Potent neuroprotective role of novel melatonin derivatives for management of central neuropathy induced by acrylamide in rats

Article abstract of DOI:10.1016/j.ejmech.2010.09.017

Acrylamide (ACR) has been shown to be a neurotoxic agent for both laboratory animals and human. The present study aimed at synthesizing new functionalized melatonin derivatives bearing promising heterocyclic moiety that could be expected to have protectiv

Full text of DOI:10.1016/j.ejmech.2010.09.017

European Journal of Medicinal Chemistry 45 (2010) 5452e5459
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Potent neuroprotective role of novel melatonin derivatives for management of
central neuropathy induced by acrylamide in rats
,
b
a
Hanaa H. Ahmed a, Gamal A. Elmegeed a , El-Sayed M. El-Sayed , Mervat M. Abd-Elhalim ,
*
Wafaa Gh. Shousha b, Reham W. Shafic a
a Hormones Department, National Research Centre, Dokki, Cairo, Egypt
b Chemistry Department, Faculty of Science, Helwan University, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2010
Received in revised form
14 August 2010
Accepted 8 September 2010
Available online 17 September 2010
Acrylamide (ACR) has been shown to be a neurotoxic agent for both laboratory animals and human. The
present study aimed at synthesizing new functionalized melatonin derivatives bearing promising
heterocyclic moiety that could be expected to have protective effect against ACR-induced neurotoxicity in
adult female rats. The novel melatonin derivatives 4, 6, 7 and 11 were synthesized and their chemical
structures were confirmed by studying their analytical and spectral data. The administration of ACR [i.p.,
50 mg kgꢀ1 body weight (b. wt.)] alone resulted in significant increase in brain malondialdehyde level
(MDA) and lactate dehydrogenase (LDH) activity whereas it caused significant decrease in brain
monoamines levels and antioxidant enzymes activity. Treatment with melatonin derivatives 4, 6, 7 and
11 (i.p., 50 mg kgꢀ1 b. wt) prior to ACR produced significant decrease in brain MDA level and LDH activity
with concomitant significant increase in brain monoamines and antioxidant enzymes activity. It could be
concluded that the new synthesized melatonin derivatives exhibited promising protective activity
against ACR-induced neurotoxicity.
Keywords:
Melatonin
Thiadiazole
Morpholine
Pyrazole
Neuroprotective
Acrylamide
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
Melatonin, the main secretory product of the pineal gland, has
been shown to be potentially effective in prevention of numerous
Acrylamide (ACR) is water soluble, vinyl monomer that has
multiple chemical and industrial applications such as waste water
management, or processing, preparing polymers or co-polymers
containing polar functional groups and also it is used extensively in
molecular laboratories for gel chromatography [1]. ACR is
frequently observed in foods that have been prepared at very high
temperatures, in potato and grain-based foods, and thus humans
consume relatively high doses of ACR during life [2]. Extensive
studies in rodents and other laboratory animals have provided
evidence that exposure to ACR causes genotoxicity, cellular damage
in both nervous and reproductive systems, and produces tumors in
certain hormonally responsive tissues. However, only neurotoxicity
has been well identified by epidemiological studies on human
population. ACR neurotoxicity has been known to affect nerve
terminal and ACR could adduct cysteine residues on functionally
important presynaptic proteins, resulting in neurotransmitter
release inhibition and eventual process degeneration [3,4].
types of neurodegenerative disorders in which free radical
processes are involved [5]. In brief, melatonin has been found to
protect the CNS from beta-amyloid toxicity, glutamate-induced
excitotoxicity, nitric oxide toxicity, cyanide toxicity and ischemia/
reperfusion injury [6]. It is well known that indole derivatives,
extensively present in natural compounds and are very important
substances in medicinal and biological aspects. The antioxidant
effect of indole ring carrier melatonin has been well described [7].
The development of synthetic compounds capable of mimicking
the effects of melatonin has progressed considerably during the
past decade. These compounds are structurally diverse, and ranged
from simple indole derivatives and its bioisosteres, to phenylalkyl
amides and constrained melatoninergic agents [8,9]. For instances,
many heterocyclic moieties have been demonstrated as neuro-
protective agents [10].
In the scope of a research program aimed at the development of
new alternatives to treat neurological disorders [11,12]. We describe
in the present study the synthesis of new functionalized melatonin
derivatives bearing promising heterocyclic moiety, various polar,
basic, flexible and/or rigid functional groups fused to the essential
pharmacophoric features of the parent molecule. Furthermore, the
* Corresponding author. Hormones Department, National Research Centre, El-
Bohose St., 12622 Dokki, Cairo, Egypt. Tel.: þ2 02 35682070; fax: þ2 02 33370931.
E-mail address: gamalae@hotmail.com (G.A. Elmegeed).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.017

H.H. Ahmed et al. / European Journal of Medicinal Chemistry 45 (2010) 5452e5459
5453
potential neuroprotective effect of these newly synthesized agents
against ACR-induced neurotoxicity in rats was investigated.
(Scheme 1) [16]. Compound 2 seemed to be a key precursor for
many interesting chemistry. The reactivity of acetonitrile moiety
of compound 2 towards the formation of pyrazoles was studied.
Thus, compound 2 was allowed to react with the acetyl hydra-
zonyl halide in sodium ethoxide solution afforded the corre-
sponding pyrazolyl thiadiazoloindole derivative 4 in 70% yield via
the intermediacy of compound 3 (Scheme 1). The mass spectrum
(EIMS) of compound 4 showed molecular ion peak at m/z 487
(Mþ$ ꢀ 1, 65%). The IR spectrum of compound 4 revealed the
2. Results and discussion
2.1. Chemistry
To improve the essential pharmacophoric features of mela-
tonin molecules, we have outlined the synthesis of several indole
derivatives containing promising heterocyclic moiety starting
with melatonin. Therapeutic agents containing thiadiazole, pyr-
azole, morpholine, have attracted the attention of researches in
pharmaceutical and biochemistry [13e15]. In a recent commu-
nication from this laboratory we have reported about the
synthesis of thiadiazoloindole derivatives 2 by the reaction of
melatonin (1) with equimolar amounts of malononitrile and
elemental sulfur in refluxing ethanolic triethylamine solution
presence of amino group stretching at
y
¼ 3375 cmꢀ1 and two
carbonyl groups at
trum showed D2O-exchangeable singlet (2H) at
y
¼ 1732 and 1695 cmꢀ1. The 1H NMR spec-
d
¼ 5.23 for the
amino group and two multiplets at
at
¼ 7.05e7.08 (5H, C6H5).
Phenyl indole derivatives significantly inhibited lipid perox-
d
¼ 6.95e7.03 (3H, C6H3) and
d
idation and exhibited important antioxidant activity [17]. The
reaction of compound 2 with acetophenon in the presence of
CH2CH2NHCOCH3
CH2CH2NHCOCH3
H3CO
H3CO
CN
EtOH / Et3N
+
S8
+
S
CN
N
H
N
N
2
CH2CN
1
fussion / amm.acetate
NaOEt
stirring
O
Cl
PhCOCH3
NNHPh
C
CH3C
CH2CH2NHCOCH3
CH2CH2NHCOCH3
H3CO
H3CO
S
S
N
N
CN
N
CN
N
H3COC
NNHPh
Ph
CH3
CN
CN
3
5
CH2CH2NHCOCH3
H3CO
CH2CH2NHCOCH3
S
NH2
N
H3CO
N
CN
S
NH2
N
Ph
N
NH
N
Ph
6
N
H3COC
4
Scheme 1.

5454
H.H. Ahmed et al. / European Journal of Medicinal Chemistry 45 (2010) 5452e5459
ammonium acetate afforded the condensed product, butyroni-
trilylthiadiazolo-indole derivative in 68% yield [18] which
melatonin derivative 7 in 80% yield as a single product as confirmed
by the analytical and spectral data (Scheme 2). The mass spectrum
(EIMS) of compound 7 showed molecular ion peak at m/z 487
(Mþ$ þ 1, 72%). The IR spectrum revealed in addition to the bands of
5
cyclized on heating with malononitrile in ethanolic triethylamine
solution to give the biphenyl thiadiazoloindole derivative 6 in 75%
yield (Scheme 1). Elucidation of the proposed structure 6 was based
on their correct elemental analyses and compatible spectroscopic
data (cf. Materials and methods).
Morpholine derivatives possess various biological activities in
addition to their powerful antioxidant and free radical scavenging
properties [13]. In addition some morpholine derivatives promote
neuroprotection and neurite elongation through protein kinase C
(PKC) signaling on motoneurons [19]. Hence it was thought of
interest to undertake the synthesis of morpholinoindole derivative.
Melatonin reacts with equimolar amounts of formaldehyde and
morpholine in tetrahydrofurane to afford 1-morpholinomethyl
melatonin moiety the presence sharp band at
y
¼ 1045 cmꢀ1 due to
the morpholine CeO group. The 1H NMR spectrum showed singlet
(2H) at
¼ 2.27e2.35 (4H) and at
pholine groups (cf. Materials and methods). The 13C NMR spectrum
showed two peaks at
d
¼ 4.94 for the CH2-N group and two multiplets at
d
d
¼ 3.67e3.72 (4H) for the CH2-mor-
d
¼ 51.2 and 67.1 for the morpholine-C.
A wide range of antioxidant activities have been attributed to
pyrazoles [20]. Thus, we would like to report here on the formation
and characterization of novel 1-pyrazolyl melatonin derivatives.
The indole NH group of melatonin reacted with
a-bromoaceto-
phenone to afford 1-benzoyl methinomelatonin derivative 8 in 78%
CH2CH2NHCOCH3
H3CO
THF /
1
+
+
O
CH2O
HN
N
H2C
N
O
7
CH2CH2NHCOCH3
H3CO
dioxan / pip
1
+
PhCOCH2Br
N
CH2COPh
8
DMFDMA
xylene
CH2CH2NHCOCH3
CH2CH2NHCOCH3
H3CO
H3CO
NH2NH2.H2O
N
N
EtOH / Et3N
Ph
H3C
N
HC
H3C
H3C
COPh
N
HC
H3C
NNH2
10
9
CH2CH2NHCOCH3
H3CO
N
Ph
N
N
H
11
Scheme 2.

H.H. Ahmed et al. / European Journal of Medicinal Chemistry 45 (2010) 5452e5459
5455
yield [21] (Scheme 2). Heating of compound 8 with dime-
thylformamide dimethylacetal (DMF-DMA) in xylene afforded the
corresponding 1-(dimethylamino)-3-oxophenyl propene indole
derivative 9 (Scheme 2) in 70% yield as confirmed by IR, 1H NMR,
MS and elemental analysis (cf. Materials and methods). Further
confirmation for the structure of compound 9 was obtained by
studying its reactivity towards nucleophilic reagents. Thus,
compound 9 reacts with hydrazine hydrate in ethanolic triethyl-
amine solution to give the pyrazolyl melatonin derivative 11
(Scheme 2). The reaction takes place via simple condensation of
hydrazine hydrate to the carbonyl group of compound 9 to give the
intermediate 10 which readily underwent cyclization via loss of
dimethylamine to afford compound 11 in 78% yield. All the
elemental analysis and spectroscopic data of compound 11 are in
accordance with the proposed structure (cf. Materials and
methods).
compounds exhibit neuroprotective effect. It was noted that
melatonin and morpholinomethyl melatonin derivative 7 were the
most potent in this respect, whereas compound 6 showed efficacy
more than that of compounds 11 and 4. Many reports have docu-
mented the neuroprotective action of melatonin in various models
of neurodegenerative diseases [22,23]. The possible mechanism by
which, melatonin could protect adrenergic, noradrenergic and
dopaminergic neurons is the ability of melatonin to distribute
readily in all subcellular compartments due to its solubility in both
water and lipids. As such, it can pass through the cell membrane
easily and enter the neurons to remove free radicals and limits
breakdown of membrane lipids [24]. Supplying proof of this action
is that melatonin interrupts the suicide program that causes
apoptotic death in such beleaguered neurons [25]. Also, melatonin
directly scavenges peroxynitrite and its metabolites peroxynitrous
acid and hydrogen peroxide [26] which plays a major role in the
degeneration of the dopaminergic neurons. In addition to the
essential pharmacophoric features of the melatonin molecule,
compound 7 containing morpholine ring which is considered as
agonist of sigma-1 receptor (Sig-1R) with significant neuro-
protective effect [19]. Similarly, the biphenyl thiadiazoloindole
derivative 6 has been shown to be sig-1R agonist, as described
before for similar biphenyl derivatives [27]. Sig-1R agonists
modulate the neuronal response to pharmacologic stimulation of
the N-methyl-D-aspartate (NMDA) receptor [28] and exert neuro-
protective action in animal and neural culture models of cerebral
ischemia [29]. Sig-1R agonists are able to stimulate the secretion of
catecholamines from adrenal chromaffin cells due to their positive
effect on acetylcholine synthesis in the brain [30]. Additionally,
a growing body of evidence indicated that Sig-1R agonists coul-
d increase DA concentration in different brain regions [31]. The
pyrazole ring of compounds 11 and 4 behaves as ligand of benzo-
diazepine receptor which improves functional recovery of ACR-in-
duced neuropathy in rat due to its neuroprotective effect [32].
Another possible mechanism for the neuroprotective activity of
pyrazole derivative is that it could inhibit neural nitric oxide syn-
thase (nNOS) activity [19] with consequent reduction in NO gener-
ation and formation of peroxynitrite that inhibit the activity of
tyrosine hydroxylase, rate limiting enzyme in the biosynthesis of the
neurotransmitter DA [33]. The thiadiazole moiety of compounds 6
and 4 functions as muscarinic agonist [34] that can augment the
release of NA from chromaffin cells.
2.2. Biochemistry assay
The in vivo potent neuroprotective effect of melatonin and its
novel synthesized derivatives, compounds 4, 6, 7 and 11, against
ACR-induced neurotoxicity in rats was investigated. Neuronal
damage due to ACR was detected by measuring: (1) The levels of
brain monoamines, adrenaline (AD), noradrenalin (NA) and dopa-
mine (DA), (2) The end product of brain lipid peroxidation,
malondialdehyde, (MDA), (3) The activity of brain antioxidative
enzymes, glutathione peroxidase (GPx), superoxide dismutase
(SOD) and glutathione content (GSH), (4) The activity of brain
enzymes lactate dehydrogenase (LDH) and creatine kinase (CK).
2.2.1. Effect of tested compounds administration on brain
monoamines level
The data reported in Table 1 demonstrated that ACR-adminis-
tration induced degenerative effect on brain tissue as manifested by
the significant decrease in the levels of the neurotransmitters, AD,
NA and DA (40%, 21%, 16% respectively) as compared to the control
group. These results are in agreement with those of Lopachin [3]
and Mannaa et al. [11]. Lopachin [3] suggested that the inhibition
of neurotransmission contributes significantly to the development
of corresponding neurological deficits as a potential mechanism of
synaptic dysfunction. Administration with melatonin or the newly
tested compounds 4, 6, 7 and 11 showed significant increase in
brain AD level (67%, 60%, 63%, 65%, 60% respectively), brain NA level
(32%, 14%, 28%, 28%, 11% respectively) and brain DA level (33%, 10%,
11%, 19%, 3% respectively) as compared to ACR-intoxicated group.
This increase led to reversing monoamines level approaching that
of the control group. These results indicated that, all tested
2.2.2. Effect of tested compounds administration on brain oxidant/
antioxidant status
The level of MDA in the brain was significantly increased (31%)
in ACR-intoxicated rats as compared to the control group (Table 2).
On the other hand, the brain antioxidant enzymes, SOD, GPx
activity and GSH content significantly decreased in ACR-intoxicated
rats, by the ratio (32%, 43% and 27% respectively), as compared to
the control group (Table 2). These results are in consistent with
those of Mannaa et al. [11] and Zhu et al. [35]. In contrast, admin-
istration of melatonin or other one of the newly tested compounds
4, 6, 7, 11 significantly decreased the brain level of MDA as
compared to ACR-intoxicated group by the percentages (22%, 19%,
20%, 21%, 17% respectively). Meanwhile, administration of mela-
tonin or its novel derivatives increased the activity of the antioxi-
dant enzyme SOD significantly by percentage (135%, 65%, 71%, 77%,
41% respectively) as compared to ACR-intoxicated group (Table 2).
Melatonin and morpholinomethyl melatonin derivative 7 revealed
the most potent effect in this concern followed by compounds 6
and 4. The brain GPX activity showed significant increase (38%) in
the group administered with melatonin as compared to ACR-
intoxicated group. Administration with all tested compounds
showed insignificant change in brain GPX activity as compared to
Table 1
Effect of melatonin tested compound on brain monoamines levels in ACR-intoxi-
cated rats.
Groups
AD (ng gꢀ1
)
NA (ng gꢀ1
)
DA (ng gꢀ1
)
Control
86 ꢁ 1.0
863 ꢁ 14.5
1508 ꢁ 37.1
Acrylamid
Melatonin
þ ACR
52 ꢁ 1.5a (ꢀ40%)
87 ꢁ 1.3b (67%)
680 ꢁ 22.7a (ꢀ21%)
894 ꢁ 4.8b (32%)
1267 ꢁ 50.3a (ꢀ16%)
1690 ꢁ 17.8b (33%)
Compound
4 þ ACR
83 ꢁ 3.7b (60%)
85 ꢁ 0.7b (63%)
86 ꢁ 0.8b (65%)
83 ꢁ 1.0b (60%)
775 ꢁ 23.4bd (14%)
869 ꢁ 12.4b (28%)
871 ꢁ 19.9b (28%)
753 ꢁ 8.8bd (11%)
1395 ꢁ 13.6bd (10%)
1412 ꢁ 27.2bd (11%)
1503 ꢁ 41bd (19%)
1305 ꢁ 12.1d (3%)
Compound
6 þ ACR
Compound
7 þ ACR
Compound
11 þ ACR
Within each column means with different superscript letters are significantly
different (p ꢂ 0.05). AD: adrenaline, NA: noradrenalin, DA: dopamine.

5456
H.H. Ahmed et al. / European Journal of Medicinal Chemistry 45 (2010) 5452e5459
Table 2
Effect of melatonin and tested compounds on brain oxidant/antioxidant status of ACR-intoxicated rats.
Groups
MDA (nmol mgꢀ1 protein)
GSH (mmol mgꢀ1 protein)
GPx (mU mgꢀ1 protein)
SOD (U mgꢀ1 protein)
Control
ACR
6.2 ꢁ 0.3
1.5 ꢁ 0.14
1.4 ꢁ 0.08
2.5 ꢁ 0.11
8.1 ꢁ 0.9a (31%)
6.3 ꢁ 0.2b (ꢀ22%)
6.6 ꢁ 0.4 (ꢀ19%)
6.5 ꢁ 0.5 (ꢀ20%)
6.4 ꢁ 0.22b (ꢀ21%)
6.7 ꢁ 0.11 (ꢀ17%)
1.1 ꢁ 0.05a (ꢀ27%)
2.0 ꢁ 0.1.0b (82%)
1.4 ꢁ 0.03bd (27%)
1.4 ꢁ 0.04bd (27%)
1.4 ꢁ 0.1bd (27%)
1.2 ꢁ 0.03d (9%)
0.8 ꢁ 0.03a (ꢀ43%)
1.1 ꢁ 0.08b (38%)
0.84 ꢁ 0.06d (5%)
0.86 ꢁ 0.06d (8%)
0.89 ꢁ 0.04d (11%)
0.82 ꢁ 0.04d (3%)
1.7 ꢁ 0.22a (ꢀ32%)
4.0 ꢁ 0.21b (135%)
2.8 ꢁ 0.12bd (65%)
2.9 ꢁ 0.26bd (71%)
3.0 ꢁ 0.06bd (77%)
2.4 ꢁ 0.19bd (41%)
Melatonin þ ACR
Compound 4 þ ACR
Compound 6 þ ACR
Compound 7 þ ACR
Compound 11 þ ACR
Within each column means with different superscript letters are significantly different (p ꢂ 0.05). MDA: malondialdehyde, GSH: glutathione content, GPx: glutathione
peroxidase, SOD: superoxide dismutase.
ACR-intoxicated group. Brain GSH content showed significant
increase in the groups administered with melatonin and
compounds 4, 6 and 7 as compared to ACR-intoxicated group.
Administration with compound 11 showed insignificant change in
brain GSH content as compared to ACR-intoxicated group. The
action of melatonin as antioxidant is well known and melatonin is
considered as a powerful biological neutralizer of the hydroxyl
radical, being protective in the different models of oxidative stress
both in vivo and in vitro [22,36]. Moreover, it has been demon-
strated that melatonin and its metabolite 6-hydroxymelatonin
significantly reduces KCN-induced superoxide anion generation,
with commensurate reduction in lipid peroxidation [37]. All the
tested compounds showed significant antilipidperoxidative influ-
ence and could significantly enhance brain SOD and GSH content.
These findings could be attributed to that the morpholine, thia-
diazol, pyrazole and biphenyl rings, attached to the essential
pharmacophoric features of the parent molecule, exerted antioxi-
dant and free radical scavenging properties weaker or similar to
that of melatonin and also they able to stimulate antioxidant
enzymes activity [13,15,38]
inhibit the intrinsic pathway of apoptosis by impairing the acti-
vation/dimerization of Bax and re-localization of Bcl2 to mito-
chondria [41]. Regarding to the effect of the newly tested
compounds on LDH activity, these results could be attributed to
the activity of the morpholine ring of compound 7 and the
biphenyl ring of compound 6 as an agonist of Sig-1R with
a significant neuroprotective effect [19,27]. The pyrazole ring of
compounds 4 and 11 acts as ligand of benzodiazepine receptor
which improves functional recovery of the neurons and promotes
neuronal survival [32].
2.3. Histopathology assay
Examination of brains of control rats showed the highly active
nerve cells that have huge nuclei with relatively pale-stained
(arrow a, Fig. 1A), disappeared nuclear chromatin and prominent
nuclei. The surrounding relatively inactive support cells have small
nuclei with densely stained (arrow b, c, Fig. 1A), condensed chro-
matin and no visible nucleoli.
Histopathological investigation of brain of ACR treated rats
showed the dark neurons (arrow d, e, Fig. 1B) with corkscrew
dendrites. Also, the brain of ACR treated rat showed neurons with
coarse clumping of hyperchromatic nuclear chromatin (arrow f, g,
Fig. 1C) and with granular or amorphous gray cytoplasm. These
results confirmed the degenerative effect of ACR on brain tissue
[11].
In rats treated with Melatonin prior to ACR, the examination of
brain tissue showed neurons that appeared more or less as normal
control one. Some vacuoles that contain the remaining neurons
(arrow h, i, Fig. 1D) were also seen. Examination of brain tissue of
rats given melatonin derivatives 4, 6, 7, 11 prior to ACR indicated
that neurons appeared more or less like normal one (Fig. 1EeH).
2.2.3. Effect of tested compounds administration on brain enzymes
activity
As illustrated in Table 3, administration of ACR increased the
brain LDH activity (67%) and decreased brain CK activity (73%)
significantly as compared to the control group. These results are in
agreement with those of Mannaa et al. [11] and Lüa et al. [39].
Treatment with melatonin or any of the tested compounds 4, 6, 7,
11 showed significant decrease in brain LDH activity (65%, 45%,
47%, 55%, 40% respectively) and insignificant change in brain CK
activity (59%, 6%, 18%, 30%, 6% respectively) as compared to ACR-
intoxicated group. The decrease in LDH activity led to normali-
zation of its value approaching that of the control group.
Concerning melatonin effect on LDH activity our finding is in
agreement with that of Roth et al. [40] who demonstrated that
melatonin prevented cell death and decreased release of LDH into
the culture media, indicating that melatonin was able to enhance
the cell survival. More recent study achieved that melatonin could
2.4. Conclusion
We have described a facile synthesis of novel promising neu-
roprotective indole derivatives and investigated also the impor-
tance of incorporating heterocyclic moiety to the melatonin
nucleus to form new effective hybrid molecules. Melatonin and its
novel derivatives 4, 6, 7 and 11 showed neuroprotective activity
with various intensities depending on the structure of each
Table 3
Effect of melatonin and tested compounds on brain enzymes activity in ACR-
intoxicated rats.
Groups
LDH (U mgꢀ1 protein)
CK (U mgꢀ1protein)
compound. Morpholinomethyl melatonin derivative
7 is the
Control
ACR
0.12 ꢁ 0.004
0.27 ꢁ 0.05
strongest one followed by compound 6 then 11 and 4. All the tested
compounds have been shown to possess antioxidant properties in
ACR model of neurotoxicity offering promising efficacy by
enhancing SOD activity as well as decreasing lipid peroxidative
marker. Finally, the neuroprotective activity displayed by these
compounds may be of interest for further derivatization, further
studies in the hope of finding more active and selective neuro-
protective agents.
0.2 ꢁ 0.012a (67%)
0.073 ꢁ 0.006a (ꢀ73%)
0.116 ꢁ 0.008 (59%)
0.077 ꢁ 0.004 (6%)
0.086 ꢁ 0.004 (18%)
0.095 ꢁ 0.007 (30%)
0.077 ꢁ 0.004 (6%)
Melatonin þ ACR
Compound 4 þ ACR
Compound 6 þ ACR
Compound 7 þ ACR
Compound 11 þ ACR
0.07 ꢁ 0.004b (ꢀ65%)
0.11 ꢁ 0.008bd (ꢀ45%)
0.106 ꢁ 0.008bd (ꢀ47%)
0.09 ꢁ 0.008b (ꢀ55%)
0.12 ꢁ 0.01bd (ꢀ40%)
Within each column means with different superscript letters are significantly
different (p ꢂ 0.05). LDH: lactate dehydrogenase, CK: creatine kinase.

H.H. Ahmed et al. / European Journal of Medicinal Chemistry 45 (2010) 5452e5459
5457
Fig. 1. Photomicrograph of a brain of control rats (A), ACR treated rats (B), (C), melatonin treated rats (D), compound 4 treated rats (E), compound 6 treated rats (F), compound 7
treated rats (G), compound 11 treated rats (H).
3. Materials and methods
mixtures were separated by preparative TLC and gravity chroma-
tography. The starting compounds N-[2-(2-(cyanomethyl)-
7-methoxy-1,3,4-thiadiazolo[3,2-a]indol-9-yl)ethyl]acetamide (2),
N-[2-(2-(1-cyano-2-phenylprop-1-enyl)-7-methoxy-1,3,4-thiadia-
zolo[3,2-a]indol-9-yl)ethyl]acetamide (5) and N-[2-(1-benzoylme-
thino-5-methoxyindol-3-yl)ethyl]acetamide (8) were prepared
according to the published procedure [16,18,21].
3.1. Synthetic methods, analytical and spectral data
All chemicals were purchased from commercials suppliers and
used directly. The appropriate precautions in handling moisture-
sensitive compounds were undertaken. All melting points were
measured using an electro-thermal capillary melting point appa-
ratus (Buchi 535, Switzerland) and were uncorrected; the IR spectra
are expressed in cmꢀ1 and recorded using KBr pellets and a Pa-9721
IR spectrometer. 1H NMR and 13C NMR spectra were recorded with
Jeol instrument (Japan), at 270 and 125 MHz respectively, in DMSO-
3.1.1. N-[2-(2-(3-Acetyl-5-amino-1phenylpyrazol-4-yl)-7-
methoxy-1,3,4-thiadiazolo[3,2-a]indol-9-yl)ethyl]acetamide (4)
To a solution of thiadiazoloindole derivative 2 (0.33 g, 1 mmol)
in sodium ethoxide solution (20 mL) [prepared by dissolving
sodium metal (0.46 g, 0.02 mol) in absolute ethanol (50 mL)], acetyl
hydrazonyl chlorides (0.19 g, 1 mmol) was added. The reaction
mixture was stirred at room temperature over night, until all
starting materials had disappeared as indicated by TLC, and then
poured into ice. The resulting solid product was collected
by filtration and crystallized from methanol to yield dark brown
d6 or CDCl3 as solvent and the chemical shifts (d) were recorded in
ppm relative to TMS. Mass spectra were recorded on a GCMS-QP
1000 Ex spectra mass spectrometer operating at 70 eV. Elemental
analyses were carried by the Micro-analytical Data Unit at the
National Research Centre, Giza, Egypt and the Micro-analytical
data Unit at Cairo University. The reactions were monitored by thin
layer chromatography (TLC) which was carried out using Merck 60
F254 aluminum sheets and visualized by UV light (254 nm). The
powder, 0.34 g (70%), of compound 4, mp 168e170 ꢃC. IR (
3428e3335 (NH, NH2), 3030 (CH aromatic), 2985e2875
y
/cmꢀ1):

5458
H.H. Ahmed et al. / European Journal of Medicinal Chemistry 45 (2010) 5452e5459
(CH-aliphatic), 1692, 1705 (2C]O), 1667 (C]N), 1643 (C]C). 1H
NMR ( ppm): 1.70, 2.03 (2s, 6H, 2COCH3), 2.72 (t, 2H, CH2), 3.43 (t,
2H, CH2), 3.82 (s, 3H, OCH3), 5.23 (s, NH2, D2O-exchangeable),
60 ꢃC). The resulting solid was collected and crystallized from
d
absolute ethanol to gave dark yellow crystals from compound 9,
yield 0.28 g (70%), mp 118e120 ꢃC, IR ( /cmꢀ1): 3387 (NH), 3030
y
6.95e7.03 (m, 3H, C6H3), 7.05e7.08 (m, 5H, C6H5), 8.42 (s, 1H, NH,
(CH aromatic), 2976e2878 (CH-aliphatic),1697 (C]O),1648 (C]C).
D2O-exchangeable). 13C NMR (
d): 23.8, 26.0 (2COCH3), 25.5, 41.2
1H NMR (
d
ppm): 2.05 (s, 3H, COCH3), 2.47 (s, 6H, 2CH3), 2.72 (t, 2H,
(2CH2), 55.7 (OCH3), 112.8 (indole-C-3), 102.0, 155.3, 109.0, 112.5,
128.9, 128.7 (indole aromatic-C), 139.2, 94.3, 147.5 (pyrazole-C),
122.0, 175.6 (thiadiazole-C), 196.2, 170.5 (2C]O). MS (EI): m/z (%):
CH2) 3.47 (t, 2H, CH2), 3.78 (s, 3H, OCH3), 6.52 (s, 1H, CH-N),
6.67e7.02 (m, 4H, C6H3 þ CH-indole), 7.45e7.76 (m, 5H, C6H5), 9.50
ꢄ
(s,1H, NH, D2O-exchangeable). MS (EI): m/z (%): 405 (Mþ , 68%), 390
ꢄ
ꢄ
487 (Mþ ꢀ 1, 65%), 200 (34), 130 (100), 77 (46). Anal. Calc. for
(Mþ ꢀ CH3, 70), 348 (34), 174 (62), 105 (100). Anal. Calc. for
C25H24N6O3S (488.562): C, 61.46, H, 4.95, N, 17.20, S, 6.56; found: C,
61.65, H, 4.78, N, 17.42, S, 6.36.
C24H27N3O3 (405.490): C, 71.09, H, 6.71, N, 10.36; found: C, 71.25, H,
6.49, N, 10.58.
3.1.2. N-[2-(2-(6-Amino-5-cyano-4-imino-2-phenylcyclohexa-1,5-
dienyl)-7-methoxy-1,3,4-thiadiazolo[3,2-a]indol-9-yl)ethyl]
acetamide (6)
3.1.5. N-[2-(5-Methoxy-1-(3-phenyl-1H-pyrazol-4-yl)-1H-indol-3-
yl)ethyl]acetamide (11)
To a solution of compound 9 (0.40 g, 1 mmol) in absolute
ethanol (25 mL) containing catalytic amount of triethylamine
(2 mL) equimolar amount of hydrazine hydrate (0.05 mL, 1 mmol)
was added. The reaction mixture 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 ꢅ 20 mL). The organic layer was dried over calcium
chloride. Removal of the solvent in vacuo afforded the corre-
sponding product, which was crystallized from dioxane to gave
brown crystals from compound 11, yield 0.29 g (78%) mp 192e194,
To a solution of propenyl indole derivative 5 (0.43 g, 1 mmol), in
absolute ethanol (20 mL) containing a catalytic amount of trie-
thylamine (1 mL), malononitrile (0.07 g, 1 mmol) was added. The
reaction mixture was heated under reflux for 4 h until all starting
materials had disappeared as indicated by TLC. The reaction
mixture was left to cool at room temperature, poured into ice/water
mixture, neutralized with dilute hydrochloric acid. The obtained
solid product was collected by filtration, dried and crystallized from
dioxane to yield 0.37 g (75%) of compound 6, pale brown crystals,
mp 268e270 ꢃC. IR (v/cmꢀ1): 3428e3335 (NH, NH2), 3035 (CH
aromatic), 2976e2886 (CH-aliphatic), 2220 (CN), 1698 (C]O), 1675
IR (
y
/cmꢀ1): 3387 (NH), 3038 (CH aromatic), 2975e2883 (CH-
ppm): 2.08 (s, 3H,
(C]N), 1647 (C]C). 1H NMR (
d
ppm): 1.76, (s, 3H, COCH3), 2.05 (s,
aliphatic), 1697 (C]O), 1645 (C]C). 1H NMR (
d
2H, CH2), 2.75 (t, 2H, CH2), 3.42 (t, 2H, CH2), 3.80 (s, 3H, OCH3), 4.83
(s, NH2, D2O-exchangeable), 6.93e7.05 (m, 3H, C6H3), 7.21e7.34 (m,
5H, C6H5), 8.42e8.97 (2s, 2H, 2NH, D2O-exchangeable). MS (EI): m/z
COCH3), 2.68 (t, 2H, CH2) 3.40 (t, 2H, CH2), 3.80 (s, 3H, OCH3),
6.68e7.06 (m, 4H, C6H3 þ CH-indole), 7.36e7.47 (m, 5H, C6H5), 7.84
(s, 1H, CH-pyrazole), 10.40 (s, 1H, NH, D2O-exchangeable). 13C NMR
ꢄ
(%): 496 (Mþ , 46%), 288 (54), 173 (100), 77 (42). Anal. Calc. for
(d): 23.7 (COCH3), 29.5, 43.2 (2CH2), 55.6 (OCH3), 124.4 (C-2), 112.8
C27H24N6O2S (496.584): C, 65.30, H, 4.87, N, 16.92, S, 6.46; found: C,
65.12, H, 5.07, N, 17.07, S, 6.27.
(C-3), 102.0 (C-4), 156.3 (C-5), 109.4 (C-6), 112.5 (C-7), 128.0, 132.2
(fused aromatic-C), 151.2, 104.1, 134.0 (pyrazole-C), 127.5, 129.3,
128.4 (fused aromatic-C), 170.9 (C]O). MS (EI): m/z (%): 373
ꢄ
3.1.3. N-[2-(5-Methoxy-1-(morpholinomethyl)-1H-indol-3-yl)
ethyl]acetamide (7)
(Mþ ꢀ 1, 28%), 297 (Mþꢄ, 37), 143 (62), 77 (100). Anal. Calc. for
C22H22N4O2 (374.436): C, 70.57, H, 5.92, O, 8.55, N, 14.96; found: C,
70.77, H, 5.73, N, 15.21.
A mixture of melatonin 1 (0.23 g, 1 mmol) and formaldehyde
(0.2 mL, 33%) in tetrahydrofurane (THF) (20 mL) was heated under
reflux for 5 h. Then the reaction mixture treated gradually with
continuous stirring with a solution of morpholine (0.1 mL, 1 mmol)
in THF (5 mL). After complete addition, the reaction mixture
refluxed again until all starting materials had disappeared as
indicated by TLC and then the solvent was removed under reduced
pressure. The semisolid residue left behind was subjected to
column chromatography over silica gel. Elution with dry diethyl
ether gave white crystals from the product 7. Yield 0.27 g (80%), mp
3.2. Biochemistry assay
3.2.1. Animals
Female Sprague Dawley rats (70 rats), weighing 160e180 g, aged
16e18 weeks, were supplied by the Animal House Colony of the
National Research Centre, Cairo, Egypt, and acclimated for one
week in a specific pathogen-free (SPF) barrier area where temper-
ature is 25 ꢁ1 ꢃC and humidity is 55%. Rats were controlled
constantly with 12 h light/dark cycle at National Research Centre
animal facility breeding colony. Rats were individually housed with
ad libitum access to standard laboratory diet and tap water. All
animal procedures were performed after approval from the Ethics
Committee of the National Research Centre and in accordance with
the recommendations for the proper care and use of laboratory
animals (NIH publication No. 85-23, revised 1985).
100e102. IR (
y
/cmꢀ1): 3435 (NH), 3032 (CH aromatic), 2980e2868
(CH-aliphatic), 1702 (C]O), 1655 (C]C), 1045 (CeO). 1H NMR (
d
ppm): 2.02 (s, 3H, COCH3), 2.27e2.35 (m, 4H, 2CH2), 2.69 (t, 2H,
CH2), 3.43 (t, 2H, CH2), 3.67e3.72 (m, 4H, 2CH2), 3.83 (s, 3H, OCH3),
4.94 (s, 2H, CH2-N), 6.64 (s, 1H, CH-indole), 6.75e6.98 (m, 3H,
C6H3), 9.12 (s, 1H, NH, D2O-exchangeable). 13C NMR (
d): 23.3
(COCH3), 28.5, 41.2 (2CH2), 55.5 (OCH3), 77.3 (CH2), 122.4 (C-2),
112.8 (C-3), 102.0 (C-4), 155.3 (C-5), 109.0 (C-6), 112.5 (C-7), 128.0,
129.2 (fused aromatic-C), 51.2, 67.1 (morpholine-C), 170.2 (C]O).
ꢄ
MS (EI): m/z (%): 333 (Mþ þ2, 72%), 288 (35), 100 (54), 115 (100).
Anal. Calc. for C18H25N3O3 (331.409): C, 65.23, H, 7.60, N, 12.68;
found: C, 65.54, H, 7.40, N, 12.93.
3.2.2. Experimental design
The animals were randomly assigned to 7 groups (n ¼ 10) as
follows: control, ACR, melatonin þ ACR, compound 4 þ ACR,
compound 6 þ ACR, compound 7 þ ACR and compound 11 þ ACR.
ACR dose [50 mg kgꢀ1 body weight (b. wt.)] dissolved in water was
injected intraperitoneally [42]. Melatonin and its derivatives were
dissolved in 2% Tween and injected intraperitoneally in a dose of
50 mg kgꢀ1 b. wt. [43], 3 days/week and ten minutes prior to ACR
injection for 3 weeks.
3.1.4. N-[2-(1-(1-(Dimethylamino)-3-oxo-3-phenylprop-1-en-
2-yl)-5-methoxy-1H-indol-3-yl)ethyl]acetamide (9)
A solution of 1-benzoyl methinomelatonin derivative 8 (0.35 g,
1 mmol) and DMF-DMA (0.12 mL, 1 mmol) in dry xylene (30 mL)
was refluxed for 12 h. Then, the solvent was removed under
vacuum and the residue triturated with petroleum ether (b.p. 40/

H.H. Ahmed et al. / European Journal of Medicinal Chemistry 45 (2010) 5452e5459
5459
_
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Acknowledgment
The authors acknowledge the financial support of the National
Research Center, Egypt (grant no.: E-8040505-2008/2009). The
authors expressed sincere appreciation to Dr. Abdel Razk Hussein
Farrag, Dept. of Pathology, National Research Centre, Egypt, for his
kind cooperation in conducting histopathological investigations in
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