Design, synthesis and melatoninergic activity of new unsubstituted and β,β′-difunctionalised 2,3-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-6-alkanamides

Article abstract of DOI:10.1016/j.ejmech.2007.01.005

A series of new 2,3-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-6-alkanamides, with and without alkyl and cycloalkyl moieties in the β-position of the alkanamido side chain, have been prepared and tested for their ability to activate pigment granule aggregation

Full text of DOI:10.1016/j.ejmech.2007.01.005

European Journal of Medicinal Chemistry 42 (2007) 1004e1013
http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and melatoninergic activity of new unsubstituted
and b,b0-difunctionalised 2,3-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-
6-alkanamides
a
a
Andrew Tsotinis a, , Maria Panoussopoulou , Andreas Eleutheriades ,
*
Kathryn Davidson b, David Sugden b
a Faculty of Pharmacy, Department of Pharmaceutical Chemistry, University of Athens, Panepistimioupoli-Zografou, 157 71 Athens, Greece
b Division of Reproduction and Endocrinology, School of Biomedical and Health Sciences, King’s College London, Guy’s Campus,
London Bridge, London SE1 1UL, UK
Received 10 October 2006; received in revised form 3 January 2007; accepted 5 January 2007
Available online 21 January 2007
Abstract
A series of new 2,3-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-6-alkanamides, with and without alkyl and cycloalkyl moieties in the b-position of
the alkanamido side chain, have been prepared and tested for their ability to activate pigment granule aggregation in Xenopus laevis melano-
phores and bind to the recombinant human MT1 and MT2 melatonin receptor subtypes expressed in NIH 3T3 cells. An increase of the spacer’s
length in the side chain by a methylene unit (from 17d to 21d) leads to a six-fold decrease in antagonistic activity. On the other hand, the
introduction of two methyl groups in the b-position of the side chain of 17a induces agonist potency (compound 24), implying thus that the
two b-methyl groups are not only tolerated by the receptor, but constitute functional probes in its dynamic agonisteantagonist conformational
equilibrium. The presence of more bulky b-substituents, regardless of the size of the R group, compounds 24a,b, seems to lead to antagonism
and to a noteworthy MT2 subtype selectivity. Last, the new N1eC7 annulated derivatives presented herein are substantially more potent than
their respective N1eC2 annulated counterparts, previously reported.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: N1eC7 annulated indoles; Synthesis; Melatoninergic activity
1. Introduction
recently been granted approval for the treatment of insomnia
associated with sleep onset in the USA [5]. Sleep problems
are common in the elderly [6] and the lack of melatonin,
which decreases with age, may be a major factor. It also has
therapeutic potential in Seasonal Affective Disorders (SAD)
[7] and as an agent in restoring circadian rhythms both in
the blind and where these have been disturbed by shift-
work or jet-lag [8]. Melatonin has also been implicated in
Alzheimer’s and other neurological disorders [9], in certain
cancers [10] and in Parkinson’s disease [11]. Furthermore,
due to its hydrophobic and hydrophilic nature [1,12] melatonin
can easily enter cells and partition itself between ‘‘shallow
subsurface’’ and intracellular compartments, where it effec-
tively scavenges both water-soluble peroxy (ROOꢀ) [13] and
lipoperoxy (LOOꢀ) radicals [14,15].
Melatonin (Fig. 1) is a hormone synthesized by the pineal
gland of mammals, including humans [1]. It plays a critical
role in the regulation of reproduction in seasonally breeding
mammals, where it has been commercially exploited [2]. It
has a major role in the regulation of circadian rhythms in
non-mammalian vertebrates and is a component of their regu-
lation in mammals [3]. Melatonin has a hypnotic action in an-
imals and humans [4] and ROZEREMÔ (ramelteon) (Fig. 1),
a potent melatonin MT1 and MT2 receptor agonist, has
* Corresponding author. Tel.: þ30 210 7274812; fax: þ30 210 7274747.
E-mail address: tsotinis@pharm.uoa.gr (A. Tsotinis).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.01.005

A. Tsotinis et al. / European Journal of Medicinal Chemistry 42 (2007) 1004e1013
NHCOCH3
1005
NHCOCH3
H
CH2CH2NHCOC2H5
O
CH3O
N
H
N
H
Melatonin
ROZEREM™ (ramelteon)
Luzindole
Fig. 1. Structures of melatonin, ROZEREMÔ (ramelteon) and luzindole.
The physiological actions of melatonin in regulating sea-
sonal and circadian rhythms are mediated through a family
of specific, high affinity, G-protein-coupled cell membrane re-
ceptors [16]. Radioligand binding studies using 2-[125I]-mela-
tonin have revealed a widespread distribution of binding sites
throughout the nervous system [17]. The distribution of bind-
ing sites shows that they are particularly abundant in tissues
that respond to melatonin, including the retina, suprachiasmatic
nucleus (SCN), the pars tuberalis of the pituitary, and cerebral
and tail arteries [18]. Two receptor subtypes have been cloned
in mammals, MT1 (Mel1a) and MT2 (Mel1b), and a third,
Mel1c, in chicken, the Xenopus laevis and zebrafish [19].
The way in which melatonin binds at these receptors and
the possible therapeutic potential of melatonin in a wide vari-
ety of clinical conditions has led to a surge of interest in the
synthesis of agonists and antagonists to its actions. The prep-
aration of compounds that can discriminate between the recep-
tor types is a major goal [20], since together with the genetic
disruption of specific receptors [21] these could provide tools
for targeting specific functions.
In our ongoing effort to probe the stereoelectronic require-
ments for optimal melatoninergic activity we have previously
reported the synthesis and biological activity of novel 2-phenyl-
tryptamines annulated on the a-face of the pyrrole moiety by the
introduction of 1, 2 or 3 methylene groups, 2: n ¼ 1, 2, 3 (Fig. 2)
[20]. This work was extended by the synthesis of the
N-[2-(6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl)ethyl]alka-
namides, 3aee and N-[2-(2-methoxy-6,7,8,9-tetrahydropyr-
ido[1,2-a]indol-10-yl)ethyl]alkanamides, 4aee (Fig. 2) [22].
In the Xenopus melanophore pigment aggregation model, the
melatoninergic ligands 2: n ¼ 1 are agonists while their conge-
ners 2: n ¼ 3 were found to be antagonists. Molecules 2: n ¼ 2
are either antagonists (R5 ¼ H) or agonists (R5 ¼ OCH3). Simi-
larly, the non-methoxy substituted compounds, 3, are antago-
nists while their 2-methoxy counterparts, 4, are full agonists,
the butyramido analog 4 (R ¼ n-C3H7) being almost as potent
(pEC50 ¼ 9.91) as melatonin (pEC50 ¼ 10.07).
Compounds, 2e4, probe the constraints at the receptor site
with regard to the lower N1eC2 region of the indole moiety.
In order to explore a possible synergistic influence on potency
upon introducing alkyl and cycloalkyl groups in the b-position
of the alkanamido side chain, we recently reported the synthe-
sis and biology of the b,b0edisubstituted 6,7,8,9-tetrahydro-
pyrido[1,2-a]indol-10-yl ethylamido melatoninergic ligands
5aed and 6e9 (Fig. 2) [23]. The results we obtained sug-
gested that the new 5-methoxy (numbering with respect to
the indole ring) substituted analogs 5c,d, like their analogous
congeners 4 (R ¼ CH3 and n-C3H7) are full agonists. Con-
versely, the activity of the new non-methoxy substituted mol-
ecules ranges from antagonists, in the case of compounds 6e9,
to partial agonists in the case of ligands 5a,b. The activity of
5a,b contrasts with that of their analogous counterparts 3
which are all antagonists.
Based on these findings we designed and synthesized the
N1eC7 annulated 2,3-dihydro-1H-pyrrolo[3,2,1-ij]quinolino-
6-alkanamides 17aee and 21aee (Scheme 1), and their b,b0-
difunctionalised counterparts 24 and 27a,b (Scheme 2).
Similarly to their a-face annelated congeners 3, the new
b-unsubstituted analogs 17 are antagonists in the Xenopus me-
lanophore assay. The antagonistic character remains unaltered
( ) n
CH
3 NHCOR
NHCOR
NHCOR
NHCOR
H3C
R5
R5
R5
N
N
N
N
( )n
5a: R5 = H, R = CH3
3a: R5 = H, R = CH3
3b: R5 = H, R = C2H5
3c: R5 = H, R = n-C3H7
3d: R5 = H, R = c-C3H5
3e: R5 = H, R = c-C4H7
4: R5 = OCH3, R = CH3,
C2H5, n-C3H7, c-C3H7,
c-C4H7
6: n = 1, R = CH3
7: n = 1, R = n-C3H7
8: n = 3, R = CH3
9: n = 3, R = n-C3H7
2: n = 1, 2, 3
5b: R5 = H, R = n-C3H7
5c: R5 = OCH3, R = CH3
5d: R5 = OCH3, R = n-C3H7
Fig. 2. Structures of melatoninergic ligands 2e9.

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A. Tsotinis et al. / European Journal of Medicinal Chemistry 42 (2007) 1004e1013
CONH2
CN
a
b
c
a
b
N
H
N
N
N
N
N
NO
NH2
10
11
12
15
22
CO2H
CO2CH3
CONH2
NHCOCH3
d
e
f
CN
N
N
N
N
c
N
13
14
15
NH2
NHCOR
23
24
g
a: R=CH3
CN
CN
N
b: R=C2H5
d
e
c: R=n-C3H7
d: R=c-C3H5
e: R=c-C4H7
N
N
16
17
22
25
CN
CHO
CO2CH3
NHCOR
NH2
j
h
i
N
N
N
N
f
N
N
14
18
19
a: R=CH3
26
27
NHCOR
NH2
b: R=C3H7
Scheme 2. (a) (CF3CO)2O, Et3N, CH2Cl2, 0 ꢁC; (b) CH3I, NaH, DMF; (c)
H2/Raney-Ni, Ac2O, AcONa; (d) 1,5-dibromopentane, NaH, DMF/Et2O; (e)
LiAlH4, C6H6/Et2O, reflux; (f) (RCO)2O, Et3N, CH2Cl2.
g
a: R=CH3
b: R=C2H5
c: R=n-C3H7
d: R=c-C3H5
e: R=c-C4H7
N
21
20
used as internal standard. All the experiments were carried
out under an atmosphere of argon. Elemental analyses (C,
H, N) were carried out by the Microanalytical Section of the
Institute of Organic and Pharmaceutical Chemistry, NHRF.
Scheme 1. (a) NaNO2, conc. HCl, H2O; (b) LiAlH4, THF; (c) HO2C(CH2)2-
COCO2H, conc. HCl, AcOH, 80 ꢁC; (d) CH3I, K2CO3, DMF; (e) NH3 (gas),
CH3OH; (f) LiAlH4, THF, reflux; (g) (RCO)2O or RCOCl, Et3N, CH2Cl2;
(h) DIBAL-H, toluene, e78 ꢁC; (i) TosMIC, t-BuOK, DME, ꢂ30 ꢁC to
60 ꢁC; (j) LiAlH4, C6H6/Et2O, reflux.
DC-Alufolien plates (Kieselgel 60 F254
,
Schichtdicke
0.2 mm, Merck) were used for analytical TLC and were visu-
alized with ultraviolet light or developed with iodine or phos-
phomolybdic acid. Flash chromatography was performed
using Sorbsil c60-A silica as the stationary phase. Spinning
plate chromatography (SPC) was performed in a Chromatotron
apparatus (Model 7924), using plates of 4 mm thickness
coated with Merck Kieselgel GF254 silica gel.
and in the case of the N-acylated propanamines 21. Within the
new b,b0-difunctionalised series, the antagonistic potency drops
on moving from R ¼ n-C3H7 (27b) to R ¼ CH3 (27a). An inter-
esting shift towards partial agonism is observed when R and b,b0
are all methyl (24). Compounds 27a,b have some MT2 subtype
selectivity.
2.2. Synthesis of 1-nitroso-1,2,3,4-tetrahydroquinoline
(11)
2. Experimental
The title compound was prepared upon nitrosation of com-
mercially available 1,2,3,4-tetrahydroquinoline (10), following
the method of Paris et al. [24].
2.1. Instrumentation and chemicals
Melting points were determined on a Buchi 530 apparatus
¨
and are uncorrected. H NMR spectra were taken in CDCl3
1
and recorded either on a Bruker AC 200 (200 MHz) or on
a Bruker DRX 400 (400 MHz) spectrometer, and the spectra
are reported in d. 13C NMR spectra were taken at 50 MHz
on a Bruker AC 200 spectrometer. Tetramethylsilane was
2.3. Synthesis of 1-amino-1,2,3,4-tetrahydroquinoline (12)
The desired compound was obtained in 80% yield upon
reduction of 1-nitroso-1,2,3,4-tetrahydroquinoline (11) with

A. Tsotinis et al. / European Journal of Medicinal Chemistry 42 (2007) 1004e1013
1007
lithium aluminum hydride in THF. The 1H NMR spectral data
are in full agreement with those reported [24].
hydride. The resulting suspension is stirred for 30 more
minutes and filtered through Celite. The filtrate is diluted
with AcOEt (20 mL), washed with H2O (2 ꢃ 20 mL) and sat-
urated aqueous NaCl (2 ꢃ 25 mL) and dried (Na2SO4). The
solvent is removed under reduced pressure and the desired
amine 16 is obtained as a pale yellow viscous liquid, which
is used as such for further reactions.
2.4. Synthesis of 2,3-dihydro-1H-pyrrolo-
[3,2,1-ij]quinolin-6-acetic acid (13)
The title compound was prepared by reacting hydrazine 12
with a-ketoglutaric acid in the presence of a mixture of conc.
HCl and acetic acid. Yield 48%; Mp 86e89 ꢁC. The 1H NMR
spectral data are in full agreement with those reported [24].
2.8. General procedure for the preparation of
amides 17aee
2.5. Synthesis of the methyl ester of 2,3-dihydro-1H-
pyrrolo[3,2,1-ij]quinolin-6-acetic acid (14)
Triethylamine (1.5 ml) and the appropriate acid anhydride
(0.88 mmol) (compounds 17aec) or acid chloride (0.88 mmol)
(compounds 17d,e) are added dropwise to a chilled (0 ꢁC)
solution of amine 16 (0.15 g, 0.76 mmol) in dichloromethane
(2.5 mL). The resulting mixture is stirred at room tempera-
ture for 30e60 min prior to being transferred to a beaker
containing H2O (5 mL). The biphasic mixture is separated
and the organic phase is diluted with AcOEt (40 mL),
washed with H2O (2 ꢃ 20 mL) and saturated aqueous NaCl
(2 ꢃ 25 mL) and dried (Na2SO4). The solvents are removed
under reduced pressure and the dark residue obtained is trit-
urated with AcOEt (1 mL). The dark brown solids produced
are then recrystallised from ethanol to give the desired am-
ides 17aee as beige powders.
Potassium carbonate (2.35 g, 17.03 mmol) is added to a so-
lution of the acid 13 (3.39 g, 15.77 mmol) in DMF (31 mL) at
ambient temperature. The mixture is sonicated for 10 min and
methyl iodide (1.2 mL, 19.55 mmol) is then added dropwise.
The resulting suspension is stirred for 60 min prior to the ad-
dition of H2O (20 mL) and then extracted with AcOEt
(3 ꢃ 50 mL). The organic phases are washed with H2O
(2 ꢃ 50 mL), saturated aqueous NaCl (2 ꢃ 50 mL) and dried
(Na2SO4). The solvent is evaporated in vacuo and the residue
obtained is triturated with AcOEt (5 mL) to give the title com-
pound as an off-white powder. Yield 67%; Mp 103e105 ꢁC
(recryst. EtOH); 1H NMR: d ¼ 2.00e2.35 (m, 2H), 2.85e
3.10 (m, 2H), 3.67 (s, 3H), 3.70 (s, 2H), 4.00 (t, 2H,
J ¼ 6.0 Hz), 6.90 (s, 1H), 7.35e7.65 (m, 3H); 13C NMR:
d ¼ 32.8, 33.3, 34.4, 50.4, 58.5, 112.4, 117.8, 119.4, 121.3,
121.8, 126.4, 132.0, 139.9, 171.0.
2.8.1. Synthesis of N-(2,3-dihydro-1H-pyrrolo[3,2,1-ij]-
quinolin-6-ethyl)acetamide (17a)
Acetamide 17a is prepared according to the general method
1
given in Section 2.8. Yield 55%; Mp 149e152 ꢁC; H NMR:
d ¼ 1.86 (s, 3H), 2.00e2.33 (m, 2H), 2.81e3.10 (m, 2H), 3.15
(m, 2H), 3.01 (t, 2H, J ¼ 6.4 Hz), 3.66 (m, 2H), 5.54 (br s,
1H), 6.88 (s, 1H), 7.35e7.63 (m, 3H); 13C NMR: d ¼ 18.0,
30.9, 33.3, 36.0, 46.0, 58.5, 112.4, 117.8, 119.4, 121.3,
121.8, 126.4, 132.0, 139.9, 170.9. Anal. Calcd for
C15H18N2O (242.32): C, 74.35; H, 7.49; N, 11.56. Found: C,
74.08; H, 7.69; N, 11.28.
2.6. Synthesis of 2,3-dihydro-1H-pyrrolo-
[3,2,1-ij]quinolin-6-acetamide (15)
A fine stream of dry ammonia gas is bubbled for 15 min
through a solution of the ester 14 (0.64 g, 2.79 mmol) in meth-
anol (10.5 mL) at 0 ꢁC. The reaction mixture is then left in the
fridge (4 ꢁC) for 16 h, chilled to 0 ꢁC and ammonia gas is bub-
bled for 15 min. After a period of 16 more hours in the fridge
the desired compound precipitates as a white solid and filtered.
The cake is washed with methanol and dried in vacuo
(0.1 mm/Hg), until a constant weight is obtained. Yield
97%; Mp 119e123 ꢁC; 1H NMR: d ¼ 2.00e2.33 (m, 2H),
2.81e3.10 (m, 2H), 3.63 (s, 2H), 3.98 (t, 2H, J ¼ 6.0 Hz),
5.80 (br s, 1H), 6.10 (br s, 1H), 6.88 (s, 1H), 7.35e7.63 (m,
3H); 13C NMR: d ¼ 32.8, 33.3, 36.0, 58.5, 112.4, 117.8,
119.4, 121.3, 121.8, 126.4, 132.0, 139.9, 172.7.
2.8.2. Synthesis of N-(2,3-dihydro-1H-pyrrolo-
[3,2,1-ij]quinolin-6-ethyl)propionamide (17b)
The title compound 17b is prepared according to the gen-
eral method given in Section 2.8. Yield 46%; Mp 167e
1
169 ꢁC; H NMR: d ¼ 1.13 (t, 3H, J ¼ 7.5 Hz), 2.16 (q, 2H,
J ¼ 7.5 Hz), 2.20e2.33 (m, 2H), 2.81e3.10 (m, 2H), 3.15
(m, 2H), 3.01 (t, 2H, J ¼ 6.4 Hz), 3.66 (m, 2H), 5.51 (br s,
1H), 6.88 (s, 1H), 7.36e7.64 (m, 3H); 13C NMR: d ¼ 9.9,
29.9, 30.9, 33.3, 36.0, 46.0, 58.5, 112.4, 117.8, 119.4, 121.3,
121.8, 126.4, 132.0, 139.9, 173.5. Anal. Calcd for
C16H20N2O (256.35): C, 74.97; H, 7.86; N, 10.93. Found: C,
75.13; H, 7.98; N, 10.75.
2.7. Synthesis of 2,3-dihydro-1H-pyrrolo-
[3,2,1-ij]quinolin-6-ethanamine (16)
A solution of the amide 15 (1.00 g, 4.67 mmol) in dry THF
(8 mL) is added dropwise to a suspension of lithium aluminum
hydride (0.36 g, 9.35 mmol) in dry THF (5 mL) at 0 ꢁC. The
reaction mixture is then left stirring at ambient temperature
for 16 h, chilled once again to 0 ꢁC and treated with H2O
(1 mL) in order to destroy the excess of lithium aluminum
2.8.3. Synthesis of N-(2,3-dihydro-1H-pyrrolo-
[3,2,1-ij]quinolin-6-ethyl)butyramide (17c)
Amide 17c is prepared according to the general procedure
given in Section 2.8. Yield 61%; Mp 158e161 ꢁC; 1H
NMR: d ¼ 0.90 (t, 3H, J ¼ 7.4 Hz), 1.57e1.65 (m, 2H), 2.09
(t, 2H, J ¼ 7.1 Hz), 2.20e2.33 (m, 2H), 2.81e3.10 (m, 2H),

1008
A. Tsotinis et al. / European Journal of Medicinal Chemistry 42 (2007) 1004e1013
3.01 (t, 2H, J ¼ 6.4 Hz), 3.05e3.15 (m, 2H), 3.66 (m, 2H),
5.51 (br s, 1H), 6.86 (s, 1H), 7.36e7.64 (m, 3H); 13C NMR:
d ¼ 13.6, 19.2, 30.9, 33.3, 36.0, 38.6, 46.0, 58.5, 112.4,
117.8, 119.4, 121.3, 121.8, 126.4, 132.0, 139.9, 172.8. Anal.
Calcd for C17H22N2O (270.37): C, 75.52; H, 8.20; N, 10.36.
Found: C, 75.43; H, 8.09; N, 10.11.
2.10. Synthesis of 3-(2,3-dihydro-1H-pyrrolo-
[3,2,1-ij]quinolin-6-yl)propionitrile (19)
A solution of p-tolouenesulfonylmethyl isocyanide (Tos-
MIC) (0.22 g, 1.13 mmol) in dimethoxyethane (DME)
(1.5 mL) is added dropwise to a suspension of potassium
tert-butoxide (0.26 g, 2.32 mmol) in DME (1.5 mL) at
ꢂ30 ꢁC. After the addition, the mixture is cooled to ꢂ60 ꢁC
and a solution of the aldehyde 18 (210 mg, 1.05 mmol) in
DME (3.5 mL) is cautiously added. The reaction is stirred at
this temperature for 1 h and methanol (2.5 mL) is added.
The mixture is allowed to thaw and then refluxed for
15 min. Upon completion of the reaction, the solvent is
removed in vacuo and H2O (5 mL) followed by addition of
acetic acid (0.1 mL). The resulting suspension is extracted
with dichloromethane (3 ꢃ 50 mL), the combined organics
washed with saturated aqueous NaHCO3 and NaCl and dried
(Na2SO4). The solvent is evaporated under reduced pressure
and the residue is purified by column chromatography (cyclo-
hexane/ethyl acetate, 60:40) to give the desired compound as
an off-white solid. Total yield of the above two reactions
41%; Mp 54e57 ꢁC (recryst. EtOH); 1H NMR: d ¼ 2.17e
2.29 (quintet, 2H, CH2CH2CH2, J ¼ 5.9 Hz), 2.64e2.71 (t,
2H, CH2CH2CN, J ¼ 7.3 Hz), 2.95e3.01 (t, 2H, CH2CH2CH2,
J ¼ 5.9 Hz), 3.08e3.15 (t, 2H, CH2CH2CN, J ¼ 7.3 Hz),
4.09e4.15 (t, 2H, CH2CH2CH2N, J ¼ 5.9 Hz), 6.91e7.11
(m, 3H, Harom), 7.33e7.38 (d, 1H, Harom, J ¼ 7.7 Hz); 13C
NMR: d ¼ 19.0, 22.0, 22.8, 24.6, 44.0, 111.1, 115.8, 118.8,
119.6, 119.9, 120.2, 122.0, 124.2, 124.5.
2.8.4. Synthesis of N-cyclopropanecarboxamido (2,3-
dihydro-1H-pyrrolo[3,2,1-ij]quinolin-6-yl)ethanamine
(17d)
The title compound is obtained according to the general
method given in Section 2.8. Yield 41%; Mp 198e202 ꢁC;
1H NMR: d ¼ 0.70e0.79 (m, 2H), 0.93e1.03 (m, 2H), 1.23
(m, 1H), 2.20e2.33 (m, 2H), 2.69 (t, 2H, J ¼ 6.4 Hz), 2.85e
3.15 (m, 2H), 3.47 (q, 2H, J ¼ 6.3 Hz), 4.00 (t, 2H,
J ¼ 6.0 Hz), 5.81 (br s, 1H), 6.86 (s, 1H), 7.36e7.64 (m,
3H); 13C NMR: d ¼ 7.2, 14.9, 30.9, 33.3, 36.0, 46.0, 58.5,
112.4, 117.8, 119.4, 121.3, 121.8, 126.4, 132.0, 139.9,
172.8. Anal. Calcd for C17H20N2O (268.36): C, 76.09; H,
7.51; N, 10.44. Found: C, 75.92; H, 7.65; N, 10.22.
2.8.5. Synthesis of N-cyclobutanecarboxamido-
(2,3-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-6-yl)-
ethanamine (17e)
Amide 17e was made following the general procedure
given in Section 2.8. Yield 46%; Mp 177e179 ꢁC; 1H
NMR: d ¼ 1.80e1.93 (m, 2H), 2.04e2.12 (m, 1H), 2.15e
2.33 (m, 5H), 2.69 (t, 2H, J ¼ 6.4 Hz), 2.85e3.15 (m, 3H),
3.47 (q, 2H, J ¼ 6.3 Hz), 4.00 (t, 2H, J ¼ 6.0 Hz), 5.45 (br s,
1H), 6.86 (s, 1H), 7.36e7.64 (m, 3H); 13C NMR: d ¼ 18.2,
25.4, 30.9, 33.3, 36.0, 40.1, 46.0, 58.5, 112.4, 117.8, 119.4,
121.3, 121.8, 126.4, 132.0, 139.9, 175.1. Anal. Calcd for
C18H22N2O (282.38): C, 76.56; H, 7.85; N, 9.92. Found: C,
76.38; H, 7.74; N, 10.05.
2.11. Synthesis of 3-(2,3-dihydro-1H-pyrrolo-
[3,2,1-ij]quinolin-6-yl)propylamine (20)
A solution of the nitrile 19 (75 mg, 0.35 mmol) in dry ben-
zene (1 mL) is added dropwise to a suspension of lithium alu-
minum hydride (0.06 g, 1.58 mmol) in dry ether (2 mL) at
0 ꢁC. The mixture is then refluxed for 1.5 h, cooled to 0 ꢁC
and treated carefully with H2O (2 mL). The resulting suspen-
sion is filtered through Celite and the filtrate is extracted with
AcOEt (3 ꢃ 50 mL). The combined organics are washed with
a saturated aqueous NaCl solution and dried (Na2SO4). The
solvent is removed in vacuo to give the desired amine as a yel-
lowish oil, pure enough to be used as such in the following
reactions.
2.9. Synthesis of 2,3-dihydro-1H-pyrrolo-
[3,2,1-ij]quinolin-6-acetaldehyde (18)
DIBAL-H (2.2 mL, 12.17 mmol) is added dropwise to
a stirred solution of the methyl ester 14 (0.20 g, 0.87 mmol)
in dry toluene (7.5 mL) at ꢂ78 ꢁC. The mixture is stirred at
this temperature for 25 min and then allowed to reach 0 ꢁC.
HCl (1 N, 2.5 mL) is added and the resulting suspension is fil-
tered through Celite. The filtrate is extracted with AcOEt
(3 ꢃ 50 mL) and the combined organic phases are washed
with saturated aqueous NaCl. After drying over Na2SO4, the
solvents are evaporated in vacuo to give the title compound
as a clear oil, pure enough to be used in the following reaction.
1H NMR: d ¼ 2.19e2.31 (quintet, 2H, CH2CH2CH2,
J ¼ 5.7 Hz), 2.98e3.11 (t, 2H, CH2CH2CH2, J ¼ 5.7 Hz),
3.79e3.80 (d, 2H, CH2CHO, J ¼ 2.3 Hz), 4.12e4.21 (t, 2H,
NCH2, J ¼ 5.7 Hz), 6.96e7.11 (m, 3H, Harom), 7.36e7.40
2.12. General procedure for the preparation of
amides 21aee
These amides were prepared by the general method, given
in Section 2.8, followed for the synthesis of their counterparts
17aee. Their purification was effected by flash column chro-
matography eluting with AcOEt.
2.12.1. Synthesis of N-[3-(2,3-dihydro-1H-pyrrolo-
[3,2,1-ij]quinolin-6-propyl]acetamide (21a)
Following the general procedure given in Section 2.12,
amide 21a is obtained as a viscous oil. Yield 42%; 1H
(d, 1H, Harom
J ¼ 2.3 Hz).
,
J ¼ 7.7 Hz), 9.75e9.77 (t, 1H, CHO,

A. Tsotinis et al. / European Journal of Medicinal Chemistry 42 (2007) 1004e1013
1009
NMR: d ¼ 1.82e1.97 (m, 5H, CH2CH2CH2NH, CH3CO),
2.14e2.25 (quintet, 2H, CH2CH2CH2, J ¼ 5.9 Hz), 2.74e
2.81 (t, 2H, IndCH2CH2CH2NH, J ¼ 7.3 Hz), 2.93e2.99 (t,
2H, CH2CH2CH2, J ¼ 5.9 Hz), 3.24e3.34 (t, 2H, CH2NHCO,
J ¼ 6.6 Hz), 4.05e4.10 (t, 2H, NCH2, J ¼ 5.9 Hz), 5.60 (br s,
1H, NHCO), 6.86e7.02 (m, 3H, Harom), 7.34e7.38 (d, 1H,
CH2CH2CH2NH, J ¼ 7.3 Hz), 2.14e2.26 (quintet, 2H,
CH2CH2CH2, J ¼ 5.9 Hz), 2.74e2.82 (t, 2H, CH2CH2CH2NH,
J ¼ 7.3 Hz), 2.92e2.98 (t, 2H, CH2CH2CH2, J ¼ 5.9 Hz),
3.28e3.38 (t, 2H, CH2NHCO, J ¼ 6.6 Hz), 4.05e4.11 (t,
2H, NCH2, J ¼ 5.9 Hz), 5.57 (br s, 1H, NHCO), 6.87e7.02
(m, 3H, Harom), 7.35e7.38 (d, 1H, Harom, J ¼ 7.7 Hz); 13C
NMR: d ¼ 9.1, 13.5, 26.9, 33.3, 34.4, 34.9, 43.0, 58.5,
114.1, 116.3, 118.4, 119.1, 121.7, 123.6, 132.0, 139.9,
180.7. Anal. Calcd for C18H22N2O (282.38): C, 76.56; H,
7.85; N, 9.92. Found: C, 76.41; H, 7.76; N, 9.81.
H
arom, J ¼ 7.7 Hz) ppm; 13C NMR: d ¼ 18.0, 26.9, 33.3,
34.4, 34.9, 42.4, 58.5, 114.1, 116.3, 118.4, 119.1, 121.7,
123.6, 125.0, 134.6, 174.8 ppm. Anal. Calcd for C16H20N2O
(256.35): C, 74.97; H, 7.86; N, 10.93. Found: C, 74.84; H,
7.78; N, 10.81.
2.12.5. Synthesis of N-cyclobutanecarboxamido-
2.12.2. Synthesis of N-[3-(2,3-dihydro-1H-pyrrolo-
[3,2,1-ij]quinolin-6-propyl]propionamide (21b)
3-(2,3-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-6-yl)-
propylamine (21e)
Amide 21b is obtained as an off-white solid, following the
general method given in Section 2.12. Yield 48%; Mp 58e
61 ꢁC (recryst. EtOH); 1H NMR: d ¼ 1.06e1.10 (t, 3H,
Amide 21e is obtained as a white solid, following the gen-
eral procedure given in Section 2.12. Yield 45%; Mp 68e
71 ꢁC (recryst. EtOH); 1H NMR: d ¼ 1.66e2.31 (m, 10H,
CH2CH2CH2NH, CH2CH2CH2, CH2 cyclobutyl), 2.74e2.81
(t, 2H, CH2CH2CH2NH, J ¼ 7.3 Hz), 2.84e2.99 (m, 3H,
CH2CH2CH2, CHCO), 3.26e3.35 (t, 2H, CH2NHCO,
J ¼ 6.6 Hz), 4.05e4.11 (t, 2H, NCH2, J ¼ 5.9 Hz), 5.34 (br
s, 1H, NHCO), 6.87e7.04 (m, 3H, Harom), 7.34e7.38 (d,
1H, Harom, J ¼ 7.7 Hz); 13C NMR: d ¼ 18.7, 22.8, 22.9, 24.7,
25.3, 29.7, 30.1, 39.3, 39.9, 43.8, 114.1, 116.3, 118.4, 119.1,
121.7, 123.6, 125.0, 134.6, 179.9. Anal. Calcd for
C19H24N2O (296.41): C, 76.99; H, 8.16; N, 9.45. Found: C,
76.85; H, 8.02; N, 9.37.
CH3CH2CO,
J ¼ 7.3 Hz),
1.86e1.94
(quintet,
2H,
CH2CH2CH2NH, J ¼ 7.3 Hz), 2.09e2.14 (q, 2H, COCH2CH3,
J ¼ 7.3 Hz), 2.17e2.23 (quintet, 2H, CH2CH2CH2,
J ¼ 5.9 Hz), 2.76e2.80 (t, 2H, CH2CH2CH2NH, J ¼ 7.3 Hz),
2.94e2.97 (t, 2H, CH2CH2CH2, J ¼ 5.9 Hz), 3.28e3.33 (t,
2H, CH2NHCO, J ¼ 6.6 Hz), 4.07e4.09 (t, 2H, NCH2,
J ¼ 5.9 Hz), 5.50 (br s, 1H, NHCO), 6.87e7.03 (m, 3H,
H
arom), 7.35e7.37 (d, 1H, Harom, J ¼ 8.1 Hz) ppm; 13C
NMR: d ¼ 9.7, 26.9, 27.1, 33.3, 34.4, 34.9, 42.7, 58.5,
114.1, 116.3, 118.4, 121.7, 123.6, 126.4, 132.0, 139.9,
175.4. Anal. Calcd for C17H22N2O (270.37): C, 75.52; H,
8.20; N, 10.36. Found: C, 75.43; H, 8.09; N, 10.11.
2.13. Synthesis of 2,3-dihydro-1H-pyrrolo-
[3,2,1-ij]quinolin-6-acetonitrile (22)
2.12.3. Synthesis of N-[3-(2,3-dihydro-1H-pyrrolo-
[3,2,1-ij]quinolin-6-propyl]butyramide (21c)
Trifluoroacetic acid anhydride (0.5 mL, 3.6 mmol) is added
in one portion to a suspension of 2,3-dihydro-1H-pyr-
rolo[3,2,1-ij]quinolin-6-acetamide (15) (0.60 g, 2.78 mmol)
in triethylamine (1.5 mL, 8.33 mmol) and dichloromethane
(17 mL) at 0 ꢁC. The mixture is stirred at this temperature
for 2 min and then transferred to a beaker containing ice-wa-
ter. The resulting suspension is stirred for 10 more minutes
and then extracted with dichloromethane (3 ꢃ 25 mL). The
combined extracts are washed with H2O (2 ꢃ 30 mL) and
dried (Na2SO4). The solvent is evaporated in vacuo to leave
a dark brown solid, which is purified by spinning plate chro-
matography (SPC) (cyclohexane/dichloromethane 80:20) to
give the desired acetonitrile as a pale yellow solid. Yield
93%; Mp 112e114 ꢁC (recryst. AcOEt); 1H NMR:
d ¼ 2.05e2.15 (m, 2H, NeCH2CH2CH2), 2.55 (t, 2H, Ne
CH2CH2CH2, J ¼ 5.9 Hz), 3.55 (s, 2H, CH2CN), 3.85 (t, 2H,
CH2CH2CH2eN, J ¼ 5.8 Hz), 6.35 (s, 1H, Harom), 6.63e6.75
(m, 1H, Harom), 7.10e7.15 (m, 1H, Harom), 7.28e7.33 (m,
1H, Harom); 13C NMR: d ¼ 17.8, 33.3, 34.4, 58.5, 112.4,
114.9, 117.8, 119.4, 121.3, 121.8, 126.4, 132.0, 139.9.
Compound 21c is obtained as a viscous yellowish oil, fol-
lowing the general procedure given in Section 2.12. Yield
38%; 1H NMR: d ¼ 0.87e0.94 (t, 3H, CH3CH2CH2CO,
J ¼ 7.3 Hz), 1.49e1.67 (sextet, 2H, CH3CH2CH2CO,
J ¼ 7.3 Hz), 1.82e1.97 (quintet, 2H, CH2CH2CH2NH,
J ¼ 7.3 Hz), 2.02e2.10 (t, 2H, COCH2CH2CH3, J ¼ 7.3 Hz),
2.14e2.26 (quintet, 2H, CH2CH2CH2, J ¼ 5.9 Hz), 2.74e
2.81 (t, 2H, CH2CH2CH2NH, J ¼ 7.3 Hz), 2.93e2.99 (t, 2H,
CH2CH2CH2, J ¼ 5.9 Hz), 3.26e3.36 (t, 2H, CH2NHCO,
J ¼ 6.6 Hz), 4.05e4.11 (t, 2H, NCH2, J ¼ 5.9 Hz), 5.55 (br
s, 1H, NHCO), 6.87e7.03 (m, 3H, Harom), 7.35e7.38 (d,
1H, Harom, J ¼ 7.7 Hz); 13C NMR: d ¼ 13.0, 18.8, 26.9, 33.3,
34.4, 34.9, 36.5, 42.7, 58.5, 114.1, 116.3, 118.4, 119.1,
121.7, 123.6, 125.0, 139.9, 174.7. Anal. Calcd for
C18H24N2O (284.40): C, 76.02; H, 8.51; N, 9.85. Found: C,
75.63; H, 8.19; N, 10.09.
2.12.4. Synthesis of N-cyclopropanecarboxamido-
3-(2,3-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-6-yl)-
propylamine (21d)
The title compound 21d was obtained as a beige solid, fol-
lowing the general procedure given in Section 2.12. Yield
47%; Mp 92e94 ꢁC (recryst. EtOH); 1H NMR: d ¼ 0.63e
0.74 (m, 2H, CH2 cyclopropyl), 0.83e0.98 (m, 2H, CH2 cyclo-
propyl), 1.18e1.28 (m, 1H, CHCO), 1.83e1.98 (quintet, 2H,
2.14. Synthesis of 2-methyl-2-(2,3-dihydro-1H-pyrrolo-
[3,2,1-ij]quinolin-6-yl)propionitrile (23)
To a stirred suspension of sodium hydride (60%) (0.08 g,
1.9 mmol) in DMF (2.0 mL) is added dropwise a mixture of

1010
A. Tsotinis et al. / European Journal of Medicinal Chemistry 42 (2007) 1004e1013
the acetonitrile 22 (0.15 g, 0.77 mmol) and methyl iodide
(0.1 mL, 1.91 mmol) in DMF (2 mL). The resulting suspen-
sion is stirred at room temperature for 18 h and then treated
with a saturated aqueous solution of ammonium chloride
(15 mL). The mixture is taken up with AcOEt (3 ꢃ 25 mL),
washed with H2O (2 ꢃ 20 mL) and a saturated aqueous solu-
tion of NaCl (2 ꢃ 20 mL). The extracts are dried (Na2SO4)
and the solvent is removed under reduced pressure to leave
a yellowish oil, which is purified by spinning plate chromatog-
raphy (SPC) (cyclohexane/AcOEt 95:5) to give the title com-
pound as a white solid. Yield 29%; Mp 134e136 ꢁC (recryst.
AcOEt); 1H NMR: d ¼ 1.90 (s, 6H, C(CH3)2), 2.05e2.111 (m,
2H, NeCH2CH2CH2), 2.53 (t, 2H, NeCH2CH2CH2,
J ¼ 5.8 Hz), 3.85 (t, 2H, CH2CH2CH2eN, J ¼ 5.6 Hz), 6.35
(s, 1H, Harom), 6.63e6.75 (m, 1H, Harom), 7.12e7.15 (m,
1H, Harom), 7.28e7.30 (m, 1H, Harom); 13C NMR: d ¼ 25.6,
33.6, 34.5, 58.9, 112.4, 117.8, 119.2, 119.4, 121.6, 121.8,
126.1, 132.1, 138.9.
chromatography (SPC) (cyclohexane/AcOEt 95:5) to give
the title compound as a clear viscous oil. Yield 78%; 1H
NMR: d ¼ 1.79e1.96 (m, 8H, 4CH2 cyclohexano), 2.16e
2.27 (quintet, 2H, CH2CH2CH2, J ¼ 5.6 Hz), 2.32e2.45 (m,
2H, CH2 cyclohexano), 2.95e3.01 (t, 2H, CH2CH2CH2N,
J ¼ 5.9 Hz), 4.08e4.13 (t, 2H, NCH2, J ¼ 5.7 Hz), 6.93e
7.09 (m, 3H, Harom), 7.60e7.64 (d, 1H, Harom, J ¼ 7.7 Hz).
2.17. Synthesis of [1-(2,3-dihydro-1H-pyrrolo-
[3,2,1-ij]quinolin-6-yl)cyclohexane]methylamine (26)
The synthesis of the title compound was accomplished by
following the method reported for the preparation of its conge-
ner 20. Amine 26 was used without any chromatographic pu-
rification in the next reaction.
2.18. Synthesis of N-[1-(2,3-dihydro-1H-pyrrolo-
[3,2,1-ij]quinolin-6-yl)cyclohexanemethyl]acetamide-
(27a) and N-[1-(2,3-dihydro-1H-pyrrolo-
[3,2,1-ij]quinolin-6-yl)cyclohexanemethyl]-
butyramide (27b)
2.15. Synthesis of N-[2-methyl-2-(2,3-dihydro-1H-
pyrrolo[3,2,1-ij]quinolin-6-yl)propyl]acetamide (24)
A mixture consisting of the nitrile 23 (0.025 g, 0.11 mmol),
acetic anhydride (1.5 mL), sodium acetate (0.014 g,
0.17 mmol) and Raney-Ni (5 mg) is hydrogenated in a Parr
apparatus for 16 h at 50 ꢁC. The catalyst is then filtered off
through Celite and the mixture concentrated in vacuo. The res-
idue obtained is diluted with dichloromethane (10 mL),
washed with H2O (3 ꢃ 15 mL) and a saturated aqueous solu-
tion of NaCl (2 ꢃ 15 mL) and dried (Na2SO4). The solvent
is removed under reduced pressure to give the desired amide
24 as a white amorphous solid. Yield 30%; Mp 132e135 ꢁC
Acetamide 27a was prepared by the general method given
in Section 2.12, reported for its counterpart 21a. Yield 52%;
1
Mp 109e111 ꢁC (recryst. EtOH); H NMR: d ¼ 1.32e1.85
(m, 11H, 4CH2 cyclohexano and CH3CO), 1.98e2.12 (m,
2H, CH2 cyclohexano), 2.20e2.28 (m, 2H, CH2CH2CH2N ),
2.96e3.02 (t, 2H, CH2CH2CH2N, J ¼ 5.9 Hz), 3.59e3.61 (d,
2H, CH2NH, J ¼ 5.9 Hz), 4.11e4.16 (t, 2H, NCH2,
J ¼ 6.6 Hz), 5.13 (br s, 1H, NHCO), 6.83e7.04 (m, 3H,
H
arom), 7.51e7.54 (d, 1H, Harom, J ¼ 7.3 Hz); 13C NMR:
d ¼ 13.7, 19.1, 22.5, 22.6, 24.8, 26.4, 34.5, 38.8, 39.9, 44.1,
48.0, 60.4, 118.5, 118.6, 119.2, 122.0, 123.7, 124.6, 135.2,
172.9. Anal. Calcd for C20H26N2O (310.44): C, 77.38; H,
8.44; N, 9.02. Found: C, 77.27; H, 8.35; N, 8.91.
1
(recryst. EtOH); H NMR: d ¼ 1.50 (s, 6H, C(CH3)2), 1.88
(s, 3H, COCH3), 2.05e2.11 (m, 2H, NeCH2CH2CH2), 2.53
(t, 2H, NeCH2CH2CH2, J ¼ 5.7 Hz), 3.63 (d, 2H, CH2NH,
J ¼ 5.8 Hz), 3.85 (t, 2H, CH2CH2CH2eN, J ¼ 5.6 Hz), 6.35
(s, 1H, Harom), 6.65e6.76 (m, 1H, Harom), 7.13e7.16 (m,
1H, Harom), 7.26e7.28 (m, 1H, Harom); 13C NMR: d ¼ 12.4,
18.0, 27.5, 33.3, 34.4, 38.4, 58.5, 58.8, 112.4, 117.8, 119.2,
119.4, 121.6, 121.8, 126.1, 132.1, 139.9, 170.9. Anal. Calcd
for C17H22N2O (270.37): C, 75.52; H, 8.20; N, 10.36. Found:
C, 75.36; H, 8.03; N, 10.18.
Butyramide 27b was prepared by the general method given
in Section 2.12, reported for its counterpart 21c. Yield 41%;
1
Mp 106e108 ꢁC (recryst. EtOH); H NMR: d ¼ 0.81e0.85
(t, 3H, CH3CH2CH2CO, J ¼ 7.3 Hz), 1.40e1.78 (m, 10H,
4CH2 cyclohexano and CH3CH2CH2CO), 1.92e2.08 (m, 4H,
CH3CH2CH2CO and CH2 cyclohexano), 2.18e2.27 (m, 2H,
ArCH2CH2CH2), 2.97e3.00 (t, 2H, CH2CH2CH2NAr,
J ¼ 5.9 Hz), 3.59e3.60 (d, 2H, CH2NH, J ¼ 5.9 Hz), 4.09e
4.13 (t, 2H, ArNCH2, J ¼ 6.6 Hz), 5.11 (br s, 1H, NHCO),
2.16. Synthesis of 1-(2,3-dihydro-1H-pyrrolo-
[3,2,1-ij]quinolin-6-yl)cyclohexanecarbonitrile (25)
6.87e6.98 (m, 3H, Harom), 7.51e7.53 (d, 1H, Harom,
J ¼ 8.1 Hz). Anal. Calcd for C22H30N2O (338.49): C, 78.06;
To a stirred suspension of sodium hydride (60%) (60 mg,
1.53 mmol) in DMSO (1.5 mL) is added dropwise a solution
2,3-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-6-acetonitrile (22)
(100 mg, 0.51 mmol) and 1,5-dibromopentane (0.1 mL,
0.62 mmol) in DMSO (1.0 mL) and diethyl ether (1.0 mL).
The mixture is stirred at ambient temperature for 2 h and
then quenched with H2O (2 mL). Extraction with AcOEt
(3 ꢃ 50 mL), followed by washing with a saturated aqueous
solution of NaCl (2 ꢃ 15 mL), drying (Na2SO4) and sol-
vent evaporation under reduced pressure, results in the forma-
tion of a dark liquid, which is purified by spinning plate
H, 8.93; N, 8.28. Found: C, 78.28; H, 8.88; N, 8.19.
2.19. Pharmacological protocols
2.19.1. Xenopus melanophore model for the evaluation of
agonist and antagonist activity
Melanophore cells were grown in 96-well tissue culture
plates and growth medium was replaced with 0.7 ꢃ L-15 cul-
ture medium 18 h before analogs were tested [20, 25e28]. Ini-
tial absorbance (Ai, 630 nm) of cells (w8000 cells/well) was
measured in each well using a Bio-Tek microtiter plate reader

A. Tsotinis et al. / European Journal of Medicinal Chemistry 42 (2007) 1004e1013
1011
(model EL3115, Anachem, U.K.), then cells were treated with
the varying concentrations of the analogs. The maximal con-
centration used was 10ꢂ4 M. All experiments used triplicate
wells at six concentrations of analog. The final absorbance
(Af) was measured after 60 min, and the fractional change in
absorbance (1 ꢂ Af/Ai) was calculated. Vehicle did not alter
pigment granule distribution itself or inhibit responses to mel-
atonin. The concentration of analog producing 50% of the
maximum agonist response (EC50) was determined from con-
centrationeresponse curves. For evaluation of antagonist po-
tency, cells were treated with vehicle (1% DMSO or
methanol) or varying concentrations (10ꢂ4 e 10ꢂ9 M) of the
analogs for 60 min before melatonin (10ꢂ9 M) was added.
The concentration of analog reducing melatonin-induced pig-
ment aggregation by 50% (IC50) was determined.
amine 16 with lithium aluminum hydride in tetrahydrofuran,
which was not purified but immediately acylated with the
appropriate reagent to give the target compounds 17aee.
The synthesis of their congeners, 21aee, involved the con-
version of ester 14 to the aldehyde 18, effected on treating the
former with DIBAL-H at ꢂ78 ꢁC in the presence of toluene,
followed by the reaction of 18 with tosylmethyl isocyanide
in the presence of potassium tert-butoxide and 1,2-dimethoxy-
ethane, reduction of the resulting nitrile 19 with lithium alumi-
num hydride in the presence of benzene and diethyl ether (1:5)
to the amine 20, and immediate acylation with the appropriate
reagent.
The synthesis of the side chain restrained analogs, 24 and
27a,b, is depicted in Scheme 2. Amide 15 was dehydrated
to the acetonitrile 22, on treatment with trifluoroacetic anhy-
dride in dichloromethane in the presence of triethylamine. Ni-
trile 22 was then reacted with excess methyl iodide in the
presence of sodium hydride in N,N-dimethylformamide to
give the b,b0-dimethyl analog 23. Treatment of nitrile 22
with 1,5-dibromopentane in the same way gave the b-carbocy-
clic analog 25. Compound 24 was obtained by a simultaneous
Raney-Nickel hydrogenation/acetylation process, whilst its
b,b0-disubstituted counterparts 27a,b on reduction of 25,
with lithium aluminum hydride, in the presence of benzene
and diethyl ether (1:5), to the amine 26 and immediate acyla-
tion with the appropriate reagent.
2.19.2. Binding affinity assay
The binding affinity of the analogs was determined in com-
petition with radioligand binding assays using 2-[125I]-iodo-
melatonin (specific activity 2200 Ci/mol, Perkin Elmer,
U.K.), as described previously [27] on the recombinant human
MT1 and MT2 subtypes expressed in NIH 3T3 cells.
3. Results and discussion
3.1. Chemistry
The synthesis of 17aee and 21aee is shown in Scheme 1.
Commercially available 1,2,3,4-tetrahydroquinoline (10) was
nitrosated with sodium nitrite and conc. HCl to the N-nitroso
analog 11, which was then converted to the hydrazine 12 on
reduction with lithium aluminum hydride. Condensation of
12 with a-ketoglutaric acid in the presence of conc. HCl and
glacial acetic acid afforded the tricyclic acetic acid 13 [24]
in 40% overall yield. Basic esterification of the latter with
methyl iodide in N,N-dimethylformamide gave ester 14, which
on treatment with gaseous ammonia in methanol was con-
verted to the primary amide 15. This was then reduced to the
3.2. Pharmacological studies
The agonist and antagonist potency of the new analogs was
assessed in a well-established, specific model of melatonin
action, the pigment aggregation response of X. laevis melano-
phores. In these cells, granules containing melanin pigment
are normally distributed evenly throughout the cell but move
towards the cell centre when melatonin receptors are activated.
The response is readily quantified by measuring the change in
light (630 nm) absorbance of the cells as the pigment concen-
trates near the cell centre.
Table 1
Melatoninergic activity of compounds 17aee and 21aee in the Xenopus laevis melanophore assay and on human MT1 and MT2 receptors expressed in NIH 3T3
cells
Compound
R
Agonist pEC50
Antagonist pIC50
Human MT1 (pKi, nm)
Human MT2 (pKi, nm)
Selectivity
(MT2/MT1)
Melatonin
Luzindole
17a
10.07
NAa
NA
NA
NA
NA
NTb
NA
NA
NA
NA
NA
NA
9.41 ꢄ 0.09
6.10 ꢄ 0.08
NT
9.45 ꢄ 0.04
7.33 ꢄ 0.06
NT
1.1
17
e
5.61 ꢄ 0.08
5.21 ꢄ 0.03
5.66 ꢄ 0.01
5.77 ꢄ 0.05
6.55 ꢄ 0.01
NT
CH3
C2H5
17b
17c
NT
NT
NT
NT
e
C3H7
e
e
17d
17e
c-C3H5
c-C4H7
CH3
NT
NT
NT
NT
e
21a
21b
5.29 ꢄ 0.02
5.33 ꢄ 0.04
5.85 ꢄ 0.04
5.82 ꢄ 0.02
5.80 ꢄ 0.03
6.72 ꢄ 0.05
7.12 ꢄ 0.01
7.38 ꢄ 0.02
6.94 ꢄ 0.05
6.43 ꢄ 0.04
7.02 ꢄ 0.04
7.76 ꢄ 0.08
7.93 ꢄ 0.05
7.15 ꢄ 0.06
6.73 ꢄ 0.07
2.0
4.4
3.5
1.6
2.0
C2H5
C3H7
21c
21d
c-C3H5
c-C4H7
21e
a
NA ¼ no agonist or antagonist effect detected at 100 mM. Agonist and antagonist data are the mean of triplicate experiments.
b
NT ¼ not tested.

1012
A. Tsotinis et al. / European Journal of Medicinal Chemistry 42 (2007) 1004e1013
Table 2
Melatoninergic activity of the side chain conformationally restrained analogs 24 and 27a,b in the Xenopus laevis melanophore assay and on human MT1 and MT2
receptors expressed in NIH 3T3 cells
Compound
R
Agonist pEC50
Antagonist pIC50
Human MT1 (pKi, nm)
Human MT2 (pKi, nm)
Selectivity
(MT2/MT1)
Melatonin
Luzindole
10.07
NAa
NA
5.61 ꢄ 0.08
9.41 ꢄ 0.09
6.10 ꢄ 0.08
NTb
9.45 ꢄ 0.04
7.33 ꢄ 0.06
NT
1.1
17
24
27a
27b
a
CH3
CH3
C3H7
5.72 ꢄ 0.03
NA
NA
w4.00
e
5.93 ꢄ 0.03
5.69 ꢄ 0.04
5.58 ꢄ 0.23
5.23 ꢄ 0.02
6.63 ꢄ 0.06
6.39 ꢄ 0.01
11.2
14.4
NA ¼ no agonist or antagonist effect detected at 100 mM.
b
NT ¼ not tested. Agonist and antagonist data are the mean of triplicate experiments.
It is apparent from the data presented in Table 1 that irre-
Interestingly, the incorporation of a six-membered (27a,b)
ring has either almost no effect in the degree of antagonism
on Xenopus melanophores (pIC50 ¼ 5.66 for 17b vs.
pIC50 ¼ 5.69 for 27b) or it increases antagonist potency by al-
most five-fold (pIC50 ¼ 5.21 for 17a vs. pIC50 ¼ 5.93 for 27a)
(Table 2). However, the new N1eC7 annulated cyclohexane
side chain substituted derivatives 27a,b, are substantially
more potent than their respective N1eC2 annulated counter-
parts 8 and 9 [23] (pIC50 ¼ 5.93 for 27a vs. pIC50 ¼ 5.10 for
8 and pIC50 ¼ 5.69 for 27b vs. pIC50 ¼ 5.02 for 9). This is an-
other example of a topographically induced enhancement of
antagonism due to steric factors.
spective of the number of methylene units in the side chain
spacer, analogs 17aed and 21aee are all antagonists in the
melanophore assay. With the exception of the acetamides
17a and 21a and the propionamide 21b, the potencies of the
other new compounds are comparable to that of luzindole
(Fig. 1), a commonly used melatonin receptor antagonist
(pIC50 ¼ 5.61). Moreover, there is a general trend of reduced
potency upon spacer’s elongation. Compound 17d is of partic-
ular interest in that by increasing the spacer’s length in the side
chain by a methylene unit (from 17d to 21d) a six-fold de-
crease in potency occurs. This may indicate that in this series
(21aee) the long side chain is not well accommodated in its
binding site and so cannot readily induce the receptor confor-
mation needed for antagonist action.
4. Conclusion
Comparison of the new N1eC7 annulated derivatives 17ae
d with their N1eC2 annulated congeners 3aee (Fig. 2) shows
that the former annulation enhances the antagonist potency in
the Xenopus melanophore as much as 691-fold in the case of
the cyclopropanamido analogs 3d (pIC50 ¼ 3.71) and 17d
(pIC50 ¼ 6.55). This remarkable difference nicely illustrates
that steric factors in the N1eC7 area increase the preference
for the active conformation [29] but, as these effects are
exerted closer to the pharmacophoric side chain the population
of the preferred conformation declines.
All five analogs belonging to the 21 series exhibit a greater
affinity at MT2 than at the MT1 receptor, indicating that the
MT2 receptor cavity is larger and/or more flexible than that
of the MT1 [29].
The MT2 selectivity is also retained in the case of the side
chain restrained analogs 27a,b. There is a subtle difference
though in the degree of selectivity, as these compounds are
11- and 14-fold MT2 selective, respectively.
Conversely to the unsubstituted analog 17a and the cyclo-
hexane side chain substituted derivatives 27a,b, the new gem-
dimethyl compound 24 is a partial agonist (pEC50 ¼ 5.72;
Table 2) with w80% of full agonist response. Thus, it seems
that the introduction of two b-methyl groups is not only toler-
ated by the receptor but it constitutes a functional probe in its
dynamic agonisteantagonist conformational equilibrium.
Similar behaviour has been noticed in the case of the analo-
The biological data presented herein for the three series of
new analogs suggest that the influence of steric factors in bind-
ing is significant, when it arises from the presence of bulky
moieties in the b-position of the alkanamidoethyl side chain
and/or elongated spacers, coupled with a suitable N1eC7 an-
nulation. This was demonstrated by the partial agonist activity
of 24 compared to the antagonist activity exhibited by its non-
substituted congeners 17aed and 21aee. The presence of
more bulky b-substituents, regardless of the size of the R
group, compounds 27a,b, seems to lead to reduced antago-
nism. Moreover, steric effects in the N1eC7 area increase
the preference for the active conformation, but when these ef-
fects are exerted closer to the pharmacophoric side chain (N1e
C2 annulation) the population of the preferred conformation
declines. However, in order to validate these hypotheses, a re-
currence of similar effects on a large number of melatonin an-
alogs, acting either as agonists or antagonists, is necessary.
The results from such comparative studies may disclose infor-
mation on important synergism between steric effects and po-
tency, which could be exploited in the design of high affinity,
specific ligands.
Acknowledgment
The University of Athens group wishes to thank EPEAEK
II Program Pythagoras II e Support of Universities Research
Groups (KA: 70/3/7993) for financial support. The King’s
gous gem-dimethyl N1eC2 annulated compound 5a (pEC50
5.86; [23]).
¼

A. Tsotinis et al. / European Journal of Medicinal Chemistry 42 (2007) 1004e1013
1013
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College London group was supported by the Wellcome Trust
(grant no. GR065816).
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