Design, synthesis and biological evaluation of novel β-substituted indol-3-yl ethylamido melatoninergic analogues

Article abstract of DOI:10.1211/0022357021771869

A series of new melatonin analogues have been synthesized. Interestingly, two of the new compounds, 11c and 11e, which did not show any appreciable affinity for the melatonin receptor, were found to be potent inhibitors of lipid peroxidation in rat liver microsomes. Analogue 11c, in particular, is a better antioxidant than melatonin.

Full text of DOI:10.1211/0022357021771869

Communication
Journal of
JPP 2002, 54: 147–156
#
2002 The Authors
Received April 28, 2001
Accepted August 1, 2001
ISSN 0022-3573
Design, synthesis and biological evaluation of novel
b-substituted indol-3-yl ethylamido melatoninergic
analogues
K. Iakovou, A. Varvaresou, A. P. Kourounakis, K. Stead, D. Sugden
and A. Tsotinis
Abstract
A series of new melatonin analogues have been synthesized. Interestingly, two of the new
compounds, 11c and 11e, which did not show any appreciable afŽ nity for the melatonin
receptor, were found to be potent inhibitors of lipid peroxidation in rat liver microsomes.
Analogue 11c, in particular, is a better antioxidant than melatonin.
Introduction
The pineal hormone melatonin (N-acetyl-5-methoxytryptamine) is present in all
mammalian species including humans. Melatonin has been shown to have a
physiological role in regulating seasonal breeding in photoperiodic species
(Bartness et al 1993) and can entrain circadian rhythms (Arendt 1995). The
hormone also increases vascular tone in rat tail artery (Ting et al 1997) and cerebral
School of Pharmacy, Department
of Pharmaceutical Chemistry,
University of Athens,
Panepistimiopolis-Zografou,
3
vascular bed (Capsoni et al 1995) and inhibits [ H]dopamine release from rabbit
157 71 Athens, Greece
retina (Dubocovich 1983). In addition, a sleep-promoting action of melatonin has
been repeatedly observed in animal and human studies (Holmes & Sugden 1982;
Dollins et al 1994). Melatonin analogues are currently being examined as a therapy
for treating circadian rhythm disturbances resulting from various causes (e.g. jet-
lag, shift-work, blindness) and the use of melatonin as a hypnotic has been
advocated (Gar®nkel et al 1995). Molecular cloning data suggest that melatonin
exerts all of these eŒects through a family of speci®c, high-a nity, G-protein-
coupled cell-membrane receptors, MT , MT and Mel , (Reppert et al 1994, 1995),
K. Iakovou, A. Varvaresou,
A. Tsotinis
Department of Pharmaceutical
Chemistry, School of Pharmacy,
Aristotelian University of
Thessaloniki, Thessaloniki
540 06, Greece
A. P. Kourounakis
1
2
1c
which are particularly abundant in tissues known to respond to melatonin (e.g. in
the retina and the suprachiasmatic nuclei of the hypothalamus). Various other
eŒects have been ascribed to melatonin including eŒects on the immune system
Endocrinology & Reproduction
Research Group, GKT School of
Biomedical Sciences, New Hunts
House, Kings College London,
Guy’s Campus, London Bridge,
London SE1 1UL, UK
(Maestroni 1993), an ability to scavenge hydroxyl and peroxy free radicals (Reiter
et al 1999) and an oncostatic action (Molis et al 1995).
D. Sugden
Notwithstanding the renewed interest in melatonin and its actions in recent years,
the pharmacology of the hormone is still in its infancy as a limited number of
structurally diverse melatonin agonists and antagonists, which can be used as
pharmacological tools, is available. Our previous work has been directed at
mapping the interaction of melatonin with its receptor by synthesizing and
evaluating a variety of melatonin analogues (Garratt et al 1994a,1994b, 1995, 1996;
Davies et al 1998). We have shown, in a structure±activity relationship study of a
Correspondence: A. Tsotinis,
School of Pharmacy, Department
of Pharmaceutical Chemistry,
University of Athens,
Panepistimiopolis-Zografou,
157 71 Athens, Greece. E-mail:
tsotinis!pharm.uoa.gr
147

148
K. Iakovou et al
Figure 1 Structures and synthesis of series 9a±e and 10a±h analogues of melatonin.
series of simple aromatic molecules, that the minimal 2-phenyltryptamines annelated on the a-face of the
structural features for receptor recognition encompass pyrrole moiety by the introduction of 1, 2 or 3 methylene
an anisole or resorcinol dimethyl ether nucleus and an groups (Faust et al2000). These molecules wereprepared
alkylamide moiety (Garratt et al 1996). Furthermore, to probe the constraints at the receptor site with regard
we and others have demonstrated that the 5-methoxyl to the lower N1±C2 region of the indole nucleus.
group of melatonin is an important site for binding to
In this study, we report the synthesis of analogues
the receptor, but it is not an essential requirement for 9a±e, 10a±h and 11a±e (Figures 1 and 2, respectively),
agonist activity (Garratt et al 1994a; Davies et al 1998). which bear a keto and a hydroxy moiety b to the C3
The active conformation of the 3-ethanamide side chain acylamido chain. The constraint conferred on the side
has also been established from studies with confor- chain by the introduction of these groups functions as a
mationally restricted indole (Spadoni et al 1993; Garratt vicarious probe for the investigation of the interaction
et al 1994b; Davies et al 1998) and non-indole analogues of the upper part of the indole nucleus and especially the
(
Copinga et al 1993; Sugden et al 1995; Grol & Jansen C3 side chain with the receptor. Furthermore, a number
996; Jansen et al 1996; Leclerc et al 1996). of reports in recent years have supported the claim that
In our ongoing eŒort to probe the stereoelectronic melatonin possesses antioxidant activity (for review see
1
requirements for optimal activity we have recently re- Reiter 1997). As there is just one report on the anti-
ported on the synthesis and biological activity of novel oxidant action of analogues of melatonin (Gozzo et al

Novel b-substituted indol-3-yl ethylamido melatoninergic analogues
149
Figure 2 Synthesis of series 11a±e analogues of melatonin.
1999), representative members of the new series were 1-(1-Methyl-1H-indol-3-yl)ethanone (2)
tested for their ability to inhibit in-vitro non-enzymatic Methyl iodide (0.1 mL, 2.1 mmol) was added to a
peroxidation of microsomal membrane lipids.
stirred mixture of 1-(1H-indol-3-yl)ethanone (1) (0.3 g,
.9 mmol) and powdered potassium hydroxide (80% )
0.28 g, 4.4 mmol) in dimethylformamide (10 mL) at
°C. The resulting suspension was then stirred at room
temperature for 2 h before being poured into ice-water.
Extraction with chloroform followed by washing of the
organic phase with brine and drying over anhydrous
sodium sulfate aŒorded a yellow solution, which was
concentrated under reduced pressure to give0.20 g,90%
of the title compound as a white solid pure enough to be
1
(
0
Materials and Methods
Chemistry
Melting points were determined on a Buchi 530 ap-
$
1
paratus and are uncorrected. H NMR spectra were
recorded either on a Bruker AC200 or a Bruker
DRX400 MHz spectrometer, and the spectra are re-
used as such in the next step, mp 92±93°C (Kurihara et
1
1
3
al 1987, 94±96°C). H NMR (DMSO-d ): 2.33 (s, 3H,
COCH ), 3.77 (s, 3H, N-CH ), 7.35 (m, 3H, arom.), 7.77
ported in d. C NMR spectra were taken at 50 MHz on
a Bruker AC200 spectrometer. Tetramethylsilane was
used as internal standard. All the experiments were
carried out under an atmosphere of Argon. The solvents
6
3
3
(
s, 1H, H2), 8.46 (m, 1H, arom).
used were dried as follows: benzene using sodium wire; General method for the preparation of the a-bromo
triethylamine over sodium hydroxide; dimethylforma- ketones (3 and 4)
mide and dichloromethane over molecular sieves (4A
diethyl ether and tetrahydrofuran over calcium hydride a stirred suspension of 1-(1H-indol-3-yl)ethanone (1)
and acetone over molecular sieves (4A). DC-Alufolien (1.9 mmol) or 1-(1-methyl-1H-indol-3-yl)ethanone (2)
I
); Bromine (0.1 mL, 1.9 mmol) was added drop-wise to
I
plates (Kieselgel 60 F254, Schichtdicke 0.2 mm, Merck) (1.9 mmol) in methanol (3 mL) at 0°C. The resulting
were used for analytical TLC and were visualized with solution was then heated under re¯ ux for 2 h, cooled to
ultraviolet light or developed with iodine or phospho- ambient temperature and the solvent wasremoved under
molybdic acid. Microanalyses were carried out by the reduced pressure. The residue formed was treated with
Microanalytical Section of the Institute of Organic and water and made alkaline by adding sodium hydrogen
Pharmaceutical Chemistry, NHRF. Analyses indicated carbonate. Extraction with ethyl acetate followed by
by the symbols of the elements were within³0.4% of drying of the organic phase over anhydrous sodium
the theoretical values.
sulfate and concentration in-vacuo gave the desired

150
K. Iakovou et al
compounds 3 and 4 as mixtures with starting materials in dichloromethane. The aqueous layer was decanted
and 2, respectively. The mixtures were not separated oŒ and the organic phase was acidi®ed with dilute
but used as such for the synthesis of compounds 5 and hydrochloric acid, washed with water, dried over an-
1
6
.
hydrous sodium sulfate and concentrated in-vacuo to
give 10e and 10f as sole products and amides 9d and
1
0 g, 9e and 10h as mixtures. Crystallization of the latter
General method for the preparation of the analogues (7
and 8)
pairs of mixtures from benzene and tetrachlorome-
thane}cyclohexane, respectively aŒorded the respective
sole compounds as white solids.
Hexamethylenetetramine (0.02 mol) was added at am-
bient temperature to a stirred solution of 3 (0.02 mol) or
4
(0.02 mol) in acetone±chloroform (50:50) (40 mL).
N-[2-(1H-Indol-3-yl)-2-oxo-ethyl]acetamide (9a)
Yield (based on 1-(1H-indol-3-yl)ethanone 1: 26% . Mp
The resulting solution was stirred at this temperature
for 3 h. At the end of this period a precipitate was
formed, which corresponds to analogues 5 and 6, re-
spectively. The precipitate was ®ltered, air-dried and
treated with conc. hydrochloric acid (2.5 mL) and etha-
nol (15 mL). The mixture was stirred at room tem-
perature for 19 h and then extracted with ethyl acetate.
The organic phase was discarded and the aqueous phase
was evaporated to dryness under reduced pressure to
1
2
20°C (dec.) (Guella et al 1994, 225°C). H NMR
CDCl ): 2.16 (s, 3H, COCH ), 4.66 (s, 2H, CH ), 6.71
(
3
3
2
(
H2), 8.32±8.36 (m, 1H, arom), 8.77 (s, 1H, NH).
brs, 1H, NHCO), 7.34±7.44 (m, 3H, arom), 7.99 (s, 1H,
3
1
C
NMR (CDCl ): 28.5, 45.2, 111.2, 113.5, 119.9, 121.4,
3
1
22.1, 124.6, 131.6, 135.8, 169.3, 188.8.
give mixtures of the corresponding analogues 7 or 8 and N-[2-(1H-Indol-3-yl)-2-oxo-ethyl]propionamide (9b)
inorganic ammonium salts.
Yield (based on 1-(1H-indol-3-yl)ethanone 1: 22% . Mp
1
2
12°C (dec.). H NMR (CDCl ): 1.22 (t, 3H, J ¯ 7.5
3
Hz, CH CH ), 2.34 (q, 2H, J ¯ 7.5 Hz, CH CH ), 4.66
2
3
2
3
General method for the preparation of the N-[2-(1H-
indol-3-yl)-2-oxo-ethyl]alkanamides (9a±e) and N-[2-
(
arom.), 7.32 (m, 1H, arom.), 7.98 (s, 1H, H2), 8.30 (m,
s, 2H, COCH NH), 6.87 (s, 1H, NHCO), 7.28 (m, 2H,
2
(
(
1-methyl-1H-indol-3-yl)-2-oxo-ethyl]alkanamides
10a±h)
1H, arom.), 11.05 (s, 1H, NH). Anal. (C H N O ) C,
13 14 2 2
H, N.
For the synthesis of analogues 9a±c and 10a±d the
appropriate acid anhydride was added to the hydro-
chloride 7 or 8 (3.1 mmol) at 0°C. The mixture was then
treated with pyridine (0.8 mL), acetone (5 mL) and left
stirring at room temperature for 45 min. The solvents
were then removed in-vacuo and the residue formed was
acidi®ed with dilute hydrochloric acid and taken up in
ethyl acetate. The aqueous phase was discarded and the
N-[2-(1H-Indol-3-yl)-2-oxo-ethyl]butyramide (9c)
Yield (based on 1-(1H-indol-3-yl)ethanone 1: 26% . Mp
1
2
2
15°C. H NMR (CDCl ): 0.99 (m, 3H, CH ), 1.73 (m,
H, CH CH ), 2.31 (m, 2H, NHCOCH ), 4.68 (s, 2H,
3
3
2
3
2
COCH NH), 6.68 (s, 1H, NHCO), 7.27 (m, 2H, arom.),
2
7
.35 (m, 1H, arom.), 7.98 (s, 1H, H2), 8.67 (s, 1H, NH).
Anal. (C H N O ) C, H, N.
1
4
16
2
2
organic layer was washed with H O and brine. In the
2
case of compound 10d the organic layer was washed
with sodium hydroxide (10% ) to remove benzoic acid,
which was formed during the work up. Drying over
anhydrous sodium sulfate and evaporation of the sol-
ventunder reduced pressure gavethe desired compounds
as oŒ-white solids.
Cyclopropanecarboxylic acid [2-(1H -indol-3-yl)-2-oxo-
ethyl]amide (9d) Yield (based on 1-(1H-indol-3-yl)-
1
ethanone 1: 29% . Mp 238°C. H NMR (CDCl ): 0.68
3
(
arom.), 7.48 (m, 1H, arom.), 8.12 (m, 1H, arom.), 8.37
m, 4H), 1.72 (m, 1H), 4.47 (s, 2H, CH ), 7.18 (m, 2H,
2
(
(
brs, 2H, 1H arom. ­1H NH indole). Anal.
C H N O ) C, H, N.
For the synthesis of analogues 9d, e and 10 g, h
triethylamine (4.7 mmol, 0.7 mL) was added drop-wise
to a chilled solution of the appropriate acid chloride
14 14
2
2
(2.5 mmol) in dichloromethane (3 mL). For the syn- Cyclobutanecarboxylic acid [2-(1H-indol-3-yl)-2-oxo-
thesis of analogues 10e, f the quantity of the triethyl- ethyl]amide (9e) Yield (based on 1-(1H-indol-yl)-
1
amine added to the above solution was 8.7 mmol, ethanone 1: 25% . Mp 198°C. H NMR (CDCl ): 1.9
3
1
amine 7 or 8 (0.5 g) was added and the resulting mixture Hz, CH ), 6.60 (brs, 1H, NHCO), 7.38 (m, 3H, arom.),
.2 mL. Then, the hydrochloric acid salt of the b-keto (m, 2H), 2.3 (m, 4H), 3.1 (m, 1H), 4.63 (d, 2H, J ¯ 4.4
2
was allowed to thaw before stirring for 45 min. The 7.95 (s, 1H, H2), 8.30 (m, 1H, arom.), 8.96 (brs, 1H,
suspension was then poured into ice-water and taken up NH). Anal. (C H N O ) C, H, N.
1
5
16
2
2

Novel b-substituted indol-3-yl ethylamido melatoninergic analogues
151
N-[2-(1-Methyl-1H-indol-3-yl)-2-oxo-ethyl]acetamide CH ), 6.64 (brs, 1H, NHCO), 7.31 (m, 1H, arom.), 7.34
(
2
10a) Yield (based on 1-(1-methyl-1H-indol-3-yl)- (m, 2H, arom.), 7.85 (s, 1H, H2), 8.32 (m, 1H, arom.).
1
ethanone 2: 20% . Mp 164°C. H NMR (CDCl ): 2.1(s, Anal. (C H N O ) C, H, N.
3
16 18
2
2
3
Hz, CH ), 6.72 (s, 1H, NHCO), 7.30 (m, 3H, arom.),
H, COCH ), 3.86 (s, 3H, N-CH ), 4.61 (d, 2H, J ¯ 4.0
3
3
2
Cyclopropanecarboxylic acid [2-(1-cyclopropanecar-
bonyl-1H-indol-3-yl)-2-oxo-ethyl]amide (10g) Yield
7
.83 (s, 1H, H2), 8.30 (m, 1H, arom.). Anal.
C H N O ) C, H, N.
(
1
3
14
2
2
(
based on 1-(1H-indol-3-yl)ethanone 1: 26% . Mp
1
1
1
93°C. H NMR (CDCl ): 0.8 (m, 2H), 1.00 (m, 2H),
3
.24 (m, 4H), 1.38 (m, 2H), 4.72 (d, 2H, J ¯ 4.2 Hz,
N-[2-(1-Methyl-1H-indol-3-yl)-2-oxo-ethyl]propion-
amide (10b) Yield (based on 1-(1-methyl-1H-indol-3-
COCH ), 6.74 (brs, 1H, NHCO), 7.40 (m, 2H, arom.),
8.32 (m, 2H, arom.), 8.48 (m, 1H, arom.). Anal.
2
1
yl)ethanone 2: 29% . Mp 135°C. H NMR (CDCl ):
3
(
C H N O ) C, H, N.
18 18 2 3
1
.23 (t, 3H, J ¯ 7.7 Hz, CH CH ), 2.34 (q, 2H, J ¯ 7.7
2
3
Hz, CH CH ), 3.88 (s, 3H, N-CH ), 4.64 (d, 2H, J ¯ 4.0
Hz, COCH ), 6.75 (brs, 1H, NHCO), 7.36 (m, 3H,
2
3
3
2
Cyclobutanecarboxylic acid [2-(1-cyclobutanecarbonyl-
H-indol-3-yl)-2-oxo-ethyl]amide (10h) Yield (based
1
3
arom), 7.85 (s, 1H, H2), 8.32 (m, 1H, arom.). C NMR
CDCl ): 9.6, 29.3, 35.5, 46.1, 109.7, 113.6, 121.9, 122.8,
1
(
1
3
on 1-(1H-indol-3-yl)ethanone 1: 24% ; Mp 108°C. H
NMR (CDCl ): 1.84±2.61 (m, 12H), 3.01±3.23 (m, 1H,
123.5, 125.8, 135.3, 173.7, 188.4. Anal. (C H N O ) C,
14 16 2 2
H, N.
3
COCH), 3.79±3.96 (m, 1H, COCH(CH ) ), 4.64 (d, 2H,
J ¯ 4.4 Hz, COCH ), 6.54 (brs, 1H, NHCO), 7.25±7.44
2
3
2
(m, 2H, arom.), 8.09 (s, 1H, H2), 8.23±8.28 (m, 1H,
arom.), 8.44±8.48 (m, 1H, arom.). Anal. (C H N O )
C, H, N.
N-[2-(1-Methyl-1H-indol-3-yl)-2-oxo-ethyl]butyra-
mide (10c) Yield (based on 1-(1-methyl-1H-indol-3-
2
0
22
2
3
1
yl)ethanone 2: 28% . Mp 147°C. H NMR (CDCl ): 0.9
3
(
t, 3H, J ¯ 7.3 Hz, CH CH ), 1.74 (m, 2H, CH CH ),
2
3
2
3
2
.27 (t, 2H, J ¯ 7.5 Hz, NHCOCH ), 3.87 (s, 3H, N- General method for the preparation of the N-[2-
2
CH ), 4.62 (d, 2H, J ¯ 4.0 Hz, COCH ), 6.72 (brs, 1H, hydroxy-2-(1-methyl-1H-indol-3-yl)ethyl]alkanamides
NHCO), 7.38 (m, 3H, arom.), 7.86 (s, 1H, H2), 8.34 (m, (11a±e)
3
2
1
H, arom.). Anal. (C H N O ) C, H, N.
A suspension of sodium hydroxide (0.015 mmol) in
water (0.1 mL) and methanol (0.6 mL) was added to a
chilled mixture of the b-keto alkanamides 10a±c, 10e, f
and sodium borohydride (0.1 mmol). The mixture was
then allowed to reach ambient temperature and left
stirring for 1 h. Upon completion of the reaction the
solvent was removed under reduced pressure and the
residue formed was acidi®ed with dilute hydrochloric
acid and extracted with ethyl acetate. The organic phase
was dried over anhydrous sodium sulfate and concen-
trated in-vacuo to give the title compounds as oŒ-white
solids, which were recrystallized from cyclohexane.
1
5
18
2
2
N-[2-(1-Methyl-1H-indol-3-yl)-2-oxo-ethyl]benzamide
10d) Yield (based on 1-(1-methyl-1H-indol-3-yl)-
(
1
ethanone 2: 24% . Mp 94°C. H NMR (CDCl ): 3.87 (s,
3
3
H, N-CH ), 4.84 (d, 2H, J ¯ 4.0 Hz, CH ), 7.26±8.17
3
2
(m, 10H arom. ­1H NHCO). Anal. (C H N O ) C, H,
1
8
16
2
2
N.
Cyclopropanecarboxylic acid [2-(1-methyl-1H-indol-3-
yl)-2-oxo-ethyl]amide (10e) Yield (based on 1-(1-
1
methyl-1H-indol-3-yl)ethanone 2: 20% . Mp 171°C. H
NMR (CDCl ): 0.82 (m, 2H), 1.02 (m, 2H), 1.56 (m,
3
N-[2-Hydroxy-2-(1-methyl-1H-indol-3-yl)ethyl]aceta-
1
1
COCH ), 6.90 (brs, 1H, NHCO), 7.37 (m, 3H, arom.),
H), 3.88 (s, 3H, N-CH ), 4.66 (d, 2H, J ¯ 4.0 Hz,
3
mide (11a) Yield: 82% . Mp 145°C. H NMR
(CDCl ): 2.1 (s, 3H, COCH ), 3.6±3.8 (m, 6H, N-CH ,
2
3
3
3
7
.85 (s, 1H, H2), 8.34 (m, 1H, arom.). Anal.
C H N O ) C, H, N.
CH(OH) and CH NH), 6.75 (brs, 1H, NHCO),
6.90±7.35 (m, 4H, arom.), 7.62 (m, 1H, arom.). Anal.
2
(
1
5
16
2
2
(
C H N O ) C, H, N.
13 16 2 2
Cyclobutanecarboxylic acid [2-(1-methyl-1H-indol-3-
yl)-2-oxo-ethyl]amide (10f) Yield (based on 1-(1- N-[2-Hydroxy-2-(1-methyl-1H-indol-3-yl)ethyl]pro-
1
1
methyl-1H-indol-3-yl)ethanone 2: 21% . Mp 150°C. H pionamide (11b) Yield: 79% . Mp 140°C. H NMR
NMR (CDCl ): 1.90 (m, 2H), 2.22 (m, 4H), 3.16 (m, 1H, (CDCl ): 1.05 (m, 3H, COCH CH ), 2.1 (q, 2H, J ¯ 7.9
3
3
2
3
CH), 3.86 (s, 1H, N-CH ), 4.62 (d, 2H, J ¯ 4.0 Hz, Hz, COCH CH ), 3.55±3.80 (m, 6H, N-CH , CH(OH)
3
2
3
3

152
K. Iakovou et al
9
and CH NH), 6.70 (brs, 1H, NHCO), 6.80±7.30 (m, 4H, the analogues for 60 min before melatonin (10­ m)
arom.), 7.61 (m, 1H, arom.). Anal. (CHN) C, H, N. was added. The concentration of analogue reducing
2
Anal. (C H N O ) C, H, N.
melatonin-induced pigment aggregation by 50% (IC50)
was determined.
1
4
18
2
2
N-[2-Hydroxy-2-(1-methyl-1H-indol-3-yl)ethyl]buty-
1
ramide (11c) Yield: 75% . Mp 115°C. H NMR
In-vitro lipid peroxidation assay
(
CDCl ): 0.64±0.90 (m, 2H, COCH CH CH ), 2.07 (s,
3
2
2
3
The hepatic microsomal fraction from untreated female
Fischer-344 rats (180±220 g) was prepared as described
elsewhere (Rekka et al 1989). The incubation mixture
contained heat inactivated (90°C for 90 s)hepatic micro-
somal fraction corresponding to 2.5 mg protein per mL
2
CH(OH) and CH NH), 6.70 (brs, 1H, NHCO),
H, COCH CH CH ), 3.55±3.80 (m, 6H, N-CH ,
2 2 3 3
2
6
.80±7.30 (m, 4H, arom.), 7.55 (m, 1H, arom.). Anal.
C H N O ) C, H, N.
(
1
5
20
2
2
(
(
®nal concentration) or 4 mm fatty acid residues
Eichenberger et al 1982), ascorbic acid (0.2 nm) in Tris-
Cyclopropanecarboxylic acid [2-hydroxy-2-(1-methyl-
1
1
1
H-indol-3-yl)ethyl]amide (11d) Yield: 52% . Mp
1
HCl}KCl buŒer (50 mm}150 mm, pH 7.4) and various
concentrations (1.0±0.5 mm) of the test compounds dis-
solved in DMSO. The reaction was started by the
21°C. H NMR (CDCl ): 0.53±1.00 (m, 4H), 1.26 (m,
H), 3.59 (brs, 2H, CH NH), 3.76 (s, 3H, N-CH ), 3.87
3
2
3
(brs, 1H, CH(OH)), 6.80 (brs, 1H, NHCO), 6.90±7.20
addition of a freshly prepared FeSO solution (10 lm)
4
(m, 4H, arom.), 7.62 (m, 1H, arom.). Anal. (C H N O )
C, H, N.
1
5
18
2
2
and the mixture was incubated at 37°C for 45 min.
Samples (0.3 mL) from the incubation mixture were
taken at various time intervals. Lipid peroxidation was
assessed spectrophotometrically (535 against 600 nm)
by the determination of the 2-thiobarbituric acid (TBA)
reactive material (Kourounakis et al 1999). Under the
above experimental conditions, all compounds, includ-
ing DMSO, were tested and found not to interfere with
the assay. Each experiment was performed at least in
duplicate.
Cyclobutanecarboxylic acid [2-hydroxy-2-(1-methyl-
1
1
1
6
H-indol-3-yl)ethyl]amide (11e) Yield: 71% . Mp
1
12°C. H NMR (CDCl ): 1.78±2.14 (m, 6H), 2.97 (m,
H), 3.44±3.69 (m, 6H, N-CH , CH(OH) and CH NH),
.74±7.63 (m, 5H, arom.). Anal. (C H N O ) C, H, N.
3
3
2
1
6
20
2
2
Pharmacology
Hydrophobicity, expressed as clogP values, of com-
pounds, was calculated with the programme CLOGP,
v4.62, Daylight Chemical Information Systems Inc.
Xenopus melanophore model for the evaluation of
agonistic and antagonistic activity
Melanophore cells were grown in 96-well tissue culture
plates, and growth medium (Daniolos et al 1990;
Potenza & Lerner 1992) was replaced with 0.7¬L-15
culture medium 18 h before analogues were tested.
Initial absorbance of cells (Ai, 630 nm) was measured
in each well using a Bio-Tek microtiter plate reader
Results and Discussion
Chemistry
(model EL3115, Anachem, UK), then cells were treated The synthetic route followed for the preparation of the
with the concentrations of the analogues indicated. All new derivatives 9a±e and 10a±h is depicted in Figure 1.
experiments used triplicate wells at six concentrations Thus, commercially available 1-(1H-indol-3-yl)etha-
9
4
(
10­ ±10­ m) of analogue (each well contains
C5000
none (1) and 1-(1-methyl-1H-indol-3-yl)ethanone (2),
cells and so is the average response of a large number prepared from 1 upon treatment with iodomethane in
of melanophores). S.e.m.s were always less than 5% the presence of potassium hydroxide, were reacted with
of the mean. The ®nal absorbance (Af ) was measured bromine in methanol to give the a-bromo ketones 3
after 60 min, and the fractional change in absorbance (Guella et al 1994) and 4, respectively. These were in
(
1®Af}Ai) was calculated. Vehicle did not alter pig- turn treated with hexamethylenetetramine in acetone±
ment granule distribution itself or inhibit responses to chloroform to aŒord the tetra-aza analogues 5 and 6.
melatonin. The concentration of analogue producing The latter were hydrolysed with concentrated hydro-
5
0% of the maximum agonist response (EC50) was chloric acid in ethanol to the corresponding ammonium
determined from concentration±response curves. For chloride salts 7 (Guella et al 1994) and 8, which were
evaluation of antagonist potency, cells were treated converted to the target molecules 9a (Guella et al 1994),
with vehicle (1% dimethyl sulfoxide (DMSO) or 9b±e and 10a±h either by reaction with the appropriate
4
9
methanol) or varying concentrations (10­ ±10­ m) of acid anhydride, compounds 9a±c and 10a±d, or with the

Novel b-substituted indol-3-yl ethylamido melatoninergic analogues
153
analogous acid chloride, compounds 9d, e and 10e±h congener alkanamide I (Figure 3) (Garratt et al 1995)
Faust et al 2000). seems to lead to antagonism. Interestingly, in the case of
The synthetic route followed for the preparation of 11e the replacement of the b-methyl substituent in II
(
the b-hydroxy amides 11a±e is shown in Figure 2. It (Figure 3) (Garratt et al 1995) by a b-hydroxyl group
involved reduction of the ketones 10a±c and 10e, f with does not enhance its antagonistic activity as for 11e no
analkalinesolution of sodium borohydride in methanol. antagonist eŒect was detected at concentrations up to
4
1
0­ m.
In the b-keto series, 9a±e and 10a±h, the in¯ uence on
Pharmacology
antagonism of the constraint imposed on the side chain
does not seem to be a critical factor. Thus, within these
series compound 10h inhibited the binding of the mela-
tonin response by 65% . Next in potency was analogue
As mentioned above, the agonist and antagonist potency
of the new analogues was assessed in the Xenopus laevis
melanophore model. None of the tested compounds
exhibited any agonist action at concentrations up to
4
9
e (37% inhibition), which, like 10h, bears an N-cyclo-
1
0­ m, while six of them (9d, 9e, 10g, 10h, 11a and 11b)
butanoyl group in its side chain, a moiety which is
known to exert a detrimental eŒect on agonistic activity
while it enhances antagonism (Leclerc et al 1998). The
marked diŒerence in potency observed between com-
pounds 10h and 9e might be attributed to the presence
of a second N-cyclobutanoyl group in the former at N-
were melatonin antagonists at this maximum concen-
tration (Table 1). The most potent antagonist was
compound 11b (pIC50 ¯ 4.68), which inhibited pigment
9
aggregation due to melatonin (10­ m) by 87% . This
compound, like its counterpart 11a, bears a b-hydroxyl
group in its side chain. The diminution in potency
observed in the case of 11a (57% inhibition) compared
with that of 11b might be due to the presence of the more
lipophilic acylamido chain in the latter, which could
enhance hydrophobic interactions with the receptor.
The introduction of a hydroxyl group in 11a in place
of the b-methyl moiety in the C-3 side chain of its
1
. Presumably the extra N-cyclobutanoyl moiety at this
position reduces further the ability of the receptor
binding pocket to recognize and bind this molecule, thus
leading to reduced antagonistic activity. However, an
analogous trend was not observed in the case of the bis
N-cyclopropyl analogue 10 g, which was equipotent
with its mono N-cyclopropanoyl counterpart, 9d.
Besides investigating the agonist and antagonist pro-
®
le of the new compounds, the free radical scavenging
Table 1 Antagonistic activity of compounds 9±11 in the Xenopus
laevis melanophore assay; clogP values of selected analogues.
properties of selected analogues of both series were also
studied. Many reports claim that melatonin exhibits
antioxidant activity. For example, it has been reported
Compound
R1
R
clogP
Antagonist
inhibition
E
that melatonin has the ability to detoxify the LOO
4
(
% ) at 10­ m
radical, which, apart from being highly reactive, re-
initiates (propagates) the process of the oxidation of
lipids (Pieri et al 1994). Lipid peroxidation is especially
devastating in the CNS, which is particularly rich in
lipids that can be readily oxidized. Besides melatonin
(Pappolla et al 1997), it has been shown that analogues
like indole-3-propionic acid (Chyan et al 1999) are
capable of reducing lipid peroxidation caused by amyl-
Melatonin
Ð
9
9
9
9
9
1
1
1
1
1
1
1
1
1
1
1
1
1
a
H
CH3
C H5
NA
NA
NA
35
b
H
2
c
H
C H7
3
1.95
1.14
d
H
c-C H5
3
e
H
c-C H7
4
37
0a
0b
0c
0d
0e
0f
0g
0h
1a
1b
1c
1d
1e
CH3
CH3
CH3
CH3
CH3
NA
NA
NA
NA
NA
NA
32
C H5
2
C H7
3
2.19
2.79
oid b-protein (A ), thus protecting primary neurons and
b
C H5
6
neuroblastoma cells against oxidative damage and con-
sequent death.
CH3
c-C H5
3
CH3
c-C H7
4
2.05
To the best of our knowledge there is only one report
on melatonin analogues as inhibitors of non-enzymatic
lipid peroxidation (Gozzo et al 1999), so we examined
the eŒect of seven representative compounds reported
herein (9c, 10a, 10c, 10d, 10f, 11c and 11e) on the in-
vitro peroxidation of rat hepatic microsomal membrane
lipids. Most of these compounds exhibited a modest
(11e, 9c, 10d, 10f) or no (10a, 10c) antioxidant activity,
with the exception of 11c, which signi®cantly inhibited
C(O)c-C H5
3
c-C H5
3
C(O)c-C H7
4
c-C H7
4
65
57
CH3
CH3
CH3
C H5
2
87
CH3
3
C H7
1.68
1.53
NA
NA
NA
CH3
c-C H5
3
CH3
c-C H7
4
4
NA, no antagonist eŒect detected at concentrations up to 10­ m.

154
K. Iakovou et al
Figure 3 Structures of compounds I and II (Garratt et al 1995).
lipid peroxidation with an IC50
0
of approximately
45 min)
(
.5 mm, (Figure 4).
The enhanced potency of 11c and, to a lesser extent,
1e might be attributed to the presence of the benzylic-
like b-hydrogen in their acylamido side chains. Ab-
1
E
straction of this hydrogen by the LOO radical may lead
to detoxi®cation of the latter and to the formation of the
indolyl radicals 12c, e (Figure 5), which are stabilized by
the presence of the hydroxyl group and their conjugation
with the aromatic system (cf. resonance structures
1
2c, e±13c, e). Conversely, the absence of benzylic-like
hydrogens in the b-keto analogues, 9c, 10a, 10c, 10d and
0f, leads to reduced or no potency. Also, the inactive
1
compounds have the tendency to participate much less
in free radical processes than the active compounds due
to the greater stability of the keto-indole system through
extended conjugation.
As hydrophobicity is known to aŒect antioxidant
activity in many cases (Ohkawa et al 1991), clogP values
were calculated for the tested compounds, Table 1. The
results obtained indicate that the b-hydroxy analogues
11c and 11e are less lipophilic than their b-keto counter-
Figure 4 Time course of lipid peroxidation as aŒected by 1 mm
(
and 0.5 mm) of compounds 9c, 10a, 10c, 10d, 10f, 11e and 11c.
E
Figure 5 Proposed mechanism of detoxi®cation of the LOO radical by analogues 11c, e.

Novel b-substituted indol-3-yl ethylamido melatoninergic analogues
155
parts, a fact that might be partially attributed to the Faust, R., Garratt, P. J., Jones, R., Yeh, L.-K., Tsotinis, A.,
Panoussopoulou, M., Calogeropoulou, T., Teh, M.-T., Sugden, D.
enhanced capability of the former to form hydrogen
bonds. Nevertheless, the diŒerence in lipophilicity of
(2000) Mapping the melatonin receptor. 6. Melatonin agonists and
antagonists derived from 6H-Isoindolo[2,1-a]indoles, 5,6-Dihydro-
indolo[2,1-a]isoquinolines, and 6,7-Dihydro-5H-benzo[c]azepino
[2,1-a]indoles. J. Med. Chem. 43: 1050±1061
1
1
1c and 11e, compared with that of 9c, 10a, 10c, 10d and
0f, appears to play a less signi®cant role towards
antioxidant activity than the presence of the benzylic- Gar®nkel, D., Laudon, M., Nof, D., Zisapel, N. (1995) Improvement
of sleep quality in elderly people by controlled-release melatonin.
Lancet 346: 541±544
like hydrogen.
Garratt, P. J., Jones, R., Rowe, S. J., Sugden, D. (1994a)Mapping the
Conclusion
melatonin receptor. 1. The 5-methoxy group of melatonin is not an
essential requirement for biological activity. Bioorg. Med. Chem.
Lett. 4: 1555±1558
Under our experimental conditions, melatonin wasequi-
potent with 11e, less potent than 11c and more potent Garratt, P. J., VonhoŒ, S., Rowe, S. J., Sugden, D. (1994b)Mapping
the melatonin receptor. 2. Synthesis and biologicalactivity of indole
than the other analogues. It is probable, however, that
derived melatonin analogues with restricted conformations of the
the antioxidant activity of 11c and 11e follows diŒerent
pathways to that of melatonin, which does not possess a
C-3 amidoethaneside chain.Bioorg. Med. Chem. Lett. 4: 1559±1564
Garratt, P. J., Jones, R., Tocher, D. A., Sugden, D. (1995) Mapping
side chain hydroxyl group. Melatonin, upon donation
of one of the two electrons of the N-1 lone pair, is
transformed to an indolyl cation radical (Reiter 1998).
Based on these ®ndings we are currently involved
with the synthesis and screening of melatonin analogues
of diverse structures.
the melatonin receptor. 3. Design and synthesis of melatonin
agonistsand antagonistsderivedfrom2-phenyltryptamines.J. Med.
Chem. 38: 1132±1139
Garratt, P. J., Travard, S., VonhoŒ, S., Tsotinis, A., Sugden, D.
(1996) Mapping the melatonin receptor. 4. Comparison of the
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Med. Chem. 39: 1797±1805
Gozzo, A., Lesieur, D., Duriez, P., Fruchart, J.-C., Teissier, E. (1999)
Structure-activity relationships in a series of melatonin analogs
with the low-density lipoprotein oxidation model. Free Rad.
BioMed. 26: 1538±1543
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