Development of new indole-derived neuroprotective agents

Article abstract of DOI:10.1016/j.bmc.2011.03.031

There is a great deal of interest in neurotrophin therapy to prevent neuronal degeneration. The present study aimed at synthesizing new functionalized indole derivatives with structures justifying neuroprotective activity using l-tryptophan (TRP)^?

Full text of DOI:10.1016/j.bmc.2011.03.031

Bioorganic & Medicinal Chemistry 19 (2011) 2966–2974
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of new indole-derived neuroprotective agents
Rafat M. Mohareb a,b, Hanaa H. Ahmed c, Gamal A. Elmegeed c, , Mervat M. Abd-Elhalim c, Reham W. Shafic c
⇑
a Organic Chemistry Department, Faculty of Pharmacy, October University of Modern Sciences and Arts (MSA), October City, Egypt
b Chemistry Department, Faculty of Science, Cairo University, Cario, Egypt
c Hormones Department, National Research Centre, Dokki 12622, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
There is a great deal of interest in neurotrophin therapy to prevent neuronal degeneration. The present
study aimed at synthesizing new functionalized indole derivatives with structures justifying neuropro-
tective activity using L-tryptophan (TRP) as starting material. The potential neuroprotective effect of these
Received 28 December 2010
Revised 6 March 2011
Accepted 14 March 2011
Available online 21 March 2011
newly synthesized agents against acrylamide (ACR) induced neurotoxicity was investigated in adult female
rats. The novel indole derivatives, indolylmethyl pyridine derivatives 9a,b, pyrimidinylindolyl propanone
derivatives 12a–c, pyrazolylindolyl propanone derivatives 14a,b, and indolyl tetrazolopropanoic acid deriv-
ative 17 were synthesized and their chemical structures were confirmed by studying their analytical and
spectral data. The administration of ACR [ip, 50 mg kgÀ1 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 the
indole derivatives 9b, 12c, 14a, and 17 (ip, 50 mg kgÀ1 b. wt.) prior to ACR produced neuroprotective activity
with various intensities depending on the structure of each compound. Compound 17 in which the tetrazole
ring was attached to the TRP moiety ranked as the strongest neuroprotective agent. All the tested com-
pounds have been shown to possess antioxidant properties offering promising efficacy against oxidative
stress induced by ACR administration.
Keywords:
Indole
Neuroprotective
Pyridine
Pyrimidine
Pyrazole
Tetrazole
Tryptophan
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
It is well known that indole derivatives, extensively present in
natural compounds and are very important substances in medici-
The nervous system is considered the most complex organ
system in the body, and many toxic chemicals can affect many dif-
ferent parts of this system. Since the nervous system innervates all
areas of the body, some toxic effects may be quite specific and oth-
ers very generalized depending upon where in the nervous system
the toxin exerts its effect. There is a great deal of interest in neuro-
trophin therapy to prevent neuronal degeneration.1 Acrylamide
(ACR) is water soluble, vinyl monomer that has multiple chemical
and industrial applications and also used extensively in molecular
laboratories for gel chromatography.2 ACR has been found to cause
a distinct central–peripheral, distal neuropathy, it also showed to
have neurotoxic effect on the central nervous system in experi-
mental rats.3 Recent research suggested that the behavioral acryl-
amide neurotoxicity, a product of nerve damage, was classified as a
central–peripheral, distal axonopathy.4
nal and biological aspects. Indole is a highly conserved molecule
that it acts as a free radical scavenger and has broad-spectrum
antioxidant activity.5 Moreover, indole and its derivatives exhibit
various therapeutic effects, such as regulation of circadian rhythm
and immune functions.6,7 Recent research has proved that the
indole ring in the melatonin molecule is the reactive center dealing
with oxidants due to its high resonance stability and very low
activation energy barrier towards free radical reactions.8,9 Indole-
3-propionamide derivatives exert higher antioxidant activity
compared to melatonin.10 Also, 2-phenylindole derivatives signifi-
cantly inhibited lipid peroxidation.11
L-Tryptophan (TRP) is the precursor amino acid for the synthesis
of serotonin (5-HT) and it has been found that 5-HT increases sig-
nificantly by TRP treatment.12 Indole ring in the TRP compound
acts as ligand of peripheral benzodiazepine receptor (PBR) which
improved functional recovery of the brain in neuro-intoxicated
animal models due to its neuroprotective effect.13 In addition,
TRP is the precursor of melatonin14 which could save both dopami-
nergic and non-dopaminergic neurons from oxidative stress and
protect all types of brain damaged neurons.15
⇑
Corresponding author. Tel.: +20 2 35682070; fax: +20 2 33370931.
E-mail address: gamalae@hotmail.com (G.A. Elmegeed).
Abbreviations: ACR, acrylamide; AD, adrenaline; b.wt., body weight; CK, creatine
kinase; DA, dopamine; GPx, glutathione peroxidase, GR, glutathione reductase; GSH,
glutathione content; 5-HT, serotonin; ip, intraperitoneal; LDH, lactate dehydroge-
nase; MDA, malondialdehyde; NA, noradrenaline; PBR, peripheral benzodiazepine
receptor; SOD, superoxide dismutase; TRP, L-tryptophan; TLC, thin layer
chromatography.
A large variety of synthetic compounds have been identified
as potent neuroprotective agents, yet many of these compounds
have not provided great clinical benefits, and some produced side
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.031

R. M. Mohareb et al. / Bioorg. Med. Chem. 19 (2011) 2966–2974
2967
O
C
CN
effects. Therefore, the main goal of the present study was to syn-
thesize new indole derivatives with structures justifying neuropro-
tective activity using TRP as starting material. Furthermore, the
potential neuroprotective effects of these newly synthesized
agents against acrylamide (ACR) induced neurotoxicity in rats
was investigated compared with those of the parent compound.
CN
CN
CH2
N
a
5
+
CN
8a
N
H
10
b
c
H2N
2. Results and discussion
2.1. Chemistry
O
N
N
NH2
C
X
O
N
N
C
CH2
N
NH2
R
In the aim of synthesis of indolyl aminopyridine derivatives of
potential neuroprotective activity,16 the amino group of TRP (1) re-
acted with formaldehyde 2 in the presence of ammonium acetate
CH2
N
NH2
N
H
12a, X = O
b, X = S
c, X = NH
N
14a, R = H
b, R = Ph
H
to afford Nb-formaltryptophan derivative
3 in 95% yield
(Scheme 1). The reaction of compound 3 with thionyl chloride 4
in anhydrous diethyl ether resulted in the corresponding chloride
derivative, indolyl methylenaminopropanyl chloride 5, in 90%
yield. The latter reacted with aniline 6 in refluxing dioxane/ trieth-
ylamine solution to give the corresponding anilide derivative 7 in
75% yield (Scheme 1). The reaction of compound 7 with either mal-
ononitrile 8a or ethyl cyanoacetate 8b in refluxing ethanolic/piper-
idine solution gave the corresponding chromatographically pure
products the indolylmethyl aminopyridine derivatives 9a and 9b
in 76% and 70% yield, respectively (Scheme 1).
The reactivity of indolyl methylenaminopropanyl chloride
derivative 5 towards some active methylene reagents was studied.
Refluxing of compound 5 with equimolar amount of malononitrile
8a in 1,4-dioxane/triethylamine solution afforded the correspond-
ing condensate product, indolyl methylenamino-3-oxopentanonit-
Scheme 2. Reagents and conditions: (a) dioxane, Et3N, reflux; (b) H2NCXNH2
(11a–c), EtOH, NaOEt, or NaOAc, reflux; (c) NH2NHR (13a,b), EtOH, reflux.
drate 13a or phenyl hydrazine 13b in refluxing absolute ethanol
to afford the aminopyrazolylindolyl propanone derivatives 14a
and 14b, respectively (Scheme 2).
The acetonitrilocarbonyl tryptophan derivative 15 was synthe-
sized via the reaction of TRP with ethyl cyanoacetate in refluxing
dimethylformamide (Scheme 3).19 The reactivity of acetonitrile
moiety of compound 15 towards the formation of tetrazole ring
was studied. In attempts of having a straightforward synthesis of
indolyl tetrazolopropanoic acid, the reaction of compound 15 with
sodium azide 16 in dimethylformamide containing a catalytic
amount of ammonium chloride has been carried out. Heating of this
mixture over oil bath at 125 °C gave one chromatographically isola-
ble product in 75% yield, whose mass spectra and elemental analy-
sis indicated a molecular formula C14H14N6O3 (m/z = 314). The IR
spectrum of the product showed the disappearance of the cyano
stretching of compound 15. The 1H NMR spectrum showed the
presence of three singlets at d = 8.05, 8.62, 9.36 (D2O-exchangeable)
for three NH groups and showed also a singlet signal at d = 11.02 for
carboxylic-OH group. The 13C NMR spectrum confirmed the pres-
ence of tetrazole-C peak at d = 153.9 ppm. Based on all the spectral
data, the indolyl tetrazolopropanoic acid derivative 17 proposed to
have the structure of this product (Scheme 3). Assignments of struc-
tures of all new compounds were obtained on the basis of their cor-
rect analytical analyses and compatible spectral data (cf. Section 3).
rile derivative 10 (Scheme 2). The reactivity of the a,b-unsaturated
nitrile moiety of compound 10 towards some nitrogen nucleo-
philes was investigated in the aim of synthesis of amino-heterocy-
clic agents with potential neuroprotective activity.17,18 Thus,
compound 10 reacted with equimolar amount of either urea 11a
or thiourea 11b in ethanolic sodium ethoxide solution to afford
the aminopyrimidinylindolyl propanone derivatives 12a and 12b,
respectively (Scheme 2). Compound 10 reacted also with guanidine
hydrochloride 11c in ethanolic sodium acetate solution to afford
the corresponding aminopyrimidinylindolyl propanone derivative
12c (Scheme 2). Also, compound 10 reacted with hydrazine hy-
COOH
2.2. Biological assays
COOH
CH2
NH2
a
N
CH2O
2
The in vivo neuroprotective effect of TRP and the novel synthe-
sized indole derivatives, 9b, 12c, 14a, and 17, against ACR-induced
neurotoxicity in rats was investigated. ACR is a well established
agent for induction of neurotoxicity.20,21 Neuronal damage due to
+
N
H
N
H
1
3
COCl
CH2
CONHPh
CH2
b
N
N
c
COOH
N
H
N
H
CN
NHCOCH2CN
a
5
7
1
+
COOEt
N
H
d
PhHN
R
15
8b
NH2
COOH
N
b
N
N
N
NHCOCH2
N
H
N
H
9a, R = CN
9b, R = COOEt
N
H
17
Scheme 1. Reagents and conditions: (a) (i) NH4OAc, fusion; (ii) EtOH, heat; (b)
SOCl2 (4), diethyl ether, ice bath; (c) PhNH2 (6), dioxane, Et3N, reflux; (d) RCH2CN
(8a,b), EtOH, pip, reflux.
Scheme 3. Reagents and conditions: (a) DMF, reflux; (b) NaN3 (16), DMF, NH4Cl,
heat.

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R. M. Mohareb et al. / Bioorg. Med. Chem. 19 (2011) 2966–2974
Table 1
as compared to ACR-intoxicated group (Table 1, Fig. 1). These find-
ings could be attributed to the fact that the indole ring in the TRP
acts as ligand of peripheral benzodiazepine receptor (PBR) which
improves functional recovery of the brain in this model of neuro-
toxicity.13 In addition, TRP is the precursor of melatonin14 which
could rescue both dopaminergic and non-dopaminergic neurons
from oxidative stress and preserve all types of brain neurons from
neurological damage.15
Treatment with TRP prior to ACR intoxication produced signifi-
cant increase in brain GPx activity accompanied with significant
decrease in brain MDA level as compared to ACR-intoxicated group
(Table 2, Fig. 2). This result could be explained as that indole pos-
sesses antioxidant and free radical scavenging properties. This
explanation is in great agreement with Suzen and Buyukbingol7
and Biradar et al.26 In addition, indole skeleton has been reported
to be a protective agent in different models of oxidative stress both
in vivo and in vitro.27 Furthermore, indoles have been found to in-
duce GSH synthesis and promote GPx activity28 as well as it could
inhibit lipid peroxidation probably by the direct scavenging of the
reactive oxygen species.27 The current results demonstrated that
the pre-administration of ACR-intoxicated rats with TRP signifi-
cantly decreased brain LDH activity (Table 3, Fig. 3). This finding
could be attributed to the fact that indole compound exhibits
ligand to PBR which promotes neuronal survival and thus preserv-
ing LDH activity.13
Effect of TRP and the new synthesized indole derivatives on brain monoamines levels
in ACR-intoxicated rats
Groups
AD (ng mgÀ1
protein)
NA (ng mgÀ1
protein)
DA (ng gÀ1 tissue)
Control
ACR
86 1.0
863 14.5
1508 37.1
52 1.5a (À40%) 680 22.7a (À21%) 1267 50.3a (À16%)
TRP + ACR
Compound
9b + ACR
Compound
12C + ACR
Compound
14a + ACR
Compound
17 + ACR
61 1.1b (17%)
59 1.7b (14%)
791 18.1b (16%)
690 2.8c (2%)
1531 24.4b (21%)
1380 14.1b,c (9%)
57 1.8b (10%)
60 2.0b (15%)
684 5.6c (1%)
722 16.5c (6%)
1237 27.3c (À2%)
1460 34.2b (15%)
1638 31.1b,c (29%)
87 1.3b,c (67%) 823 15.6b (21%)
Within each column means with different superscript letters are significantly dif-
ferent (p 6 0.05).
a
Difference changes in relation to control group.
Difference changes in relation to ACR group.
b
c
Difference changes in relation to TRP + ACR group. ACR: acrylamide, TRP:
tryptophan, AD: adrenaline, NA: noradrenalin, DA: dopamine.
L-
ACR was detected by measuring: (1) the levels of brain monoam-
ines, adrenaline (AD), noradrenalin (NA) and dopamine (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), and (4) the activity of brain enzymes lactate dehy-
drogenase (LDH) and creatine kinase (CK).
AD
NA
DA
80
70
60
50
40
30
20
10
0
The data depicted in Table 1 demonstrated that ACR administra-
tion induced degenerative effect on brain tissue as manifested by
the significant decrease in the levels of the neurotransmitters,
AD, NA, and DA (40%, 21%, and 16%, respectively) as compared to
the control group. These results are in agreement with those of
LoPachin22 and Mannaa et al.23 LoPachin22 suggested that the inhi-
bition of neurotransmission contributes significantly to the devel-
opment of corresponding neurological deficits as a potential
mechanism of synaptic dysfunction. The level of MDA in the brain
was significantly increased (31%) in ACR-intoxicated rats as com-
pared to the control group (Table 2). On the other hand, the brain
antioxidant enzymes, SOD, GPx activity, and GSH content signifi-
cantly decreased in ACR-intoxicated rats, by 32%, 43%, and 27%,
respectively, as compared to the control group (Table 2). These re-
sults are in consistent with those of Mannaa et al.23 and Zhu et al.24
As illustrated in Table 3, administration of ACR increased the brain
LDH activity (67%) and decreased brain CK activity (73%) signifi-
cantly as compared to the control group. These results are in agree-
ment with those of Mannaa et al.23 and Lüa et al.25
-10
TRP
9b
12c
14a
17
Figure 1. Levels of AD, NA, and DA as % difference from ACR intoxication in TRP and
the newly synthesized derivatives-treated groups. ACR: acrylamide, TRP:
phan, AD: adrenaline, NA: noradrenalin, DA: dopamine.
L-trypto-
In the present study, pre-treatment with TRP in ACR-intoxicated
rats resulted in significant increase in brain AD, NA, and DA levels
Table 2
Effect of TRP and the new synthesized indole derivatives on brain oxidant/antioxidant status of ACR-intoxicated rats
Groups
MDA (nmol mgÀ1 protein)
GSH (mmol mgÀ1 protein)
GPx (mU mgÀ1 protein)
SOD (U mgÀ1 protein)
Control
ACR
TRP + ACR
Compound 9b + ACR
Compound 12C + ACR
Compound 14a + ACR
Compound 17 + ACR
6.2 0.3
1.5 0.14
1.4 0.08
2.5 0.11
8.1 0.9a (31%)
6.1 0.3b (À25%)
7.3 1.1 (À10%)
7.7 0.75 (À5%)
6.3 0.6b (À22%)
6.0 0.7b (À26%)
1.1 0.05a (À27%)
1.2 0.12 (9%)
0.8 0.03a (À43%)
1.1 0.02b (38%)
0.98 0.04b (23%)
0.81 0.03c (1%)
1.0 0.04b (25%)
1.2 0.03b (50%)
1.7 0.22a (À32%)
2.0 0.06 (18%)
1.8 0.11 (6%)
1.75 0.02 (3%)
1.9 0.12 (12%)
3.3 0.17b,c (94%)
0.9 0.04b,c (À18%)
0.9 0.08b,c (À18%)
1.2 0.08 (9%)
1.5 0.07b,c (36%)
Within each coulmn means with different superscript letters are significantly different (p 6 0.05).
a
Difference changes in relation to control group.
Difference changes in relation to ACR group.
b
c
Difference changes in relation to TRP + ACR group. ACR: acrylamide, TRP:
SOD: superoxide dismutase.
L-tryptophan, MDA: malondialdehyde, GSH: glutathione content, GPx: glutathione peroxidase,

R. M. Mohareb et al. / Bioorg. Med. Chem. 19 (2011) 2966–2974
2969
In the present study, pre-administration with the newly synthe-
sized indole derivatives 9b, 12c, 14a, or 17 in ACR-intoxicated rats
resulted in significant increase in brain AD level and pre-adminis-
tration with compounds 9b, 17, or 14a led to significant increase in
brain DA level while pre-administration with compound 17 pro-
duced significant increase in brain NA level as compared to ACR-
intoxicated group (Table 1, Fig. 1). These results indicated that,
all the newly tested compounds exhibited neuroprotective effect.
In addition to the essential pharmacophoric features of the TRP
molecule, compounds 9b and 14a containing aminopyridine and
aminopyrazole rings, respectively, that behaved as ligand to
PBR.16,29 Therefore, these compounds exerted more or less similar
effect as TRP on brain AD level.
Our results demonstrated that the indolyl tetrazolopropanoic
acid derivative 17 in which tetrazole ring was attached to the
TRP moiety ranked as the strongest neuroprotectant as indicated
by the significant increase in each of brain AD, DA, and NA levels.
The powerful neuroprotective action of compound 17 might be
attributed to the highly antioxidant activity of both the indole
and tetrazole moieties.30 Pyrimidinylindolyl propanone derivative
12c that contains indole and aminopyrimidine rings has been
found to have moderate neuroprotective activity as indicated by
the significant increase only in brain DA level.
MDA
GSH
GPx
SOD
100
80
60
40
20
0
-20
-40
TRP
9b
12c
14a
17
Figure 2. Values of MDA, GSH, GPx and SOD as % difference from acrylamide
intoxication in TRP and the newly synthesized derivatives-treated groups. ACR:
acrylamide, TRP: L-tryptophan, MDA: malondialdehyde, GSH: glutathione content,
GPx: glutathione peroxidase, SOD: superoxide dismutase.
The current results (Table 2, Fig. 2) demonstrated that pre-
administration with the newly synthesized compounds 9b, 14a,
or 17 induced significant increases in brain GPx activity and com-
pounds 9b, 17, or 12c led to significant increase in brain GSH level.
Pre-administration of compound 17 or 14a resulted in significant
decrease in brain MDA level and compound 17 only showed signif-
icant increase in brain SOD activity. These findings could be attrib-
uted to the indole moiety which has remarkable antioxidant
properties. In addition, the newly synthesized compounds 9b, 17,
12c, and 14a containing pyridine,31 tetrazole,30 pyrimidine,32 and
pyrazole33 rings, respectively, possess antioxidant and free radical
scavenging properties. These compounds could suppress oxidative
stress with consequent inhibition of lipid peroxidation product
(MDA), enhance antioxidant enzyme activities (SOD, GPx) and in-
duce GSH synthesis.
Compounds 9b, 12c, 14a, or 17 administered 10 min before
ACR intoxication revealed significant decrease in brain LDH activ-
ity and compound 17 only showed significant increase in brain
CK activity (Table 3, Fig. 3). These results indicated that, our
compounds exhibit neuroprotective effect and confirmed the
powerful neuroprotective action of compound 17. The tetrazole
ring of compound 17 acted as protecting agent for neuronal
cells from oxidative stress due to its superior pharmacokinetic
properties.34,35
Table 3
Effect of TRP and the new synthesized indole derivatives on brain enzymes activity in
ACR-intoxicated rats
Groups
LDH (U mgÀ1protein)
CK (U mgÀ1protein)
Control
ACR
TRP + ACR
Compound 9b + ACR
Compound 12C + ACR
Compound 14a + ACR
Compound 17 + ACR
0.12 0.004
0.27 0.05
0.2 0.012a (67%)
0.073 0.006a (À73%)
0.093 0.007 (27%)
0.08 0.005 (14%)
0.078 0.01 (7%)
0.089 0.003 (22%)
0.120 0.007b (64%)
0.13 0.008b (À35%)
0.16 0.005b,c (À20%)
0.18 0.014b,c (À10%)
0.16 0.005b,c (À20%)
0.13 0.011b (À35%)
Within each column means with different superscript letters are significantly dif-
ferent (p 6 0.05).
a
Difference changes in relation to control group.
Difference changes in relation to ACR group.
b
c
Difference changes in relation to TRP + ACR group. ACR: acrylamide, TRP:
tryptophan, LDH: lactate dehydrogenase, CK: creatine kinase.
L-
LDH
CK
80
60
40
20
0
2.3. Histological examination
Microscopic investigation of brain section of control rat (Fig. 4
A) showed the highly active nerve cells that have huge nuclei with
relatively pale-stained (arrow a), disappeared nuclear chromatin
and prominent nuclei. The surrounding relatively inactive support
cells have small nuclei with densely-stained (arrows b and c), con-
densed chromatin and no visible nucleoli.
Photomicrograph of brain section of ACR-intoxicated rats
showed the dark neurons (arrows d and e) with corkscrew den-
drites (Fig. 4 B). Also, the brain section of ACR exposed rats showed
neurons with coarse clumping of hyperchromatic nuclear chroma-
tin (arrows f and g) and with granular or amorphous cytoplasm
(Fig. 4 C). These findings confirmed the degenerative effect of
ACR on brain tissue.23
-20
-40
TRP
9b
12c
14a
17
The examination of brain section of rats treated with TRP prior
to ACR showed neurons that appeared more or less as the normal
control rats (Fig. 4 D). Examination of the brain section of rats gi-
Figure 3. LDH and CK enzymes activity as % difference from acrylamide intoxica-
tion in TRP and the newly synthesized derivatives-treated groups. ACR: acrylamide,
TRP: L-tryptophan, LDH: lactate dehydrogenase, CK: creatine kinase.

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R. M. Mohareb et al. / Bioorg. Med. Chem. 19 (2011) 2966–2974
3. Materials and methods
3.1. Chemistry
All chemicals were purchased from commercials suppliers and
were used directly. The appropriate precautions in handling mois-
ture-sensitive compounds were undertaken. All melting points
were measured using an electro-thermal capillary melting point
apparatus (Buchi 535, Switzerland) and were uncorrected; the IR
spectra expressed in cmÀ1 and recorded using KBr pellets and a
Pa-9721 IR spectrometer. 1H and 13C NMR spectra were recorded
with Jeol instrument (Japan), at 270 and 125 MHz, respectively,
in DMSO-d6 or CDCl3 as solvent and the chemical shifts (d) were re-
corded in ppm relative to TMS. Mass spectra were recorded on a
GC–MS-QP 1000 Ex spectra mass spectrometer operating at
70 eV. Elemental analyses were carried by the Micro-analytical
Data Unit at the National Research Center, Giza, Egypt and the Mi-
cro-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 mixtures were separated by preparative TLC
and gravity chromatography. The starting compound a-amino(ace-
tonitrilocarbamido)-3-indolopropionic acid (15) was prepared
according to the published procedure.19
3.1.1. Synthesis of 3-(1H-indol-3-yl)-2-(methylenamino)-
propanoic acid (3)
A suspension of TRP 1 (0.2 g, 1 mmol), formaldehyde 2 (0.03 mL,
1 mmol) and ammonium acetate (0.08 g, 1 mmol) was heated in an
oil bath at 120 °C for 20 min and then left to cool at room temper-
ature. The reaction mixture triturated with absolute ethanol
(10 mL) and heated again under reflux for 15 min. Then the reac-
tion mixture was left to cool at room temperature, poured over
ice/water mixture and neutralized with dilute hydrochloric acid.
The solid product obtained was collected by filtration, dried, and
crystallized from absolute ethanol.
Figure 4. Photomicrograph of a brain section of control rats (A), ACR-intoxicated
rats (B and C), TRP pre-treated rats (D), compound 9b pre-treated rats (E),
compound 12c pre-treated rats (F), compound 14a pre-treated rats (G), and
compound 17 pre-treated rats (H).
Compound 3: Yellow crystals yield 0.21 g (95%), mp 207–209 °C.
IR
(CH-aromatic), 2984, 2887 (CH-aliphatic), 1728 (C@O), 1660
(C@N), 1645 (C@C). 1H NMR (DMSO-d6,
ppm): 2.68 (t,
J = 2.07 Hz, 1H, CH), 2.84 (d, J = 2.07 Hz, 2H, CH2), 4.56 (s, 2H,
N@CH2), 6.78 (s, 1H, CH-indole), 7.16–7.27 (m, 4H, C6H4),
10.40 (s, 1H, NH, D2O-exchangeable), 11.21 (s, 1H, COOH, D2O-
exchangeable). MS (EI): m/z (%): 215 (M+ꢀÀ1, 42%), 188 (M+ÀNCH2,
18), 171 (M+ÀCOOH, 50), 116 (100). Anal. Calcd for C12H12N2O2
(216.236): C, 66.65; H, 5.59; N, 12.96. Found: C, 66.46; H, 5.79;
N, 13.21.
(m
/cmÀ1): 3424 (br, OH, carboxylate), 3396 (NH), 3027
ven compound 9b prior to ACR (Fig. 4 E) indicated that neurons ap-
peared more or less like normal one but some dark neurons (arrow
h) were also seen. Examination of brain section of ACR-intoxicated
rats pre-treated with compound 12c (Fig. 4 F) revealed that neu-
rons appeared more or less like normal one with the appearance
of some small vacuoles (arrow i) that contain the remaining of
dead neurons. Examination of brain section of rats received com-
pounds 14a (Fig. 4 G) or 17 (Fig. 4 H) prior to ACR indicated that
neurons appeared more or less like normal one.
d
2.4. Conclusion
3.1.2. Synthesis of 3-(1H-indol-3-yl)-2-(methylenamino)-
propanoyl chloride (5)
The present study described a facile synthesis of novel promis-
ing neuroprotective indole derivatives and investigated also the
importance of incorporating heterocyclic moiety to the TRP nu-
cleus to form new effective hybrid molecules. TRP and its novel in-
dole derivatives 9b, 12c, 14a, and 17 showed neuroprotective
activity with various intensities depending on the structure of each
compound. Compound 17 in which the tetrazole ring was attached
to the TRP moiety ranked as the strongest neuroprotective agent.
All the tested compounds have been shown to possess antioxidant
properties offering promising efficacy against oxidative stress in-
duced by ACR administration. Finally, the neuroprotective activity
displayed by these compounds may be of interest for further study
of the toxicity profile of promising tested compounds before appli-
cation in phase 1 of clinical study in the hope of finding more ac-
tive and selective neuroprotective agents.
To a solution of compound 3 (0.22 g, 1 mmol) in anhydrous
diethyl ether (20 mL) in ice bath, equimolar amount of thionyl
chloride 4 (0.119 g, 1 mmol) was added dropwise with stirring.
After complete addition, the reaction mixture was stirred in ice
bath for 5 h until all starting materials had disappeared as indi-
cated by TLC. Then, the reaction mixture was left at room tem-
perature over night. The solid product that formed was filtered
off, dried and crystallized from absolute ethanol.
Compound 5: Yellow crystals, yield 0.24 g (90%), mp 252–254 °C,
IR (m
/cmÀ1): 3385 (NH), 3035 (CH-aromatic), 2987, 2890 (CH-ali-
phatic), 1715 (C@O), 1657 (C@N), 1642 (C@C). 1H NMR (CDCl3, d
ppm): 2.72 (t, J = 3.01 Hz, 1H, CH), 2.89 (d, J = 3.01 Hz, 2H, CH2),
4.73 (s, 2H, N@CH2), 6.65 (s, 1H, CH-indole), 7.18–7.29 (m, 4H,
C6H4), 10.12 (s, 1H, NH, D2O-exchangeable). MS (EI): m/z (%): 234
(M+ꢀ, 34%), 206 (M+ÀNCH2, 12), 171 (M+ÀCOCl, 25), 116 (100).

R. M. Mohareb et al. / Bioorg. Med. Chem. 19 (2011) 2966–2974
2971
Anal. Calcd for C12H11ClN2O (234.682): C, 61.41; H, 4.72; N, 11.94.
Found: C, 61.19; H, 4.95; N, 12.15.
167.3 (C@O), 126.4, 143.2, 109.2, 145.0, 135.9 (pyridine-C), 143.2,
116.9, 129.7, 118.0 (phenyl-C). MS (EI): m/z (%): 386 (M+ꢀ, 65),
371 (M+ꢀÀCH3, 28), 294 (M+ꢀÀNHPh, 20), 130 (100), 92 (56). Anal.
Calcd for C23H22N4O2 (386.446): C, 71.48; H, 5.74; N, 14.50. Found:
C, 71.24; H, 5.97; N, 14.22.
3.1.3. Synthesis of 3-(1H-indol-3-yl)-2-(methylenamino)-N-
phenylpropanamide (7)
Equimolar amounts of compound 5 (0.24 g, 1 mmol) and aniline
6 (0.09 mL, 1 mmol) in dioxane (30 mL) containing a catalytic
amount of triethylamine (2 mL) were heated under reflux for 6–
7 h. The reaction was monitored by TLC, concentrated under vac-
uum and poured into crushed ice and then left at 4 °C over night.
The formed solid product was filtrated off, dried and crystallized
from methanol.
3.1.5. Synthesis of 2-cyano-5-(1H-indol-3-yl)-4-(methyl-
enamino)-3-oxopentanonitrile (10)
To a solution of compound 5 (0.24 g, 1 mmol) in dioxane
(20 mL) containing a catalytic amount of triethylamine (0.5 mL)
equimolar amount of malononitrile 8a (0.07 g, 1 mmol) was added.
The reaction mixture was heated under reflux for 3 h until all the
reactants had disappeared as indicated by TLC. Then, the reaction
mixture was left to cool at room temperature, poured over ice/
water mixture, neutralized with dilute hydrochloric acid and ex-
tracted with diethyl ether (3 Â 20 mL). The organic layer was dried
over anhydrous calcium chloride. Removal of the solvent in vacuo
afforded the corresponding product, which was crystallized from
dioxane.
Compound 7: Pale brown crystals, yield 0.22 g (75%), mp 233–
235 °C, IR
(m
/cmÀ1): 3435–3376 (2NH), 3043 (CH-aromatic),
2987, 2878 (CH-aliphatic), 1712 (C@O), 1667 (C@N), 1639 (C@C).
1H NMR (CDCl3, d ppm): 2.57 (t, J = 2.56 Hz, 1H, CH), 2.80 (d,
J = 2.56 Hz, 2H, CH2), 4.69 (s, 2H, N@CH2), 6.65 (s, 1H, CH-indole),
7.16–7.34 (m, 4H, C6H4), 7.60–7.85 (m, 5H, C6H5), 8.70, 10.12 (2s,
2H, 2NH, D2O-exchangeable). 13C NMR (CDCl3, d ppm): 122.9 (C-
2, indole), 110.8 (C-3), 119.0 (C-4), 122.3 (C-5), 120.0 (C-6), 112.0
(C-7), 127.0, 136.2 (fused aromatic-C), 32.2, 96.3 (CH2, CH), 163.0
(CH2@N), 139.2, 121.9, 129.7, 124.3 (C-phenyl), 172.2 (C@O). MS
(EI): m/z (%): 291 (M+ꢀ, 74), 199 (M+ÀNHPh, 12), 92 (100). Anal.
Calcd for C18H17N3O (291.347): C, 74.20; H, 5.88; N, 14.42. Found:
C, 73.95; H, 6.08; N, 14.17.
Compound 10: Brown crystals, yield 0.21 g (78%), mp 261–
263 °C, IR (m
/cmÀ1): 3382 (NH), 3040 (CH-aromatic), 2987, 2875
(CH-aliphatic), 2232, 2225 (2CN), 1706 (C@O), 1658 (C@N), 1649
(C@C). 1H NMR (DMSO-d6, d ppm): 2.60 (d, J = 4.02 Hz, 2H, CH2),
2.87 (t, J = 4.02 Hz, 1H, CH), 4.35 (s, 1H, CH), 5.06 (s, 2H, N@CH2),
6.82 (s, 1H, CH-indole), 7.20–7.45 (m, 4H, C6H4), 8.43 (s, 1H, NH,
D2O-exchangeable). MS (EI): m/z (%): 265 (M+ꢀ+1, 32), 199 (38),
116 (23), 84 (100), 65 (12). Anal. Calcd for C15H12N4O (264.282):
C, 68.17; H, 4.58; N, 21.20. Found: C, 68.42; H, 4.29; N, 21.37.
3.1.4. Synthesis of indolylmethylpyridine derivatives (9a,b)
General procedure: To a solution of compound 7 (0.29 g, 1 mmol)
in absolute ethanol (25 mL) containing a catalytic amount of piper-
idine (0.5 mL) either malononitrile 8a (0.07 g, 1 mmol) or ethyl
cyanoacetate 8b (0.11 g, 1 mmol) was added. The reaction mixture
was heated under reflux for 4–6 h until all the reactants had disap-
peared as indicated by TLC. The reaction mixture after cooling at
room temperature was poured into ice/water mixture and neutral-
ized with dilute hydrochloric acid. The formed solid product, in
each case, was filtered off, dried and crystallized from the appro-
priate solvent.
3.1.6. Synthesis of pyrimidinylindolylmethylenamino-propan-
1-one derivatives (12a,b)
General procedure: Either urea 11a (0.12 g, 2 mmol) or thiourea
11b (0.15 g, 2 mmol) was added to a suspension of compound 10
(0.52 g, 2 mmol) in sodium ethoxide (10 mmol) [prepared by dis-
solving sodium metal (0.23 g, 10 mmol) in absolute ethanol
(35 mL)]. The reaction mixture, in each case, was heated in a boil-
ing water bath for 3–4 h, and then left to cool at room temperature.
The reaction was monitored by TLC, concentrated under vacuum,
poured into crushed ice and neutralized with dilute hydrochloric
acid. The formed solid product, in each case, collected by filtration,
dried and crystallized from the appropriate solvent.
3.1.4.1. 2-[(1H-Indol-3-yl)methyl]-5-amino-3-(phenylamino)-
pyridine-4-carbonitrile (9a).
yield 0.26 g (76%), mp 310–312 °C, IR (
Yellow crystals from dioxane,
m
/cmÀ1): 3438–3396 (2NH,
NH2), 3032 (CH-aromatic), 2985, 2883 (CH-aliphatic), 2228 (CN),
1660 (C@N), 1630 (C@C). 1H NMR (CDCl3, d ppm): 4.87 (s, 2H,
CH2), 5.23 (s, 2H, NH2, D2O-exchangeable), 6.68 (s, 1H, CH-indole),
6.76–7.04 (m, 4H, C6H4), 7.16–7.25 (m, 5H, C6H5), 8.72 (s, 1H, pyr-
idine-H), 9.80, 10.45 (2s, 2H, 2NH, D2O-exchangeable). 13C NMR
(CDCl3, d ppm): 123.4 (C-2, indole), 109.2 (C-3), 119.2 (C-4),
121.8 (C-5), 120.2 (C-6), 111.0 (C-7), 127.6, 136.4 (fused aro-
matic-C), 36.2 (CH2), 137.4, 146.2, 91.2, 147.0, 126.9 (pyridine-C),
117.2 (CN), 143.2, 116.9, 129.7, 118.0 (phenyl-C). MS (EI): m/z
(%): 338 (M+ꢀÀ1, 35), 247 (M+ꢀÀNHPh, 22), 218 (32), 130 (100),
92 (46). Anal. Calcd for C21H17N5 (339.393): C, 74.32; H, 5.05; N,
20.63. Found: C, 74.54; H, 5.28; N, 20.89.
3.1.6.1. 1-(4,6-Diamino-2-oxo-5H-pyrimidin-5-yl)-3-(1H-indol-
3-yl)-2-(methylenamino)-propan-1-one (12a).
crystals from absolute ethanol, yield 0.24 g (75%), mp 281–
283 °C, IR (
/cmÀ1): 3385–3348 (2NH2, NH), 3036 (CH-aromatic),
Pale yellow
m
2979, 2864 (CH-aliphatic), 1705, 1698 (2C@O), 1645 (C@N), 1620
(C@C). 1H NMR (DMSO-d6, d ppm): 2.35 (s, 1H, pyrimidine-H),
2.63 (d, J = 3.61 Hz, 2H, CH2), 2.74 (t, J = 3.61 Hz, 1H, CH), 4.03 (s,
4H, 2NH2, D2O-exchangeable), 5.12 (s, 2H, N@CH2), 6.80 (s, 1H,
CH-indole), 7.18–7.35 (m, 4H, C6H4), 10.03 (s, 1H, NH, D2O-
exchangeable). 13C NMR (DMSO-d6, d ppm): 121.8 (C-2, indole),
111.2 (C-3), 119.0 (C-4), 122.3 (C-5), 120.9 (C-6), 111.7 (C-7),
127.2, 135.3 (fused aromatic-C), 34.2 (CH2), 74.0 (CH), 164.0
(N@CH2), 198.5 (C@O), 49.4, 163.2, 164.0, 158.2 (pyrimidine-C).
MS (EI): m/z (%): 325 (M+ꢀ+1, 22), 199 (59), 125 (43), 116 (100).
Anal. Calcd for for C16H16N6O2 (324.337): C, 59.25; H, 4.97; N,
25.91. Found: C, 59.45; H, 4.76; N, 25.63.
3.1.4.2. Ethyl 2-[(1H-indol-3-yl)methyl]-5-amino-3-(phenylami-
no)-pyridine-4-carboxylate (9b).
anol, yield 0.27 g (70%), mp 233–234 °C, IR (
Brown crystals from meth-
m
/cmÀ1): 3442–3392
(2NH, NH2), 3036 (CH-aromatic), 2987, 2879 (CH-aliphatic), 1732
(C@O), 1664 (C@N), 1638 (C@C). 1H NMR (CDCl3, d ppm): 1.38 (t,
J = 7.01 Hz, 3H, CH3-ester), 4.28 (q, J = 7.01 Hz, 2H, CH2-ester),
4.76 (s, 2H, CH2), 5.03 (s, 2H, NH2, D2O-exchangeable), 6.73 (s,
1H, CH-indole), 6.75–7.07 (m, 4H, C6H4), 7.16–7.25 (m, 5H, C6H5),
8.67 (s, 1H, pyridine-H), 9.92, 10.25 (2s, 2H, 2NH, D2O-exchange-
able). 13C NMR (CDCl3, d ppm): 122.9 (C-2, indole), 109.8 (C-3),
119.2 (C-4), 122.3 (C-5), 121.0 (C-6), 111.3 (C-7), 127.4, 136.7
(fused aromatic-C), 30.2 (CH2), 61.0 (CH2-ester), 15.0 (CH3-ester),
3.1.6.2.
indol-3-yl)-2-(methylenamino)-propan-1-one (12b).
low crystals from dioxane, yield 0.26 g (75%), mp 302–304 °C, IR
/cmÀ1): 3429–3356 (2NH2, NH), 3037 (CH-aromatic), 2987,
1-(4,6-Diamino-2-thioxo-5H-pyrimidin-5-yl)-3-(1H-
Yel-
(m
2874 (CH-aliphatic), 1696 (C@O), 1654 (C@N), 1627 (C@C), 1197
(C@S). 1H NMR (CDCl3, d ppm): 2.30 (s, 1H, pyrimidine-H), 2.58
(d, J = 2.98 Hz, 2H, CH2), 2.75 (t, J = 2.98 Hz, 1H, CH), 4.23 (s, 4H,

2972
R. M. Mohareb et al. / Bioorg. Med. Chem. 19 (2011) 2966–2974
2NH2, D2O-exchangeable), 5.17 (s, 2H, N@CH2), 6.80 (s, 1H, CH-in-
dole), 7.18–7.32 (m, 4H, C6H4), 10.11 (s, 1H, NH, D2O-exchange-
able). MS (EI): m/z (%): 340 (M+ꢀ, 16), 199 (52), 141 (43), 116
(100). Anal. Calcd for C16H16N6OS (340.403): C, 56.45; H, 4.74; N,
24.69. Found: C, 56.68; H, 4.53; N, 24.88.
N@CH2), 4.57, 5.03 (2s, 4H, 2NH2, D2O-exchangeable), 6.58–7.06
(m, 6H, C6H5 + CH-indole), 7.23–7.38 (m, 4H, C6H4), 10.22 (s, 1H,
NH, D2O-exchangeable). MS (EI): m/z (%): 374 (M+ꢀ, 13), 259 (19),
199 (36), 116 (83), 77 (100). Anal. Calcd for
C21H22N6O
(374.439): C, 67.36; H, 5.92; N, 22.44. Found: C, 67.62; H, 6.09;
N, 22.72.
3.1.7. Synthesis of 1-(4,6-diamino-2-imino-5H-pyrimidin-5-yl)-
3-(1H-indol-3-yl)-2-(methylenamino)propan-1-one (12c)
A mixture of compound 10 (0.52 g, 2 mmol), guanidine hydro-
chloride 11c (0.2 g, 2 mmol) and sodium acetate (0.16 g, 2 mmol)
in absolute ethanol (30 mL) was refluxed for 7 h. The reaction mix-
ture was concentrated in vacuo and the residue was triturated with
ethyl acetate. The solid that formed was filtered off, dried and crys-
tallized from DMF.
3.1.9. Synthesis of 2-(1H-tetrazole-5-methylencarboxamido)-3-
(1H-indol-3-yl)propanoic acid (17)
To a solution of compound 15 (1.35 g, 5 mmol) in dimethyl
formamide (10 mL) containing ammonium chloride (0.027 g,
0.5 mmol), sodium azide 16 (0.05 g) was added with stirring. After
complete addition, the reaction mixture was heated in an oil bath
at 120 °C with stirring for 7 h. The reaction was monitored by TLC,
concentrated under vacuum, poured into 100 mL cold water and
acidified with dilute hydrochloric acid to pH 2. Then, the reaction
mixture was left to cool at 5 °C overnight. The solid product that
formed was filtered off, washed with water several times, dried
and crystallized from absolute ethanol.
Compound 12c: Pale brown powder, yield 0.26 g (80%) mp
247–249 °C, IR (m
/cmÀ1): 3435–3358 (2NH2, 2NH), 3030 (CH-aro-
matic), 2985, 2874 (CH-aliphatic), 1698 (C@O), 1645 (C@N), 1595
(C@C). 1H NMR (DMSO-d6, d ppm): 2.50 (s, 1H, pyrimidine-H),
2.62 (d, J = 3.04 Hz, 2H, CH2), 2.84 (t, J = 3.04 Hz, 1H, CH), 5.07 (s,
2H, N@CH2), 5.53 (s, 4H, 2NH2, D2O-exchangeable), 6.86 (s, 1H,
CH-indole), 7.20–7.37 (m, 4H, C6H4), 9.76, 10.11 (2s, 2H, 2NH,
D2O-exchangeable). 13C NMR (DMSO-d6, d ppm): 122.3 (C-2, in-
dole), 110.8 (C-3), 119.2 (C-4), 122.8 (C-5), 121.6 (C-6), 111.0 (C-
7), 127.6, 136.7 (fused aromatic-C), 31.2 (CH2), 74.0 (CH), 163.5
(N@CH2), 202.3 (C@O), 50.4, 164.2, 160.2, 164.8 (pyrimidine-C).
MS (EI): m/z (%): 323 (M+ꢀ, 62), 199 (24), 116 (100), 84 (76). Anal.
Calcd for C16H17N7O (323.352): C, 59.43; H, 5.30; N, 30.32. Found:
C, 59.17; H, 5.53; N, 30.12.
Compound 17: Dark brown crystals from absolute ethanol, yield
0.23 g (75%), mp 208–210 °C, IR (m
/cmÀ1): 3392–3353 (3NH), 3042
(CH-aromatic), 2987, 2874 (CH-aliphatic), 1655 (C@N), 1618
(C@C). 1H NMR (DMSO-d6, d ppm): 2.84 (d, J = 3.44 Hz, 2H, CH2),
4.23 (s, 2H, CH2), 4.85 (t, J = 3.44 Hz, 1H, CH), 6.81 (s, 1H, CH-in-
dole), 7.06–7.20 (m, 4H, C6H4), 8.05, 8.62, 9.36 (3s, 3H, 3NH,
D2O-exchangeable), 11.10 (s, 1H, OH, D2O-exchangeable). 13C
NMR (CDCl3, d ppm): 122.4 (C-2, indole), 110.2 (C-3), 119.2 (C-4),
122.2 (C-5), 120.3 (C-6), 111.2 (C-7), 127.6, 135.7 (fused aro-
matic-C), 30.4 (CH2), 55.9 (CH), 162.7, 175.2 (2C@O), 153.9 (tetra-
zole-C), 143.2, 116.9, 129.7, 118.0 (phenyl-C). MS (EI): m/z (%): 300
(M+ꢀ, 45), 255 (M+ꢀÀCOOH, 38), 231 (23), 69 (100). Anal. Calcd for
3.1.8. Synthesis of pyrazolylindolylmethylenaminopropan-1-
one derivatives (14a,b)
General procedure: To a solution of compound 10 (0.26 g,
1 mmol) in absolute ethanol (20 mL) either hydrazine hydrate
13a (0.05 g, 1 mmol) or phenyl hydrazine 13b (0.11 g, 1 mmol)
was added. The reaction mixture, in each case, was heated under
reflux for 6–8 h until all starting materials had disappeared as indi-
cated by TLC. Then the reaction mixture was concentrated under
vacuum, whereby the resulted oily product was triturated with
cold water/ice mixture. The solid product so formed in each case
was filtered off, dried and crystallized from the appropriate
solvent.
C14H14N6O3 (314.299): C, 53.50; H, 4.49; N, 26.74. Found: C,
53.78; H, 4.28; N, 27.01.
3.2. Biological assays
3.2.1. Animals
Sprague–Dawley female rats (70 rats), weighing 160–180 g,
aged 16–18 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 tem-
perature (25 1 °C) and humidity (55%). Rats were controlled con-
stantly 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 Eth-
ical Committee of the National Research Centre and in accordance
with the recommendations for the proper care and use of labora-
tory animals (NIH publication No. 85–23, revised 1985).
3.1.8.1.
1-(3,5-Diamino-4,5-dihydro-1H-pyrazol-4-yl)-3-(1H-
indol-3-yl)-2-(methylenamino)-propan-1-one (14a).
low crystals, yield 0.22 g (75%), mp 248–250 °C, IR (
Yel-
m
/cmÀ1):
3442–3365 (2NH2, 2NH), 3042 (CH-aromatic), 2989, 2869 (CH-ali-
phatic), 1687 (C@O), 1652 (C@N), 1605 (C@C). 1H NMR (CDCl3, d
ppm): 2.64 (d, J = 4.05 Hz, 2H, CH2), 2.72 (t, J = 4.05 Hz, 1H, CH),
2.85, 3.86 (2d, J = 5.22 Hz, 2H, pyrazole-2H), 5.12 (s, 2H, N@CH2),
4.46, 5.23 (2s, 4H, 2NH2, D2O-exchangeable), 6.85 (s, 1H, CH-in-
dole), 7.23–7.38 (m, 4H, C6H4), 8.74, 10.00 (2s, 2H, 2NH, D2O-
exchangeable). 13C NMR (CDCl3, d ppm): 123.0 (C-2, indole),
110.9 (C-3), 119.2 (C-4), 122.5 (C-5), 120.6 (C-6), 111.2 (C-7),
127.6, 137.0 (fused aromatic-C), 30.8 (CH2), 74.6 (CH), 163.5
(N@CH2), 208.3 (C@O), 50.4, 64.3, 155.2 (pyrazole-C). MS (EI): m/
z (%): 300 (M+ꢀ+2, 32), 199 (26), 182 (17), 116 (83), 99 (100). Anal.
Calcd for C15H18N6O (298.343): C, 60.39; H, 6.08; N, 28.17. Found:
C, 60.61; H, 5.86; N, 28.43.
3.2.2. Chemicals
ACR (99%) was purchased from Merck-Schuchardt Chemical Co.
(Hohenbrunn, Germany) and the starting pure powder of
phan was purchased from Sigma Company, USA.
L-trypto-
3.2.3. Experimental design
Chart 1 is intended to demonstrate the experimental design.
Animals were randomly assigned to seven groups (n = 10) as fol-
lows: control, ACR, TRP + ACR, compound 9b + ACR, compound
12c + ACR, compound 14a + ACR, and compound 17 + ACR. ACR
dose (50 mg kgÀ1 b. wt.) dissolved in water was injected intraper-
itoneally.21,36 TRP and its derivatives were dissolved in 2% Tween
80 and injected intraperitoneally in a dose of 50 mg kgÀ1 b. wt.37
3 days/week and 10 min prior to ACR injection for a period of
3 weeks.
3.1.8.2.
1-(3,5-Diamino-4,5-dihydro-1-phenyl-pyrazol-4-yl)-
3-(1H-indol-3-yl)-2-(methylenamino)propan-1-one
Brown crystals, yield 0.28 g (75%), mp 236–238 °C IR (
(14b).
m
/cmÀ1):
3439–3375 (2NH2, NH), 3035 (CH-aromatic), 2987, 2865 (CH-ali-
phatic), 1695 (C@O), 1658 (C@N), 1614 (C@C). 1H NMR (DMSO-
d6, d ppm): 2.60 (d, J = 2.68 Hz, 2H, CH2), 2.74 (t, J = 2.68 Hz, 1H,
CH), 2.89, 3.73 (2d, J = 4.92 Hz, 2H, pyrazole-2H), 5.09 (s, 2H,

R. M. Mohareb et al. / Bioorg. Med. Chem. 19 (2011) 2966–2974
2973
Experimental Groups
(7groups, n=10)
(1) Control Group
(3) TRP + ACR
(2) ACR Group
Treated Groups*
(50 mg kg-1 b.wt, i.p)
(4) Compound
(5) Compound
(6) Compound
(7) Compound
9b + ACR
12c + ACR
14a + ACR
17 + ACR
Chart 1. Experimental design for the in vivo neuroprotective effect of TRP and the novel synthesized indole derivatives. ACR: acrylamide, TRP:
L
-tryptophan, ⁄TRP and all
tested compounds injected in a dose of 50 mg kgÀ1 b. wt., ip, 3 days/week and 10 min prior to ACR injection for a period of 3 weeks.
3.2.4. Sample collection
authors expressed sincere appreciation to Dr. Abdel Razk Hussein
Farrag, Dept. of Pathology, National Research Centre, Egypt, for
his kind cooperation in conducting histological investigations
incorporated in this study.
The rats were killed and the whole brain of each animal was
rapidly dissected, thoroughly washed with isotonic saline, dried,
and then weighed. The first portion of each brain was homogenized
immediately to give 10% (w/v) homogenate in ice-cold medium
containing 50 mM Tris–HCl (pH 7.4) and 300 mM sucrose.38 The
homogenate was centrifuged at 1800 Â g for 10 min at 4 °C. The
supernatant (10%) was used for the determination of AD, NA,
MDA, GSH, GPx, SOD, LDH, CK, and total protein. The second por-
tion was stored at À80 °C for analysis of DA. The third portion of
the brain was fixed in formalin buffer 10% for histological
examination.
References and notes
1. Shimohama, S. Biol. Pharm. Bull. 2009, 32, 332.
2. Kopp, E. K.; Dekant, W. Toxicol. Appl. Pharmacol. 2009, 235, 135.
3. Crofton, K. M.; Padilla, S.; Tilson, H. A.; Anthony, D. C.; Raymer, J. H.; MacPhail,
R. C. Toxicol. Appl. Pharmacol. 1996, 139, 163.
4. Park, H. R.; Kim, M. S.; Kim, S. J.; Park, M.; Kong, K. H.; Kim, H. S.; Kwack, S. J.;
Kang, T. S.; Kim, S. H.; Kim, H. S.; Lee, J. Toxicol. Lett. 2010, 193, 86.
5. Sreejith, P.; Beyo, R. S.; Divya, L.; Vijayasree, A. S.; Manju, M.; Oommen, O. V.
Indian J. Biochem. Biophys. 2007, 44, 164.
6. Guerrero, J. M.; Reiter, R. J. Curr. Top. Med. Chem. 2002, 2, 167.
7. Suzen, S.; Buyukbingol, E. Farmaco 2000, 55, 246.
8. Tan, D. X.; Reiter, R. J.; Manchester, L. C.; Yan, M. T.; El-sawi, M.; Sainz, R. M.;
Mayo, J. C.; Kohen, R.; Allegra, M.; Hardeland, R. Curr. Top. Med. Chem. 2002, 2,
181.
9. Bozkaya, P.; Dogan, B.; Suzen, S.; Nebioglu, D.; Ozkan, S. A. Can. J. Anal. Sci.
Spectros. 2006, 51, 125.
10. Ates-Alagoz, Z.; Coban, T.; Suzen, S. A. Med. Chem. Res. 2005, 14, 169.
11. Suzen, S.; Bozkaya, P.; Coban, T.; Nebioglu, D. J. Enzyme Inhib. Med. Chem. 2006,
21, 405.
12. Ozer, C.; Gönül, B.; Ercan, Z. S.; Take, G.; Erdogan, D. Amino Acids 2007, 32, 453.
13. Ferzaz, B.; Brault, E.; Bourliaud, G.; Robert, J. P.; Poughon, G.; Claustre, Y.;
Marguet, F.; Liere, P.; Schumacher, M.; Nowicki, J. P.; Fournier, J.; Marabout, B.;
Sevrin, M.; George, P.; Soubrie, P.; Benavides, J.; Scatton, B. J. Pharmacol. Exp.
Ther. 2002, 301, 1067.
14. Bitzer-Quintero, O. K.; Dávalos-Marín, A. J.; Ortiz, G. G.; Meza, A. R.; Torres-
Mendoza, B. M.; Robles, R. G.; Huerta, V. C.; Beas-Zárate, C. Biomed.
Pharmacother. 2010, 64, 77.
15. Iacovitti, L.; Stull, N. D.; Johnston, K. Brain Res. 1997, 768, 317.
16. Serra, M.; Madau, P.; Chessa, M. F.; Caddeo, M.; Sanna, E.; Trapani, G.; Franco,
M.; Liso, G.; Purdy, R. H.; Barbaccia, M. L.; Biggio, G. B. J. Pharmacol. 1999, 127,
177.
17. Leoón, R.; Rios, C. D.; Marco-Contelles, J.; Huertas, O.; Barril, X.; Luque, F. J.;
López, M. G.; Garcia, A. G.; Villarroya, M. Bioorg. Med. Chem. 2008, 16, 7759.
18. Maurice, T.; Lockhart, B. P. Prog. Neuropsychopharmacol. Biol. Psychiatry 1997,
21, 69.
3.2.5. Biochemical analyses
Brain AD and NA levels were determined by enzyme linked
immunosorbent assay using kits purchased from Labor Diagnostic
Nord Gmbh Company (Germany). Brain DA level was determined
spectrofluorimetrically according the method of Ciarlone.39 Brain
MDA, GSH, GPx, and SOD levels were assayed by spectrophotome-
ter using kits purchased from Biodiagnostic Company (Egypt)
according the published methods.40–43 Brain LDH level was esti-
mated by spectrophotometer using kit purchased from Biosystems
Company (Spain) according to the method described by Friedman
and Young.44 Brain CK level was determined by spectrophotometer
using kit purchased from Biostic Company (Germany) according to
the method described by DGKC.45 Brain total protein determined
by spectrophotometer using folin reagent and according to the
method described by Lowry et al.46
3.3. Histopathology assay
After fixation of brain tissues in buffered formalin solution for
1 week, the brain tissues were washed in tap water for 24 h,
dehydrated in ascending series of alcohol. The samples were
then cleared in xylene and immersed in paraffin. The paraffin
19. Mohareb, R. M.; Louca, N. A.; Elmegeed, G. A.; Hana, H. Y. J. Chilean Chem. Soc.
2009, 54, 175.
20. LoPachin, R. M.; Lehning, E. J.; Opanashuk, L. A.; Jortner, B. S. Toxicol. Appl.
Pharmacol. 2000, 167, 75.
21. LoPachin, R. M.; Ross, J. F.; Lehning, E. J. Neurotoxicology 2002, 23, 43.
22. LoPachin, R. M. Neurotoxicology 2004, 25, 617.
23. Mannaa, F.; Abdel-Wahab, M. A.; Ahmed, H. H.; Park, M. H. J. Appl. Toxicol. 2006,
26, 198.
blocks were sectioned at 5 lm thickness and mounted on clean
glass slides and the ordinary hematoxylin and eosin stain was
used.47
24. Zhu, Y.; Zeng, T.; Zhu, Y.; Yu, S.; Wang, Q.; Zhang, L.; Guo, X.; Xie, K. Neurochem.
Res. 2008, 33, 2310.
3.4. Statistical analysis
25. Lüa, Z.; Zoua, H.; Parkc, S. J.; Parkc, D.; Shia, L.; Ohc, S. H.; Parka, Y.; Bhakc, J.;
Zoua, F. Int. J. Biol. Macromol. 2009, 44, 128.
26. Biradar, J. S.; Sasidhar, B. S.; Parveen, R. Eur. J. Med. Chem. 2010, 45, 4074.
27. Poeggeler, B.; Pappolla, M. A.; Hardeland, R.; Rassoulpour, A.; Hodgkins, P. S.;
Guidetti, P.; Schwarcz, R. Brain Res. 1999, 815, 382.
In the present study, all results were expressed as mean + SE of
the mean. Data were analyzed by one way analysis of variance
(ANOVA) using the Statistical Package for the Social Sciences (SPSS
)
program, version 11 followed by least significant difference (LSD)
to compare significance between groups.48 Difference was consid-
ered significant when P value was 6 0.05.
28. Topalca, N.; Yegin, E.; Celik, I. Food Chem. Toxicol. 2009, 47, 2441.
29. Campagna, F.; Palluotto, F.; Carotti, A.; Maciocco, E. Farmaco 2004, 59, 849.
30. Koufaki, M.; Kiziridi, C.; Nikoloudaki, F.; Alexis, M. N. Bioorg. Med. Chem. Lett.
2007, 17, 4223.
31. El-Ebiary, N. M.; Swellem, R. H.; Mossa, A. T.; Nawwar, G. A. Arch. Pharm.
(Weinheim) 2010, 343, 528.
32. Azam, F.; Alkskas, I. A.; Ahmed, M. A. Eur. J. Med. Chem. 2009, 44, 3889.
33. Padmavathi, V.; Reddy, S. N.; Mahesh, K. Chem. Pharm. Bull. 2009, 57, 1376.
34. Lima, L. M.; Barreiro, E. J. Curr. Med. Chem. 2005, 12, 23.
35. Koufaki, M.; Theodorou, E.; Alexi, X.; Alexis, M. N. Bioorg. Med. Chem. 2010, 18,
3898.
Acknowledgments
The authors acknowledge the financial support of the National
Research Centre, Egypt (Grant No.: E-8040505-2008/2010). The

2974
R. M. Mohareb et al. / Bioorg. Med. Chem. 19 (2011) 2966–2974
36. Fournier, J.; Steinberg, R.; Gauthier, T.; Keane, P. E.; Guzzi, U.; Coude, F. X.;
Bougault, I.; Maffrand, J. P.; Soubrie, P.; Le Fur, G. Neuroscience 1993, 55, 629.
37. Hayashi, M.; Bandoh, T.; Ushizawa, D.; Takada, S.; Hoshi, K. Alcohol 2004, 34,
225.
38. Tsakiris, S.; Schulpis, K. H.; Marinou, K.; Behrakis, P. Pharmacol. Res. 2004, 49,
475.
43. Ohkawa, H.; Ohishi, N.; Yagi, K. Anal. Biochem. 1979, 95, 351.
44. Effects of Disease on Clinical Laboratory Tests; Friedman, R. B., Young, D. S., Eds.,
3rd ed.; AACC press, 1997.
45. German Clinical Chemistry Society (DGKC), J. Clin. Chem. Clin. Biochem. 1977,
15, 255.
46. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. 1951, 193, 265.
47. Drury, R. A. B.; Wallington, E. A. Preparation and Fixation of Tissues in Carleton’s
Histological Technique, 4th ed.; Oxford University Press: Oxford, 1980.
48. Statistical Method in Medical Research; Armitage, P., Berry, G., Eds., 2nd ed.;
Blackwell Significant Publication: Oxford, 1987; pp 186–213.
39. Ciarlone, E. A. Am. J. Physiol. 1978, 125, 731.
40. Beutler, E.; Duron, O.; Kelly, M. B. J. Lab. Clin. Med. 1963, 61, 882.
41. Paglia, D. E.; Valentine, W. N. J. Lab. Clin. Med. 1967, 70, 158.
42. Nishikimi, M.; Roa, N. A.; Yogi, K. Biochem. Biophys. Res. Commun. 1972, 46, 849.