2-N-acylaminoalkylindoles: Design and quantitative structure - Activity relationship studies leading to MT2-selective melatonin antagonists

Article abstract of DOI:10.1021/jm001125h

Several indole analogues of melatonin (MLT) were obtained by moving the MLT side chain from C₃ to C₂ of the indole ring. Binding and in vitro functional assays were performed on cloned human MT₁ and MT₂ receptors, stably transfected in NIH3T3 cells. Quantitative structure-activity relationship studies showed that 4-methoxy-2-(N-acylaminomethyl)indoles, with a benzyl group in position 1, were selective MT₂ antagonists and, in particular, N-[(1-p-chlorobenzyl-4-methoxy-1H-indol-2-yl)methyl]propanamide (12) behaved as a pure antagonist at MT₁ and MT₂ receptors, with a 148-fold selectivity for MT₂. We present a topographical model that suggests a lipophilic group, located out of the plane of the indole ring of MLT, as the key feature of the MT₂ selective antagonists.

Full text of DOI:10.1021/jm001125h

2900
J . Med. Chem. 2001, 44, 2900-2912
2-N-Acyla m in oa lk ylin d oles: Design a n d Qu a n tita tive Str u ctu r e-Activity
Rela tion sh ip Stu d ies Lea d in g to MT2-Selective Mela ton in An ta gon ists
Gilberto Spadoni,* Cesarino Balsamini, Giuseppe Diamantini, Andrea Tontini, and Giorgio Tarzia
Istituto di Chimica Farmaceutica e Tossicologica, Universita` degli Studi di Urbino, Piazza Rinascimento 6,
I-61029 Urbino, Italy
Marco Mor, Silvia Rivara, and Pier Vincenzo Plazzi
Dipartimento Farmaceutico, Universita` degli Studi di Parma, Parco Area delle Scienze 27A I-43100 Parma, Italy
Romolo Nonno, Valeria Lucini, 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 November 29, 2000
Several indole analogues of melatonin (MLT) were obtained by moving the MLT side chain
from C3 to C2 of the indole ring. Binding and in vitro functional assays were performed on
cloned human MT1 and MT2 receptors, stably transfected in NIH3T3 cells. Quantitative
structure-activity relationship studies showed that 4-methoxy-2-(N-acylaminomethyl)indoles,
with a benzyl group in position 1, were selective MT2 antagonists and, in particular, N-[(1-p-
chlorobenzyl-4-methoxy-1H-indol-2-yl)methyl]propanamide (12) behaved as a pure antagonist
at MT1 and MT2 receptors, with a 148-fold selectivity for MT2. We present a topographical
model that suggests a lipophilic group, located out of the plane of the indole ring of MLT, as
the key feature of the MT2 selective antagonists.
In tr od u ction
putative melatonin binding site called MT3 was hypoth-
esized from the observation of a binding site in hamster
brain membranes15 and in some peripheral hamster
tissues including intestine, liver, kidney, lung muscle,
and heart; this binding site has recently been character-
ized as the hamster homologue of the human enzyme
quinone reductase 2.16
Despite these advances and the recent studies with
chimeric receptors17 and with MT1-receptor-deficient
mice,18 there are still important issues, such as the exact
interaction of agonists and antagonists with the amino
acids of the binding site and the physiological role of
the two receptor subtypes, that remain to be settled.
These questions could be better answered if specific,
potent, and subtype-selective agonists and antagonists
were available.
The search for novel high-affinity MLT ligands led to
the synthesis of several indole19 and non-indole MLT
analogues20 and the establishment of a structure-
activity relationship (SAR) for MLT binding affinity; the
field was recently reviewed.21
On the basis of the MLT ligand affinity data on native
receptors and on the amino acid sequence of the cloned
MLT receptors, some three-dimensional receptor-ligand
interaction models were proposed.22 However, the mo-
lecular basis for the subtype selectivity is still unknown.
Comparison of the potencies and binding affinities of
MLT agonists, both to native MLT receptors23 and to
MT1 and MT2 receptors expressed in COS-7, COS-1,
CHO, and NIH3T3 cellular lines, showed that most of
these ligands exhibit little or no subtype selectivity.15,24
Lack of subtype-selective agonists for MLT receptors has
made it impossible to critically evaluate the contribution
Melatonin (N-acetyl-5-methoxytryptamine, MLT) is a
neurohormone with a well-defined circadian nocturnal
secretion by the pineal gland, being also producted by
the photoreceptor cells of the retina.1 Considerable
attention is being devoted to the pharmacology of MLT
in view of its potential applications in several thera-
peutic areas, but a clear depiction of the therapeutic
applications still has to be established.2 The circadian
rhythms of many species, including human, is synchro-
nized by MLT. This raises many possibilities for the use
of MLT as a supplement to combat jet lag3 or as a
treatment for certain types of circadian-based sleeping
disorders.4 The pharmacological effects of MLT in
humans have also been investigated in psychiatric
disorders5 and in the cardiovascular system.6 The mo-
lecular mechanisms underlying the reported antioxi-
dant,7 neuroprotective,8 anticarcinogenic,9 and immu-
nostimulatory10 properties of melatonin are not yet fully
understood.
Many of the MLT effects are mediated through high-
affinity G-protein-coupled receptors expressed primarily
in the brain, retina, pituitary, and blood vessels.11
Recent cloning of several G-protein-coupled melatonin
receptor genes revealed at least three melatonin recep-
tor subtypes, two of which (MT1 and MT2) have been
found in mammals.12 These receptor subtypes are
coupled to Gi/o proteins associated with inhibition of
adenylyl cyclase;13 MT1 receptors were also shown to
activate other signal transduction pathways.14 Another
* To whom correspondence should be addressed. Phone: +39-0722-
2545. Fax: +39-0722-2737. E-mail: gilberto@uniurb.it.
10.1021/jm001125h CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/02/2001

2-N-Acylaminoalkylindoles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2901
F igu r e 1. Chemical structures of melatonin antagonists or partial agonists.
of MT1 and MT2 to the various MLT effects in the body.
Moreover, no selective MLT antagonists were reported
for the h-MT1 receptor. Recently, partial agonists/
antagonists with some degree of selectivity for MT2
subtype appeared in the literature (Figure 1). Some of
these compounds were successfully used to dissect MT1
and MT2-mediated melatonin effects in tissues coex-
pressing both subtypes.25
The first approach to design MLT antagonists in-
volved variation of the 5-methoxy substituent. Some of
these compounds, including N-acetyltryptamine,26 4-
phenyl-2-acylaminotetralins,13 luzindole,27 and its con-
gener DH97,24g are structurally characterized by the
absence of the methoxy group. Substitution of the
5-OMe group with methyl or halogens did not yield
antagonist activity.24i The 5-hydroxyethoxy derivative
(5-HEAT)28 was a full agonist at the h-MT1 and an
antagonist/weak partial agonist at the h-MT2 receptor
subtype. Other approaches lead to modification of the
amide function such as in GR 135533 and GR 128107,24b
where a tertiary amide replaces the secondary amide
of the 3-ethylamido side chain. Other modifications of
the amide side chain seem to depress intrinsic activity,
such as the N-cyclobutylcarbonyl group of N-cyclobu-
tylcarbonyl-2-phenyltryptamine (CBCPT)29 or of the
naphthalene derivative S 20928.30
reclassified as partial agonists when tested in different
tissues or recombinant systems31 or were partially
characterized, with the notable exception of K 185.24h
Recently we discovered a new series of h-MT1 partial
agonists and antagonists by transposing the MLT side
chain from C3 to C2 of the indole nucleus (i.e., 2-acy-
laminomethyl-4-methoxyindole, 2-AMI), and we re-
ported a preliminary Free-Wilson analysis of group
contributions to MT1 affinity and intrinsic activity.32 The
study showed that, whereas the N1-benzyl group had a
negative effect on MT1 affinity, the phenyl group in the
same position had a positive, statistically significant
effect. This observation and the strong reduction of
intrinsic activity caused by the shortening of the 2-acy-
laminoalkyl chain led us to predict for 1-phenyl-2-
propionylaminomethyl-4-methoxyindole (10b) a pKi value
of 6.8 and an intrinsic activity of 0.3 in the test based
on GTPγS binding. These predictions were considered
encouraging because in the old series a pKi around 7
could only be obtained at the expense of an increase of
intrinsic activity to 0.5 or higher. This prompted us to
pursue further this strategy to obtain relationships
explaining selective receptor binding and activation,
looking for new melatonin antagonists. Thus, to inves-
tigate in detail the influence of structural variation
(length of the side chain, position of the methoxy group,
N1 and C3 indole substitution) on affinity and intrinsic
activity, the synthesis of additional compounds (5b-c,
7b, 10a ,b, 11, 12, and 15-18) has been performed.
Furthermore, we evaluated the affinity and efficacy of
these and of the previously synthesized 2-N-acylami-
noalkyl derivatives (5d -n , 7c, 10c-f)32 on human MT2
Another approach called for a cycloheptane ring fused
to the C2-C3 [N-ethylcarbonyl-10-(aminomethyl)-2-
methoxy-5-methylhexahydrocyclohept[b]indole; 10-AM-
HCI]19e or to the N1-C2 (K 185)24h indole positions, and
the compounds were claimed to possess an antagonist
profile. Some of the putative antagonists, however, were

2902 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Spadoni et al.
Sch em e 1a
a
Reagents: (a) LiAlH4, THF, room temperature, 45 min; (b) MnO2, CH2Cl2, room temperature, 7 h; (c) CH3NO2, NH4OAc, reflux, 1.5
h; (d) LiAlH4, THF, room temperature, 5 h; (e) cyclopropanecarbonyl or cyclobutanecarbonyl chloride, TEA, THF, room temperature, 2 h;
(f) (Ph)3P+CH2CNCl-, DBU, toluene, reflux, 30 min; (g) Raney nickel, H2, 4 atm, (EtCO)2O, THF, 50 °C, 6 h; (h) 3 N KOH, MeOH, THF,
room temperature, 16 h; (i) SOCl2, THF, 50 °C, 4 h; (j) NH3, THF, room temperature, 16 h; (k) LiAlH4, THF, reflux, 3 h; (l) (EtCO)2O,
TEA, THF, room temperature, 8 h.
Sch em e 2a
a
Reagents: (a) NBS, AcOH, room temperature, 2 h; (b) NaH, DMF, p-chlorobenzyl or benzyl chloride, room temperature, 16 h.
receptors, stably transfected in NIH3T3 cells, to delin-
eate the structural features for h-MT1/h-MT2 subtype
selectivity.
chloride in the presence of triethylamine (TEA) to afford
the corresponding target amides 5b,c. The 2-acylami-
nopropyl derivative 7b was prepared by hydrogenation
over Raney nickel of the nitrile 6b and concomitant
N-acylation with propionic anhydride. The propeneni-
trile 6b was obtained by subjecting the aldehyde 3b32
to Wittig reaction conditions33 using cyanomethyltriph-
enylphosphonium chloride34 in the presence of 1,7-
diazabicyclo[4.5.0]undec-6-ene (DBU). The 2-acylami-
nomethyl derivatives 10a ,b were prepared by LiAlH4
reduction of the corresponding amides 9a ,b followed by
N-acylation with propionic anhydride. These amides
were obtained by alkaline ester hydrolysis of indoles
1a ,b, followed by acylation of the acids 8a ,b with thionyl
chloride and treatment of the crude acid chloride with
a saturated solution of ammonia in dichloromethane.
N-alkylation of the 2-(N-acylaminoalkyl)indoles 10a ,
10c, 5g, and 10d was carried out with sodium hydride
and benzyl bromide or p-chlorobenzyl chloride to give
Ch em istr y
Melatonergic derivatives 5d -n , 7c, and 10c-f were
prepared as previously described.32 The synthesis of the
novel melatonin analogues 5b,c, 7b, 10a ,b, 11, 12, and
15-18 was achieved following the routes described in
Schemes 1 and 2 and according to our previous methods
for the synthesis of similar compounds.32 Briefly, the
methyl ester 1b32 was reduced with lithium aluminum
hydride (LiAlH4), followed by oxidation with MnO2 to
give the aldehyde 3b.32 Condensation of the aldehyde
3b with nitromethane in the presence of NH4OAc gave
the nitroethenyl derivative 4b,32 which was then re-
duced with LiAlH4 to give (4-methoxy-1-phenyl-1H-
indol-2-yl)ethanamine. This crude amine was subse-
quently acylated with cyclopropanoyl or cyclobutanoyl

2-N-Acylaminoalkylindoles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2903
Ta ble 1. Binding Affinitya and Intrinsic Activity of 2-(Acylaminoalkyl)indole Derivatives 5b-n , 7b,c, 10b-f, 11, 12, and 15-18 for
the Human MT1 and MT2 Melatonin Receptors Stably Expressed in NIH3T3 Cells
human MT1
pKi ( SEM
human MT2
pKi ( SEM
b
b
c
compd
R1
R2
R3
R4
n
IAr
1
IAr
1
pKi1 - pKi2
MLT
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
9.54 ( 0.02
6.48 ( 0.03
6.16 ( 0.12
6.87 ( 0.07
5.61 ( 0.03
5.42 ( 0.05
5.07 ( 0.05
4.79 ( 0.11
7.20 ( 0.03
7.06 ( 0.05
6.20 ( 0.02
5.74 ( 0.01
5.40 ( 0.07
6.08 ( 0.05
6.51 ( 0.02
5.53 ( 0.03
6.65 ( 0.01
6.39 ( 0.06
5.91 ( 0.09
6.28 ( 0.02
6.20 ( 0.04
5.89 ( 0.04
5.89 ( 0.09
6.56 ( 0.01
7.30 ( 0.05
6.62 ( 0.05
5.50 ( 0.06
9.55 ( 0.02
7.34 ( 0.12
6.94 ( 0.11
6.65 ( 0.02
5.79 ( 0.04
5.37 ( 0.01
5.84 ( 0.02
5.03 ( 0.04
8.78 ( 0.16
8.11 ( 0.08
6.03 ( 0.09
5.91 ( 0.05
6.45 ( 0.04
7.75 ( 0.08
7.89 ( 0.12
3.99 ( 0.04
7.99 ( 0.05
6.54 ( 0.02
6.09 ( 0.01
6.19 ( 0.05
8.12 ( 0.08
6.56 ( 0.01
8.06 ( 0.11
6.61 ( 0.02
6.84 ( 0.15
6.70 ( 0.01
6.85 ( 0.16
-0.01
-0.86
-0.78
0.22
-0.18
0.05
c-Pr
c-Bu
CH2CH3
CH3
CH3
CH3
CH3
CH2CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
Br
H
H
H
H
H
H
H
H
H
H
Br
Br
Br
Br
4-OCH3
4-OCH3
4-OCH3
H
Ph
Ph
H
H
H
H
H
Ph
Ph
H
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
1
1
1
1
1
1
1
1
2
2
1
0.19
0.08
0.49
0.09
-0.06
-0.04
-0.01
0.47
0.61
0.60
0.31
-0.07
0.47
0.10
0.44
0.22
0.05
0.08
-0.01
0.03
0.00
0.04
0.20
0.61
0.16
0.04
0.26
0.31
0.71
0.48
0.02
0.02
0.18
0.58
0.59
0.62
0.50
-0.13
0.56
0.40
0.16
0.49
0.30
0.42
0.26
0.18
0.16
0.01
0.01
0.75
0.01
0.04
7-OCH3
6-OCH3
5-OCH3
4-OCH3
4-OCH3
4-OCH3
6-OCH3
6-OCH3
4-OCH3
4-OCH3
5-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
6-OCH3
4-OCH3
6-OCH3
4-OCH3
6-OCH3
4-OCH3
-0.77
-0.24
-1.58
-1.05
0.17
-0.17
-1.05
-1.67
-1.38
1.54
-1.34
-0.15
-0.18
0.09
-1.92
-0.67
-2.17
-0.05
0.46
CH3
CH3
CH3
H
CH2CH3
CH2CH3
CH3
CH2CH3
CH2CH3
CH3
Bnd
Ph
H
7b
7c
10b
10c
10d
10e
10f
11
12
15
16
17
Ph
H
H
c-Bu
H
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH3
Bn
Bn
4-Cl-Bn
H
H
Bn
Bn
-0.08
-1.35
18
CH3
a
pKi values were calculated from IC50 values obtained from competition curves by the method of Cheng and Prusoff40 and are the
b
means of at least three independent determinations performed in duplicate. The relative intrinsic activity values (IAr) were obtained by
dividing the maximal analogue-induced G-protein activation by that of MLT. c The difference (pKi1 - pKi2) represents selectivity toward
d
the MT1 (positive values) or the MT2 (negative values) subtype. Bn ) benzyl.
the desired compounds 11-14. The 3-bromoderivatives
15-18 were synthesized by direct bromination of the
3-unsubstituted compounds 5d , 10a , 13, and 14.
of basal [35S]GTPγS binding with a maximal stimula-
tion, above basal levels, of 370% and 250% in MT1 and
MT2, respectively. In the case of MLT analogues the
amount of bound [35S]GTPγS is proportional to the level
of the analogue-induced G-protein activation, and it is
related to the intrinsic activity of the compounds. Full
agonists increased the basal [35S]GTPγS binding in a
concentration-dependent manner, like the natural ligand
MLT, whereas partial agonists increased it to an extent
much less than that of MLT, and antagonists were
without effect. 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, and the results are summarized in Table
1.
P h a r m a cology
The biological evaluation of the compounds was
performed by measuring the h-MT1and h-MT2 binding
affinity and in vitro functional activity.
The binding affinity of compounds 5b-n , 7b,c 10b-
f, 11, 12, and 15-18 was determined using 2-[125I]-
iodomelatonin (100 pM) as a labeled ligand in compe-
tition binding analyses on cloned human MT1 and MT2
receptor subtypes stably expressed in NIH3T3 rat
fibroblast cells, and the results are summarized in Table
1. The characterization of NIH3T3-MT1 and MT2 cells
was described in detail elsewhere.24j,31c
The relative intrinsic activity (IAr) of the compounds
was measured by the direct activation of the G protein
after the binding of the compounds to MT1 and MT2
receptor subtypes. IAr was estimated by means of the
[35S]-guanosine-5′-O-(3-thiotriphosphate) ([35S]GTPγS)
method in NIH3T3 cells expressing human cloned MT1
or MT2 melatonin receptors. The system provides a
functional measure for the interaction between mela-
tonin receptors and pertussis-toxin-sensitive G proteins.
The detailed description and validation of this method
was reported elsewhere.24j,31c,35
The interaction between ligands and melatonin was
investigated by competition experiments in which in-
creasing concentrations of an antagonist inhibit the
maximum melatonin-induced stimulation of [35S]GTPγS
binding.
Resu lts
The affinity and intrinsic activity data for human MT1
and MT2 melatonin receptor subtypes of the new
compounds 5b,c, 7b, 10b, 11, 12, and 15-18 are
reported in Table 1. Also included in Table 1 are the
previously reported h-MT1 pharmacological data32 of
5d -n , 7c, and 10c-f for comparison with h-MT2 results
of the same compounds obtained in the present study.
In cell lines expressing human MT1 or MT2 receptors,
MLT produced a concentration-dependent stimulation

2904 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Spadoni et al.
The data now available permit the study of the
contribution to pKi and IAr of the following fragments
(italic entities are those of the reference compound): (a)
the methoxy in two different positions of the indole ring,
R3 ) 4-OCH3 or 6-OCH3; (b) the acylamino group from
acetic, propionic, or cycloalkanecarboxylic acids (R1 )
CH3, CH2CH3, or c-Bu); (c) lipophilic substituents on the
indole nitrogen (R4 ) H, phenyl, or benzyl); (d) meth-
ylene or ethylene alkyl chains (n ) 1, 2); (e) bromine in
C3 (R2 ) H or Br). Other minor structural modifications
have also been occasionally tested, such as the elonga-
tion of the alkyl chain (compounds 7b and 7c), a methyl
group on the indole nitrogen (5l), or a cyclopropanecar-
boxylic derivative (5b), but their effect does not deserve
further exploration.
We have selected a set of 18 compounds among those
of Table 1 (subset A, reference standard 5k ), character-
ized by features “a-e” appearing more than once. The
data matrix for the Free-Wilson analysis is reported
in Table 2, where each column corresponds to an X
variable of the correlation equations. The multiple
regression analysis (MRA) equations with less than the
seven contemporary X variables give a poor fit in the
case of the MT1 receptor. This lack of correlation
between the Free-Wilson variables, and the MT1-
affinity and MT1-intrinsic activity data, may be due to
nonadditivity of the group contributions or to a high
ratio of noise to variation in the dependent variables.
The inapplicability of the Free-Wilson analysis to this
set might also be explained assuming different modes
of interaction at the MT1 receptor for the different
compounds.
F igu r e 2. Plot of pKi data on h-MT1 and h-MT2 receptors.
Points on the straight line correspond to compounds having
the same affinity for both receptors.
The affinities of all compounds are lower than those
of MLT for both receptor subtypes. Nonetheless, it is
apparent from past32 and present studies that the
shifting of the melatonin side chain from the C3 to the
C2 indole position provides exclusively MT1 and MT2
receptor antagonists or partial agonists.
A plot of the MT2 vs MT1 affinity values reveals
(Figure 2) that most of the compounds have some
selectivity for the MT2 subtype. Compound 12, in
particular, has the highest selectivity for the h-MT2
receptor but not the highest affinity in the series.
With respect to relative intrinsic activity (IAr; see
Experimental Section), the effort to get antagonists with
relatively high affinity gave different results for the two
subtypes, as shown in Figure 3. At the bottom of the
plot representing IAr vs pKi for the MT1 subtype, there
are no points at pKi values greater 6.5, and compounds
with the highest affinity behave as partial agonists. In
constrast, the analogous plot for the MT2 is more
scattered and some compounds (e.g., 10f and 12) have
very low IAr but maintain good pKi values. This could
lead to the hypothesis that these compounds engage in
additional interactions, leading to antagonist activity
and high affinity at the MT2 subtype but not at the MT1
subtype. In the case of 5m a small reduction of the
baseline level of the [35S]GTPγS binding is observed,
suggesting a propensity to act as an inverse agonist.
The analysis of the pharmacological data, reported in
Table 1, allows some preliminary considerations about
the SAR for this series. Comparison of the simple
acetylaminoethyl derivatives substituted on the benzene
ring of indole (R3), 5f-h and 5k , indicates that relative
to 5e the position of the methoxy group does not
dramatically influence receptor affinity. Within the
small variation observed, a 5- or a 7-methoxy group
lowers receptor affinity on both MT1 and MT2, while a
6-methoxy one leads to lower affinity on MT1 and no
change on MT2. Only a 4-methoxy group results in a
small increase in affinity for both subtypes.
The affinity data for the MT2 receptor correlate with
the structure of the compounds better than those for
the MT1 receptor, and it is possible to obtain a statisti-
cally significant equation:
pKi ) 0.73((0.25) (R4 ) benzyl) + 1.56((0.26)
(R4 ) phenyl) + 0.66((0.21) (R1 ) CH2CH3) + 6.07
((0.17)
n ) 18, R2 ) 0.78, s ) 0.44, F ) 16.5,
Q2 ) 0.60, SDEP ) 0.52
According to this model, the contribution of R3
)
6-OCH3 is not significant despite the different MT2
affinity of 10f and 11. This is likely due to the nonad-
ditive properties of this contribution, as is argued from
the similar affinities of 5k and 5g.
Assuming that 4- and 6-methoxy derivatives interact
differently at the MT2 receptor when R4 ) benzyl (see
10f and 11), we have considered the series of 4-methoxy
compounds as a separate subset (subset B, 13 com-
pounds, Table 2), and new quantitative structure-
activity relationships (QSARs) were built. It was man-
datory, in this case, to consider MRA equations with a
small number of variables to avoid excessive risk of
chance correlation.
The pKi and IAr values on MT1 of compound 10b are
6.65 and 0.22, respectively, confirming the prediction
of our previous Free-Wilson analysis.32 It is interesting
to observe that its pKi for the MT2 receptor is 7.99,
indicating that 10b can discriminate between the two-
receptor subtypes.
When the Free-Wilson analysis was limited to the
4-methoxy derivatives, some interesting QSARs are
observed (see Table 3). For MT1 affinity, significant
contributions are those of R1 ) CH2CH3 (positive) and
of R4 ) benzyl and n ) 1 (negative). The contribution
of the phenyl group is positive but statistically not

2-N-Acylaminoalkylindoles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2905
F igu r e 3. Affinity (pKi) vs intrinsic activity (IAr) on h-MT1 (left) and h-MT2 (right) receptors for the tested compounds.
Ta ble 2. Data Matrix for the Free-Wilson Analysis
R1 R2 R3 R4
compd subset CH2CH3 c-Bu Br 6-OCH3 phenyl benzyl
A good MRA equation could be obtained for the pKi
data on MT2 (R2 ) 0.87, Q2 ) 0.71), including the three
variables corresponding to R1 ) CH2CH3, R4 ) benzyl,
and R4 ) phenyl, all showing a positive contribution to
receptor affinity (Table 3). The IAr on the same receptor
subtype generally decreased when n ) 1, R4 ) benzyl,
and R1 ) cyclobutyl, as indicated from the coefficients
reported in Table 3.
Compound 10f, according to these equations, has the
best balance among receptor affinity (pKi ) 8.12), MT2
selectivity (∼100-fold), and functional antagonism, al-
though the IAr ) 0.18 on MT2 is that of a partial agonist.
The introduction of a chlorine atom into the para
position of the benzyl group gives compound 12, a pure
antagonist with increased MT2 selectivity, thus confirm-
ing the previous findings.
n
1
5c
A, B
A, B
A
0
1
0
1
0
0
0
1
1
1
0
0
1
1
1
1
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
1
1
1
0
0
1
0
0
0
1
0
0
0
0
0
0
1
1
0
1
0
1
0
0
1
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
1
0
0
1
1
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
0
0
1
5d
5g
5i
5j
5k
5m
5n
10b
10c
10d
10e
10f
11
A, B
A, B
A, B
A
A, B
A, B
A, B
A, B
A, B
A, B
A
15
A
16
17
A, B
A
Discu ssion
18
A, B
2-Acylaminoalkylindoles proved to be a suitable scaf-
fold for the design of potent and selective MT2 antago-
nists, whereas the same result could not be obtained
for MT1 antagonism because of the low affinity (pKi <
7.5) or high relative intrinsic activity (approximately 0.5
for the most potent compounds). Compound 12 showed
some interesting characteristics, such as 148-fold se-
lectivity for h-MT2 receptor and lack of significant effect
on basal [35S]GTPγS binding to NIH3T3 MT1 or MT2
receptors at any concentration tested (Figure 4). 4-Phen-
yl-2-propionylaminotetralin (4-P-PDOT)24b showed a
very high selectivity but behaved as a partial agonist
at the h-MT2 receptor;31b,c other compounds, such as the
pentanoyl derivative of luzindole (DH97)24g and the
tetracyclic compound K185,24h,k (Figure 1) showed the
same level of selectivity of 12.
The role of the methoxy group remains undefined
within the compounds of this work. The 4- and 6-meth-
oxy derivatives, however, appear to be the most promis-
ing series because of the MT1 affinity of compound 5k
and of the MT2 selectivity of 5g, with respect to 5e. The
set composed by the two series (subset A) leads to poor
QSAR equations on the MT1 receptor, even if it is
possible to observe that the 4-methoxy derivatives are
generally endowed with higher affinity. Restriction of
the QSAR study to this last series (subset B) permits
us to calculate the group contributions to receptor
affinity and intrinsic activity. Thus, the change of the
Ta ble 3. Results of the Multiple Regression Analysis for the
13 Compounds in Subset B
MT1
MT2
pKi
IAr
pKi
IAr
n ) 1
R4 ) benzyl -0.66 ( 0.22 a
-0.38 ( 0.18 -0.40 ( 0.06 a
-0.26 ( 0.06
1.05 ( 0.27 -0.22 ( 0.07
1.57 ( 0.24 a
a
R4 ) phenyl
R2 ) Br
a
a
a
a
a
a
R1 ) c-Bu
R1 ) CH2CH3 0.54 ( 0.18
-0.28 ( 0.08 a
-0.24 ( 0.08
a
0.77 ( 0.21 a
intercept
6.48 ( 0.16 0.53 ( 0.04 6.00 ( 0.19 0.65 ( 0.04
R2
s
0.74
0.32
8.3
0.46
0.38
0.86
0.11
29.9
0.52
0.17
0.87
0.37
20.3
0.71
0.47
0.82
0.10
14.1
0.47
0.15
F
Q2
SDEP
a
Not significant.
significant (5i vs 5d ). The resulting equation (Table 3)
gives a sufficient explanation of the pKi values (R2
)
0.74, s ) 0.32), considering the uncertainty of the data
and the simplified description of the structures inherent
to the method. For what concerns MT1 IAr, chain
shortening (n ) 1) leads to a significant lowering of
intrinsic activity, and the same is observed for the
cyclobutyl group in the acylamino fragment. Other
groups did not provide statistically significant contribu-
tions to IAr on MT1 receptor subtype.

2906 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Spadoni et al.
some geometric features of the selected conformations
and shows how, in the three selective MT2 antagonists,
the external phenyl ring is located out of the plane of
the indole (or tetralin) nucleus, with an orthogonal
orientation. It should be noted that the chirality of the
models is not yet defined. Table 4 also indicates that
both the trans and cis isomers of 4-P-PDOT fit these
models (see Experimental Section).
Some results can be further discussed. While the
different effect of the benzyl group on the two receptor
subtypes could be attributed to the occupancy of a
lipophilic pocket in the MT2 receptor, the Free-Wilson
analysis also pointed out a positive contribution of the
N1-phenyl group, statistically significant for the MT2
receptor and not significant, although positive, for the
MT1 receptor. This contribution is expressed, for ex-
ample, by the high MT2 and moderate MT1 affinity (pKi
) 8.78 and 7.20, respectively) shown by compound 5i,
which maintains a medium intrinsic activity. Compari-
son of the hypothesized hydrophobic cavity for MT2
antagonists with the lipophilic pocket allocating the
phenyl group of 2-phenyl-MLT previously evidenced by
a 3D-QSAR investigation36 helps us interpret these
results. Figure 6 depicts the volumes occupied by the
phenyl groups of the three MT2 antagonists (cyan) and
of 2-phenyl-MLT (yellow). Compound 5i (magenta,
right-hand side of the figure), once superposed on MLT
structure, places the N1-phenyl group at the boundary
between the cyan and the yellow volumes, and this could
explain its intermediate behavior, considering that
2-phenyl-MLT is a potent, nonselective agonist.
In conclusion, this work has identified new, readily
available melatonin antagonists with subtype specificity
for the h-MT2 receptor. They are obtained by moving
the MLT side chain from C3 to C2 indole position. The
2-(N-acylaminomethyl)indole derivative 12 appears as
a promising new tool for investigating the role for the
MT2 receptor. We postulate that the N1-benzyl group is
the key feature to confer MT2-selective antagonism on
the basis of SAR and molecular modeling studies in this
limited series of compounds.
F igu r e 4. Effects of MLT and of selected analogues on the
stimulation of the [35S]GTPγS binding to NIH3T3-h-MT1 and
NIH3T3-h-MT2 membranes.
ethylene into the methylene chain leads to a small but
significant decrease of MT1 affinity, whereas this is not
observed for MT2 affinity. On the other hand, a benzyl
group on the indole nitrogen (R4) has opposite effects
on the two receptor subtypes, being favorable for MT2
affinity and unfavorable for MT1 affinity; in the same
position, a phenyl group leads to an evident improve-
ment of MT2 affinity but not MT1 affinity. The positive
effect of the propionamido group (R1 ) CH2CH3) is
shared by the two subtypes. Relative to the intrinsic
activity, the only difference observed between the two
subtypes relates to the contribution of the benzyl group,
which is significant for the MT2 only. It can be argued,
from the coefficients reported in Table 3, that, in the
case of the 4-methoxy-2-acylaminoalkylindole deriva-
tives, a benzyl group discriminates the two receptor
subtypes, favoring the interaction with the MT2 one,
although decreasing the intrinsic activity.
To provide an interpretation of these findings, we
hypothesize that the benzyl group interacts with a
lipophilic pocket of the MT2 receptor whose occupation
leads to antagonist activity. To test this hypothesis, the
minimum-energy conformations of selective MT2 an-
tagonists and partial agonists were mutually compared,
looking for the common arrangements of a lipophilic
group (benzyl or phenyl), and added to the pharma-
cophore elements previously defined for nonselective
melatonin receptor ligands.22a,36 Initially, 4-P-PDOT and
luzindole conformers were mutually superposed, search-
ing for the best fit of the known pharmacophore ele-
ments (the condensed benzene ring and the amido
group) and of the phenyl and benzyl groups in position
4 of the tetralin nucleus and in position 2 of the indole
ring, respectively. The best superposition presents the
phenyl group above or under the plane of the indole and
above or under the plane of the tetralin nuclei, thus
being on the same or on the opposite side relative to
the amide chain. For this reason, we have named “syn”
and “anti” the corresponding models. Subsequently, the
selected conformations of luzindole have been super-
posed to the minimum-energy conformations of 10f, thus
permitting the selection of the best ones for the last
molecule (see Experimental Section).
Exp er im en ta l Section
(a ) Ch em ica l Ma ter ia ls a n d Meth od s. 2-[125I]iodomela-
tonin (specific activity ) 2000 Ci mmol-1) and [35S]-GTPγS
(specific activity ) 1070 mmol-1) were purchased from Am-
ersham (Buckinghamshire, U.K.). 2-Iodomelatonin was ob-
tained from RBI (Natick, MA). Compounds 1a ,37 1b-4b,32 and
8a 37 were synthesized as described elsewhere. 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 (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. 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
for C, H, and N of the tested compounds were performed on a
Carlo Erba analyzer.
2-(4-Meth oxy-1-ph en yl-1H-in dol-2-yl)eth ylam in e.4-Meth-
oxy-2-(2-nitroethenyl)-1-phenyl-1H-indole (4b)32 (0.294 g, 1
mmol) was added portionwise 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. The
reaction mixture was cooled to 0 °C, then water was added
dropwise to destroy the excess hydride. The resulting mixture
was filtered on Celite, and the filtrate was concentrated in
The chosen conformations of the three compounds are
represented in Figure 5, which depicts the out-of-plane
arrangement of the lipophilic groups. Table 4 quantifies

2-N-Acylaminoalkylindoles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2907
F igu r e 5. Superposition of 4-P-PDOT (orange carbons), luzindole (yellow carbons), and 10f (white carbons), according to the
models named syn (on the left) and anti (on the right), as described in the text.
Ta ble 4. Superposition of MT2 Selective Antagonists
raphy (silica gel, cyclohexane/EtOAc, 6:4, as eluent) gave 0.175
g (50%) of the desired product 5b as a white solid: mp 125 °C
(EtOAc/n-hexane). MS (EI): m/z 334 (M+), 249 (100). 1H NMR
(CDCl3): δ 0.64-0.73 (m, 2H), 0.90-0.96 (m, 2H), 1.17-1.32
(m, 1H), 2.85 (t, 2H, J ) 6.7), 3.47 (m, 2H), 4.00 (s, 3H), 5.81
(br s, 1H), 6.58 (d, 1H, J ) 8.1)), 6.60 (s, 1H), 6.72 (d, 1H, J )
distance
between
angle
between
height
rmsa centroidsb planesc
above the
compd
(Å)
(Å)
(deg)
planed (Å)
syn Modele
luzindole
(2S,4S)-cis-4-P-PDOT
(2R,4S)-trans-4-P-PDOT 0.67
6.26
82.0
69.9
82.5
96.2
2.21
2.00
2.64
2.62
8.3), 7.06 (t, 1H, J ) 8.1), 7.31-7.51 (m, 5H). IR (cm-1
,
0.57
4.82
4.86
4.95
Nujol): 3298, 1647. Anal. (C21H22N2O2) C, H, N.
N-[2-(4-Meth oxy-1-p h en yl-1H-in d ol-2-yl)eth yl]cyclobu -
ta n a m id e (5c). 5c was obtained following the above procedure
by using cyclobutanecarbonyl chloride (0.12 g, 1 mmol) instead
of cyclopropanecarbonyl chloride; white solid (45% yield), mp
109 °C (EtOAc/n-hexane). MS (EI): m/z 348 (M+), 249 (100).
1H NMR (CDCl3): δ 1.75-2.32 (m, 6H), 2.84 (t, 2H, J ) 6.6),
2.86 (m, 1H), 3.44 (m, 2H), 3.99 (s, 3H), 5.48 (br s, 1H), 6.55
(s, 1H), 6.57 (d, 1H, J ) 6.9), 6.71 (d,1H, J ) 8.3), 7.05 (t, 1H,
J ) 8.0), 7.29-7.58 (m, 5H). IR (cm-1, Nujol): 3303, 1650.
Anal. (C22H24N2O2) C, H, N.
10f
0.66
anti Modele
6.30
luzindole
(2R,4R)-cis-4-P-PDOT
98.5
69.9
81.3
79.9
2.59
2.00
2.63
2.54
0.63
4.82
4.86
4.86
(2S,4R)-trans-4-P-PDOT 0.88
10f
0.61
a
Value obtained by superposition of the four atoms of the amido
group and of the two aromatic centroids onto those of luzindole.
b
Distance between the centroid of the benzene fragment of the
indole or tetralin ring and the centroid of the aromatic substituent.
c Angle between the plane of the benzene fragment of the indole
or tetralin ring and the plane of the phenyl ring of the aromatic
(E)-3-(4-Met h oxy-1-p h en yl-1H -in d ol-2-yl)p r op en en i-
tr ile (6b). Cyanomethyltriphenylphosphonium chloride34 (0.71
g, 2.1 mmol) was added to a stirred solution of 4-methoxy-1-
phenyl-1H-indole-2-carboxaldehyde (3b)32 (0.377 g, 1.5 mmol)
and DBU (0.337 mL, 2.26 mmol) in toluene (25 mL), and the
mixture was refluxed for 30 min. The mixture was cooled to
room temperature, dichloromethane was added, and the
organic phase was washed with water, dried (Na2SO4), and
concentrated under reduced pressure. Purification by flash
chromatography (silica gel; cyclohexane/EtOAc, 7:3, as eluent)
and crystallization from dichloromethane/n-hexane gave 0.22
g (74% yield) of the desired compound 6b; yellow solid (73%
yield); mp 136 °C (dichloromethane/n-hexane). MS (EI): m/z
274 (M+, 100). 1H NMR (CDCl3): δ 3.99 (s, 3H), 5.57 (d, 1H, J
) 16.5), 6.56 (d, 1H, J ) 7.8), 6.73 (d, 1H, J ) 8.2), 7.14 (d,
1H, J ) 16.5), 7.10-7.60 (m, 7H). IR (cm-1, Nujol): 2215, 1622.
N-[3-(4-Met h oxy-1-p h en yl-1H -in d ol-2-yl)p r op yl]p r o-
p a n a m id e (7b). A solution of 6b (0.14 g, 0.52 mmol) in THF
(10 mL) and propionic anhydride (1.1 mL) was hydrogenated
over Raney nickel at 4 atm of H2 for 6 h at 50 °C. The catalyst
was filtered on Celite, the filtrate was concentrated in vacuo,
and the residue was partitioned between ethyl acetate and 2
N NaOH. The organic layer was washed with brine, dried (Na2-
SO4), and evaporated under reduced pressure. Purification by
flash chromatography (silica gel; EtOAc as eluent) and crystal-
lization from ethyl acetate/n-hexane gave 0.245 g (79% yield)
of the desired compound 7b as a white solid; mp 83 °C. MS
(EI): m/z 336 (M+), 250 (100). 1H NMR (CDCl3): δ 1.07 (t, 3H,
J ) 7.6), 1.70-1.84 (m, 2H), 2.09 (q, 2H, J ) 7.6), 2.67 (t, 2H,
d
substituent. Distance between the plane of the benzene fragment
of the indole or tetralin ring and the centroid of the aromatic
substituent. e The names of the models refer to the apparent
torsion angle defined by the following nonconsecutive atoms or
pseudoatoms of luzindole: (1) CR in the ethylamido chain, (2) Câ
in the ethylamido chain, (3) methylene C of the benzyl group, and
(4) the centroid of the phenyl group (see Figure 5).
vacuo and then acidified with 2 N HCl and partitioned between
water and ethyl acetate. The aqueous phase was made alkaline
with NaOH and then extracted with ethyl acetate. The
combined organic layers were washed with brine, dried (Na2-
SO4), and then concentrated under reduced pressure to give a
crude orange oily amine that was used without further
purification. MS (EI): m/z 266 (M+), 237 (100). 1H NMR
(CDCl3): δ 1.90 (br s, 2H), 2.70-3.00 (m, 4H), 3.99 (s, 3H),
6.57 (d, 1H, J ) 7.1), 6.59 (s, 1H), 6.71 (d, 1H, J ) 8.1), 7.04
(t, 1H, J ) 7.8), 7.29-7.33 (m, 5H).
N-[2-(4-Met h oxy-1-p h en yl-1H -in d ol-2-yl)et h yl)]cyclo-
p r op a n a m id e (5b). Cyclopropanecarbonyl chloride (0.091 mL,
1 mmol) was added to an ice-cooled solution of the crude amine
in dry THF (5 mL) and TEA (0.13 mL, 1 mmol), and the
mixture was stirred at room temperature for 2 h. The solvent
was evaporated in vacuo, the residue was dissolved in ethyl
acetate, and the solution was washed with
a saturated
NaHCO3 aqueous solution and then with brine, dried (Na2-
SO4), and evaporated again. Purification by flash chromatog-

2908 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Spadoni et al.
F igu r e 6. Representation of the volume occupied by the phenyl rings of 4-P-PDOT, luzindole, and 10f (cyan), superposed according
to the model syn, and of the volume occupied by the phenyl ring of 2-phenyl-MLT (yellow). 2-Phenyl-MLT is represented with
green carbons and superposed onto 10f (left, white carbons) or onto 5i (right, magenta carbons).
J ) 7.6), 3.23 (m, 2H), 3.98 (s, 3H), 5.27 (br s, 1H), 6.52 (s,
1H), 6.56 (d, 1H, J ) 7.3), 6.70 (d, 1H, J ) 8.2), 7.03 (t, 1H, J
) 8.0), 7.30-7.55 (5H). IR (cm-1, Nujol): 3318, 1644. Anal.
(C21H24N2O2) C, H, N.
After the solution was cooled to 0 °C, water was added
dropwise to destroy the excess hydride, the mixture was
filtered on Celite, and the filtrate was concentrated in a
vacuum and then acidified with 2 N HCl and partitioned
between water and ethyl acetate. The aqueous phase was made
alkaline with 6 N NaOH and then extracted (3×) with
dichloromethane. The combined organic layers were washed
with brine, dried (Na2SO4), and concentrated under reduced
pressure to give the crude amine, which was purified by
trituration with diethyl ether; orange solid (yield 90%). MS
(EI): m/z 252 (M+, 100). 1H NMR (acetone-d6): δ 3.93 (s, 3H),
4.39 (s, 2H), 6.55-7.59 (m, 9H). IR (cm-1, CDCl3): 3465, 1587.
4-Meth oxy-1-p h en yl-1H-in d ol-2-ca r boxylic a cid (8b). A
solution of 1b32 (2.81 g, 10 mmol) in THF (20 mL), MeOH (20
mL), and 3 N KOH (10 mL) was stirred at room temperature
for 16 h. The solvent was removed in vacuo, the residue was
dissolved in water, and the solution was acidified with 6 N
HCl and extracted with ethyl acetate. The organic phase was
washed with brine, dried (Na2SO4), and evaporated in vacuo
to give 8b as white solid (92% yield); mp 218-219 °C
1
(dichloromethane/n-hexane). MS (EI): m/z 267 (M+, 100). H
N-[(6-Meth oxy-1H-in dol-2-yl)m eth yl]pr opan am ide (10a).
A solution of 9a (0.95 g, 5 mmol) in dry THF (25 mL) was
added dropwise to a stirred ice-cooled suspension of LiAlH4
(1.14 g, 30 mmol) in dry THF (25 mL) under nitrogen. Upon
completion of the addition, the mixture was refluxed for 3 h.
After the mixture was cooled to 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 dichloromethane. The
organic layer was washed with brine, dried (Na2SO4), and
concentrated under reduced pressure to give crude (6-meth-
oxyindol-2-yl)methanamine, which was then used without any
further purification. Propionic anhydride (0.64 mL, 5 mmol)
was added to a solution of the crude amine in THF (15 mL)
and TEA (0.65 mL, 5 mmol), and the mixture was stirred at
room temperature for 6 h. The solvent was evaporated in
vacuo, and the residue was dissolved in ethyl acetate. The
solution was washed with a saturated NaHCO3 aqueous
solution and with brine, then it was dried (Na2SO4) and
evaporated to give a solid residue that was purified by
crystallization from ethyl acetate/hexane; white solid (80%
NMR (CDCl3): δ 3.99 (s, 3H), 6.55 (d, 1H, J ) 7.8), 6.69 (d,
1H, J ) 8.4), 7.21 (m, 1H J ) 8.3 and J ) 7.9), 7.25-7.51 (m,
5H), 7.67 (s, 1H).
6-Meth oxy-1H-in d ol-2-ca r boxa m id e (9a ). Thionyl chlo-
ride (1.29 mL) was added to a solution of the acid 8a 32 (1.91 g,
10 mmol) in dry THF (30 mL), and the mixture was stirred at
room temperature for 15 min under nitrogen, then heated at
50 °C for 4 h, and finally allowed to stand at room temperature
for 2 h. The solvent and excess thionyl chloride were removed
under reduced pressure, and the residue was dissolved in dry
THF (40 mL). A saturated solution of ammonia in dichlo-
romethane was poured into the ice-cooled solution of the acid
chloride, and the mixture was stirred at room temperature for
16 h. Petroleum ether was added to the reaction mixture, and
the brown solid that precipitated upon cooling to 0 °C was
collected, washed with water, and dried (1.62 g; 85% yield).
An analytical sample was prepared by recrystallization from
ethyl acetate/petroleum ether; mp 194 °C. MS (EI): m/z 190
1
(M+), 173 (100). H NMR (DMSO-d6): δ 3.49 (s, 3H), 6.16 (br
s, 2H), 6.39 (d, 1H, J ) 8.7), 6.57 (s, 1H), 6.72 (s, 1H), 7.13 (d,
1H, J ) 8.5). IR (cm-1, Nujol): 3369, 1650, 1628.
yield), mp 138-139 °C. MS (EI): m/z 232 (M+, 100). H NMR
1
(CDCl3): δ 1.17 (t, 3H, J ) 7.6), 2.24 (q, 2H, J ) 7.6), 3.83 (s,
3H), 4.42 (d, 2H, J ) 5.9), 6.06 (br s, 1H), 6.24 (s, 1H), 6.73-
4-Meth oxy-1-p h en yl-1H-in d ol-2-ca r boxa m id e (9b). 9b
was obtained following the above procedure by using the acid
8b (2.67 g, 10 mmol) instead of the acid 8a ; beige solid (83%
yield), mp 172 °C (ethyl acetate/petroleum ether). MS (EI): m/z
266 (M+, 100). 1H NMR (CDCl3): δ 3.99 (s, 3H), 5.78 (br s,
2H), 6.57 (d, 1H, J ) 7.8), 6.75 (d, 1H, J ) 8.4), 7.19 (t, 1H, J
) 7.9), 7.28-7.57 (m, 6H). IR (cm-1, Nujol): 3375, 1642, 1629.
(4-Meth oxy-1-ph en yl-1H-in d ol-2-yl)m eth a n a m in e. A so-
lution of the amide 9b (1.33 g, 5 mmol) in dry THF (25 mL)
was added dropwise to a stirred ice-cooled suspension of LiAlH4
(1.14 g, 30 mmol) in dry THF (25 mL) under nitrogen. Upon
completion of the addition, the mixture was refluxed for 3 h.
6.83 (m, 2H), 7.40 (d, 1H, J ) 8.5), 8.92 (br s, 1H). IR (cm-1
CDCl3): 3400, 1650.
,
N-[(4-Meth oxy-1-p h en yl-1H-in d ol-2-yl)m eth yl]p r op a n -
a m id e (10b). Propionic anhydride (0.128 mL, 1 mmol) was
added to a solution of (4-methoxy-1-phenyl-1H-indol-2-yl)-
methanamine (0.27 g, 1 mmol) in THF (5 mL) and TEA (0.13
mL, 1 mmol), and the mixture was stirred at room temperature
for 6 h. The solvent was evaporated in vacuo, and the residue
was dissolved in ethyl acetate. The solution was washed with
a saturated NaHCO3 aqueous solution and with brine, then it

2-N-Acylaminoalkylindoles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2909
1
was dried (Na2SO4) and evaporated to give a solid residue that
was purified by crystallization from dichloromethane/n-hexane;
white solid (90% yield), mp 120 °C. MS (EI): m/z 308 (M+,
326 (M+), 173 (100). H NMR (CDCl3): δ 1.14 (t, 3H, J ) 7.5),
2.19 (q, 2H, J ) 7.5), 3.02 (t, 2H, J ) 6.5), 3.63 (m 2H), 3.95
(s, 3H), 5.77 (br s, 1H), 6.51-7.21 (m, 3H), 9.32 (br s, 1H). IR
(cm-1, Nujol): 3320, 1649. Anal. (C14H17BrN2O2) C, H, N.
N-[2-(1-Ben zyl-3-br om o-6-m eth oxy-1H-in d ol-2-yl)eth y-
l)]a ceta m id e (17). 17 was obtained following the above
procedure for compound 15 by using 13 (0.322 g, 1 mmol)
instead of 10a ; white solid (40% yield), mp 125 °C dec
(dichloromethane/n-hexane). MS (EI): m/z 400, 402 (M+), 91
1
100). H NMR (CDCl3): δ 1.08 (t, 3H, J ) 7.6), 2.11 (q, 2H, J
) 7.5), 3.98 (s, 3H), 4.50 (d, 2H, J ) 5.0), 5.51 (br s, 1H), 6.57
(d, 1H, J ) 7.7), 6.69 (s, 1H), 6.75 (d, 1H, J ) 8.2), 7.08 (t, 1H,
J ) 7.7), 7.31-7.57 (m, 5H). IR (cm-1, Nujol): 3390, 3323,
1658. Anal. (C19H20N2O2) C, H, N.
N-[(1-Ben zyl-6-m eth oxy-1H-in d ol-2-yl)m eth yl]p r op a n -
a m id e (11). A solution of 10a (0.232 g, 1 mmol) in dry DMF
(4 mL) was added dropwise to a stirred suspension of sodium
hydride (0.033 g of an 80% dispersion in mineral oil, 1.1 mmol)
in dry DMF (2 mL) at 0 °C under a N2 atmosphere. The
mixture was stirred at 0 °C for 30 min, benzyl chloride (0.115
mL, 1 mmol) was then added, and the resulting mixture was
stirred at room temperature for 16 h. After this time the
reaction mixture was poured into ice/water (40 g) and extracted
with ethyl acetate. The organic phase was washed with brine,
dried (Na2SO4), and concentrated under reduced pressure to
give a residue that was purified by crystallization from CH2-
Cl2/n-hexane; white solid (51% yield), mp 160 °C. MS (EI): m/z
1
(100). H NMR (CDCl3): δ 1.91 (s, 3H), 2.99 (t, 2H, J ) 6.6),
3.40 (m, 2H), 3.79 (s, 3H), 5.38 (s, 2H), 5.65 (br s, 1H), 6.70 (s,
1H), 6.84-7.26 (m, 6H), 7.43 (d, 1H, J ) 8.3). IR (cm-1
,
Nujol): 3307, 1650. Anal. (C20H21BrN2O2) C, H, N.
N-[(1-Ben zyl-3-br om o-4-m eth oxy-1H-in d ol-2-yl)m eth y-
l)]a ceta m id e (18). 18 was obtained following the above
procedure for compound 15 by using 14 (0.308 g, 1 mmol)
instead of 10a ; white solid (47% yield), mp 150 °C (dichlo-
romethane/n-hexane). MS (EI): m/z 386-388 (M+), 91 (100).
1H NMR (CDCl3): δ 1.67 (s, 3H), 3.97 (s, 3H), 4.64 (d, 2H, J )
5.8), 5.45 (s, 2H), 5.56 (br s, 1H), 6.58 (d, 1H, J ) 7.7), 6.87-
7.23 (m, 7H). IR (cm-1, Nujol): 3298, 1645. Anal. (C19H19
BrN2O2) C, H, N.
-
1
322 (M+), 175 (100). H NMR (CDCl3): δ 0.92 (t, 3H, J ) 7.6),
1.73 (q, 2H, J ) 7.6), 3.79 (s, 3H), 4.58 (d, 2H, J ) 5.4), 5.30
(br s, 1H), 5.32 (s, 2H), 6.45 (s, 1H), 6.71 (m, 1H), 6.81 (dd,
1H, J ) 2.0 and J ) 8.5), 6.92-7.25 (m, 5H), 7.49 (d, 1H, J )
8.3). IR (cm-1, Nujol): 3305, 1642. Anal. (C20H22N2O2) C, H,
N.
N-[(1-p-Ch lor oben zyl-4-m eth oxy-1H-in d ol-2-yl)m eth yl]-
p r op a n a m id e (12). 12 was prepared by N-alkylation of 10c32
(0.232 g, 1 mmol) with 4-chlorobenzyl chloride (0.16 g, 1 mmol)
following the procedure described for compound 11; white
amorphous solid (73% yield), mp 218 °C (chloroform). MS
(EI): m/z 356 (M+), 175 (100). 1H NMR (CDCl3): δ 0.97 (t, 3H,
J ) 7.8), 1.91 (q, 2H, J ) 7.8), 3.97 (s, 3H), 4.58 (d, 2H, J )
5.4), 5.33 (s, 2H), 5.40 (br s, 1H), 6.56 (d, 1H, J ) 7.8), 6.62 (s,
1H), 6.83 (1H, J ) 8.2), 6.85 (d, 2H, J ) 8.5), 7.10 (m, 1H, J
) 7.8 and J ) 8.2), 7.21 (d, 2H, J ) 8.5). IR (cm-1, Nujol):
3307, 1639. Anal. (C20H21ClN2O2) C, H, N.
N-[2-(1-Ben zyl-6-m et h oxy-1H -in d ol-2-yl)et h yl]a cet a -
m id e (13). 13 was obtained following the above procedure for
compound 11 by using the starting material 5g32 (0.232 g, 1
mmol) instead of 10a ; white solid (66% yield), mp 102 °C (ethyl
acetate/n-hexane). MS (EI): m/z 322 (M+), 263 (100). 1H NMR
(CDCl3): δ 1.91 (s, 3H), 2.87 (t, 3H, J ) 6.8), 3.52 (m, 2H),
3.79 (s, 3H), 5.30 (s, 2H), 5.63 (br s, 1H), 6.32 (s, 1H), 6.72 (d,
1H, J ) 2.1), 6.80 (dd, 1H, J ) 2.2 and, J ) 8.5), 6.95-7.29
(m, 5H), 7.47 (d, 1H, J ) 8.2). IR (cm-1, Nujol): 3311, 1642.
(b) P h a r m a cology. Mem br a n e P r ep a r a tion . The prepa-
ration of membranes was described elsewhere.24j, 31c In short,
NIH3T3 cells stably expressing the cloned human MT1 or MT2
receptor subtypes were grown to confluence. On the day of
assay the cells were detached from the flasks with ethylene-
diaminetetraacetic acid (EDTA) (4 mM)/Tris-HCl (50 mM), pH
7.4, room temperature, and collected by centrifugation at
1000g for 10 min at 4 °C. The cells were suspended in EDTA
(2 mM)/Tris-HCl (50 mM), homogenized in 10-15 volumes of
ice-cold EDTA (2 mM)/Tris-HCl (50 mM) with an ultra-Turrax
apparatus and centrifuged at 50000g at 4 °C for 25 min. The
final pellet was then resuspended in ice-cold Tris-HCl (50 mM)
assay buffer.
Membrane protein level was determined according to a
previously reported method.38
2-[125I]Iod om ela ton in Bin d in g Assa ys. Affinities of com-
pounds were determined in competition binding assays by the
displacement of 2-[125I]iodomelatonin from MT1 and MT2
receptors expressed in NIH3T3 cells.
2-[125I]iodomelatonin (100 pM) and the compound under test
at 10 different concentrations were incubated with the receptor
preparation for 90 min at 37 °C. The final membrane concen-
tration was 5-10 µg protein per tube. The binding conditions
were described in detail elsewhere.39 IC50 values were deter-
mined by nonlinear fitting strategies, and pKi values were
calculated from the IC50 values using the Cheng-Prusoff
equation.40 In saturation studies 2-[125I]iodomelatonin was
added at concentrations ranging from 10 to 1000 pM.
Deter m in a tion of th e In tr in sic Activity: [35S]GTP γS
Bin d in g Assa ys. [35S]GTPγS binding studies in NIH3T3 cells
expressing human cloned MT1 or MT2 receptors were per-
formed as previously described.24j,31c,32 The final pellet, obtained
as described above (membrane preparation), was resuspended
in ice-cold Tris-HCl assay buffer (50 mM) to give a final
membrane concentration of 20-30 mg/mL. The membranes
(15-25 µg of protein) were then 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). The final
incubation volume was 100 µL. The incubation was terminated
by the addition of ice-cold Tris-HCl buffer, pH 7.4 (1 mL), rapid
vacuum filtration through Whatman GF/B glass-fiber filters,
and three (3 mL) washes with ice-cold Tris-HCl buffer, pH 7.4.
Bound radioactivity was determined by liquid scintillation
spectrophotometry after overnight extraction in 4 mL of Filter-
Count scintillation fluid. Basal binding was assessed in the
absence of ligands, and nonspecific binding was defined using
GTPγS (10 µM). 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
N-[(1-Ben zyl-4-m et h oxy-1H -in d ol-2-yl)m et h yl]a cet a -
m id e (14). 14 was obtained following the above procedure for
compound 11 by using 10d 32 (0.218 g, 1 mmol) instead of 10a ;
white solid (71% yield); mp 186-187 °C (dichloromethane/n-
1
hexane). MS (EI): m/z 308 (M+), 175 (100). H NMR (CDCl3):
δ 1.60 (s, 3H), 3.98 (s, 3H), 4.58 (d, 2H), 5.33 (br s, 1H), 5.35
(s, 2H), 6.56 (d, 1H, J ) 7.3), 6.62 (s, 1H), 6.87-7.26 (m, 7H).
IR (cm-1, Nujol): 3420, 1658. Anal. (C19H20N2O2) C, H, N.
N-[(3-Br om o-6-m eth oxy-1H-in d ol-2-yl)m eth yl]p r op a n -
a m id e (15). NBS (1.05 equiv) was added portionwise to an
ice-cooled solution of 10a (0.232 g, 1 mmol) in AcOH (4 mL),
and the mixture was stirred at room temperature for 2 h. The
mixture was then poured into an ice-cooled 30% NaOH
solution and extracted (3×) with EtOAc. The combined extracts
were washed with brine and dried (Na2SO4). Evaporation of
the solvent gave a residue that was purified by chromatogra-
phy (cyclohexane/EtOAc, 3:7, as eluent) to give 15 as a white
solid (0.134 g, 45%); mp 131 °C dec (dichloromethane/n-
hexane). MS (EI): m/z 312, 310 (M+), 175 (100). 1H NMR
(CDCl3): δ 1.17 (t, 3H, J ) 7.5), 2.24 (q, 2H, J ) 7.5), 3.83 (s,
3H), 4.50 (d, 2H, J ) 5.8), 6.13 (br s, 1H), 6.80-6.84 (m, 2H),
7.36 (d, 1H, J ) 8.8), 9.24 (br s, 1H). IR (cm-1, Nujol): 3450,
1648. Anal. (C13H15BrN2O2) C, H, N.
N-[2-(3-Br om o-4-m eth oxy-1H-in d ol-2-yl)eth yl]p r op a n -
a m id e (16). 16 was obtained following the above procedure
for compound 15 by using 5d 32 (0.246 g, 1 mmol) instead of
10a ; white amorphous solid (41% yield). MS (EI): m/z 324,

2910 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Spadoni et al.
equivalent to 100 nM of 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 occupies the same
number of receptors as MLT, 100 nM. All measurements were
performed in triplicate. The SEM values were below 15% of
the mean.
(c) QSAR. The Fujita-Ban modification41 of the Free-
Wilson analysis42 was applied to affinity and intrinsic activity
data. In this approach, starting from a reference compound,
the contribution of each chemical modification to biological
activity (y) was calculated as
4-P-PDOT deserved particular attention because of its low
conformational flexibility that can be very useful for the
definition of a pharmacophore model for MT2 receptor antago-
nists. 4-P-PDOT has a cis and a trans isomer, each isomer
being a mixture of two enantiomers. Unfortunately, the
literature does not report which isomer is the active one or if
one of the enantiomers has selective affinity, and the authors
do not specify the stereochemistry of the compound tested.24b
For this reason all the isomers were studied.
For both the cis and the trans compounds, the conformers
with the acylamino group in axial conformation were discarded
on the basis of our published pharmacophore model for
melatonin receptor ligands,22a where the acylamino group of
4-methoxytetralin derivatives was aligned in equatorial dis-
position. In the cis and trans isomers with an equatorial
acylamino group, the 4-phenyl substituent adopts a pseu-
doequatorial or a pseudoaxial conformation, respectively. The
minimum-energy conformations obtained by the Tripos force
field were in good agreement with the crystal structures of
2-substituted 4-phenyltetralins in the Cambridge Structural
Database.49 The 2S,4R isomer of 4-P-PDOT was strictly
superposable onto the 1R,3S-1-phenyl-3-amino-1,2,3,4-tetrahy-
dronaphthalene (reference code: PEXLUZ)50 for their common
part, with a root mean square (rms) of 0.09 Å calculated for
the carbon atoms of the tetralin and the phenyl rings.
y )
bjXj + c
∑
j
where bj is the contribution of the chemical modification j
calculated as the regression coefficient for the variable Xj,
indicating the presence of that modification in each compound,
and the intercept c is the calculated activity y for the reference
compound. In this work the reference compound was defined
by the following substituents: R1 ) CH3, R2 ) H, R3 ) 4-OCH3,
R4 ) H, n ) 2.
For both the cis and trans isomers of 4-P-PDOT two
enantiomers exist in which, maintaining a fixed orientation
for the tetralin nucleus, the phenyl group is positioned above
or beneath the plane of the nucleus. When the 2-acylamino
chain is kept in equatorial conformation, it was possible to get
a strict superposition of the 2S,4S cis isomer on the 2R,4S
trans one, and the same was possible for the respective
enantiomers. Only one out of each couple of diasteroisomers
was therefore selected for the superposition with other com-
pounds; in particular, the two cis enantiomers are represented
in Figure 5.
The affinity for each receptor subtype was expressed as pKi,
whereas the efficacy was expressed as relative intrinsic activity
(IAr), relating the maximal G-protein activation induced by
each compound to that induced by MLT (see Table 1).
Multiple regression analysis (MRA) calculations were per-
formed by an Excel (Microsoft Co., version 97) spreadsheet,
employing the built-in statistical functions and automated
macro procedures. For each subset of compounds, MRA models
were calculated by including all the independent variables
from Table 2, giving a nonsingular matrix, and all the
combinations of smaller numbers of those variables. Typical
statistical parameters43 for some of these models are reported
in Table 3. Standard deviation of the errors in prediction
(SDEP) and the predictivity parameter Q2 were calculated by
cross-validation, omitting one compound at a time from the
set according to the leave-one-out technique (LOO).44 The best
models, from a statistical point of view, were selected by
looking for the best F (absence of nonsignificant X variables)
value.
Statistical significance of the MRA models was considered
to confirm the assumptions of the Free-Wilson analysis, i.e.,
that the change of biological activity resulting from a change
of chemical structure is independent of all other modifications
in the molecule and that these contributions are additive
within congener series.45
(d ) Molecu la r Mod elin g. Molecular modeling studies were
performed by Sybyl 6.646 running on an SGI O2 workstation.
Molecules were energy-minimized, using the standard Tripos
force field47 with the Powell method,48 to an energy gradient
of 0.01 kcal mol-1 Å-1; the electrostatic contribution was
ignored.
Five conformers were found for luzindole, 27 for 10f, and
23 for 5i. In the following superposition, the four atoms of the
amido group, the centroid of the benzene ring in the indole or
tetralin nuclei, and the centroid of the aromatic substituent
were taken as the points to be fitted. The lowest rms values
were considered as the criterion for conformer selection.
The superposition of luzindole and 4-P-PDOT allowed the
selection of three conformers of luzindole, one with the benzyl
group above and two beneath the plane of the indole ring,
considering the nitrogen atom on the bottom-right. The
conformers of 10f allowed the selection of two conformers of
luzindole, one having the phenyl group above and the other
beneath the plane.
Compounds 5i and 2-phenyl-MLT were superposed to luz-
indole, fitting the four atoms of the amido group and the
centroid of the benzene ring of the indole nucleus.
Ack n ow led gm en t. This work was supported by
grants from the Italian Ministero della Ricerca Scien-
tifica e Tecnologica (MURST), Consiglio Nazionale delle
Ricerche (CNR), and Faculty of Pharmacy of the Uni-
versity of Urbino. The authors from the University of
Urbino thank Mr. Francesco Palazzi for technical as-
sistance.
Compounds submitted to conformational analysis (luzindole,
4-P-PDOT, 5i, and 10f) were investigated by the systematic
search routine of Sybyl. Rotatable bonds were rotated by steps
of 30°, and the generated conformations were optimized to
obtain minimum-energy geometry. The resulting conformers
where then matched to reject duplicates.
For luzindole, the two single bonds of the benzyl group were
the only bonds to be rotated because the acylaminoethyl chain
was kept in the conformation proposed by a previously
reported pharmacophore model for melatonin receptor ligands
(model B in ref 22a). The conformational analysis of 5i was
limited to the three rotatable bonds corresponding to the
ethylamino portion of the side chain in position 2 of the indole
nucleus. In 10f, four bonds were rotated: the two single bonds
of the benzyl group and the two bonds around the methylene
fragment of the side chain in position 2. The amido group was
kept in trans conformation.
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