Synthesis, characterization & anticonvulsant activity of amide derivatives of 4-amino-1,2-naphthoquinone

Article abstract of DOI:10.1007/s00044-013-0531-6

In the present study, 4-amino-1,2-naphthoquinone analogues were synthesized and characterized by spectroscopic (FT-IR, ¹H NMR and ¹³C NMR) and elemental analysis. The synthesized compounds were evaluated for anticonvulsant activity b

Full text of DOI:10.1007/s00044-013-0531-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0531-6
ORIGINAL RESEARCH
Synthesis, characterization & anticonvulsant activity of amide
derivatives of 4-amino-1,2-naphthoquinone
•
•
•
Mukesh Bansal Bharat Goel Shubhanjali Shukla
Radhey Shyam Srivastava
Received: 4 December 2012 / Accepted: 1 February 2013
Ó Springer Science+Business Media New York 2013
Abstract In the present study, 4-amino-1,2-naphthoqui-
none analogues were synthesized and characterized by
Introduction
1
spectroscopic (FT-IR, H NMR and 13C NMR) and ele-
Epilepsy is the chronic disorder of the central nervous
system (CNS) and is characterized by recurrent seizures
(Leppik, 1996) affecting more than 50 million people world-
wide (Dalkara and Karakurt, 2012). Latest survey indicates
that approximately 0.5 million new cases of epilepsy are
diagnosed every year in India and beside that majority of
people cannot afford or access appropriate medical care due
mental analysis. The synthesized compounds were evalu-
ated for anticonvulsant activity by the maximal electroshock
(MES) test and subcutaneous pentylenetetrazole (sc. PTZ)
test, the most widely employed seizure models for early
identification of anticonvulsant candidates, whereas their
neurotoxicity was examined by rotarod test. Compounds
were administered to animals at different concentrations
(10, 20 & 40 mg/kg) by intraperitonial (i.p.) route and the %
seizure protection was measured. The pharmacological
results revealed that majority of compounds were effective
in MES and sc. PTZ tests. Compounds N-(1,2-dihydro-1,2-
dioxonaphthalen-4-yl)butyramide (4) and N-(1,2-dihydro-
1,2-dioxonaphthalen-4-yl)-3-methylbutanamide (6) were
active at the dose 40 & 20 mg/kg, respectively, while
compounds N-(1,2-dihydro-1,2-dioxonaphthalen-4-yl)hex-
anamide (7) and 4-acetamido-N-(1,2-dihydro-1,2-diox-
onaphthalen-4-yl)benzamide (10) were active at the dose
10 mg/kg and emerged as the most active compounds of the
series. These compounds showed anticonvulsant effect
comparable to phenytoin which was used as reference
antiepileptic drug. The above-mentioned compounds have
diminutive neurotoxic effects so they can move on next
phase of anticonvulsant drug development.
´
to high treatment cost (Bonifacio et al., 2001; Krishnan and
Ritvik, 2007). Seizures occur because small numbers of
neurons discharge abnormally. Anything that disrupts the
normal homeostasis of the neuron and disturbs its stability
may trigger abnormal activity and seizures (Gidal and
Garnett, 2005). Inhibition of neuronal cation conductance
via blockage of voltage gated sodium channels is one of the
proven mechanisms to regulate the seizures activity for
many anti-epileptic drugs (AEDs) like Phenytoin, Carbam-
azepine, Valproic acid, etc. (Fig. 1). Despite strong antiep-
ileptic capabilities many marketed antiepileptic drugs, i.e.
Ethosuximide, Valproic acid, Phenytoin, Carbamazepine
and Barbiturates, are also producing many adverse effects
such as ataxia, hepatotoxicity, gingival hyperplasia (Brodie
and Dichter, 1996) and megaloblastic anaemia (Perucca and
Gilliam, 2012). Moreover, Drug-resistant epilepsy with
uncontrolled severe seizures continues to be a major clinical
problem for up to one in three epileptic patients despite state-
of-the-art medical treatment and is associated with an
increased risk of death and other consequences (Das et al.,
2012). Thus, it is necessary to explore a unique antiepileptic
agent that is highly efficacious and safe that not only abol-
ishes the manifestation of seizures but also lead to a self-
sustained life.
Keywords 4-Amino-1,2-naphthoquinone ꢀ
Anticonvulsant activity ꢀ Maximal electroshock test ꢀ
Subcutaneous pentylenetetrazole test ꢀ Neurotoxicity
M. Bansal ꢀ B. Goel ꢀ S. Shukla ꢀ R. S. Srivastava (&)
Department of Pharmaceutics, Indian Institute of Technology
(Banaras Hindu University), Varanasi 221005, U.P., India
e-mail: rssriitbhu@yahoo.com
The quinone derivatives attracted our interest due to
their wide biological and medicinal applicability. They
123

Med Chem Res
Experimental
The melting points were determined in an open capillary
melting point apparatus using BARNSTEAD/ELECTRO-
THERMAL STUART-SMP10 and were uncorrected. Spec-
troscopic data were recorded on SHIMADZU RF-1501-UV
Spectro-fluorophotometer, IR spectra were recorded on KBr
discs using SHIMADZU Infrared-spectrophotometer, FTIR-
1
8400S. The H-NMR and 13C-NMR spectra’s were mea-
sured by JEOL, AL300, FT-NMR Spectrophotometer
(300 MHz) at temperature 25 °C, compounds were solubi-
lized in DMSO-d6/CDCl3 solvents. All the chemical shifts
were reported in d (ppm) values using tetramethylsilane
(TMS) as internal standard. Elemental analysis has been
performed using Exeter Analytical Inc., USA, CE-440 ele-
mental analyzer. The homogeneity of the compounds was
monitored by ascending thin layer chromatography (TLC)
on silica gel-G (0.2-mm thickness Merck, India) coated
aluminium plates, visualized in UV light.
Fig. 1 Chemical structure of the antiepileptic drugs (AEDs)
have already been reported with anticonvulsant (Sousa DPd
et al., 2011), anticancer (Kennedy et al., 2011; Shukla
et al., 2012a; Shukla et al., 2012b), trypanocidal (Pinto and
de Castro, 2009), leishmanicidal (Mori-Yasumoto et al.,
2012) and antibacterial (Chung et al., 2009) activities.
Amide derivatives also received considerable attention
due to their anticonvulsant efficacy (Pessah et al., 2011;
Tripathi et al., 2012). We aimed to synthesize amide
derivatives of 4-amino-1,2-naphthoquinone and their dose-
dependant anticonvulsant efficiency was determined by the
MES test and sc. PTZ test. Further, compounds were tested
for the impairment of motor coordination by the rotarod
test.
Procedure for the synthesis of 4-Amino-1,2-Naphtho-
quinone (ANQ, 1)
A solution of NQ (0.01 M) in 15 ml glacial acetic acid at
40 °C was treated with a solution of sodium azide
(0.017 M) in 5 ml water. Gas was evolved, and the solution
became reddish brown in colour; after 2 h, the reaction
product was crystallized from water and deep red needle
shape crystals were observed.
Its spectral data has been described in our previous
research paper (Shukla et al., 2012a).
Chemistry
The target compounds, i.e. N-(1,2-dihydro-1,2-dioxonaphth-
alen-4-yl)alkylamide/arylamide (2–11) were prepared by
following the synthetic pathway shown in scheme 1.
General procedure for synthesis of amide derivatives
of ANQ (2–11)
The starting material 4-amino-1,2-naphthoquinone (1)
was prepared in good yield according to previously
reported method (Fieser and Hartwell, 1935) by refluxing
solution of 1,2-naphthoquinone in glacial acetic acid with
the aqueous solution of sodium azide. Then, amide deriv-
atives (2–11) were prepared by condensing acyl chlorides
with 4-amino-1,2-naphthoquinone in methanol (solvent).
The chemical structures of the synthesized compounds
Acyl chlorides (1.0 mmol) was added to a suspension of
ANQ (1.0 mmol) in hot ethanol (50 ml). The mixture was
refluxed on a water bath for 4–8 h. The reaction progress was
monitored by thin layer chromatography (TLC). On com-
pletion of reaction, solvent was evaporated to dryness and the
resultant crude product was recrystallized using ethanol.
Physicochemical data and results of elemental analysis
of the synthesized compounds are listed in Table 1. The
spectral data of all the compounds are given below:
1
were established by spectroscopic (FT-IR, H NMR and
13C NMR) and elemental analysis.
Scheme 1 Scheme for
synthesis of 4-amino-1,2-
naphthoquinone derivatives
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Med Chem Res
Table 1 Analytical and physicochemical data of the synthesized compounds
O
O
NHCOR
Compound
R
Molecular formula M.W.a M.P. (°C)b Yield (%) % analysis of C, H and N found (calc.)
2
3
4
5
6
7
8
–CH3
C12H9NO3
C13H11NO3
C14H13NO3
C14H13NO3
C15H15NO3
C16H17NO3
C17H11NO3
215.2
229.2
243.2
243.2
257.2
271.3
277.3
156–157
160–161
165–166
153–154
174–175
179–180
148–150
40
25
60
48
56
78
89
66.97 (66.41), 4.22 (4.02), 6.51 (6.22)
68.11 (67.8), 4.84 (4.5), 6.11 (5.91)
69.12 (68.6), 5.39 (5.12), 5.76(5.87)
69.12 (68.4), 5.39(4.82), 5.76 (5.42)
70.02 (69.4), 5.88 (5.53), 5.44 (5.21)
70.83 (69.6), 6.32 (6.1), 5.16 (4.9)
73.64 (72.8), 4.00 (3.6), 5.05 (4.75)
–CH2CH3
–CH2CH2CH3
–CH(CH3)2
–CH2CH(CH3)2
–(CH2)4CH3
3'
2'
4'
1'
6'
5'
9
–CH2NHCOCH3
C14H12N2O4
C19H14N2O4
272.2
334.3
177–178
187–188
78
56
61.76 (61.3), 4.44 (4.15), 10.29 (9.6)
68.26 (68.1), 4.22 (3.9), 8.38 (7.92)
3'
2'
10
NHCOCH3
1'
4'
6'
5'
C16H10N2O3
278.2
159–160
58
69.06 (68.8), 3.62 (3.45), 10.07 (9.62)
11
N
a
Molecular weight (M.W.) of the compound
b
Melting point (M.P.) of the compounds at their decomposition
Ar–H), 2.22 (m, 2H, CH2), 1.15 (t, 3H, CH3); 13C NMR
(CDCl3) (ppm): 105.2 (C3, ethylene proton),
N-(1,2-dihydro-1,2-dioxonaphthalen-4-yl)acetamide (2)
d
TLC (chloroform/methanol, 9:1) Rf: 0.61; IR (cm-1, KBr):
3230 (N–H str.), 1587 (N–H bend.), 1680, 1575 (C=O str.),
3110 (aromatic C–H str.), 1338 (C–N str.); 1H NMR (CDCl3,
300 MHz) d (ppm): 6.5 (s, 1H, NH), 5.8 (s, 1H, ethylene
proton), 7.48–7.76 (m, 4H, Ar–H), 2.02 (s, 3H, CH3); 13C
NMR (CDCl3) d (ppm): 91.2 (C3, ethylene proton),
126.9–135.34 (aromatic C, C5–C10), 177.64, 181.7 (C1–C2,
C=O), 177.8 (C4, Ar–C–N), 168.4 (NH–C=O), 23.7 (Ali-
phatic C); UV–Vis kmax: 265 nm in ethanol (cf. Scheme 1).
125.5–135.00 (aromatic C, C5–C10), 173.64, 183.7 (C1–C2,
C=O), 175.8 (C4, Ar–C–N), 164.6 (NH—C=O), 10.1–30.1
(Aliphatic C); UV–Vis kmax: 267 nm in ethanol (cf.
Scheme 1).
N-(1,2-dihydro-1,2-dioxonaphthalen-4-yl)butyramide
(4)
TLC (chloroform/methanol, 9:1) Rf: 0.46; IR (cm-1, KBr):
3230 (N–H str.), 1580 (N–H bend.), 1685, 1576 (C=O str.),
3150 (aromatic C–H str.), 2920 (aliphatic C–H str.), 1420
N-(1,2-dihydro-1,2-dioxonaphthalen-4-yl)propionamide
(3)
1
(C–N str.); H NMR (CDCl3, 300 MHz) d (ppm): 6.2 (s,
1H, NH), 5.8 (s, 1H, ethylene proton), 7.48–7.76 (m, 4H,
Ar–H), 0.9–2.2 (m, 7H, aliphatic H); 13C NMR (CDCl3) d
(ppm): 107.5 (C3, ethylene proton), 120.4–139.20 (aro-
matic C, C5–C10), 176.64, 181.6 (C1–C2, C=O), 179.7
(C4, Ar–C–N), 165.9 (NH–C=O), 13.1–39.2 (Aliphatic C);
UV–Vis kmax: 268 nm in ethanol (cf. Scheme 1).
TLC (chloroform/methanol, 9:1) Rf: 0.71; IR (cm-1, KBr):
3213 (N–H str.), 1590 (N–H bend.), 1684, 1587 (C=O str.),
3153 (aromatic C–H str.), 2854 (aliphatic C–H str.), 1380
1
(C–N str.); H NMR (CDCl3, 300 MHz) d (ppm): 6.8 (s,
1H, NH), 5.8 (s, 1H, ethylene proton), 7.48–7.76 (m, 4H,
123

Med Chem Res
N-(1,2-dihydro-1,2-dioxonaphthalen-4-yl)isobutyramide
(5)
2-acetamido-N-(1,2-dihydro-1,2-dioxonaphthalen-4-yl)
acetamide (9)
TLC (chloroform/methanol, 9:1) Rf: 0.59; IR (cm-1, KBr):
3248 (N–H str.), 1598 (N–H bend.), 1685, 1576 (C=O str.),
3155 (aromatic C–H str.), 2915 (aliphatic C–H str.), 1370
TLC (chloroform/methanol, 9:1) Rf: 0.82; IR (cm-1, KBr):
3250 (N–H str.), 1540 (N–H bend.), 1610, 1540 (C=O str.),
3185 (aromatic C–H str.), 1368 (C–N str.); 1H NMR
(CDCl3, 300 MHz) d (ppm): 6.2 (s, 1H, NH), 5.8 (s, 1H,
ethylene proton), 7.4–7.8 (m, 4H, Ar–H), 4.1 (s, 2H, =C–
CH2–NH), 2.02 (s, 3H, CH3); 13C NMR (CDCl3) d (ppm):
106.47 (C3, ethylene proton), 123.7–148.60 (aromatic C,
C5–C10), 172.57, 186.3 (C1–C2, C=O), 175.8 (C4, Ar–C–
N), 168.5 (NH–C=O), 43.6 (C, CH2), 171.1 (C, O=C–
CH3), 22.6 (C, CH3); UV–Vis kmax: 278 nm in ethanol (cf.
Scheme 1).
1
(C–N str.); H NMR (CDCl3, 300 MHz) d (ppm): 6.76 (s,
1H, NH), 5.8 (s, 1H, ethylene proton), 7.48–7.76 (m, 4H,
Ar–H), 1.2–2.8 (m, 7H, aliphatic H); 13C NMR (CDCl3) d
(ppm): 104.7 (C3, ethylene proton), 122.5–146.60 (aro-
matic C, C5–C10), 175.84, 183.5 (C1–C2, C=O), 176.9 (C4,
Ar–C–N), 167.2 (NH–C=O), 19.8–35.9 (Aliphatic C);
UV–Vis kmax: 270 nm in ethanol (cf. Scheme 1).
N-(1,2-dihydro-1,2-dioxonaphthalen-4-yl)-3-methylbu-
tanamide (6)
4-acetamido-N-(1,2-dihydro-1,2-dioxonaphthalen-4-yl)
benzamide (10)
TLC (chloroform/methanol, 9:1) Rf: 0.67; IR (cm-1, KBr):
3250 (N–H str.), 1543 (N–H bend.), 1610, 1540 (C=O str.),
3180 (aromatic C–H str.), 2958 (aliphatic C–H str.), 1386
TLC (chloroform/methanol, 9:1) Rf: 0.76; IR (cm-1, KBr):
3240 (N–H str.), 1560 (N–H bend.), 1610, 1540 (C=O str.),
3085 (aromatic C–H str.), 1420 (C–N str.); 1H NMR (CDCl3,
300 MHz) d (ppm): 6.3 (s, 1H, NH), 5.8 (s, 1H, ethylene
proton), 7.4–8.1 (m, 8H, Ar–H), 2.02 (s, 3H, CH3); 13C NMR
(CDCl3) d (ppm): 97.5 (C3, ethylene proton), 121.6–149.50
1
(C–N str.); H NMR (CDCl3, 300 MHz) d (ppm): 5.86 (s,
1H, NH), 5.8 (s, 1H, ethylene proton), 7.48–7.76 (m, 4H,
Ar–H), 1.0–2.2 (m, 9H, aliphatic H); 13C NMR (CDCl3)
d (ppm): 103.7 (C3, ethylene proton), 125.5–148.70 (aro-
matic C, C5–C10), 176.54, 182.9 (C1–C2, C = O), 178.2
(C4, Ar–C–N), 161.9 (NH–C=O), 22.2–46.7 (Aliphatic C);
UV–Vis kmax: 271 nm in ethanol (cf. Scheme 1).
0
0
(aromatic C, C5–C10, C1 –C6 ), 176.57, 183.4 (C1–C2, C=O),
180.6 (C4, Ar–C–N), 163.6 (NH–C=O), 22.9 (C, CH3);
UV–Vis kmax: 293 nm in ethanol (cf. Scheme 1).
N-(1,2-dihydro-1,2-dioxonaphthalen-4-yl)nicotinamide (11)
N-(1,2-dihydro-1,2-dioxonaphthalen-4-yl)hexanamide (7)
TLC (chloroform/methanol, 9:1) Rf: 0.70; IR (cm-1, KBr):
3315 (N–H str.), 1564 (N–H bend.), 1610, 1540 (C=O str.),
3110 (aromatic C–H str.), 1405 (C–N str.); 1H NMR
(CDCl3, 300 MHz) d (ppm): 6.2 (s, 1H, NH), 5.8 (s, 1H,
ethylene proton), 7.4–9.2 (m, 8H, Ar–H); 13C NMR
(CDCl3) d (ppm): 94.5 (C3, ethylene proton), 121.5–147.60
(aromatic C, C5–C10), 177.8, 186.5 (C1–C2, C=O), 179.7
(C4, Ar–C–N), 163.6 (NH–C=O), 125–153.7 (pyridinyl C);
UV–Vis kmax: 268 nm in ethanol (cf. Scheme 1).
TLC (chloroform/methanol, 9:1) Rf: 0.79; IR (cm-1, KBr):
3257 (N–H str.), 1540 (N–H bend.), 1610, 1540 (C=O str.),
3185 (aromatic C–H str.), 2968 (aliphatic C–H str.), 1350
1
(C–N str.); H NMR (CDCl3, 300 MHz) d (ppm): 6.9 (s,
1H, NH), 5.8 (s, 1H, ethylene proton), 7.48–7.76 (m, 4H,
Ar–H), 0.90–2.2 (m, 11H, aliphatic H); 13C NMR (CDCl3)
d (ppm): 110.4 (C3, ethylene proton), 121.5–149.60 (aro-
matic C, C5–C10), 178.75, 183.56 (C1–C2, C=O), 177.9
(C4, Ar–C–N), 162.6 (NH–C=O), 14.1–36.9 (Aliphatic C);
UV–Vis kmax: 274 nm in ethanol (cf. Scheme 1).
Pharmacology
N-(1,2-dihydro-1,2-dioxonaphthalen-4-yl)benzamide (8)
Anticonvulsant screening of the synthesized compounds
was performed by maximal electroshock (MES) test and
subcutaneous pentylenetetrazole (sc. PTZ) test. These
compounds were also evaluated for neurotoxicity (rotorod
method) in mice. Pharmacological experiments were carried
out on Swiss albino mice of either sex weighing 20–30 g.
The animals were obtained from Central Animal House,
Institute of Medical science, Banaras Hindu University
(Regd. No. Dean/12-13/CAEC/50). The animals were ran-
domly housed in polypropylene cages with free access to
TLC (chloroform/methanol, 9:1) Rf: 0.73; IR (cm-1, KBr):
3250 (N–H str.), 1540 (N–H bend.), 1610, 1540 (C=O str.),
3185 (aromatic C–H str.), 1368 (C–N str.); 1H NMR (CDCl3,
300 MHz) d (ppm): 7.1 (s, 1H, NH), 5.8 (s, 1H, ethylene
proton), 7.4–8.0 (m, 9H, Ar–H); 13C NMR (CDCl3) d (ppm):
105.2 (C3, ethylene proton), 122.5–144.50 (aromatic C,
0
0
C5–C10, C1 –C6 ), 179.34, 184.5 (C1–C2, C=O), 176.9 (C4,
Ar–C–N), 163.6 (NH–C=O); UV–Vis kmax: 278 nm in eth-
anol (cf. Scheme 1).
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Med Chem Res
standard pellets (Unilever Limited India) and water ad libi-
tum. The experiments were conducted according to the
norms of committee for the purpose of control the supervi-
sion of the experiments in Animals (CPCSEA) New Delhi,
India, and Institutional Animal Ethical Committee (IAEC).
After 7 days of adaptation to laboratory conditions, the
animals were randomly assigned to experimental groups
(consisting of five animals). Each animal was used only
once. The experiments were conducted between 9:00 am and
4:00 pm. The test compounds were dissolved in water and
polyethylene glycol (PEG-200) depending on their solubil-
ity. In anticonvulsant screening, each compound was
administered at three dose levels 10, 20 and 40 mg/kg-1
intraperitonealy (i.p.), five mice for each dose. In MES test,
all compounds were evaluated at 1, 2, 3 & 4 h intervals after
administration; whereas in sc. PTZ test, compounds were
evaluated at 0.5 and 4 h interval. In neurotoxicity test, ani-
mals were dosed at 30, 100 and 300 mg/kg-1 i.p. (five mice
per dose) and neurotoxicity assessed at 1- and 4-h intervals.
impairment of motor performance, ataxia and loss of skeletal
muscular strength and acute neurotoxicity produced by drugs
in preclinical studies (Pandey and Srivastava, 2011).
Results and discussion
Currently, about 30 % of patients with epilepsy are not
seizure free with the existing medications (Hen et al.,
2012). In addition, AED therapy particularly with Valproic
acid is associated with severe side effects, including tera-
togenicity, which restrict the clinical use of major AEDs in
women of child-bearing age or in children due to hepato-
toxicity (Molgaard-Nielsen and Hviid, 2011; Vajda et al.,
2012). Therefore, there is a substantial need to design
anticonvulsants for the development of more effective and
safer AEDs.
Chemistry
Maximal electro shock (MES) test
In the present study, the amide derivatives from 4-amino-
1,2-naphthoquinone with various acyl chlorides were syn-
thesized according to reaction scheme. The synthesized
compounds were characterized by spectroscopic (FT-IR, 1H
and 13C NMR) and elemental analysis. A singlet was
observed at 5.8–7.1 ppm in 1H NMR spectra of the 4-amino-
1,2-naphthoquinone amide derivatives which is the charac-
teristic peak of amides. In 13C NMR, the presence of peaks at
155–170 ppm also confirmed the presence of amide group
in synthesized compounds. Absorption bands at 1630–
1680 cm-1 were characteristic of stretching vibrations of
carbonyl group m(C=O) present in amide linkage. All these
data were well in accordance with those of the reported
amides (Pradidphol et al., 2012; Yang et al., 2012).
In the MES test (Kupferberg, 1989) seizures were elicited
with a 60-Hz alternating current of 50-mA intensity in
mice. The current was applied via ear electrodes for 0.2 s.
Abolition of the hind-leg tonic-extensor component of the
seizure indicated protection against the spread of MES-
induced seizures. In the MES test, one can readily preselect
compounds that are effective in suppression of tonic–clonic
seizures and to a certain extent, of partial seizures with or
without secondary generalization in humans.
Subcutaneous pentylenetetrazole (sc. PTZ) test
This test involved subcutaneous injection of pentylenetet-
razol at the dose of 60 mg/kg in mice. After dosing, each
animal was placed into an individual plastic cage for
observation after 0.5 and 4 h. Seizures and tonic–clonic
convulsions were recorded. The number of animals con-
vulsing out of the total number of mice tested was noted for
each treatment condition (Vogel, 2002).
Anticonvulsant activity
A reasonable estimation of a compound’s potential as a
new AED candidate is based on the characterization of the
compound’s anticonvulsant profile in a variety of anti-
convulsant animal models (White et al., 2002). Although
there is a variety of animal models for epilepsy, the MES
model remains the ‘‘gold standard’’ in early stages of dis-
covery of new AEDs and has gained an appreciable degree
of predictability since the MES was first utilized in the
discovery of the anticonvulsant activity of phenytoin
(1938) (Schmidt and Rogawski, 2002). The systemic
administration of Pentylenetetrazole (PTZ) is also one of
the most commonly employed methods for anticonvulsant
screening (Gupta et al., 1999).
Neurotoxicity screening
The rotorod test was executed to gauge the minimal motor
impairment in mice. The mice were trained to stay on a
revolving rod of diameter 3.2 cm, and rotating at the speed
of 6 revolutions per min. The trained mice were given
test compounds intraperitonealy in dose 30, 100 and
300 mg kg-1 bodyweight. The neurotoxicity was measured
by the inability of the animal to maintain equilibrium on the
revolving rod for at least 1 min in each of the three trails.
Worth mentioning feature of rotorod test is to gauge the
The synthesized compounds were investigated for anti-
convulsant activity by intraperitoneal (i.p.) administration
by two seizure models maximal electroshock seizures
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Med Chem Res
Table 2 Anticonvulsant and neurotoxicity screening data in mice dosed i.p. with the compounds
Test
MES testa
sc. PTZ testb
Neurotoxicity testc
1 h 4 h
Time
1 h
2 h
3 h
4 h
0.5 h 4 h
Dose (mg/kg)
10 20 40 10 20 40 10 20 40 10 20 40 10 20 40 10 20 40 30 100 300 30 100 300
1/5 5/5 5/5 1/5 2/5 2/5 0/5 1/5 1/5 0/5 0/5 0/5 2/5 4/5 4/5 0/5 0/5 1/5 2/5 1/5 3/5 1/5 2/5 1/5
2/5 5/5 4/5 1/5 2/5 3/5 1/5 1/5 2/5 0/5 0/5 1/5 1/5 1/5 2/5 0/5 0/5 1/5 2/5 3/5 1/5 0/5 1/5 1/5
4/5 5/5 5/5 2/5 3/5 4/5 2/5 2/5 5/5 0/5 2/5 4/5 4/5 5/5 5/5 1/5 3/5 5/5 2/5 1/5 1/5 2/5 3/5 4/5
4/5 5/5 5/5 1/5 3/5 4/5 2/5 2/5 4/5 1/5 2/5 4/5 4/5 4/5 5/5 2/5 2/5 4/5 2/5 1/5 2/5 2/5 2/5 2/5
4/5 5/5 5/5 4/5 4/5 4/5 5/5 5/5 5/5 1/5 4/5 4/5 4/5 5/5 5/5 2/5 4/5 4/5 1/5 1/5 3/5 2/5 2/5 2/5
5/5 5/5 4/5 5/5 5/5 4/5 4/5 5/5 3/5 4/5 4/5 5/5 4/5 5/5 5/5 4/5 4/5 5/5 2/5 3/5 2/5 3/5 1/5 3/5
1/5 2/5 2/5 0/5 2/5 1/5 2/5 1/5 1/5 0/5 0/5 1/5 0/5 2/5 2/5 0/5 1/5 1/5 2/5 1/5 4/5 2/5 2/5 2/5
5/5 4/5 1/5 1/5 2/5 3/5 1/5 2/5 0/5 1/5 2/5 0/5 1/5 2/5 2/5 0/5 1/5 1/5 1/5 2/5 1/5 1/5 1/5 1/5
5/5 5/5 5/5 5/5 5/5 5/5 4/5 4/5 4/5 4/5 5/5 5/5 4/5 5/5 5/5 4/5 4/5 5/5 1/5 1/5 1/5 1/5 2/5 2/5
1/5 2/5 2/5 0/5 2/5 1/5 2/5 1/5 1/5 0/5 0/5 1/5 1/5 1/5 2/5 1/5 0/5 1/5 1/5 2/5 2/5 2/5 3/5 3/5
2
3
4
5
6
7
8
9
10
11
Phenytoin (5 mg/kg) 5/5
4/5
5/5
4/5
5/5
4/5
2/5
1/5
a
MES test: Maximal electroshock seizure test (number of animal protected/number of animal tested)
sc. PTZ test: Subcutaneous pentylenetetrazole test (number of animal protected/number of animal tested)
Neurotoxicity test: rotarod test (Number of animal exhibiting toxicity/number of animal tested)
b
c
(MES) test and subcutaneous pentylenetetrazole (sc. PTZ)
drastically enhances the activity and at the dose of
10 mg/kg, 4 animals out of 5 were found to be protected.
Incorporation of heterocyclic pyridine ring (11) abolishes
the activity.
test. Neurotoxicity was determined by rotorod ataxia test
(performed to know the potential of motor impairment in
coordination of movement by the test compounds) in mice.
For the determination of anticonvulsant activity and
neurotoxicity, the amide derivatives were administered i.p.
at three doses level 10, 20 and 40 mg kg-1 (Table 2) and the
responses were compared with the reference drug Phenyt-
oin. As a performance of the compounds in the MES and sc.
PTZ tests, few compounds, i.e. N-(1,2-dihydro-1,2-diox-
onaphthalen-4-yl)acetamide (2), N-(1,2-dihydro-1,2-diox-
onaphthalen-4-yl)propionamide (3) and N-(1,2-dihydro-1,
2-dioxonaphthalen-4-yl)benzamide (8), were found to be
inactive at all; while compounds N-(1,2-dihydro-1,2-diox-
onaphthalen-4-yl)butyramide (4) and N-(1,2-dihydro-1,2-
dioxonaphthalen-4-yl)-3-methylbutanamide (6) exhibited
90 % protection at dose 40 and 20 mg/kg. Compounds
N-(1,2-dihydro-1,2-dioxonaphthalen-4-yl)hexanamide (7)
and 4-acetamido-N-(1,2-dihydro-1,2-dioxonaphthalen-4-yl)-
benzamide (10) showed seizures protection at dose 10 mg/kg
and found to be the most potent compounds of the series. In
neurotoxicity screening, compounds were tested for the
impairment of motor coordination by the rotarod test and
were found to have very less neurotoxic effects.
Conclusion
The amide derivatives of 4-amino-1,2-naphthoquinone were
synthesized and characterized. All the synthesized com-
pounds gave acceptable analytical and spectroscopic data,
which were in full accordance with their depicted structures.
The activity of synthesized compounds as anticonvulsant
agents has been investigated by the MES and sc. PTZ test
and further evaluated for the neurotoxicity by the rotarod
test. Compounds N-(1,2-dihydro-1,2-dioxonaphthalen-4-yl)-
butyramide (4) and N-(1,2-dihydro-1,2-dioxonaphthalen-
4-yl)-3-methylbutanamide (6) were active at the dose 40 &
20 mg/kg, respectively, while compounds N-(1,2-dihydro-
1,2-dioxonaphthalen-4-yl)hexanamide (7) and 4-acetamido-
N-(1,2-dihydro-1,2-dioxonaphthalen-4-yl)benzamide (10)
were active at the dose 10 mg/kg and emerged as the most
active compounds of the series. These compounds showed
anticonvulsant effect comparable to phenytoin so they can
move on next phase of anticonvulsant drug development.
Structure–Activity Relationship (SAR) studies showed
that compounds having lower alkyl side chain like methyl
(2), ethyl group (3) are inactive. Increasing the carbon chain
length like propyl (4), isopropyl (5), isobutyl (6) and pentyl
(6) increases the activity. Compounds bearing unsubstituted
phenyl ring (8) was found to be inactive, but substitution
of acetamide group at 4th position in phenyl ring (10)
Acknowledgments The authors are thankful to The Head, Depart-
ment of Chemistry, Faculty of Science, Banaras Hindu University
1
(BHU), Varanasi, India, for providing the facilities of H NMR and
13C NMR spectroscopy. The authors gratefully acknowledge the
financial assistance given by University Grants Commission (UGC),
New Delhi, to Mr. Mukesh Bansal [Grant No. F38-3/2007(SRIII)].
123

Med Chem Res
Perucca P, Gilliam FG (2012) Adverse effects of antiepileptic drugs.
Lancet Neurol 11(9):792–802. doi:10.1016/S1474-4422(12)
70153-9
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