Synthesis of nitroindole derivatives with high affinity and selectivity for melatoninergic binding sites MT3

Article abstract of DOI:10.1021/jm011053+

The aim of this study was to synthesize selective ligands for melatoninergic subtype receptors that could elucidate the physiological role of melatonin (N-acetyl-5-methoxytryptamine, 1). So, we first investigated the role of a nitro substituent in the 4-,

Full text of DOI:10.1021/jm011053+

J . Med. Chem. 2002, 45, 1853-1859
1853
Syn th esis of Nitr oin d ole Der iva tives w ith High Affin ity a n d Selectivity for
Mela ton in er gic Bin d in g Sites MT3
Ve´ronique Leclerc,† Sa¨ıd Yous,† Philippe Delagrange,‡ J ean A. Boutin,§ Pierre Renard,‡ and Daniel Lesieur*,†
Institut de Chimie Pharmaceutique Albert Lespagnol, 3 rue du Professeur Laguesse, BP83, 59006 Lille Cedex, France, Institut
de Recherches Internationales Servier, 1 rue Carle He´bert, 92415 Courbevoie Cedex, France, and Division de Pharmacologie
Mole´culaire et Cellulaire, Institut de Recherches Servier, 125 Chemin de Ronde, 78290 Croissy-sur-Seine, France
Received October 8, 2001; Revised Manuscript Received J anuary 21, 2002
The aim of this study was to synthesize selective ligands for melatoninergic subtype receptors
that could elucidate the physiological role of melatonin (N-acetyl-5-methoxytryptamine, 1). So,
we first investigated the role of a nitro substituent in the 4-, 6-, or 7-position of the indole
heterocycle. Comparatively to melatonin, its analogues that nitrated in the 6- or 7-position (6
and 22) lose MT3 but retain good MT1 and MT2 affinities, whereas the 4-nitro isomer (5) shows
very high affinity (nanomolar) and selectivity for the MT3 binding sites. N-Methylation of the
indole nucleus of compound 5 potentiates these effects and affords the most potent and selective
MT3 ligand (17). The 2-iodo derivatives (12 and 10) of compounds 5 and 17 have also been
synthesized to evaluate their binding profile with a view to further develop MT3 selective
radioligands.
Ch a r t 1. Chemical Structures of Melatonin and Nitro
Derivatives
In tr od u ction
Melatonin (N-acetyl-5-methoxytryptamine, 1) (Chart
1) is a neurohormone produced by the pineal gland
during the dark period, whatever the species considered,
including humans.1 The synthesis of melatonin is
regulated by circadian and seasonal variations in day
length through a polysynaptic neuronal pathway from
the retina to the pineal gland.
Melatonin, which is released in the blood circulation
and in the cerebrospinal fluid, transmits the information
on the photoperiod to central and peripheral structures
that express melatonin binding sites. Melatonin has
been detected in numerous central and peripheral
tissues using the specific radioligand 2-[125I]-iodomela-
tonin.2,3 Three melatonin receptors have been cloned.
The first identified was the Mel1C receptor, which has
been cloned from Xenopus laevis4 and is not expressed
in mammals.5 The two others are expressed in human5,6
and defined as MT1 and MT2.7 These two receptors
subtypes belong to the family of seven transmembrane
G-protein coupled receptors, and they are involved in
the chronobiotic properties of melatonin8,9 and in the
vasoconstrictor activities on animal vessels.10,11
Beside these two receptors that both present a high
affinity for melatonin (Ki ≈ 0.1 nM), another melatonin
binding site with lower affinity (Ki ≈ 60 nM) has been
identified.12-14 This binding site has been named MT3,
according to the IUPHAR nomenclature.7 MT3 displays
very fast kinetics of ligand association/dissociation,12,15,16
which explain that binding studies are performed at 4
°C. Nevertheless, improvement of the filtration proce-
dures allowed us to determine the pharmacological
profile of MT3 at 20 °C.17 MT3 presents a specific
pharmacological profile as compared to MT1/MT2. In-
deed, prazosin (Ki ≈ 8 nM) and N-acetylserotonin (Ki ≈
30 nM, NAS) are good ligands;14,15 5-methoxycarbony-
lamino-N-acetyltryptamine (MCA-NAT)15 has been de-
scribed as a selective ligand (Ki ≈ 28 nM).
The MT3 binding site has been mainly reported in
hamster central and peripheral tissues using the selec-
tive radioligand 2-125I-MCA-NAT, but it is also ex-
pressed in mice, rat, rabbit, chicken, pig, dog, and
monkey.15,18 The physiological function of this melatonin
binding site has not been characterized to a great extent
due to the lack of more selective MT3 ligands. The recent
identification of MT3 as the quinone reductase 2 (QR2)18
opens new therapeutic perspectives although the physi-
ological function of this enzyme is not well-character-
ized. Moreover, complementary experiments must be
performed to determine if QR2 represents only a part
of MT3 binding sites. Some MT3 compounds have
already been synthesized.19
The aim of the present study was to synthesize
subtype selective melatonin receptor ligands to further
elucidate the physiological role of melatonin and to
provide highly effective therapeutic agents. Within the
indolic series, a lot of melatonin analogues bearing
various substituents in the 6- and/or in the 2-position
have been studied whereas only a few derivatives
substituted in the 4- or 7-position have been described.
So, we decided to investigate the role of substituents in
these later positions and we first chose the nitro (NO2)
group on account of its chemical accessibility and its
interest as a starting material for many substitution
* To whom correspondence should be addressed. Tel.:
+33.03.20.96.40.20. Fax: +33.03.20.96.43.61. E-mail: dlesieur@
phare.univ-lille2.fr.
† Institut de Chimie Pharmaceutique Albert Lespagnol.
‡ Institut de Recherches Internationales Servier.
§ Institut de Recherches Servier.
10.1021/jm011053+ CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/27/2002

1854 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Sch em e 1. Synthesis of 4- and 6-Nitro Melatonin (5 and 6)a
Leclerc et al.
a
Conditions: (i) NaOH, Bu4N+HSO4-, C6H5SO2Cl for 2a or (Boc)2O for 2b; (ii) HNO3, Ac2O; (iii) K2CO3, MeOH, THF, ∆ for 3-4a or
NaOMe, MeOH, THF for 3-4b.
Sch em e 2. Synthesis of Compounds 8a and 9a a
a
Conditions: (i) TMEDA, LDA, THF, I2, -45 °C; (ii) HNO3, Ac2O.
reactions via its reduction and diazotation of the cor-
responding amine. Furthermore, the introduction of an
electroattractive group such as nitro close to the meth-
oxy pharmacophore should probably influence affinity
and/or activity of the compounds and it was there-
fore interesting to test them on the three melatonin
receptor subtypes. The first results obtained with these
nitro analogues of melatonin led us to synthesize and
study some other derivatives (1-methyl and/or 2-iodo)
(Chart 1).
The synthesis of the corresponding 2-iodo derivatives
(12 and 13) was first considered from the 4- or 6-nitro-
N-protected compounds (3a and 4a ) (Scheme 2). Iodi-
nation21 of 3a by action of lithium diisopropylamide and
I2 at -45 °C in the presence of N,N,N′,N′-tetramethyl-
ethylenediamine led to compound 8a in good yield
(68%), but the same conditions applied on 4a only led
to 17% of 9a . So, we decided to reverse the synthetic
pathway. Iodination of 2a afforded 7a in good yield
(60%). Nitration of 7a also led to the two isomers 8a
(4-nitro) and 9a (6-nitro) in equal amounts.
We report here the synthesis of new indolic deriva-
tives that are very potent MT3 ligands, with high
selectivity toward the MT1/MT2 melatonin receptors.
N-Deprotection using the previously described condi-
tions (potassium carbonate in MeOH/THF) did not give
access to compounds 12 and 13 (Scheme 3) but to the
corresponding N-methyl derivatives 10 and 11. That
methylation would be due to the action of in situ-formed
methyl benzenesulfonate, which could come from the
methylate ion attack on 8a and 9a . Indeed, compounds
12 and 13 were, respectively, obtained from 8a and 9a
by action of sodium hydroxide in refluxing dioxane.
Ch em istr y
Starting from melatonin 1, compounds 2a and 2b
were, respectively, prepared by action of benzenesulfo-
nyl chloride and di-tert-butyl dicarbonate in the pres-
ence of sodium hydroxide and tetrabutylammonium
hydrogensulfate in dichloromethane20 (Scheme 1). Ni-
tration of 2a and 2b using nitric acid in acetic anhydride
led to the formation of the two isomers, 4-nitro (3a ,b)
and 6-nitro (4a ,b). Analyses by 1H nuclear magnetic
resonance (NMR) indicated that the resulting mixtures
were, respectively, composed of 66:33% of 3a :4a and 50:
50% of 3b:4b. These isomers were separated by column
chromatography with ethyl acetate/methanol as eluant.
The N-deprotection was then realized in MeOH/tetrahy-
drofuran (THF) using basic conditions (potassium car-
bonate for benzenesulfonyl derivatives (3a and 4a ) or
sodium methoxide for Boc derivatives (3b and 4b)) and
afforded the 4- and 6-nitro derivatives of melatonin (5
and 6, respectively).
The N-methyl derivatives 16 and 17 were, respec-
tively, obtained from 2-iodomelatonin (14) and 4-ni-
tromelatonin (5) by action of sodium hydride and methyl
iodide or dimethyl sulfate in the presence of sodium
hydroxide (Scheme 4). N-Methylmelatonin (15) has been
previously described.22
The 7-nitro derivative of melatonin (22) was obtained
from 5-methoxy-7-nitroindole (18)23,24 (Scheme 5).20
A
Mannich reaction with dimethylamine and formalde-
hyde in acetic acid gave 19; methylation followed by
displacement of the resulting ammonium ion with
potassium cyanide afforded 20. Reduction of the cyano
group to the corresponding amine 21 was performed

Synthesis of Nitroindole Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1855
Sch em e 3. Synthesis of Nitrated and Iodinated Compounds 10-13a
a
Conditions: (i) K2CO3, MeOH, THF, ∆; (ii) NaOH, dioxane, ∆.
Sch em e 4. Synthesis of Compounds 16 and 17a
get MT3 ligands with very good affinity (∼nM) and high
selectivity. Considering that the 6 and 7 isomers do not
induce the same effects, we can assume that the steric
parameters could be more important than the electronic
ones.
Because compound 5 was one of the best MT3 selective
ligands known at that time, we planned pharmaco-
modulations (i) to improve its binding profile and (ii) to
obtain a new potential radioligand.
(i) Replacing the NH indolic group of melatonin (1)
by a N-methylated one (15) leads to a 20-fold higher MT3
affinity (1.1 nM) together with a 10-fold decrease on MT1
and MT2 affinities. It was therefore interesting to test
the same pharmacomodulation with compound 5. The
resulting compound 17 shows a 3-fold higher affinity
(0.31 nM) on the MT3 subtype, an 8-fold lower affinity
on the MT1 receptor, but a 4-fold increased affinity on
the MT2 receptor. The selectivity ratios toward MT1 and
MT2 subtypes are, respectively, 27 612 and 625.
(ii) It is well-known that introduction of an iodine
atom in the 2-position of the indole heterocycle of
melatonin leads to a 10 times higher affinity on all three
receptor subtypes (1 vs 14); 2-[125I]-iodomelatonin is the
nonselective radioligand used for autoradiography and
binding studies. The 2-iodo derivatives of compounds 5
and 17 could exhibit a binding profile for the develop-
ment of potential MT3 selective radioligands and were
synthesized. Compound 12 and its N-methylated de-
rivative 10 have the same and the greatest affinity on
the MT3 receptor (∼0.1 nM) but strongly differ in their
MT1 affinity and MT1/MT3 selectivity. The selectivity
ratios MT1/MT3 and MT2/MT3, respectively, vary from
14 462 and 300 for 10 to 107 and 131 for 12. These data
highlight the role of both a 2-iodo and a 1-methyl
substituent on the indole ring of melatonin.
a
Conditions: (i) NaH, CH3I, THF, ∆; (ii) NaOH, (CH3O)2SO2,
(CH3)2CO.
with borane-THF complex. Finally, acetylation of 21
by acetyl chloride and potassium carbonate in a biphasic
medium afforded the desired compound 22.
Resu lts a n d Discu ssion
The affinities of the compounds for the melatonin
receptor subtypes were evaluated in vitro in binding
assays using 2-[125I]-iodomelatonin, human embryonic
kidney cell line HEK293 stably expressing MT1 or MT2
human melatonin receptors, and hamster brain mem-
brane preparations for the MT3 binding site according
to a previously described method.15
The results of the binding assays are presented in
Table 1. Introduction of a NO2 group on the indole
heterocycle strongly influences affinity according to the
receptor subtype and to the position of the substituent.
Comparatively to melatonin 1, the 6- and 7-nitro
derivatives (6 and 22) lose MT3 but retain good MT1 and
MT2 affinities, the 6 isomer being slightly better than
its 7 analogue. The most interesting result is obtained
with the 4-nitro isomer (5), which shows a considerable
loss of MT1 and MT2 binding affinity but a 60-fold higher
affinity on the MT3 subtype, leading to great selectivity
ratios (964 toward MT1 and 745 toward MT2). It seems
therefore that the presence of a nitro group in the 4-
position of the indole heterocycle, in the proximity of
the two pharmacophores, the methoxy group, and the
acetamido ethyl side chain, is an essential element to
In conclusion, introducing a nitro substituent on the
4-position of the indole heterocycle of melatonin leads
to a considerable increase of MT3 binding affinity, which
rises to the nanomolar range. This effect is accompanied
by a loss of MT1 and MT2 binding affinity giving to very
high selectivity ratios. Parallel N-methylation of the
indole nucleus potentiates these effects and affords the
most MT3 potent and selective ligand. Finally, the
Sch em e 5. Synthesis of 7-Nitro Melatonin (22)a
a
Conditions: (i) (CH3)2NH, HCHO, AcOH; (ii) CH3I, KCN, DMF, H2O, THF, ∆; (iii) BH3-THF, ∆; (iv) CH3COCl, K2CO3, ethyl acetate,
water.

1856 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Ta ble 1. Binding Affinity of Derivatives 1, 5, 6, 10-17, and 22
Leclerc et al.
Ki ( SEM (nM)
selectivity
a
b
b
compd
X
R
Y
MT3
MT1
MT2
MT1/MT3
MT2/MT3
1
15
14
16
5
17
12
10
6
H
CH3
H
CH3
H
CH3
H
CH3
H
H
CH3
H
H
H
I
56.9 ( 0.400
2.8 ( 0.241
6.5 ( 0.031
0.088 ( 0.004
1.1 ( 0.071
0.31 ( 0.003
0.18 ( 0.014
0.13 ( 0.006
∼1000
0.12 ( 0.020
2.6 ( 0.253
0.31 ( 0.050
3.1 ( 0.196
0.16 ( 0.014
0.047 ( 0.002
820 ( 25.156
194 ( 2.820
23.7 ( 2.133
39 ( 0.008
5.3 ( 0.354
0.17 ( 0.005
0.77 ( 0.047
29 ( 0.192
0.0020
0.9286
0.0020
0.9091
964
0.005
1.100
0.020
0.530
745
625
131
300
∼0.005
0.002
0.120
∼0.029
H
0.013 ( 0.000
0.08 ( 0.001
1060 ( 14.919
8560 ( 427.075
19.2 ( 0.191
1880 ( 67.353
14.6 ( 1.037
0.3 ( 0.026
I
H
H
I
I
H
I
4-NO2
6-NO2
27612
107
14462
∼0.0140
0.0043
0.1803
∼0.049
13
11
22
70 ( 2.311
6.1 ( 0.527
∼1000
I
H
1.1 ( 0.038
49 ( 0.215
7-NO2
a
b
Syrian brain. HEK-human.
water, dried (MgSO4), filtered, and evaporated under reduced
pressure. The crude solid was recrystallized from ethanol to
give 2b (11.45 g, 80%) as a white solid; mp 133-134.5 °C. H
NMR (CDCl3): δ 1.67 (s, 9H), 2.01 (s, 3H), 2.89 (t, J ) 6.6 Hz,
2H), 3.59 (m, 2H), 3.89 (s, 3H), 5.62 (br s, 1H), 6.95 (dd, J )
2.4 and 9.1 Hz, 1H), 7.00 (d, J ) 2.4 Hz, 1H), 7.40 (s, 1H),
8.02 (d, J ) 9.1 Hz, 1H).
Gen er a l P r oced u r e for th e Syn th esis of th e Nitr o
Com p ou n d s (3a -4a , 3b-4b, a n d 8a -9a ). The method
adopted for the synthesis of N-[2-(1-benzenesulfonyl-5-meth-
oxy-4 and 6-nitro-1H-indol-3-yl)ethyl]acetamides (3a and 4a )
is described. To a suspension of 2a (5 g, 13.4 mmol) in glacial
acetic acid (25 mL) at -5 °C was added dropwise 1.1 equiv of
a 68% solution of nitric acid (0.97 mL, 14.8 mmol). The medium
was progressively allowed to warm to room temperature. After
it was stirred for 2 days, the solution was poured into ice-water
and a precipitate containing a mixture of 3a and 4a was
isolated by filtration. The two isomers were separated by
column chromatography (silica gel), first with ethyl acetate
as eluant to yield 3a (3.14 g, 58%) and then with ethyl acetate/
methanol (9/1) to give 4a (1.68 g, 30%).
1-methyl-2-iodo-4-nitro derivative of melatonin could be
radiomarked with [125I]iodine and so could serve as a
novel and selective radioligand for the characterization
of MT3 binding sites in both central and peripheral
tissues.
1
Exp er im en ta l Section
Ch em istr y. Melting points were determined using a Bu¨chi
SMP-535 apparatus. Column chromatography was carried out
using silica gel (silica gel 60, 70-230 Mesh, ASTM, Merck)
with an appropriate solvent. IR spectra were recorded on
Perkin-Elmer 297 or BRUKER Vector 22 spectrometers, using
KBr pellets. 1H NMR spectra were recorded on a BRUKER
AC 300 P (300 MHz) spectrometer using dimethyl sulfoxide
(DMSO)-d6 or CDCl3 as solvents. Chemical shifts are expressed
downfield from the internal standard tetramethylsilane. Cou-
pling constants (J ) are expressed in Hertz (key: br ) broad, d
) doublet, dd ) double doublet, m ) multiplet, s ) singlet, t
) triplet). Elemental analysis (C, H, I, N) was determined by
the CNRS center of analysis, in Vernaison (France), and agrees
with the proposed structures within (0.4% of the theoretical
values.
N-[2-(1-Ben zen esu lfon yl-5-m eth oxy-1H-in dol-3-yl)eth yl]-
a ceta m id e (2a ). A solution of melatonin 1 (10 g, 43 mmol) in
dichloromethane (500 mL) was cooled to 0 °C. NaOH (6.89 g,
172.2 mmol) and tetrabutylammonium hydrogensulfate (0.7
g, 2.1 mmol) were added. The mixture was stirred at 0 °C for
30 min, and then, benzenesulfonyl chloride (8.24 mL, 64.5
mmol) was added dropwise. The solution was kept at 0 °C for
a further 1 h and allowed to warm to room temperature
overnight. The medium was filtered, and the precipitate was
twice washed with dichloromethane. The organic filtrate was
washed with 3 N HCl and water, dried (MgSO4), filtered, and
evaporated under reduced pressure to give a crude solid that
was triturated in diethyl ether and collected by filtration.
Recrystallization from 2-propanol yielded 2a as a white solid
(14.4 g, 88%); mp 139.5-140.5 °C. 1H NMR (DMSO-d6): δ 1.81
(s, 3H), 2.77 (t, J ) 6.7 Hz, 2H), 3.35 (m, 2H), 3.77 (s, 3H),
6.95 (dd, J ) 2.4 and 9.0 Hz, 1H), 7.12 (d, J ) 2.4 Hz, 1H),
7.54-7.60 (m, 3H), 7.67 (m, 1H), 7.82 (d, J ) 9.0 Hz, 1H), 7.92
(m, 2H), 8.01 (br t, J ) 5.5 Hz, 1H).
N-[2-(1-Ben zen esu lfon yl-5-m eth oxy-4-n itr o-1H-in d ol-
3-yl)eth yl]a ceta m id e (3a ). Yellow solid; mp 177-178 °C (2-
propanol or ethanol). 1H NMR (DMSO-d6): δ 1.78 (s, 3H), 2.50
(m, 2H), 3.30 (m, 2H), 3.90 (s, 3H), 7.37 (d, J ) 9.5 Hz, 1H),
7.62 (m, 2H), 7.74 (m, 1H), 7.84 (s, 1H), 7.95 (br t, J ) 5.5 Hz,
1H), 8.01 (m, 2H), 8.13 (d, J ) 9.5 Hz, 1H).
N-[2-(1-Ben zen esu lfon yl-5-m eth oxy-6-n itr o-1H-in d ol-
3-yl)eth yl]a ceta m id e (4a ). Yellow solid; mp 209-210 °C (2-
propanol). 1H NMR (DMSO-d6): δ 1.77 (s, 3H), 2.80 (t, J )
6.9 Hz, 2H), 3.35 (m, 2H), 3.93 (s, 3H), 7.51 (s, 1H), 7.60 (m,
2H), 7.71 (m, 1H), 7.91 (s, 1H), 7.98-8.02 (m, 3H), 8.40 (s, 1H).
N-[2-(1-ter t-Bu toxyca r bon yl-5-m eth oxy-4-n itr o-1H-in -
d ol-3-yl)eth yl]a ceta m id e (3b). Yield, 43% from 2b; yellow
1
solid; mp 155-156 °C (2-propanol). H NMR (CDCl3): δ 1.67
(s, 9H), 1.98 (s, 3H), 2.71 (t, J ) 7.1 Hz, 2H), 3.47 (m, 2H),
3.96 (s, 3H), 5.67 (br s, 1H), 7.05 (d, J ) 9.1 Hz, 1H), 7.51 (s,
1H), 8.30 (d, J ) 9.1 Hz, 1H).
N-[2-(1-ter t-Bu toxyca r bon yl-5-m eth oxy-6-n itr o-1H-in -
d ol-3-yl)eth yl]a ceta m id e (4b). Yield, 45% from 2b; yellow
solid; mp 152-153 °C (diisopropyl ether). 1H NMR (CDCl3): δ
1.69 (s, 9H), 2.00 (s, 3H), 2.92 (t, J ) 7.1 Hz, 2H), 3.56 (m,
2H), 4.02 (s, 3H), 5.66 (br s, 1H), 7.22 (s, 1H), 7.61 (s, 1H),
8.67 (s, 1H).
N-[2-(1-ter t-Bu toxyca r bon yl-5-m eth oxy-1H-in d ol-3-yl)-
eth yl]a ceta m id e (2b). The procedure was the same described
above for the preparation of 2a except that di-tert-butyl
dicarbonate (14.38 g, 64.6 mmol) was used instead of benze-
nesulfonyl chloride. After it was stirred overnight, the mixture
was filtered, the precipitate was washed twice with dichlo-
romethane, and the organic filtrate was washed only with
N-[2-(1-Ben zen esu lfon yl-2-iod o-5-m eth oxy-4-n itr o-1H-
in d ol-3-yl)eth yl]a ceta m id e (8a ). Yield, 41% from 7a ; yellow
1
solid; mp 177-178 °C (ethanol). H NMR (DMSO-d6): δ 1.71
(s, 3H), 2.56 (t, J ) 6.8 Hz, 2H), 2.97 (m, 2H), 3.93 (s, 3H),

Synthesis of Nitroindole Derivatives
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1857
7.36 (d, J ) 9.3 Hz, 1H), 7.64 (m, 2H), 7.76 (m, 1H), 7.84-
7.87 (m, 3H), 8.39 (d, J ) 9.3 Hz, 1H).
3H), 7.35 (s, 1H), 7.96 (br t, J ) 6.1 Hz, 1H), 8.20 (s, 1H). Anal.
(C14H16IN3O4) C, H, I, N.
Meth od C. The method adopted for the synthesis of N-[2-
(2-iodo-5-methoxy-4-nitro-1H-indol-3-yl)ethyl]acetamide (12) is
described. A solution of 8a (3 g, 5.5 mmol) was refluxed for 5
h in dioxane (60 mL) with 5 equiv of aqueous NaOH 10% (11
mL). After it was cooled, the mixture was hydrolyzed and the
precipitate was collected by filtration and recrystallized to
afford 12 (1.54 g, 69%). Yellow solid; mp 191-193 °C (ethanol).
1H NMR (DMSO-d6): δ 1.77 (s, 3H), 2.55 (t, J ) 6.9 Hz, 2H),
2.99 (m, 2H), 3.87 (s, 3H), 7.08 (d, J ) 9.0 Hz, 1H), 7.48 (d, J
) 9.0 Hz, 1H), 7.85 (br t, J ) 5.3 Hz, 1H), 12.08 (br s, 1H).
Anal. (C13H14IN3O4) C, H, I, N.
N-[2-(2-Iod o-5-m et h oxy-6-n it r o-1H -in d ol-3-yl)et h yl]-
a ceta m id e (13). Yield, 75% from 9a (method C); yellow solid;
mp 209-211 °C (ethanol 70°). 1H NMR (DMSO-d6): δ 1.77 (s,
3H), 2.77 (t, J ) 7.0 Hz, 2H), 3.20 (m, 2H), 3.91 (s, 3H), 7.33
(s, 1H), 7.85 (s, 1H), 7.95 (br t, J ) 5.7 Hz, 1H), 11.98 (br s,
1H). Anal. (C13H14IN3O4) C, H, I, N.
N -[2-(2-Iod o-5-m e t h oxy-1-m e t h ylin d ol-3-yl)e t h yl]-
a cet a m id e (16). To a solution of 14 (0.5 g, 1.4 mmol) and
methyl iodide (0.11 mL, 1.8 mmol) in dry THF (5 mL) was
added portionwise NaH (0.07 g, 1.8 mmol). The mixture was
stirred for 4 h at room temperature, and then, it was
hydrolyzed. The precipitate that was formed in water was
filtered and recrystallized from toluene to afford 16 (0.29 g,
56%). White solid; mp 129-131 °C. 1H NMR (DMSO-d6): δ
1.78 (s, 3H), 2.77 (t, J ) 7.1 Hz, 2H), 3.18 (m, 2H), 3.69 (s,
3H), 3.77 (s, 3H), 6.75 (dd, J ) 2.0 and 8.9 Hz, 1H), 7.04 (d, J
) 2.0 Hz, 1H), 7.36 (d, J ) 8.9 Hz, 1H), 7.96 (br t, J ) 5.6 Hz,
1H). Anal. (C14H17IN2O2) C, H, I, N.
N-[2-(1-Ben zen esu lfon yl-2-iod o-5-m eth oxy-6-n itr o-1H-
in d ol-3-yl)eth yl]a ceta m id e (9a ). Yield, 42% from 7a ; yellow
solid; mp 191-193 °C (ethanol). H NMR (DMSO-d6): δ 1.70
(s, 3H), 2.81 (t, J ) 7.0 Hz, 2H), 3.18 (m, 2H), 3.96 (s, 3H),
7.49 (s, 1H), 7.62 (m, 2H), 7.74 (m, 1H), 7.87 (m, 2H), 7.94 (br
t, J ) 5.6 Hz, 1H), 8.65 (s, 1H).
1
Gen er a l P r oced u r e for th e Syn th esis of th e 2-Iod o
Com p ou n d s (7a , 8a , a n d 9a ). The method adopted for the
synthesis of N-[2-(1-benzenesulfonyl-2-iodo-5-methoxy-1H-in-
dol-3-yl)ethyl]acetamide (7a) is described. To a solution of 2a
(10 g, 26.8 mmol) in dry THF (60 mL) was added under
nitrogen N,N,N′,N′-tetramethylethylenediamine (10.2 mL, 67.1
mmol) with a syringe, and then, the mixture was cooled to
-22 °C and stirred at this temperature for 30 min. A solution
of lithium diisopropylamide (2 M in THF, 40.3 mL, 80.5 mmol)
was then added dropwise with a syringe. After the mixture
was stirred for a further 1 h at -22 °C, it was cooled to -45
°C and then a solution of iodine (20.5 g, 80.5 mmol) in dry
THF (30 mL) was slowly added so that the temperature of the
medium did not exceed -35 °C. The mixture was stirred at
this temperature for a further 1 h, allowed to warm to room
temperature for 4 h, and poured into ice-water. The precipitate
was collected by filtration and recrystallized from ethanol to
1
yield 7a (7.76 g, 58%). White solid; mp 143-145 °C. H NMR
(DMSO-d6): δ 1.73 (s, 3H), 2.74 (t, J ) 6.9 Hz, 2H), 3.13 (m,
2H), 3.79 (s, 3H), 6.91 (dd, J ) 2.5 and 9.2 Hz, 1H), 7.11 (d, J
) 2.5 Hz, 1H), 7.57 (m, 2H), 7.68 (m, 1H), 7.76 (m, 2H), 7.96
(br t, J ) 5.6 Hz, 1H), 8.03 (d, J ) 9.2 Hz, 1H).
This procedure allowed us to obtain 8a and 9a , respectively,
from 3a and 4a with 63 and 17% yields.
N -[2-(5-Me t h oxy-1-m e t h yl-4-n it r oin d ol-3-yl)e t h yl]-
a ceta m id e (17). To a solution of 5 (0.5 g, 1.8 mmol) in acetone
(10 mL) was added a solution of sodium hydroxide 10% (1.44
mL, 3.6 mmol) and then dropwise dimethyl sulfate (0.26 mL,
2.7 mmol). The mixture was stirred for 2 h and then hydro-
lyzed. After acidification, the medium was filtered, and the
precipitate was recrystallized from toluene to afford 17 (0.4 g,
Gen er a l P r oced u r es for th e N-Dep r otection of th e
In d ole Nu cleu s (5, 6, a n d 10-13). Methods adopted for the
synthesis of N-[2-(5-methoxy-4-nitro-1H-indol-3-yl)ethyl]ac-
etamide (5) are described from 3a (method A) and from 3b
(method B).
Meth od A. To a solution of 3a (7 g, 16.8 mmol) in a mixture
(300 mL) of THF/MeOH (1/1) was added 100 mL of an aqueous
solution of K2CO3 (10.89 g, 78.8 mmol). The mixture was
refluxed overnight, and after it was cooled, the mixture was
concentrated under reduced pressure and then poured into
water. The resulting precipitate was filtered and recrystallized
from ethyl acetate to afford 5 (3.3 g, 71%). Yellow solid; mp
1
70%). Yellow solid; mp 144-146 °C. H NMR (CDCl3): δ 1.95
(s, 3H), 2.76 (t, J ) 7.0 Hz, 2H), 3.44 (m, 2H), 3.77 (s, 3H),
3.95 (s, 3H), 5.65 (br s, 1H), 6.99 (d, J ) 8.9 Hz, 1H), 7.04 (s,
1H), 7.37 (d, J ) 8.9 Hz, 1H). Anal. (C14H17N3O4) C, H, N.
3-Dim e t h yla m in om e t h yl-5-m e t h oxy-7-n it r o-1H -in -
d ole (19). A solution of acetic acid (35 mL), dimethylamine
(1.4 mL, 12.5 mmol), and formaldehyde (0.83 mL, 10 mmol)
was stirred at 0 °C for 30 min. To this solution was added
5-methoxy-7-nitroindole 18 (1.71 g, 8.9 mmol). After it was
stirred for 3 days at room temperature, the mixture was
hydrolyzed with a solution of sodium hydroxide 15% (180 mL)
and then extracted with dichloromethane. The organic layer
was dried over MgSO4, filtered, and concentrated under
reduced pressure. The oily residue was eluted on silica gel
chromatography with ethyl acetate and then with a mixture
of acetone/toluene/cyclohexane/triethylamine (5/3/2/1) to afford
19 as a yellow-orange-colored solid (1.9 g, 86%); mp 155-156
1
187-189 °C. H NMR (DMSO-d6): δ 1.78 (s, 3H), 2.58 (t, J )
7.5 Hz, 2H), 3.22 (m, 2H), 3.87 (s, 3H), 7.11 (d, J ) 8.9 Hz,
1H), 7.38 (s, 1H), 7.56 (d, J ) 8.9 Hz, 1H), 7.92 (br t, J ) 5.4
Hz, 1H), 11.41 (br s, 1H). Anal. (C13H15N3O4) C, H, N. This
method allowed us to obtain the N-methyl derivatives 10 and
11, respectively, from 8a and 9a .
Meth od B. To a solution of 3b (0.5 g, 1.32 mmol) in dry
THF (10 mL) under nitrogen was added a methanolic solution
of NaOMe (0.15 g in 0.35 mL of MeOH, 2.65 mmol). After it
was stirred for 1 h, the mixture was hydrolyzed and then the
precipitate was isolated by filtration to afford 66% of 5 after
recrystallization.
N-[2-(5-Met h oxy-6-n it r o-1H -in d ol-3-yl)et h yl]a cet -
a m id e (6). Yields, 49 and 65%, respectively, from 4a (method
A) and from 4b (method B); yellow solid; mp 197-198 °C (2-
propanol). 1H NMR (DMSO-d6): δ 1.79 (s, 3H), 2.84 (t, J )
7.3 Hz, 2H), 3.32 (m, 2H), 3.91 (s, 3H), 7.33 (s, 1H), 7.50 (s,
1H), 7.95-7.99 (m, 2H), 11.26 (br s, 1H). Anal. (C13H15N3O4)
C, H, N.
N-[2-(2-Iodo-5-m eth oxy-1-m eth yl-4-n itr oin dol-3-yl)eth yl]-
a ceta m id e (10). Yield, 90% from 8a (method A); yellow solid;
mp 215-217 °C (ethanol). 1H NMR (DMSO-d6): δ 1.77 (s, 3H),
2.59 (t, J ) 7.2 Hz, 2H), 3.00 (m, 2H), 3.81 (s, 3H), 3.89 (s,
3H), 7.16 (d, J ) 9.1 Hz, 1H), 7.76 (d, J ) 9.1 Hz, 1H), 7.87
(br t, J ) 5.6 Hz, 1H). Anal. (C14H16IN3O4) C, H, I, N.
N-[2-(2-Iodo-5-m eth oxy-1-m eth yl-6-n itr oin dol-3-yl)eth yl]-
a ceta m id e (11). Yield, 65% from 9a (method A); yellow solid;
mp 200-202 °C (ethanol). 1H NMR (DMSO-d6): δ 1.76 (s, 3H),
2.82 (t, J ) 6.8 Hz, 2H), 3.20 (m, 2H), 3.79 (s, 3H), 3.92 (s,
1
°C. H NMR (DMSO-d6): δ 2.22 (s, 6H), 3.68 (s, 2H), 3.87 (s,
2H), 7.44 (s, 1H), 7.66 (d, J ) 2.0 Hz, 1H), 7.73 (d, J ) 2.0 Hz,
1H), 11.65 (br s, 1H).
(5-Meth oxy-7-n itr o-1H-in dol-3-yl)aceton itr ile (20). Com-
pound 19 (1.9 g, 7.6 mmol) was dissolved in a mixture of
dimethylformamide (DMF; 3.2 mL), water (3.2 mL), THF (150
mL), and methyl iodide (2.4 mL, 38 mmol). This mixture was
refluxed for 10 min, and then, potassium cyanide (4.0 g, 61
mmol) was added and the reflux kept for 5 h. After it was
cooled, the mixture was filtered and the filtrate was evaporated
under reduced pressure to give a crude residue that was
triturated with methanol and collected by filtration. Recrys-
tallization from toluene yielded 20 (1.19 g, 68%). Yellow solid;
1
mp 188-189 °C. H NMR (DMSO-d6): δ 3.89 (s, 3H), 4.13 (s,
2H), 7.52 (s, 1H), 7.72 (s, 1H), 7.75 (s, 1H), 11.77 (br s, 1H).
N-2-(5-Meth oxy-7-n itr o-1H-in d ol-3-yl)eth yla m in e h y-
d r och lor id e (21). To a solution of nitrile 20 (1 g, 4.3 mmol)
in dry THF (20 mL) was added a solution 1 M of borane-THF
complex. The mixture was warmed at reflux under nitrogen

1858 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Leclerc et al.
for 24 h. After the medium was cooled, an aqueous solution of
3 N HCl (17 mL) was added dropwise. The mixture was
evaporated under reduced pressure. The crude solid was
recrystallized in DMF to give yellow-green crystals (0.46 g,
39%); mp >250 °C. 1H NMR (DMSO-d6): δ 3.01-3.07 (m, 4H),
3.90 (s, 3H), 7.44 (s, 1H), 7.67 (d, J ) 2.1 Hz, 1H), 7.78 (d, J
) 2.1 Hz, 1H), 8.04 (br s, 3H), 11.65 (br s, 1H).
N -[2-(5-Me t h o x y -7-n it r o -1H -in d o l-3-y l)e t h y l]a c e t -
a m id e (22). The hydrochloride salt of amine 21 (0.46 g, 1.7
mmol) was dissolved in water (55 mL). Ethyl acetate (55 mL)
and potassium carbonate (0.7 g, 5 mmol) were then added.
Acetyl chloride (0.18 mL, 2.55 mmol) was added dropwise, and
stirring was continued for 2 h. The organic layer was dried
over MgSO4, filtered, and evaporated. The crude residue was
recrystallized from ethyl acetate to afford 22 as a yellow-orange
solid (0.31 g, 67%); mp 155-156 °C. 1H NMR (DMSO-d6): δ
1.79 (s, 3H), 2.84 (t, J ) 7.3 Hz, 2H), 3.32 (m, 2H), 3.89 (s,
3H), 7.33 (s, 1H), 7.65 (d, J ) 2.1 Hz, 1H), 7.69 (d, J ) 2.1 Hz,
1H), 7.97 (br t, J ) 5.4 Hz, 1H), 11.51 (br s, 1H). Anal.
(C13H15N3O4) C, H, N.
filtration with a cell harvester (Brandel M-48; Gaithersburg,
MD) connected to a vacuum pump (Edwards 18; Gennevilliers,
France) through glass-fiber filters (GF/B; Brandel) soaked in
0.5% (v/v) polyethylenimine. Filters were washed three times
with 1 mL of ice-cold 50 mM Tris-HCl buffer. Total filtration
time was less than 5 s. Radioactivity was measured in
γ-counter (Auto-Gamma 5000 series, Packard). Nonspecific
binding was estimated as binding in the presence of 30 µM
melatonin.15,26 For competition studies, 2-[125I]iodomelatonin
was incubated in the presence of increasing concentrations of
our compounds from 10-11 to 10-6 M. For saturation binding
assays, 0.125-20 nM of 2-[125I]iodomelatonin was used. Protein
contents were determined with Coomassie blue dye reagent
(Bio-Rad Laboratories, Inc., Richmond, CA) as previously
described,25 with BSA as standard.
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a
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Synthesis of Nitroindole Derivatives
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