notable exceptions. Compound 18a , with a
dimethylene spacer, evidenced a partial agonist behav-
ior, particularly marked at the MT2 receptor. In this
case the methoxy group, which had been related to an
increase in intrinsic activity in other series of lig-
ands,28,29,37 had an opposite effect, lowering IAr and
leading to an antagonist behavior for 18b. Compounds
4a and 4b, having a monomethylene spacer, behaved
as inverse agonists at the MT2 receptor, being charac-
terized by relative intrinsic activity values of -0.32 and
-0.55, respectively. These compounds can be considered
among the few inverse agonists reported so far40,52 that
could be interesting as pharmacological tools or in the
adjustment of the circadian clock.52 On the other hand,
the N-propionyl and N-butyryl derivatives (4c and 4d )
showed a very low effect on GTPγS binding at the MT2
receptor, practically behaving as antagonists. Both a
double bond between atoms 10 and 11 and a carbonyl
group in position 5 of the tricyclic scaffold shifted
intrinsic activity toward antagonist behavior, although
the resulting compounds were less potent (5) or less
selective (9) than the corresponding parent compounds
(4a and 4b, respectively).
Our previous investigations into MLT receptor ligands
led to a set of 3D-QSAR models42 supporting the
hypothesis that occupation of the out-of-the-plane space
corresponding to positions 1 and 2 of MLT was a key
feature for obtaining selective MT2 antagonists. The
novel compounds here described fulfilled this require-
ment. They are in fact characterized by a tricyclic
scaffold in which the two benzene rings are maintained
in a noncoplanar arrangement and at a distance similar
to those of other selective antagonists. In fact, the
centroids of the benzene portion of the tetralin ring and
human MT1
pKi IAr ( SEM pKi IAr ( SEM
9.54 1 ( 0.01 9.55 1 ( 0.02
human MT2
compd
R
R1
X
n
MLT
4a
4b
4c
4d
H
Me
1
1
1
1
6.62 0.03 ( 0.02 7.52 -0.32 ( 0.07
8.21 -0.18 ( 0.04 9.18 -0.55 ( 0.08
8.87 -0.10 ( 0.02 9.79 -0.15 ( 0.08
8.89 -0.09 ( 0.06 9.61 -0.17 ( 0.05
6.26 0.02 ( 0.03 7.07 -0.09 ( 0.03
8.52 -0.14 ( 0.02 9.05 -0.08 ( 0.03
6.41 0.21 ( 0.03 7.05 0.69 ( 0.02
6.79 -0.07 ( 0.02 7.40 0.01 ( 0.01
6.26 0.03 ( 0.01 6.70 0.13 ( 0.01
6.95 0.01 ( 0.01 6.90 -0.03 ( 0.06
7.24 -0.04 ( 0.02 7.42 -0.10 ( 0.02
OCH3 Me
OCH3 Et
OCH3 n-Pr
5
9
18a
18b
21
25a
25b
28
H
Me
2
2
OCH3 Me
H
Me CH2
OCH3 Et CH2
Me CdO
H
4.69 ndc
4.96 ndc
a
pKi values were calculated from IC50 values obtained from
competition curves by the method of Cheng and Prusoff56 and are
the mean of at least three determinations performed in duplicate;
b
SEM of pKi values were lower than 0.06. The relative intrinsic
activity values were obtained by dividing the maximum analogue-
induced G-protein activation by that of MLT. c Not determined.
N-acetyl one (4c,d versus 4b), confirming the positive
effect of longer side chains observed in the case of
other MLT receptor antagonists.28,42 The presence of a
10-11 double bond in compound 5 slightly reduced the
binding affinity at both receptor subtypes compared
with the corresponding compound 4a whereas the
carbonyl function at position 5 was tolerated, leading
to affinity values for compound 9 comparable to those
of 4b.
Longer side chains were only considered for diben-
zoazepine derivatives (21, 25a ,b, 28), following the
aforementioned superposition models, and these com-

Tricyclic Alkylamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17 4207
F igu r e 2. Superposition of cis-4P-PDOT (orange carbons),
UCM 454 (white carbons), and 4c (yellow carbons).
F igu r e 3. Compound 4c surrounded by the CoMFA coef-
ficients for MT1 (cyan) or MT2 (red) selectivity, in accordance
with the 3D-QSAR models described in ref 42.
Ta ble 2. Root Mean Square Distances (RMSD) of the
Pharmacophore Elements (Four Amide Atoms and Two
Aromatic Centroids) of the Newly Synthesized Compounds from
Those of cis-(S,S)-4P-PDOT in Their Best-Fitting
Conformations
considered a promising result if associated with the MT2
binding affinity observed for compounds 4b-d , among
the highest ever reported for melatonin receptor an-
tagonists.
compd
chirality
RMSD
4a -d
R
S
0.50
0.37
0.52
0.42
0.51
0.70
0.93
0.62
0.42
0.27
A CoMFA model based on intrinsic activity data,
homogeneously evaluated by means of the GTPγS test,
had shown that steric occupancy of the out-of-plane
region led toward antagonist behavior.42 Tricyclic de-
rivatives generally confirmed this trend, although their
intrinsic activity was quantitatively predicted to be
slightly higher than observed when an 8-methoxy group
was present. In fact, while the 5-methoxy group in MLT
derivatives had a significantly positive effect on intrinsic
activity, the corresponding 8-methoxy group in the
dibenzocycloheptene series showed a negative effect, if
any (see 4a versus 4b).
Considering the results obtained for the present series
of compounds, the reported CoMFA models revealed
limited ability to quantitatively predict the observed
values of receptor affinity and intrinsic activity, as
generally happens with extrapolations of QSARs. On the
other hand, the qualitative information provided by
them proved to be useful for the design of this new
series, which comprises some valuable compounds.
These indications cannot be applied to all the MLT
receptor ligands reported so far. For example, S24601
(Figure 1) is a potent and selective MT2 antagonist
whose phenyl substituent in position 3 is coplanar with
the naphthalene ring. However, it is possible to super-
pose the amide function, the phenyl substituent, and
the methoxy oxygen of S24601 onto those of the other
selective antagonists fulfilling the cited pharmacophore,
resulting in a slightly different orientation of the
naphthalene ring with respect to the aromatic scaffold
of the other compounds. The 3D-QSAR model of ref 42
for S24601 predicted an MT2 selectivity 46 times that
of MLT, in accordance with experimental data.31
In conclusion, tricyclic compounds belonging to the
dibenzocycloheptene series represent a novel class of
selective MT2 antagonists, having the core scaffold
fulfilling a key feature outlined by our previous 3D-
QSAR models, i.e., two aromatic rings in a noncoplanar
arrangement. In particular, compounds 4c and 4d
displayed binding affinities among the highest reported
5
9
R
S
R
S
18a ,b
21
25
28
of the phenyl substituent in cis-4P-PDOT are at a
distance of 4.82 Å, similar to that of 4.81 Å between the
centroids of the two benzene rings of 4c. Moreover, the
amide function of 4c can adopt the same arrangement
as in known selective MT2 antagonists, as depicted in
Figure 2. Similar superpositions could be obtained for
the other compounds here described, with the weighted
Root Mean Square Distances (RMSD) from the phar-
macophoric elements of cis-4P-PDOT reported in Table
2. For compounds containing a chiral center (4a -d , 9,
18a ,b), similar RMSD values were observed when
different conformations of the two enantiomers were
fitted on the same enantiomer of cis-4P-PDOT, suggest-
ing poor enantioselectivity. For this reason, these
compounds were only tested as a racemic mixture.
The steric features of the tricyclic compounds of this
study are compatible with those predicted to be suitable
for MT2 selectivity, as illustrated by the comparison of
the CoMFA coefficients for MT1/MT2 selectivity with the
structure of 4c depicted in Figure 3. It can be observed
that the two red regions, corresponding to positive
correlation between steric field and MT2 selectivity, are
occupied by a portion of the bent tricyclic nucleus and
the methoxy group, respectively. It is noted, however,
that in the present series of compounds the methoxy
group does not seem to significantly influence receptor
selectivity (see above). On the basis of the 3D-QSAR
selectivity model reported in ref 42, a selectivity ratio
of between 10 and 100 times was predicted for the
tricyclic compounds here reported. Indeed, the most
interesting dibenzocycloheptene derivatives displayed
a selectivity ratio of about 10 times, which can be

4208 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17
Lucini et al.
(M+, 100). 1H NMR (CDCl3): δ 3.47 (dd, 1H, J ) 15.3, 7.9),
3.60 (dd, 1H, J ) 15.3, 2.2), 3.92 (s, 3H), 4.44 (dd, 1H, J ) 7.9,
2.2), 6.97-7.57 (m, 5H), 7.02 (s, 1H), 7.28-7.56 (m, 3H), 8.04
so far for MT2 antagonists and comparable to those of
compound A, K185, and S24601 in Figure 1.
(dd, 1H, J ) 7.26, 1.34), 8.21 (d, 1H, J ) 8.34). IR (cm-1
,
Exp er im en ta l Section
Nujol): 2237, 1632, 1602, 1584.
Melting points were determined on Buchi SMP-510 capillary
melting point apparatus and are uncorrected. 1H NMR spectra
were recorded on a Bruker AC 200 spectrometer; chemical
shifts (δ scale) are reported in parts per million (ppm) relative
to the central peak of the solvent. Coupling constants (J
values) are given in hertz (Hz). EI-MS spectra (70 eV) were
taken on a Fisons Trio 1000 instrument. Only molecular ions
(M+) and base peaks are given. Infrared spectra were obtained
on a Nicolet Avatar 360 FT-IR spectrometer; absorbances are
reported in ν (cm-1). Elemental analyses for C, H, and N of
8-Meth oxy-10,11-dih ydr o-5H-diben zo[a ,d]cycloh epten e-
10-ca r b on it r ile (7) a n d 8-Met h oxy-10,11-d ih yd r o-5H -
d iben zo[a ,d ]cycloh ep ten e-10-ca r boxylic Acid Am id e (8).
Solid sodium borohydride (0.289 g, 7.65 mmol) was added to
a stirred solution of 6 (3 g, 11.4 mmol) in dry MeOH (60 mL)
under nitrogen. After being stirred for 30 min, the reaction
mixture was diluted with EtOAc, washed once with water and
once with brine, and then dried (Na2SO4). The solvent was
evaporated and the residue was dissolved in acetonitrile (40
mL) to give a solution to which were added NaI (4.6 g, 30.6
mmol) and dichlorodimethylsilane (2 mL, 16.5 mmol). The
reaction mixture was stirred at room temperature for 1 h and
then diluted with EtOAc and washed with water, aqueous
NaHCO3, aqueous Na2S2O3, and brine. After the mixture was
dried over Na2SO4, the solvent was evaporated under reduced
pressure to give a mixture of crude compounds 7 and 8 that
were separated by flash chromatography (silica gel; cyclohex-
ane/EtOAc 8:2 and then EtOAc as eluent).
the tested compounds were performed on
a Carlo Erba
analyzer, and the results are within 0.4% of the calculated
values. Column chromatography purifications were performed
under “flash” conditions using Merck 230-400 mesh silica gel.
Analytical thin-layer chromatography (TLC) was carried out
on Merck silica gel 60 F254 plates.
The two radioligands 2-[125I]iodomelatonin (specific activity,
2000 Ci/mmol) and [35S]GTPγS ([35S]guanosine-5′-O-(3-thio-
triphosphate); specific activity, 1000 Ci/mmol) were purchased
from Amersham Pharmacia Biotech (Italy).
8-Meth oxy-10,11-dih ydr o-5H-diben zo[a ,d]cycloh epten e-
10-ca r bon itr ile (7). 10% yield, white solid, mp 115 °C. MS
3-Methoxyphenylacetonitrile, 2-carboxybenzaldehyde, trans-
10,11-dibromodibenzosuberone, 10a , 19, and 23a were com-
mercially available (Aldrich).
1
(EI): m/z 249 (M+), 222 (100). H NMR (CDCl3): δ 3.41 (dd,
1H, J ) 15.0, 9.7), 3.57 (dd, 1H, J ) 15.0, 4.0), 3.79 (s, 3H),
3.95 (d, 1H, J ) 15.4), 4.18 (d, 1H, J ) 15.4), 4.51 (dd, 1H, J
) 9.7, 4.0), 6.77 (dd, 1H, J ) 8.34, 2.7), 7.99 (d, 1H, J ) 2.7),
7.13-7.19 (m, 5H). IR (cm-1, Nujol): 2237, 1615, 1571.
8-Meth oxy-10,11-dih ydr o-5H-diben zo[a ,d]cycloh epten e-
10-ca r boxylic Acid Am id e (8). 56% yield, white solid, mp
(Z)-2-[2-Cyan o-2-(3-m eth oxyph en yl)vin yl]ben zoic Acid.
3-Methoxyphenylacetonitrile (0.845 g, 5.74 mmol) was added
to an ice-cooled solution of sodium (0.124 g, 5.4 mmol) in dry
MeOH (4 mL) under nitrogen. After the mixture was stirred
for 20 min, 2-carboxybenzaldehyde (0.735 g, 4.9 mmol) was
added at room temperature, and the mixture was refluxed for
1 h, cooled to room temperature, and poured into ice/water
(50 mL) containing concentrated HCl (0.74 mL). After the
mixture was stirred for 10 min, the solid was collected by
filtration, washed with water, and after dissolution in EtOAc,
washed once with brine and dried (Na2SO4). Removal of the
solvent afforded a yellowish solid that was purified by crystal-
lization from EtOAc (82% yield), mp 173-174 °C. MS (EI): m/z
279 (M+), 133 (100). 1H NMR (CDCl3): δ 3.87 (s, 3H), 6.97 (m,
1H), 7.25-7.41 (m, 3H), 7.56 (dd, 1H), 7.73 (dd, 1H), 7.91 (d,
1H), 8.22 (d, 1H), 8.35 (s, 1H).
2-[2-Cya n o-2-(3-m et h oxyp h en yl)et h yl]b en zoic Acid .
Solid sodium borohydride (2 g, 53 mmol) was added portion-
wise in 1 h to a boiling suspension of (Z)-2-[2-cyano-2-(3-
methoxyphenyl)vinyl]benzoic acid (6.23 g, 22.3 mmol) in dry
i-PrOH (92 mL) under nitrogen, and the mixture was refluxed
for 6 h. The reaction mixture was cooled to room temperature,
poured into ice/water, acidified with 2 N HCl, and extracted
with EtOAc. The combined organic phases were washed with
brine, dried (Na2SO4), and evaporated to afford a white solid
that was purified by trituration with diethyl ether (72% yield),
mp 145 °C. MS (EI): m/z 281 (M+), 135 (100). 1H NMR
(CDCl3): δ 3.31 (dd, 1H, J ) 12.9, 10.2), 3.76 (dd, 1H, J )
12.9, 5.3), 3.81 (s, 3H), 4.33 (dd, 1H, J ) 10.1, 5.3), 6.88 (dd,
1H, J ) 7.8, 1.9), 7.02 (m, 2H), 7.30-7.63 (m, 4H), 8.18 (d,
1H). IR (cm-1, Nujol): 3417, 2241, 1678, 1598.
1
202 °C. MS (EI): m/z 267 (M+), 223 (100). H NMR (CDCl3):
δ 3.44 (dd, 1H, J ) 14.5, 7.2), 3.55 (dd, 1H, J ) 14.5, 5.0), 3.75
(s, 3H), 3.98 (d, 1H, J ) 15.2), 4.08 (dd, 1H, J ) 7.2, 5.0), 4.18
(d, 1H, J ) 15.2), 4.93 (brs, 1H), 5.20 (brs, 1H), 6.68 (d, 1H, J
) 2.8), 6.75 (dd, 1H, J ) 8.1, 2.8), 7.15-7.19 (m, 5H). IR (cm-1
Nujol): 3429, 3394, 1645.
,
(5H-Diben zo[a ,d ]cycloh ep ten -10-yl)a ceton itr ile (17a ).
Diethyl (cyanomethyl)phosphonate (0.35 mL, 2.16 mmol) was
added dropwise under a nitrogen atmosphere to a stirred
suspension of NaH (80% in mineral oil, 0.065 g, 2.16 mmol)
in dry THF (3 mL), and the mixture was stirred for 40 min at
room temperature. A solution of the ketone 16a 47 (0.15 g, 0.72
mmol) in dry THF (2 mL) was added, and the resulting
mixture was stirred at room temperature for 24 h. To the
reaction mixture was added water, and the mixture was
extracted with diethyl ether. The combined organic phases
were dried (Na2SO4) and the solvent was evaporated to give a
crude residue that was purified by flash chromatography (silica
gel; cyclohexane/EtOAc 8:2 as eluent): oil (60% yield). MS
(EI): m/z 231 (M+), 191 (100). 1H NMR (CDCl3): δ 3.71 (s,
2H), 3.86 (s, 2H), 7.17-7.36 (m, 9H).
(8-Met h oxy-5H -d ib en zo[a ,d ]cycloh ep t en -10-yl)a cet o-
n itr ile (17b). 17b was obtained following the above procedure
by using the ketone 16b48 instead of 16a : oil (48% yield). MS
(EI): m/z 261 (M+, 100). 1H NMR (CDCl3): δ 3.65 (s, 2H), 3.79
(s, 3H), 3.83 (s, 2H), 6.90 (m, 2H), 7.18-7.31 (m, 6H). IR (cm-1
neat): 2250, 1606, 1565.
,
8-Meth oxy-5-oxo-10,11-d ih yd r o-5H-d iben zo[a ,d ]cyclo-
h ep ten e-10-ca r bon itr ile (6). Thionyl chloride (2.4 mL, 33
mmol) was added to a solution of 2-[2-cyano-2-(3-methoxy-
phenyl)ethyl]benzoic acid (6.2 g, 22.3 mmol) in dry 1,2-di-
chloroethane (30 mL), and the mixture was refluxed for 1 h.
Thionyl chloride and the solvent were removed under vacuo,
the residue was dissolved in dry 1,2-dichloroethane (30 mL),
the mixture was cooled to 0 °C, and then tin tetrachloride (3.45
mL, 29.5 mmol) was added. The reaction mixture was stirred
for 1 h at room temperature, poured into ice/water, and
extracted with EtOAc. The combined organic phases were
washed once with brine and dried (Na2SO4), and the solvent
was removed under reduced pressure. The crude product was
purified by flash chromatography (silica gel; cyclohexane/
EtOAc, 7:3, as eluent) and crystallization from diethyl ether/
petroleum ether (82% yield), mp 79-80 °C. MS (EI): m/z 263
(10,11-Dih ydr odiben zo[b,f]azepin -5-yl)aceton itr ile (20).
A solution of iminodibenzyl (0.5 g, 2.56 mmol) in bromoaceto-
nitrile (1.4 mL) was stirred at 80 °C for 24 h. The excess
bromoacetonitrile was removed by bulb to bulb distillation, and
the residue was purified by flash chromatography (silica gel;
cyclohexane/EtOAc, 9:1 as eluent) and crystallization from
diethyl ether/petroleum ether: white solid (27% yield), mp 92
°C. MS (EI): m/z 234 (M+), 194 (100). 1H NMR (CDCl3): δ 3.16
(s, 4H), 4.58 (s, 2H), 6.99-7.31 (m, 8H). IR (cm-1, Nujol): 2256,
1586.
Syn th esis of Tr icyclic Alk yla m id es (4a -d , 5, 9, 18a ,b,
21) by Red u ction of Su ita ble Nitr iles or Am id es. P r oce-
d u r e A. Ra n ey Nick el Hyd r ogen a tion of Nitr iles a n d
Con com ita n t N-Acyla tion w ith Su ita ble An h yd r id e. A
solution of a suitable nitrile (3),44 17a ,b, and 20 (1 mmol) in

Tricyclic Alkylamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17 4209
THF (10 mL) and acetic or propionic anhydride (3 mL) was
hydrogenated over Raney nickel at 4 atm of H2 for 5 h at 60
°C. The catalyst was filtered on Celite, the filtrate was
concentrated in vacuo, and the residue was partitioned be-
tween ethyl acetate and 2 N NaOH. The organic layer was
washed with brine, dried (Na2SO4), and evaporated under
reduced pressure. Purification by flash chromatography (silica
gel; EtOAc as eluent) and crystallization gave the desired
tricyclic amide 4a , 18a ,b, and 21.
(100). 1H NMR (CDCl3): δ 1.94 (s, 3H), 3.00 (dd, 1H, J ) 15.3,
8.9), 3.28-3.62 (m, 4H), 3.76 (s, 3H), 3.9 (d, 1H, J ) 15.0),
4.17 (d, 1H, J ) 15.0), 5.4 (brs, 1H), 6.68 (dd, 1H, J ) 8.3,
2.4), 6.75 (d, 1H, J ) 2.4), 7.1-7.2 (m, 5H). IR (cm-1, Nujol):
3298, 1645, 1559. Anal. (C19H21NO2) C, H, N.
N-(8-Meth oxy-10,11-d ih yd r o-5H-d iben zo[a ,d ]cycloh ep -
ten -10-ylm eth yl)p r op ion a m id e (4c). Mp 114 °C (diethyl
ether/petroleum ether), 74% yield. MS (EI): m/z 309 (M+),
1
236 (100). H NMR (CDCl3): δ 1.11 (t, 3H, J ) 7.6), 2.17 (q,
N-(10,11-Dih yd r o-5H -d ib en zo[a ,d ]cycloh ep t en -10-yl-
m eth yl)a ceta m id e (4a ). White crystalline solid, 69% yield,
mp 147-8 °C (diethyl ether). MS (EI): m/z 265 (M+), 206 (100).
1H NMR (CDCl3): δ 1.94 (s, 3H), 3.00 (dd, 1H, J ) 14.6, 9.0),
3.31-3.64 (m, 4H), 3.94 (d, 1H, J ) 15.0), 4.25 (d, 1H, J )
15.0), 5.40 (brs, 1H), 7.10-7.23 (m, 8H). IR (cm-1, Nujol): 3274,
1642, 1564. Anal. (C18H19NO) C, H, N.
2H, J ) 7.6), 3.00 (dd, 1H, J ) 15.1, 8.9), 3.29-3.62 (m, 4H),
3.76 (s, 3H), 3.89 (d, 1H, J ) 15.0), 4.17 (d, 1H, J ) 15.0), 5.4
(brs, 1H), 6.68 (dd, 1H, J ) 8.5, 2.6), 6.75 (d, 1H, J ) 2.6),
7.1-7.2 (m, 5 H). IR (cm-1, Nujol): 3292, 1644, 1561. Anal.
(C20H23NO2) C, H, N.
N-(8-Meth oxy-10,11-d ih yd r o-5H-d iben zo[a ,d ]cycloh ep -
ten -10-ylm eth yl)bu tyr a m id e (4d ). Mp 102-103 °C (diethyl
ether/cyclohexane), 57% yield. MS (EI): m/z 323 (M+), 71 (100).
1H NMR (CDCl3): δ 0.92 (t, 3H, J ) 7.26), 1.62 (m, 2H), 2.12
(t, 2H, J ) 7.3), 3.00 (dd, 1H, J ) 14.9, 8.5), 3.28-3.66 (m,
4H), 3.76 (s, 3H), 3.89 (d, 1H, J ) 15.2), 4.16 (d, 1H, J ) 15.2),
5.44 (brs, 1H), 6.68 (dd, 1H, J ) 8.3, 2.7), 6.74 (d, 1H, J )
2.7), 7.1-7.21 (m, 5H). IR (cm-1, Nujol): 3283, 1640, 1561.
Anal. (C21H25NO2) C, H, N.
N-[2-(10,11-Dih yd r o-5H -d ib en zo[a ,d ]cycloh ep t en -10-
yl)eth yl]a ceta m id e (18a ). White crystalline solid, 60% yield,
mp 98 °C (diethyl ether/petroleum ether). MS (EI): m/z 279
1
(M+), 73 (100). H NMR (CDCl3): δ 1.84 (m, 2H), 1.95 (s, 3H),
2.99 (dd, 1H, J ) 15.4, 10.3), 3.38 (m, 4H), 3.95 (d, 1H, J )
15.0), 4.25 (d, 1H, J ) 15.0), 5.38 (brs, 1H), 7.08-7.20 (m, 8H).
IR (cm-1, Nujol): 3259, 1638, 1561. Anal. (C19H21NO) C, H,
N.
Tr icyclic a lk yla m id es 4b-d were prepared following
Procedure B or Procedure C depending on the starting nitrile
(7) or amide (8) employed.
N-[2-(8-Meth oxy-10,11-d ih yd r o-5H-d iben zo[a ,d ]cyclo-
h ep ten -10-yl)eth yl]a ceta m id e (18b). Oil, 67% yield. MS
(EI): m/z 309 (M+), 73 (100). 1H NMR (CDCl3): δ 2.0 (m, 2H),
2.10 (s, 3H), 3.12 (dd, 1H, J ) 15.8, 6.2), 3.52 (m, 4H), 3.91
(s, 3H), 4.03 (d, 1H, J ) 15.0), 4.33 (d, 1H, J ) 15.0), 5.61
(brs, 1H), 6.80 (dd, 1H, J ) 8.3, 2.5), 6.85 (d, 1H, J ) 2.5),
7.22-7.41 (m, 5H). IR (cm-1, Nujol): 3281, 1649, 1551. Anal.
(C20H23NO2) C, H, N.
P r oced u r e C. LiAlH4/AlCl3 Red u ction of th e Am id e 8
F ollow ed by N-Acyla tion of Cr u d e Am in e w ith Su ita ble
An h yd r id e. A solution of aluminum trichloride (0.198 g, 1.5
mmol) in dry THF (6 mL) was added dropwise to an ice-cooled
suspension of LiAlH4 (0.164 g, 4.3 mmol) in dry THF (6 mL).
After the mixture was stirred for 10 min, a solution of the
amide 8 (0.75 mmol) in dry THF (6 mL) was added dropwise,
and the resulting mixture was heated at reflux for 4 h. The
reaction mixture was cooled to 0 °C, and the excess hydride
was cautiously destroyed with water. The resulting mixture
was filtered on Celite, and the filtrate was concentrated in
vacuo and partitioned between EtOAc and 2 N NaOH (pH 10).
The combined organic phases were washed once with brine,
dried (Na2SO4), and evaporated to afford a crude oily amine
(80% yield) that was N-acylated, without any further purifica-
tion, using the above-described procedure B.
N-[2-(10,11-Dih yd r od iben zo[b,f]a zep in -5-yl)eth yl]p r o-
p ion a m id e (21). White crystalline solid. Purification by flash
chromatography (silica gel; cyclohexane/EtOAc 1:1 as eluent),
64% yield, mp 111-2 °C (diethyl ether). MS (EI): m/z 294 (M+),
1
208 (100). H NMR (CDCl3): δ 1.10 (t, 3H, J ) 7.54), 2.15 (q,
2H, J ) 7.54), 3.18 (s, 4H), 3.45 (m, 2H), 3.90 (t, 2H), 5.59
(brs, 1H), 6.92-7.20 (m, 8H). IR (cm-1, Nujol): 3289, 1637,
1572. Anal. (C19H22N2O) C, H, N.
P r oced u r e B. Ra n ey Nick el Hyd r ogen a tion of Nitr iles
F ollow ed by N-Acyla tion of Cr u d e Am in e w ith Su ita ble
An h yd r id e. A solution of the suitable cyano derivative 6 or 7
(1.6 mmol) in THF (7 mL) and 2 N NH3 in EtOH (5 mL) was
hydrogenated over Raney nickel at 4 atm of H2 for 6 h at 60
°C. The catalyst was filtered on Celite, the filtrate was
concentrated in vacuo, and the residue was partitioned be-
tween EtOAc and water. The organic phase was washed with
brine, dried (Na2SO4), and evaporated under reduced pres-
sure to give a crude oily amine that was purified by flash
chromatography (silica gel; CH2Cl2/MeOH 95:5 as eluent) and
then used without any further purification (60-65% yields).
TEA (1.1 equiv) and the appropriate anhydride (1.1 equiv)
were added to a cold solution of the above amine (1 mmol) in
THF (4 mL), and the resulting reaction mixture was left
stirring at room temperature for 6 h. The solvent was
evaporated under reduced pressure, and the residue was taken
up in ethyl acetate and washed with a saturated aqueous
solution of NaHCO3 followed by brine. After the mixture was
dried over Na2SO4, the solvent was distilled off in vacuo to
give a crude product, which was purified by flash chromatog-
raphy on silica gel (cyclohexane/EtOAc 3:7 as eluent) and
crystallization.
N-(5H -Dib e n zo[a ,d ]cycloh e p t e n -10-ylm e t h yl)a ce t a -
m id e (5). A solution of aluminum trichloride (0.305 g, 2.27
mmol) in dry Et2O (2 mL) was added dropwise to a stirred
ice-cooled suspension of LiAlH4 (0.085 g, 2.22 mmol) in dry
Et2O (2 mL) under nitrogen, and the mixture was stirred for
10 min. A solution of the nitrile (3)44 (0.2 g, 0.92 mmol) in dry
Et2O (2 mL) was added dropwise to the above mixture, and
the resulting mixture was stirred for 1 h at room temperature.
Standard workup and N-acetylation (see procedure C) gave
the desired title compound. Mp 146 °C (diethyl ether/petroleum
1
ether), 7% yield. MS (EI): m/z 263 (M+), 204 (100). H NMR
(CDCl3): δ 1.99 (s, 3H), 3.68 (brs, 2H), 4.65 (brd, 2H), 5.58
(brt, 1H), 7.13-7.46 (m, 9H). IR (cm-1, Nujol): 3272, 1642,
1561. Anal. (C18H17NO) C, H, N.
3-Meth oxy-5H-d iben zo[b,f]a zep in e (23b). PPA (17 g)
was brought to 100 °C under N2 and mechanically stirred for
1 h. The temperature was set to 80 °C, and N-phenyl-6-
methoxyindole (0.5 g, 2.24 mmol) was added in small portions
in 30 min. After being stirred at 80 °C for 20 h, the reaction
mixture was cooled and poured into crushed ice and NaHCO3
solution to set the pH at 6. The aqueous solution was extracted
three times with CH2Cl2, and the emulsion was broken by
filtration. The combined organic phases were dried (Na2SO4)
and evaporated to give a residue that was purified by flash
chromatography (silica gel; cyclohexane/EtOAc 9:1 as eluent)
and crystallization from diethyl ether/petroleum ether. Yellow
N-(8-Meth oxy-5-oxo-10,11-d ih yd r o-5H-d iben zo[a ,d ]cy-
cloh epten -10-ylm eth yl)acetam ide (9). Mp 97-98 °C (EtOAc/
diethyl ether), 30% yield. MS (EI): m/z 309 (M+), 237 (100).
1H NMR (CDCl3): δ 1.96 (s, 3H), 3.1 (dd, 1H, J ) 15.1, 6.0),
3.25 (m, 2H), 3.54 (m, 2H), 3.88 (s, 3H), 5.42 (brs, 1H), 6.78
(d, 1H, J ) 2.3), 6.91 (dd, 1H, J ) 8.9, 2.3), 7.21-7.51 (m,
3H), 8.0 (dd, 1H), 8.24 (d, 1H, J ) 8.9). IR (cm-1, Nujol): 3268,
1637, 1597. Anal. (C19H19NO3) C, H, N.
1
solid (10% yield), mp 182 °C. MS (EI): m/z 223 (M+, 100). H
NMR (DMSO-d6): δ 3.64 (s, 3H), 5.85 (d, 1H, J ) 11.8), 5.96
(d, 1H, J ) 11.8), 6.23 (m, 2H), 6.51-6.69 (m, 3H), 6.90 (m,
2H). IR (cm-1, Nujol): 3330, 1616, 1595.
(Diben zo[b,f]a zep in -5-yl)a ceton itr ile (24a ). Bromoac-
etonitrile (1.5 mL, 21.5 mmol) and Na2CO3 (0.587 g, 5.53 mmol)
N-(8-Meth oxy-10,11-d ih yd r o-5H-d iben zo[a ,d ]cycloh ep -
ten -10-ylm eth yl)a ceta m id e (4b). Mp 124-125 °C (diethyl
ether/petroleum ether), 75% yield. MS (EI): m/z 295 (M+), 236

4210 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17
Lucini et al.
were added to a solution of iminostilbene (0.147 g, 0.76 mmol)
in CH2Cl2 (6 mL), and the resulting mixture was heated at
reflux for a week. The reaction mixture was poured into ice/
water, extracted twice with CH2Cl2, and dried (Na2SO4). After
evaporation of the solvent we obtained a crude residue that
was purified by flash chromatography (silica gel, cyclohexane/
EtOAc, 85:15 as eluent): oil (52% yield). MS (EI): m/z 232
(M+), 192 (100). 1H NMR (CDCl3): δ 4.47 (s, 2H), 6.77 (s, 2H),
7.09-7.39 (m, 8H). IR (cm-1, neat): 2250, 1708, 1595.
(3-Meth oxyd iben zo[b,f]a zep in -5-yl)a ceton itr ile (24b).
This product was obtained following the above procedure by
using the dibenzoazepine 23b instead of iminostilbene: oil
(50% yield). MS (EI): m/z 262 (M+), 222 (100). 1H NMR
(CDCl3): δ 3.82 (s, 3H), 4.44 (s, 2H), 6.64 (d, 1H, J ) 11.5),
6.69 (dd, 1H, J ) 8.3, 2.4), 6.71 (d, 1H, J ) 11.5), 6.81 (d, 1H,
J ) 2.4), 7.05-7.32 (m, 6H). IR (cm-1, neat): 2260, 1605.
N-(2-(Diben zo[b,f]a zep in -5-yleth yl)a ceta m id e (25a ). A
solution of 24a (0.27 g, 1.16 mmol) in dry THF (6 mL) was
added dropwise to a stirred ice-cooled suspension of LiAlH4
(0.088 g, 2.3 mmol) in dry THF (11 mL) under nitrogen. Upon
completion of the addition, the mixture was refluxed for 30
min. Standard workup (see procedure C) and N-acetylation of
the crude amine (see procedure B) gave the desired title
compound. Mp 127-8 °C (EtOAc/petroleum ether), 40% yield.
MS (EI): m/z 278 (M+), 206 (100). 1H NMR (CDCl3): δ 1.89
(s, 3H), 3.38 (m, 2H), 3.89 (t, 2H), 6.05 (brs, 1H), 6.81 (s, 2H),
7.00-7.25 (m, 8H). IR (cm-1, Nujol): 3262, 1635, 1570. Anal.
(C18H18N2O) C, H, N.
P h a r m a cology. Tricyclic MLT derivatives were character-
ized by evaluating their binding affinity at h-MT1 and h-MT2
receptors and their in vitro functional activity.
Binding affinities of compounds at each receptor were
determined using 2-[125I]iodomelatonin as the labeled ligand
in competition experiments on cloned human MT1 and MT2
receptors expressed in NIH3T3 rat fibroblast cells. The
characterization of NIH3T3-MT1 and MT2 cells had already
been described in detail.53,54 Membranes were incubated for 2
h at 37 °C in binding buffer (Tris-HCl, 50 mM, pH 7.4). The
final membrane concentration was 5-10 µg of protein per tube.
Membrane protein level was determined in accordance with a
previously reported method.55 2-[125I]iodomelatonin (100 pM)
and different concentrations of the new compounds were
incubated with the receptor preparation for 90 min at 37 °C.
Nonspecific binding was assessed with 10 µM melatonin. IC50
values were determined by nonlinear fitting strategies with
the program PRISM (GraphPad SoftWare Inc., San Diego, CA).
pKi values were calculated from the IC50 values in accordance
with the Cheng-Prusoff equation:56 Ki ) IC50/[1 + (L/KD)]
where IC50 is the 50% inhibitory concentration, KD is the
dissociation constant of the radioligand, and L is the concen-
tration of 2-[125I]iodomelatonin. pKi values are the mean of at
least three independent determinations performed in dupli-
cate; SEM of pKi values was lower than 0.06.
To define the functional activity of the new compounds at
each melatonin receptor subtype, [35S]GTPγS binding assays
in NIH3T3 cells expressing human cloned MT1 or MT2 recep-
tors were performed. The amount of bound [35S]GTPγS is
proportional to the level of the analogue-induced G-protein
activation and is related to the intrinsic activity of the
compound under study. The detailed description and validation
of this method have been reported elsewhere.40,53,54 Membranes
(15-25 µg of protein, final incubation volume of 100 µL) were
incubated at 30 °C for 30 min, in the presence and in the
absence of melatonin analogues, in an assay buffer consisting
of [35S]GTPγS (0.3-0.5 nM), GDP (50 µM), NaCl (100 mM),
and MgCl2 (3 mM). Nonspecific binding was defined using
GTPγS (10 µM). In cell lines expressing human MT1 or MT2
receptors, MLT produced a concentration-dependent stimula-
tion of basal [35S]GTPγS binding with a maximal stimulation,
above basal levels, of 370% and 250% for MT1 and MT2
receptors, respectively. Basal stimulation is the amount of
[35S]GTPγS specifically bound in the absence of compounds
and was taken as 100%. The maximal G-protein activation was
measured in each experiment by using MLT (100 nM).
Compounds were added at three different concentrations (one
concentration equivalent to 100 nM MLT, a second one 10
times smaller, and a third one 10 times larger), and the percent
stimulation above basal was determined. The equivalent
concentration was estimated on the basis of the ratio of the
affinity of the test compound to that of MLT. It was assumed
that at the equivalent concentration the test compound oc-
cupies the same number of receptors as 100 nM MLT. All
measurements were performed in triplicate.
N-[2-(3-Met h oxyd ib en zo[b,f]a zep in -5-yl)et h yl]p r op i-
on a m id e (25b). 25b was obtained following the above pro-
cedure by using 24b instead of 24a . Purification by flash
chromatography (cyclohexane/EtOAc 7:3 as eluent): oil (47%
1
yield). MS (EI): m/z 322 (M+), 236 (100). H NMR (CDCl3): δ
1.08 (t, 3H, J ) 7.52), 2.13 (q, 2H, J ) 7.52), 3.4 (m, 2H), 3.80
(s, 3H), 3.87 (t, 2H), 6.10 (brs, 1H), 6.61 (m, 2H), 6.67 (d, 1H,
J ) 11.5), 6.75 (d, 1H, J ) 11.5), 7.00-7.11 (m, 4H), 7.23-
7.30 (m, 1H). Anal. (C20H22N2O2) C, H, N.
2-(2-Diben zo[b,f]a zep in -5-yl-2-oxoeth yl)isoin d ole-1,3-
d ion e (27). Potassium phthalimide (0.17 g, 0.91 mmol) was
added to a stirred solution of 2-bromo-1-dibenzo[b,f]azepin-5-
ylethanone (26)50 (0.28 g, 0.89 mmol) in dry DMF (5 mL), and
the mixture was heated at reflux for 30 min. The mixture was
cooled to room temperature, H2O was added, and the aqueous
solution was extracted twice with EtOAc. The combined
organic phases were dried (Na2SO4), and the solvent was
partially evaporated. Hexane was added, and the precipitated
solid was filtered, dried, and used without any further
purification (75% yield), mp 262-265 °C. MS (EI): m/z 380
(M+), 192 (100). H NMR (acetone-d6): δ 3.78 (d, 1H, J ) 16.6),
4.50 (d, 1H, J ) 16.6), 7.08 (d, 1H, J ) 11.5), 7.16 (d, 1H, J )
11.5), 7.36-7.88 (m, 12H).
N-(2-(Diben zo[b,f]azepin -5-yl-2-oxoeth yl)acetam ide (28).
Hydrazine hydrate (30 µL) was added to a suspension of 2-(2-
dibenzo[b,f]azepin-5-yl-2-oxoethyl)isoindole-1,3-dione (0.1 g,
0.26 mmol) in 2 mL of EtOH, and the resulting mixture was
refluxed for 30 min. The solvent was removed by distillation
under reduced pressure to give a residue that was dissolved
in EtOAc and washed twice with water. The organic phase
was dried (Na2SO4) and evaporated to give the intermediate
oily amine that was directly used in the next step without any
further purification (76% yield). MS (EI): m/z 250 (M+), 193
(100).
Molecu la r Mod elin g. Molecular modeling studies were
performed with Sybyl 6.8 57 running on a Silicon Graphics O2
workstation. Molecules were built using the standard sketch
option of Sybyl, and their geometries were optimized using the
Tripos force field58 with the Powell method59 to an energy
gradient of 0.01 kcal mol-1 Å-1, ignoring the electrostatic
contribution. Only minimum energy conformations were con-
sidered for compound superposition.
The final conformations of the tricyclic scaffolds accurately
reproduced the geometry found in the Cambridge Structural
Database;60 for example, the root mean square distance value,
derived from the superposition of heavy atoms, for 10,11-
dihydro-5H-dibenzo[a,d]cycloheptene (reference code HBZCHP)
was 0.06 Å and for 5H-dibenzo[b,f]azepine (reference code
BZAZPO) it was 0.11 Å.
TEA (0.134 mL, 0.95 mmol) and acetic anhydride (0.09 mL,
0.95 mmol) were added to a solution of the above amine (0.2
g, 0.8 mmol) in THF (3 mL), and the mixture was stirred at
room temperature for 6 h. After evaporation of the solvent
under reduced pressure, we obtained a crude residue that was
purified by flash chromatography (silica gel; EtOAc as eluent)
and crystallization from CHCl3/hexane (47% yield), mp 207-
1
208 °C. MS (EI): m/z 193 (100). H NMR (CDCl3): δ 1.97 (s,
3H), 3.38 (dd, 1H, J ) 18.3, 4.2), 4.13 (dd, 1H, J ) 18.3, 4.2),
6.36 (brs, 1H), 6.92 (dd, 1H, J ) 11.5), 6.99 (dd, 1H, J ) 11.5),
7.32-7.53 (m, 8H). IR (cm-1, Nujol): 3308, 1657, 1545. Anal.
(C18H16N2O2) C, H, N.
The superposition of molecules was performed by means of
a rigid-fit procedure, considering the four atoms of the amide
function, the centroid of the benzene portion of the indole or
of the tricyclic ring, and the centroid of the substituent

Tricyclic Alkylamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17 4211
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positioned out of the plane of the molecule (for tricyclic
compounds the centroid of the second benzene ring), to those
of cis-4P-PDOT.
Selectivity and intrinsic activity predictions were performed
by means of the QSAR/CoMFA module of Sybyl, applying PLS
models previously described (models 5, 9, and 10 in Table 4 of
ref 42) and following the alignment rules employed to derive
the models.
Ack n ow led gm en t. This work was supported by the
Italian M.I.U.R. (Ministero dell’Istruzione, dell’Universita`
e della Ricerca). The C.I.M. (Centro Interfacolta` Misure)
and C.C.E. (Centro di Calcolo Elettronico) of the Uni-
versity of Parma are gratefully acknowledged for pro-
viding the Sybyl software license.
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