Evaluation of the anti-inflammatory and anti-nociceptive activities of novel synthesized melatonin analogues

Article abstract of DOI:10.1016/j.ejmech.2007.01.027

This study aimed at evaluation of the reactivity of melatonin (1) towards various chemical reagents to produce new melatonin analogues containing heterocyclic moieties which would provide basic pharmacological activities. The pyrrolo[1,2-a]indole derivati

Full text of DOI:10.1016/j.ejmech.2007.01.027

European Journal of Medicinal Chemistry 42 (2007) 1285e1292
http://www.elsevier.com/locate/ejmech
Short communication
Evaluation of the anti-inflammatory and anti-nociceptive
activities of novel synthesized melatonin analogues
b
b
Gamal A. Elmegeed a, , Ayman R. Baiuomy , Omar M.E. Abdel-Salam
*
a Hormone Department, National Research Centre, Dokki, Giza 12622, Cairo, Egypt
b Pharmacology Department, National Research Centre, Dokki, Cairo, Egypt
Received 12 November 2006; received in revised form 22 January 2007; accepted 25 January 2007
Available online 23 February 2007
Abstract
This study aimed at evaluation of the reactivity of melatonin (1) towards various chemical reagents to produce new melatonin analogues
containing heterocyclic moieties which would provide basic pharmacological activities. The pyrrolo[1,2-a]indole derivatives 3, 5, 12, 14 and
pyrido[1,2-a]indole derivatives 9a, b were synthesized via straightforward and efficient methods and their structures were established based on
the analytical and spectral data. Also, this work was extended to study the potential role of the novel synthesized melatonin analogues 3, 5, 9a
and 12 as anti-inflammatory and anti-nociceptive agents in comparison with melatonin. After s.c. administration all compounds induced sig-
nificant anti-inflammatory activity, inhibiting the paw oedema response compared with the control group at all time points in the test. Com-
pound 5 has the strongest anti-inflammatory activity which exceeds that of the parent reference, melatonin, followed by compounds 9a and
12, at the first 2 h of administration. Effect of melatonin analogues on thermal pain, acetic acid-induced writhing and gastric lesions caused by
indomethacin was also investigated. Compounds 5 and 12 were more potent as anti-nociceptive drugs; they are more potent in this respect
than melatonin.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Melatonin; Pyrrole; Indole; Pyridine; Anti-inflammatory; Anti-nociceptive
1. Introduction
synthetic melatonin analogues capable of mimicking the ef-
fects of melatonin has progressed considerably during the
past decade [10e13]. Pyrrole and pyridine compounds are
a promising starting materials in drug research in view of their
various pharmacological activities [14e17]. Derivatives of
pyrrole proved as non-steroidal anti-inflammatory drugs
(NSAIDs) are tolmetin (CAS 64490-92-2) and zomepirac
(CAS 64092-48-4). Other pyrrole and pyridine compounds
have been recently reported as potent selective COX-2 inhibi-
tors and antioxidants [18e22].
Based on these considerations, we investigate herein
a straightforward and efficient synthesis of novel melatonin
analogues containing pyrrole or pyridine nucleus fused to the
essential pharmacophoric features of the melatonin molecule.
Also the systemic anti-inflammatory and anti-nociceptive
activities of the novel synthesized melatonin analogues were
investigated in comparison with melatonin.
There is growing evidence for the important role of the
pineal hormone melatonin. A number of reports have been
published concerning the medicinal importance of melatonin
[1,2]. Melatonin has been shown to possess marked anti-
inflammatory [3,4], analgesic [5], and anti-nociceptive [6]
properties. Prostaglandin levels in the inflammatory exudates
and cyclooxygenase-2 (COX-2) expression from carra-
geenan-treated rats were reported to be completely inhibited
by melatonin [7]. Melatonin was recently shown to attenuate
acute gastric lesions induced by strong irritants because of
the scavenging of free radicals [8,9]. The development of
* Corresponding author. Tel.: þ2 02 5682070; fax: þ2 02 3370931/7601614.
E-mail address: gamalae@hotmail.com (G.A. Elmegeed).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.01.027

1286
G.A. Elmegeed et al. / European Journal of Medicinal Chemistry 42 (2007) 1285e1292
NHCOCH3
NHCOCH3
CH3O
2. Results and discussion
2.1. Chemistry
CH3O
dioxane/Et3N
PhCOCH2Br
+
N
H
reflux / -HBr
N
6
COPh
1
7
We have attempted a straightforward synthesis of pyr-
rolo[1,2-a]indole derivatives by the reaction of melatonin
(1) with equimolar amount of diethylmalonate 2 in refluxing
absolute ethanol containing piperidine (2 mL) which pro-
vided a colorless chromatographically pure product in 82%
yield. The 1,3-dioxo-2H-pyrrolo[1,2-a]indole derivative 3 is
assigned to this product through elemental analyses and spec-
troscopic data (Scheme 1). The mass spectrum (MS) of com-
pound 3 showed molecular ion peak at m/z ¼ 300 (35%)
which corresponds to the molecular formula C16H16N2O4.
The IR spectrum revealed absorption bands at y 1745,
1738, and 1685 cmꢀ1 for the three carbonyl groups and the
1H NMR spectrum revealed, in addition to the expected sig-
nals of melatonin moiety, the presence of singlet (2H) at
d 6.15 ppm for b-protons of the pyrrole nucleus. Moreover,
the 13C NMR (ppm) data showed the characteristic signals
of the pyrrole ring at 111.6 (C-2) and 118.3 (fused pyrrole-
C). Similarly, under the same experimental conditions, mela-
tonin also reacted with ethyl acetoacetate 4 to afford the
corresponding 1-oxo-3-methylpyrrolo[1,2-a]indole derivative
5 in 78% yield (Scheme 1).
NHCOCH3
NH
X
EtOH/Et3N
reflux
CH3O
CN
8a,x=CN
b,X=CO2Et
N
X
Ph
9a,x=CN
b,X=CO2Et
Scheme 2.
was assigned through the elemental analyses and spectro-
scopic data. The reaction takes place via simple addition of
the cyanoacetylhydrazide to the NH group of melatonin to
give the intermediate 11 which readily underwent cyclization
via loss of H2O molecule to afford compound 12 in 80% yield.
Confirmation for the structure of compound 12 was obtained
by studying its reactivity towards diketones. Therefore, com-
pound 12 reacted with equimolar amount of pentan-2,4-dione
(acetyl acetone) 13 in boiling absolute ethanol to afford the
corresponding 3-pyrazolylpyrrolo[1,2-a]indole derivative 14
in 72% yield (Scheme 3).
The structures of all the novel synthesized melatonin ana-
logues were assigned based on their spectroscopic and elemen-
tal analyses data (cf. Section 3)
Awide range of pharmacological activities have been attrib-
uted to fused pyridines [17,23]. In view of this information, we
would like to report here on the formation and characterization
of novel pyridomelatonin derivatives. The indole NH group of
melatonin reacted readily with phenacyl bromide 6 via elimina-
tion of HBr by refluxing in dioxane/triethylamine solution to
afford 1-benzoyl methinomelatonin derivative 7 in 78% yield
2.2. Pharmacology
In the present work, the effect of the tricyclic pyrrolo- and
pyrido-indole derivatives 3, 5, 9a and 12 was compared with
that of melatonin on inflammation, thermal and visceral pain
and on the development of gastric mucosal lesions caused
by indomethacin.
1
as the only isolated product as confirmed by IR, H NMR,
MS and elemental analyses (cf. Section 3). Further confirma-
tion for the structure of compound 7 was obtained by studying
its reactivity towards some active methylene reagents to give
pyrido[1,2-a]indole derivatives with potential biological activ-
ities. Thus, the reaction of compound 7 with either malononi-
trile 8a or ethyl cyanoacetate 8b in refluxing ethanolic
triethylamine solution resulted in the corresponding chromato-
graphically pure products, iminopyrido[1,2-a]indole deriva-
tives 9a and 9b in 82% and 75% yield, respectively (Scheme 2).
The reaction of melatonin (1) with cyanoacetylhydrazide
10 in absolute ethanol under reflux resulted in 1-imino-3-hy-
drazinopyrrolo[1,2-a]indole derivative 12 (Scheme 3) which
2.2.1. Tests of inflammation: effect of melatonin analogues
on carrageenan-induced paw oedema
The effects of systemic injections of the tested compounds
on oedema formation were studied using the carrageenan-
induced paw inflammation in comparison with melatonin. A
previous study [6] showed that melatonin has comparable
NHCOCH3
NHNH2
NHCOCH3
CH2CONHNH2
CH3O
CH3O
EtOH/ Et N
3
+
O
reflux
N
N
H
NHCOCH3
CN
CH3O
CO2Et
10
HN
1
O
11
+
N
O
CH2CO2Et
2
-H2O
NHCOCH3
3
CH3O
EtOH/Pip.
reflux
NHCOCH
NHCOCH3
NHNH2
3
NHCOCH3
O
O
CH2COCH3
N
H
CH3O
CH3O
CH3O
+
+
13
CO2Et
4
N
N
Me
N
1
N
EtOH/ reflux
N
O
HN
HN
5
14
12
Scheme 1.
Scheme 3.

G.A. Elmegeed et al. / European Journal of Medicinal Chemistry 42 (2007) 1285e1292
1287
180
160
140
120
100
80
potency to indomethacin in inhibiting carrageenan-induced
paw oedema response (29.6 vs 27.4% inhibition, respectively,
4 h post-carrageenan). After s.c. administration, all com-
pounds induced significant anti-inflammatory activity, inhibit-
ing the paw oedema response compared with the control group
at all time points in the test (two-way ANOVA: treatment
effect: F5,120 ¼ 58.4; P < 0.001; time effect: F3,120 ¼ 88.9;
P < 0.001) (Fig. 1). The percentages of inhibition were
ꢀ42.9, ꢀ35.7, ꢀ30 and ꢀ27.1% for compound 3; ꢀ67.3,
ꢀ59.4, ꢀ43, and ꢀ28.8% for compound 5; ꢀ52.2, ꢀ55.2,
ꢀ48 and ꢀ36.3% for compound 9a; ꢀ54.8, ꢀ52.6, ꢀ44.2
and ꢀ36.8% for compound 12 compared with e50.8, ꢀ50.1,
ꢀ44.4 and e30% for melatonin at 1, 2, 3 and 4 h time points.
Post-hoc comparison revealed more significant inhibition of
oedema formation by compound 5 compared with compounds
3, 9a, 12 or melatonin, at 1 h post-carrageenan. Compound 3
less significantly inhibited oedema formation than compound
9a at 2e3 h post-carrageenan and also less significantly in-
hibited oedema formation compared with compounds 5, 12
and melatonin at 2 h post-carrageenan.
Carrageenan control
3
5
9a
12
*
Melatonin
*
*
*
60
40
20
0
0
1
2
3
4
Time (h)
Fig. 1. The anti-inflammatory effect of compounds 3, 5, 9a, 12 and melatonin
administered at equimolar doses of 5.16, 5.13, 6.88, 5.40 and 4 mg/kg, respec-
tively. Results are expressed as a percentage change from control (pre-drug)
values, each point represents mean ꢁ S.E. of 6 rats/group. Asterisks indicate
significant change from control value at respective time points (ANOVA and
Duncan’s multiple comparison test).
2.2.2. Tests of nociception
that compound 5 was more effective in inhibiting inflamma-
tion and visceral pain induced by acetic acid injection in
mice, whereas compound 12 showed efficacy in inhibiting
both thermal and visceral pain. The development of gastric
mucosal lesions evoked by the non-steroidal anti-inflamma-
tory drug indomethacin in rats was much reduced by the
administration of compound 5 or 12, indicating safe gastric
profile for these two compounds, similar to that of melatonin
[24,6]. This is of particular importance in the development
of new drugs to target pain conditions, since most of the avail-
able NSAIDs are well known for their propensity to evoke gas-
tric mucosal injury [25,26]. Data obtained with compounds 5
and 12 thus encourage the development of melatonin deriva-
tives which could have particular role in the management of
pain, especially that of visceral origin.
2.2.2.1. Effect of melatonin analogues on thermal pain. Hot-
plate latency was increased after the administration of all
test compounds (Table 1, Fig. 2). One hour after drug injec-
tion, the increase in hot-plate latency by all test compounds
and melatonin was significantly less than that induced by indo-
methacin (IND). Two hours after injection of test compounds,
the increase in hot-plate latency by IND and compound 12 was
significantly more prolonged than that induced by compounds
3, 5, 9a or melatonin. Compound 9a induced significantly less
prolongation of hot-plate latency than compound 3 or 5.
2.2.2.2. Effect of melatonin analogues on acetic acid-induced
writhing. All test compounds significantly reduced the number
of abdominal writhes induced by i.p. injection of acetic acid in
mice (Fig. 3). Compounds 5 and 12 were the most potent in
this respect, inhibiting the number of abdominal writhes by
68.7 ꢁ 4.9 and 81.1 ꢁ 6.1%, respectively, compared with the
control group. Meanwhile, compounds 3, 9a and melatonin in-
hibited the number of abdominal writhes by 24.4 ꢁ 1.2,
40.7 ꢁ 3.6 and 55.6 ꢁ 3.9%, respectively. Post-hoc comparison
revealed that compounds 5 and 12 inhibited the writhing re-
sponsemoresignificantlythan3, 9aandmelatonin, whereasmel-
atonin was more potent than compounds 3 and 9a in this respect.
From the structureeactivity relationship point of view, the
oxomethylpyrrolo[1,2-a]indole derivative 5 has the strongest
anti-inflammatory activity. Replacement of the methyl group
by either carbonyl group in compound 3 or hydrazone group
in compound 12 decreased the anti-inflammatory activity of
the indolopyrrole moiety. The presence of imine and
Table 1
Percentage increase in hot-plate latency in rats treated with melatonin ana-
logues and melatonin in comparison with indomethacin, compared with their
corresponding pre-drug basal level
2.2.2.3. Effect of melatonin analogues on gastric lesions
caused by indomethacin. Compounds 5, 12 or melatonin sig-
nificantly reduced the number of gastric lesions induced by
the administration of indomethacin in rats (Table 2, Fig. 4).
Compound 5 was more potent in this respect than melatonin.
Meanwhile, all test compounds and melatonin significantly re-
duced the severity of indomethacin-induced gastric lesions.
Compound 5 was the most potent in this respect (Fig. 4).
These results indicated that all compounds exhibited anti-
inflammatory and pain alleviating properties. It was noted
Compound
Hot-plate latency (% increase)
1 h
2 h
3
103.6 ꢁ 11.3
116.6 ꢁ 10.3
31.70 ꢁ 4.70
43.70 ꢁ 5.0
39.70 ꢁ 3.80
176.7 ꢁ 21.3
132.3 ꢁ 14.7
144.6 ꢁ 16.4
56.20 ꢁ 6.6
243.7 ꢁ 27.4
96.0 ꢁ 9.2
5
9a
12
Melatonin
Indomethacin
286.6 ꢁ 29.5

1288
G.A. Elmegeed et al. / European Journal of Medicinal Chemistry 42 (2007) 1285e1292
Table 2
Percentage inhibition of the number and severity of indomethacin-induced
gastric lesions in rats treated with melatonin analogues and melatonin
2h
40
30
20
10
0
1h
243.7%
286.6%
Compound
Number of lesions
(% inhibition)
Severity of lesions
(% inhibition)
96%
103.6%
116.6%
132.3%
176.7%
3
43.5 ꢁ 3.1
72.6 ꢁ 5.7
30.2 ꢁ 3.0
53.9 ꢁ 5.0
56.3 ꢁ 4.1
54.8 ꢁ 5.3
82.6 ꢁ 4.2
53.6 ꢁ 3.8
57.7 ꢁ 4.0
69.0 ꢁ 5.2
144.6%
#
*
5
9a
#
#56.2%
*
*
39.7%
+
+
31.7%
*
*
*
12
Melatonin
43.7%
*
*
*
than melatonin. It indicates that the fusion of pyrrole ring to
melatonin moiety is a profitable modification as it increased
its anti-inflammatory and anti-nociceptive activities.
In search for NSAIDs, that can relieve pain and at the same
time do not harm gastric mucosal, selective COX-2 inhibition
have been published [27,28]. Directed by the structure of com-
pounds 3, 5, 9a and 12, it is probably that these compounds
have a dual mechanism for their anti-inflammatory and anti-
nociceptive activities: the selective inhibition of COX-2 by
melatonin moiety [7] or by the fused pyrrole or pyridine
ring [18e20] and the free radical scavenging properties of
melatonin moiety [3,29] and the fused pyrrole or pyridine
ring [21,22]. Further studies should be made to establish the
mechanism of action with the possibility to formulate a potent
anti-inflammatory and anti-nociception prescription.
Fig. 2. Reaction time on the hot-plate in seconds after the administration of
compounds 3, 5, 9a, 12 and melatonin at equimolar doses of 5.16, 5.13,
6.88, 5.40 and 4 mg/kg, respectively. The percentage change from basal
(pre-drug) values is shown (n ¼ 6/group). Asterisks indicate significant change
from the indomethacin (IND)-treated group at the respective time point. The
plus sign (þ) indicates significant change from the melatonin-treated group
at the respective time point. The sign # indicates significant change from
the compound 12-treated group at the respective time point (ANOVA and
Duncan’s multiple comparison tests).
hydrazone groups attached to the indolopyrrole moiety in
compound 12 increased its activity as anti-nociceptive agent
compared to the other tested compounds. The pyrrole moiety
in compounds 5 and 12 was more potent as anti-inflammatory
and anti-nociceptive agent than the pyridine moiety in com-
pound 9a. Compounds 5, 12 are more potent in this respect
2.3. Conclusion
In this study we have described a straightforward and effi-
cient synthesis of novel melatonin analogues containing fused
pyrrole or pyridine nucleus in addition to the pharmacophoric
features of the melatonin moiety. Melatonin and its novel
75
15
-24.4%
Severity of lesions
+
*
-40.7%
50
25
0
Number of lesions
10
+
*
-55.6%
-53.6%
*
-54.8%
-57.7%
-68.7%
-56.3%
*
-30.2%
*
*
-43.5%
-69%
*
-82.6%
-81.1%
5
0
-53.9%
*
-72.6%
*
*
*
*
*
Fig. 3. Effect of compounds 3, 5, 9a, 12 and melatonin, administered at equi-
molar doses of 5.16, 5.13, 6.88, 5.40 and 4 mg/kg, respectively, on the number
of abdominal writhes induced by i.p. injection of acetic acid in mice (mean
S.E. of 6e8 mice/group). The percent decrease in the number of writhes
from the saline control group is represented above the respective group bar.
Asterisks indicate significant change from the saline control group, whereas
the plus sign (þ) indicates significant change from compound 5- or 12-treated
group (ANOVA and Duncan’s multiple comparison tests).
Fig. 4. Effect of compounds 3, 5, 9a, 12 and melatonin, administered at equi-
molar doses of 5.16, 5.13, 6.88, 5.40 and 4 mg/kg, respectively, on the number
and severity of gastric mucosal lesions caused by s.c. injection of indometha-
cin in rats. The percent decrease in the number or severity of gastric lesions
from the indomethacin control group is represented above the respective group
bar. Asterisks indicate significant change from the corresponding indometha-
cin (IND) control group (ANOVA and Duncan’s multiple comparison test).

G.A. Elmegeed et al. / European Journal of Medicinal Chemistry 42 (2007) 1285e1292
1289
synthesized derivatives 3, 5, 9a and 12 showed anti-inflamma-
tory and anti-nociception activities with various intensities.
The oxomethylpyrrolo[1,2-a]indole derivative 5 has the stron-
gest anti-inflammatory activity which exceeds that of the
parent reference, melatonin, followed by iminopyrido[1,2-a]in-
dole derivative 9a and iminohydrazinopyrrolo[1,2-a]indole de-
rivative 12, at the first 2 h of administration. Compounds 5 and
12 were more potent as anti-nociceptive drugs; they are more
potent in this respect than melatonin.
100.3 (C-8), 112.8 (C-9), 118.3 (fused pyrrole-C), 127.5,
ꢄ
131.6 (fused-aromatic-C). MS (EI): m/z (%): 300 (Mþ , 35),
269 (22), 242 (100), 225 (50). Anal. Calcd. for C16H16N2O4
(300.312): C, 63.99; H, 5.37; N, 9.32; found: C, 64.11; H,
5.54; N, 9.08.
3.1.1.2. N -[2-(3-Oxo-7-methoxy-1-methyl-1H-pyrrolo[1,2-a]
indol-9-yl)ethyl]acetamide (5). Pale yellow crystals, from
1,4-dioxane, yield 1.16 g (78%), mp 148e150 ꢃC. IR (y/
cmꢀ1): 3369 (NH), 3035 (CH-aromatic), 2978 (CH3), 2850
1
3. Experimental section
(CH2), 1740, 1695 (2C]O). H NMR (d ppm): 1.82 (s, 3H,
COCH3), 2.32 (s, 3H, CH3), 3.02 (t, J ¼ 7.1 Hz, 2H, CH2),
3.45 (t, J ¼ 7.1 Hz, 2H, CH2), 3.85 (s, 3H, OCH3), 4.97 (brs,
1H, NH, D2O-exchangeable), 6.23 (s, 1H, pyrrole H-b),
7.57e7.90 (m, 3H, aromatic-H). 13C NMR (d): 23.0
(COCH3), 173.4 (COCH3), 23.8, 37.2 (2CH2), 27.6 (CH3),
55.6 (OCH3), 134.2 (C-1), 115.0 (C-2), 196.7 (C-3, C]O),
109.7 (C-5), 110.3 (C-6), 150.9 (C-7), 102.0 (C-8), 112.4
(C-9), 118.7 (fused pyrrole-C), 127.2, 130.8 (fused-aromatic-
3.1. Synthesis
All chemicals were purchased from commercial suppliers
and used directly. The appropriate precautions in handling
moisture-sensitive compounds were undertaken. All melting
points were measured using an electrothermal capillary melt-
ing point apparatus and are uncorrected. The IR spectra
were recorded (KBr discs) on a Shimadzu FT-IR 8201 PC
ꢄ
C). MS (EI): m/z (%): 298 (Mþ , 27), 283 (34), 267 (22),
spectrophotometer and expressed in cmꢀ1. H and 13C NMR
240 (100), 214 (42). Anal. Calcd. for C17H18N2O3
(298.342): C, 68.44; H, 6.08; N, 9.38; found: C, 68.17; H,
6.27; N, 9.20.
1
spectra were recorded on a Varian EM-390 90 MHz spectrom-
eter in DMSO-d6 as solvent, using TMS as internal reference
and chemical shifts (d) are expressed in ppm. Mass spectra
were recorded on a GCMS-QP 1000 Ex spectra mass spec-
trometer operating at 70 eV. Elemental analyses were carried
out by Microanalytical Data Unit at National Research Centre,
Cairo, Egypt. Analytical thin-layer chromatography (TLC)
was carried out using Merck 60 F254 aluminum sheets and
visualized by UV light (254 nm).
3.1.2. N-[2-(1-Benzoylmethino-5-methoxyindol-3-yl)ethyl]-
acetamide (7)
To a mixture of melatonin (1) (1.16 g, 0.005 mol) and phe-
nacyl bromide 6 (1 g, 0.005 mol) in 1,4-dioxane (30 mL),
a catalytic amount of triethylamine was added. The reaction
mixture was heated under reflux for 7 h, cooled at room tem-
perature, poured into ice and acidified with dilute hydrochloric
acid. The solution was extracted with chloroform (3 ꢂ 20 mL),
the organic extract was dried over calcium chloride and con-
centrated under vacuum to dryness to yield 1.36 g (78%) of
compound 7 as oil, IR (y/cmꢀ1): 3384 (NH), 3050 (CH-
aromatic), 2985 (CH3), 2830 (CH2), 1720, 1690 (2C]O).
1H NMR (d ppm): 1.87 (s, 3H, COCH3), 2.98 (t, J ¼ 7.2 Hz,
2H, CH2), 3.35 (t, J ¼ 7.2 Hz, 2H, CH2), 3.85 (s, 3H,
OCH3), 4.43 (s, 2H, CH2), 4.95 (brs, 1H, NH, D2O-exchange-
able), 6.75 (s, 1H, indole H-2), 7.46e7.98 (m, 8H, aromatic-
3.1.1. General method for preparation of compounds
(3) and (5)
To a solution of melatonin (1) (1.16 g, 0.005 mol) in etha-
nol (25 mL) containing an amount of piperidine (2 mL) either
diethylmalonate (0.80 g, 0.005 mol) or ethyl acetoacetate
(0.65 g, 0.005 mol) was added. The reaction mixture, in each
case, was heated under reflux for 5 h until all starting materials
had disappeared as indicated by TLC, and then left to cool at
room temperature, poured over ice/water mixture, neutralized
with dilute hydrochloric and extracted with diethyl ether
(3 ꢂ 20 mL). The organic layer, in each case, was dried over
calcium chloride. Removal of the solvent in vacuo afforded
the corresponding product, which was crystallized from the
appropriate solvent.
ꢄ
H). MS (EI): m/z (%): 351 (MHþ , 52), 231 (24), 200 (43),
120 (100), 105 (22), 77 (37). Anal. Calcd. for C21H22N2O3
(350.424): C, 71.98; H, 6.32; N, 7.99; found: C, 72.25; H,
6.12; N, 8.21.
3.1.3. General method for preparation of compounds
(9a) and (9b)
3.1.1.1. N-[2-(1,3-Dioxo-dihydro-7-methoxy-1H-pyrrolo[1,2-a]-
indol-9-yl)ethyl]acetamide (3). Yellow crystals, from MeOH,
yield 1.23 g (82%), mp 189e190 ꢃC. IR (y/cmꢀ1): 3380 (NH),
3030 (CH-aromatic), 2970 (CH3), 2853 (CH2), 1745, 1738,
1685 (3C]O). 1H NMR (d ppm): 1.78 (s, 3H, COCH3),
3.04 (t, J ¼ 7.2 Hz, 2H, CH2), 3.43 (t, J ¼ 7.2 Hz, 2H, CH2),
3.82 (s, 3H, OCH3), 4.92 (brs, 1H, NH, D2O-exchangeable),
6.15 (s, 2H, pyrrole H-b), 7.45e7.85 (m, 3H, aromatic-H).
13C NMR (d): 22.6 (COCH3), 172.5 (COCH3), 23.5, 38.2
(2CH2), 54.7 (OCH3), 196.7 (C-1, C]O), 111.6 (C-2),
214.8 (C-3, C]O), 109.6 (C-5), 110.7 (C-6), 152.9 (C-7),
To a solution of compound 7 (0.7 g, 0.002 mol) in absolute
ethanol (25 mL) containing a catalytic amount of triethyl-
amine either malononitrile 8a (0.13 g, 0.002 mol) or ethyl
cyanoacetate 8b (0.23 g, 0.002 mol) was added. The reaction
mixture, in each case, was heated under reflux for 4 h until
all starting materials had disappeared as indicated by TLC,
and then left to cool at room temperature. The solution was
concentrated under vacuum, whereby the resulted oily product
was triturated with ice/water mixture and neutralized with di-
lute hydrochloric acid. The formed solid product, in each case,

1290
G.A. Elmegeed et al. / European Journal of Medicinal Chemistry 42 (2007) 1285e1292
1
was filtered off, washed several times with water, dried and
crystallized from the appropriate solvent.
2865 (CH2), 1692 (C]O). H NMR (d ppm): 1.87 (s, 3H,
COCH3), 3.05 (t, J ¼ 7.0 Hz, 2H, CH2), 3.47 (t, J ¼ 7.0 Hz,
2H, CH2), 3.80 (s, 3H, OCH3), 4.95 (brs, H, NH, D2O-
exchangeable), 5.25 (s, 2H, NH2, D2O-exchangeable), 6.17
(s, 1H, pyrrole H-b), 7.67e7.88 (m, 3H, aromatic-H), 8.58,
8.72 (2brs, 2H, 2NH, D2O-exchangeable). 13C NMR (d):
22.8 (COCH3), 171.5 (COCH3), 23.5, 38.2 (2CH2), 54.9
(OCH3), 134.8 (C-1), 108.7 (C-2), 133.6 (C-3), 109.6 (C-5),
110.7 (C-6), 152.9 (C-7), 100.3 (C-8), 112.8 (C-9), 126.9,
131.4 (fused-aromatic-C), 118.3 (fused pyrrole-C). MS (EI):
3.1.3.1. 10-[2-(Acetylamino)ethyl]-9-imino-2-methoxy-7-phenyl-
6,9-dihydropyrido[1,2-a]indole-8-carbonirile(9a). Brown pow-
der, from EtOH, yield 0.65 g (82%), mp 181e182 ꢃC. IR (y/
cmꢀ1): 3356e3375 (2NH), 3040 (CH-aromatic), 2974 (CH3),
2853 (CH2), 2225 (CN), 1695 (C]O). 1H NMR (d ppm):
1.87 (s, 3H, COCH3), 3.07 (t, J ¼ 7.2 Hz, 2H, CH2), 3.47 (t,
J ¼ 7.2 Hz, 2H, CH2), 3.79 (s, 3H, OCH3), 4.94 (brs, 1H,
NH, D2O-exchangeable), 7.55e8.04 (m, 8H, aromatic-H),
8.35 (s, 2H, pyridine H-a), 8.75 (brs, 1H, NH, D2O-exchange-
able). 13C NMR (d): 22.7 (COCH3), 173.2 (COCH3), 23.4, 37.8
(2 CH2), 54.8 (OCH3), 122.6 (CN), 101.3 (C-1), 152.5 (C-2),
110.2 (C-3), 109.8 (C-4), 115.0 (C-6), 151.6 (C-7), 114.8 (C-
8), 133.5 (C-9), 113.2 (C-10), 149.3 (fused pyridine-C),
128.3, 132.2 (fused-aromatic-C), 148.3, 113.2, 113.8, 129.3,
ꢄ
m/z (%): 314 (MHþ , 52), 282 (25), 176 (30), 145 (100).
Anal. Calcd. for C16H19N5O2 (313.362): C, 61.32; H, 6.11;
N, 22.34; found: C, 61.15; H, 6.31; N, 22.57.
3.1.5. N-[2-(1-(3,5-Dimethylpyrazol-1-yl)-3-imino-7-
methoxy-1H-pyrrolo[1,2-a]indol-9-yl)ethyl]acetamide (14)
A mixture of compound 12 (1.56 g, 0.005 mol) and acetyl
acetone 2 (0.5 g, 0.005 mol) in absolute ethanol (25 mL)
was boiled under reflux for 3 h until all starting materials
had disappeared as indicated by TLC. Then the reaction mix-
ture was concentrated under vacuum, whereby the resulted oily
product was triturated with petroleum ether (bp 60e80 ꢃC).
The formed solid product was filtered off, dried and crystal-
lized from MeOH to yield 1.35 g (72%) of compound 14,
brown crystals, mp 239e241 ꢃC. IR (y/cmꢀ1): 3380e3295
(2NH), 3035 (CH-aromatic), 2977 (CH3), 2875 (CH2), 1695
ꢄ
129.5, 131.4 (phenyl-C). MS (EI): m/z (%): 398 (Mþ , 40),
367 (38), 321 (48), 292 (100), 77 (67). Anal. Calcd. for
C24H22N4O2 (398.462): C, 72.34; H, 5.56; N, 14.06; found:
C, 72.52; H, 5.29; N, 13.90.
3.1.3.2. Ethyl 10-[2-(acetylamino)ethyl]-9-imino-2-methoxy-7-
phenyl-6,9-dihydropyrido[1,2-a]indole-8-carboxylate (9b).
Yellow powder, from MeOH, yield 0.66 g (75%), mp 217e
218 ꢃC. IR (y/cmꢀ1): 3350e3373 (2NH), 3050 (CH-aromatic),
2975 (CH3), 2869 (CH2), 1735, 1698 (2C]O). 1H NMR
(d ppm): 1.13 (t, J ¼ 6.8 Hz, 3H, ester CH3), 1.79 (s, 3H,
COCH3), 2.95 (t, J ¼ 7.2 Hz, 2H, CH2), 3.40 (t, J ¼ 7.2 Hz,
2H, CH2), 3.85 (s, 3H, OCH3), 4.25 (q, J ¼ 6.8 Hz, 2H, ester
CH2), 4.95 (brs, 1H, NH, D2O-exchangeable), 7.54e8.10
(m, 8H, aromatic-H), 8.45 (s, 2H, pyridine H-a), 8.82 (s,
1H, NH, D2O-exchangeable). 13C NMR (d): 22.1 (COCH3),
174.8 (COCH3), 23.8, 36.3 (2 CH2), 55.3 (OCH3), 15.8
(CO2CH2CH3), 24.2 (CO2CH2CH3), 172.6 (CO2CH2CH3),
102.0 (C-1), 153.7 (C-2), 110.2 (C-3), 109.2 (C-4), 115.7
(C-6), 152.4 (C-7), 114.5 (C-8), 134.5 (C-9), 113.5 (C-10),
148.4 (fused pyridine-C), 128.7, 131.9 (fused-aromatic-C),
148.5, 113.7, 114.3, 129.3, 129.7, 131.4 (phenyl-C). MS
1
(C]O). H NMR (d ppm): 1.75 (s, 3H, COCH3), 2.26 (s,
6H, 2CH3), 3.03 (t, J ¼ 7.0 Hz, 2H, CH2), 3.38 (t,
J ¼ 7.0 Hz, 2H, CH2), 3.85 (s, 3H, OCH3), 4.78 (brs, 1H,
NH, D2O-exchangeable), 6.12 (s, 1H, pyrrole H-b), 6.65 (s,
1H, pyrazole H-4), 7.68e7.92 (m, 3H, aromatic-H), 8.62
(brs, 1H, 1NH, D2O-exchangeable). 13C NMR (d): 22.3
(COCH3), 173.5 (COCH3), 23.5, 37.3 (2 CH2), 54.7 (OCH3),
25.3, 26.2 (2CH3), 134.8 (C-1), 108.6 (C-2), 133.6 (C-3),
109.2 (C-5), 110.3 (C-6), 152.8 (C-7), 101.3 (C-8), 112.8
(C-9), 125.3, 132.2 (fused-aromatic-C), 118.7 (fused pyrrole-
C), 135.2, 106.3, 136.7 (pyrazole-C). MS (EI): m/z (%): 377
ꢄ
(Mþ , 35), 356 (42), 282 (54), 271 (30), 145 (100), 95 (63).
Anal. Calcd. for C21H23N5O2 (377.452): C, 66.82; H, 6.14;
N, 18.55; found: C, 67.05; H, 6.32; N, 18.76.
ꢄ
(EI): m/z (%): 445 (Mþ , 24), 339 (35), 308 (100), 77 (56).
Anal. Calcd. for C26H27N3O4 (445.523): C, 70.09; H, 6.10;
N, 9.43; found: C, 70.25; H, 5.91; N, 9.65.
3.2. Pharmacological assay
3.1.4. N-[2-(3-Imino-1-hydrazino-7-methoxy-1H-pyrrolo
[1,2-a]indol-9-yl)ethyl]acetamide (12)
3.2.1. Animals
SpragueeDawley strain rats weighing 120e130 g or Swiss
albino mice 20e25 g body weight was used throughout the
experiments. Food and water were provided ad libitum. Exper-
iments were performed between 0900 and 1500 h.
Cyanoacetylhydrazide 10 (0.5 g, 0.005 mol) was added to
a solution of melatonin (1) (1.16 g, 0.005 mol) in absolute eth-
anol (30 mL) containing a catalytic amount of triethylamine.
The reaction mixture was refluxed for 3 h and then evaporated
to dryness under reduced pressure. The residue was dissolved
in diethyl ether and the solution was washed with saturated
sodium carbonate. The organic layer was separated, dried
over anhydrous magnesium sulfate and the solvent was re-
moved under reduced pressure. The resulting solid was recrys-
talized from 1,4-dioxane to give 1.25 g (80%) of compound
12, pale brown crystals, mp 219e220 ꢃC. IR (y/cmꢀ1):
3458e3295 (2NH, NH2), 3043 (CH-aromatic), 2970 (CH3),
3.2.2. Tests of inflammation: carrageenan-induced
paw oedema assay
All tested compounds were screened for anti-inflammatory
activity using the carrageenan-induced paw oedema assay in
rats. This model is widely used as a screening tool for evaluation
of putative anti-inflammatory agents. The activity of the com-
pounds was compared with melatonin at a dose of 4 mg/kg.
The dose of melatonin was chosen based on previous studies

G.A. Elmegeed et al. / European Journal of Medicinal Chemistry 42 (2007) 1285e1292
1291
[30]. The equimolar doses (0.0172 mol) 5.16, 5.13, 6.88 and
5.40 mg/kg of compounds 3, 5, 9a and 12, respectively, were
used.
a percentage of change from control (pre-drug) values. Differ-
ences between vehicle control and treatment groups were
tested using one- and two-way ANOVA followed by multiple
comparison by the Duncan’s multiple comparison test. A prob-
ability value less than 0.05 was considered statistically
significant.
Paw oedema was induced by sub-plantar injection of 100 mL
of 1% sterile carrageenan lambda in saline into the right hind
paw [31]. Contra-lateral paw received an equal volume of
saline. Paw volume was determined immediately before carra-
geenan injection and at selected times thereafter using a
plethysmometer (Ugo Basile, Milan, Italy). The oedema
component of inflammation was quantified by measuring the
increase in paw volume (mL) before carrageenan injection
and at 1, 2, 3 and 4 h after carrageenan injection with respect
to the pre-injection value for each animal. Oedema was
expressed as a percentage of change from control (pre-drug)
values. The effect of systemic administration of each of the
test compounds 3, 5, 9a or 12 at doses of 5.16, 5.13, 6.88 or
5.40 mg/kg, respectively (0.5 mL, s.c., n ¼ 6/group) or mela-
tonin (4 mg/kg, s.c., 0.5 mL) given as a 30 min pretreatment
was studied. The control groups received saline (0.5 mL,
n ¼ 6/group; s.c.) instead.
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