Design, Synthesis, and Pharmacological Screening of Pyridazinone Hybrids as Anticonvulsant Agents

Article abstract of DOI:10.1002/ardp.201700135

A series of new hybrid benzimidazole containing pyridazinones derivatives were designed and synthesized in accordance with the pharmacophoric requirements essential for the anticonvulsant activity. The synthesized compounds were evaluated for anticonvulsant activity on mice by the gold standard maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ)-induced seizure models. Among the compounds tested, SS-4F showed significant anticonvulsant activity in both the screens with ED₅₀ values of 25.10 and 85.33 mg/kg in the MES and scPTZ screens, respectively. Compound SS-4F emerged as safer and effective anticonvulsant due to its several-fold higher protective indices. Further, the gamma-aminobutyric acid (GABA) estimation result showed a marked increase in the GABA level (1.7-fold) as compared to the control, which was further confirmed by good binding properties with the GABA_A receptor.

Full text of DOI:10.1002/ardp.201700135

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Full Paper
Design, Synthesis, and Pharmacological Screening of Pyridazinone
Hybrids as Anticonvulsant Agents
Sangh Partap1, Mohammad Shahar Yar1, Md. Zaheen Hassan1,2, Md. Jawaid Akhtar1,
1
and Anees A. Siddiqui
1
Department of Pharmaceutical Chemistry, School of Pharmaceutical Education and Research, Jamia
Hamdard University, New Delhi, India
School of Chemical Science, University of Sains Penang, Penang, Malaysia
2
A series of new hybrid benzimidazole containing pyridazinones derivatives were designed and
synthesized in accordance with the pharmacophoric requirements essential for the anticonvulsant
activity. The synthesized compounds were evaluated for anticonvulsant activity on mice by the gold
standard maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ)-induced seizure
models. Among the compounds tested, SS-4F showed significant anticonvulsant activity in both the
screens with ED50 values of 25.10 and 85.33 mg/kg in the MES and scPTZ screens, respectively.
Compound SS-4F emerged as safer and effective anticonvulsant due to its several-fold higher
protective indices. Further, the gamma-aminobutyric acid (GABA) estimation result showed a marked
increase in the GABA level (1.7-fold) as compared to the control, which was further confirmed by
good binding properties with the GABAA receptor.
Keywords: Anticonvulsant / Benzimidazole / GABA / Pharmacophore / Pyridazinone
Received: April 26, 2017; Revised: July 31, 2017; Accepted: August 7, 2017
DOI 10.1002/ardp.201700135
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
acid receptors and/or voltage-activated sodium channels; (ii)
enhancementofinhibitoryprocessesbymodulatingtheactivity
of the gamma-aminobutyric acid (GABA) as the brain’s
Introduction
Epilepsy is a serious and common neurological condition which
is characterized by seizures due to hyper synchronous
discharges in the brain [1]. According to an epidemiological
study, epilepsy is the third most destructive neurological
disorder, affecting more than 50 million people worldwide
with almost 90% of these patients being in developing
countries [2]. The main epilepsy treatment includes strategy
of pharmacotherapy with anticonvulsant drugs [3]. The
antiepileptic drugs act through different mechanism like: (i)
inhibition of excitatory processes by blocking excitatory amino
inhibitory neurotransmitter; and (iii) stabilization of thalamic
neuronsbyinhibitingT-typecalciumchannels [4, 5]. Despitethe
large numbers of medicines available for treatment of
epilepsy [6], still there is no complete cure for epilepsy.
Unfortunately, current AEDs suppress the seizure of 70%
epileptic patients and rest of the epileptic patients suffers from
refractory epilepsy. Moreover, all available AEDs suffer from
serious disorder, such as minimal brain impairment, anemia,
and hepatic failure [7]. Thus, there is an urgent demand for
development of more efficacious, safe, and non-toxic agents in
the area of antiepileptic drug development.
Pyridazinone is an important pharmacophore possessing
the wide range of biological applications. A number of
pyridazinone analogs have been reported as anticonvulsant
lead compounds [8]. Apart from antiepileptic activity of
pyridazinone nucleus, other activity includes antidepres-
sant [9], antihypertensive [10–12], antithrombotic [13], car-
diotonic [14], anti-inflammatory [15–17], antimicrobial [18],
Correspondence: Prof. Anees A. Siddiqui, Department of
Pharmaceutical Chemistry, School of Pharmaceutical Education
and Research, Jamia Hamdard University, New Delhi ꢀ 110062,
India.
E-mail: prof.anees1@gmail.com
Fax: þ91-11-26059663
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anticancer [19–21], antioxidant [22, 23], antiviral [24, 25],
diuretics, etc. [26]. Moreover, several benzimidazole deriva-
tives are also shown to have seizure protective effects [27].
Based on these observations and in continuation of our
research work on pyridazinone [28], we have designed some
hybrid benzimidolyl-pyridazinone derivatives as anticonvul-
sant agents which possess all the essential pharmacophoric
elements necessary for anticonvulsant activity [29]. The hybrid
molecule is expected to demonstrate the pyridazinone
nucleus as hydrophobic-domain (HBD), –NH of benzimidazole
ring as electron donor system (D) and varying distal aryl ring
(R) which influences the blood brain barrier (BBB) diffusion
and pharmacokinetic properties of the anticonvulsants.
Moreover, pyridazinones are cyclized derivatives of b-aroyl
propionic acids which contain GABA like pharmacophore [30].
Thus benzimidazole pharmacophore is being introduced to
pyridazinone nucleus (cyclized GABA) in search of potent
anticonvulsant agents (Fig. 1).
Result and discussion
Chemistry
The b-aroyl propionic acids (SS-1a to SS-19a) were synthesized
according to the Lewis acid-catalyzed Friedel–Crafts acylation
of appropriate hydrocarbon with succinic anhydride as per
our previously reported method [31]. The condensation of
compounds SS1a–SS19a with hydrazine hydrate afforded
different
6-substitued-4,5-dihydropyridazine-3(2H)-one
(SS1b–SS19b) [32]. The synthetic route of synthesized
derivatives is outlined in Scheme 1. Subsequently, these 6-
substitued-4,5-dihydropyridazine-3(2H)-ones on treatment
with ethyl bromoacetate in the presence of potassium
carbonate in dry acetone produce ethyl 2-(6-oxo-3-aryl-5,6-
dihyropyidazin-1(4H)-yl acetate (SS1C–SS19C) [33]. Finally,
the synthesized (SS1C–SS19C) compounds were cyclized on
treatment with ortho phenylene diamine in acidic condition
to yield the final product (SS-1F to SS-19F).
The present work describes the synthesis and anticonvul-
sant activity of pyridazinone derivatives. The anticonvulsant
activities were evaluated by using two standard animal
seizure models such a maximal electroshock (MES) seizure and
subcutaneous pentylenetetrazole (scPTZ) seizure test. The
neurotoxicity of synthesized derivatives was assessed through
rotarod test. To calculate pharmacokinetic properties and
docking parameters of these pyridazinone derivatives compu-
tational studies were carried out. GABA assay were performed
to check the anti-PTZ potential of compound as PTZ produces
seizure by inhibiting GABA neurotransmitters. Since all
antiepileptic drugs affect liver functions therefore the most
potent compound was also measured for liver enzymes
transaminases: serum glutamate oxaloacetate transaminase
(SGOT) and serum glutamate pyruvate transaminase (SGPT)
levels.
The purity of final compounds was checked by TLC using
solvent system (ethyl acetate/hexane: 4:6) and melting point.
The structures of the title compounds were characterized by
elemental analyses and spectral data. The IR spectra of the
–
most active compound showed the appearance of C O and
–
N–H absorption bands in the range of 1664 and 3321 cmꢀ1
,
respectively. The 1H NMR spectra showed a broad singlet
proton signal (–NH D2O exchangeable) at d 12.23 ppm. The
methylene proton of compound displayed a characteristic
singlet signal at d 4.10 ppm, while triplet peaks at d 2.59 and
3.87 ppm confirmed the pyridazinone proton. The 13C NMR
spectrum of a prototype compound SS-4F revealed two
upfield peaks at d 51.05 and d 54.95 ppm due to a pair of
methylene carbons and one methoxy carbon, respectively,
while carbonyl carbons groups appeared downfield at d
166.96 ppm (Table 1).
Figure 1. Design of title compounds bearing all the essential anticonvulsant pharmacophoric structures.
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of the compounds SS-17F was found to be active at 100 mg/kg
at only 0.5 h which reveal drug of short duration. Rest of the
compounds were found to be active at higher dose level of
300 mg/kg at 0.5 h. Thus the seizure protection of compounds
might be due to GABA-mediated mechanisms as in the scPTZ
model, seizures were blocked predominantly by AEDs acting
at the GABAA receptor (benzodiazepine, barbiturates,
valproate).
Selected compounds which had shown activity at 100 mg/kg
dose level were chosen for their neurotoxicity. All the
compounds were found to be devoid of neurotoxicity except
compound SS-16F which was found to be neurotoxic at higher
dose of 300 mg/kg.
In phase II study, selected compound SS-4F was found to
have significant anticonvulsant potential. In MES and scPTZ
test, compound SS-4F demonstrated effective median dose
(ED50) 25.10 and 85.33 and higher protective index (PI) of
27.03 and 7.95 as compared to standard drug phenytoin and
ethosuximide (Table 3). These results revealed that compound
SS-4F possessing p-methoxy phenyl is essential for better
anticonvulsant activity.
Hepatotoxic reaction limits the use of much therapeutics.
Table 4 showed the liver function test. Literature studies
showed that carbamazepine and valproic acid were associ-
ated with hepatotoxicity, whereas phenytoin may be
possible cause of acetaminophen toxicity. In this study,
selective compound SS-4F was found to be most active at
30 mg/kg in both the test, administered chronically to
animals for 15 days and biochemical in parameters were
Scheme 1. Synthesis route to the synthesized title compound
SS-1a–19a to SS-1F–19F.
Pharmacology
All the synthesized compounds were evaluated for MES and
scPTZ screen tests. The compounds were administered by
intraperitoneal (i.p.) route. The mice using dose of 30, 100,
and 300 mg/kg as the observations were taken at two
different time intervals (0.5 and 4 h). Neurotoxicity was
evaluated by rotarod method and results are presented in
Table 2.
estimated. Table
4 showed that SGOT, SGPT, alkaline
phosphatase, total albumin, and total protein of compound
SS-4F do not show any significant increase or decrease
parameters. Further, the histopathological evaluation of
liver tissue of the groups treated with compound SS-4F was
also carried out using hematoxylin and eosin staining
technique. Compound SS-4F showed normal histological
features (Fig. 2).
CNS bioavailability of drug is greatly affected by
physicochemical descriptors such as log P (partition coeffi-
cient), molecular weight (MW), hydrogen bond acceptors
In MES screen all the compounds showed some degree of
protection, indicative of these compounds to prevent seizure
threshold. The synthesized compounds SS-3F, SS-5F, SS-12F,
SS-17F, and SS-19F had shown activity at a dose level of
100 mg/kg, at both the time interval demonstrating rapid
onset and longer duration of action, whereas compounds SS-
2F, SS-6F, and SS-15F had shown protection at a dose level of
300 mg/kg after 4 h, indicating rapid onset but shorter
duration of action. Compounds SS-1F, SS-7F to SS-11F, SS-
13F to SS-14F, SS-16F, and SS-18F exhibited protection at the
time interval of 0.5 h after the dose level of 300 mg/kg. The
most active compound SS-4F was found to be active at a dose
level of 30 mg/kg at both the time intervals, thus having rapid
onset and longer duration of anticonvulsant action at lower
dose.
scPTZ screens were used to identify compounds that elevate
the seizure threshold. Synthesized compounds that showed
protection at 0.5 and 4 h at a dose level of 100 mg/kg are SS-3F
and SS-4F demonstrating of rapid onset and longer duration
of action, whereas compounds SS-5F, SS-12F, and SS-15F were
active at 4 h at a dose of 300 mg/kg indicative of longer
duration of action of these compounds at a higher dose. One
and donor counts in
a molecule. Therefore, partition
coefficients of all the compounds were determined experi-
mentally using octanol-phosphate buffer method and the
data are presented in Table 5. All the compounds were
found to be lipophilic enough (>2) and have potential to
cross the BBB to have potent anticonvulsant action. Rapid
onset and shorter duration of action may be reasoned due to
high lipophilicity of the drugs. More importantly, the log P
values were found to be in the optimal range of 2.28–4.49
(ꢁ5), otherwise it may cause CNS toxicities like motor
impairment, etc. The topological polar surface area (TPSA)
was calculated with Molinspiration and the results are
presented in Table 5. All the synthesized compounds show
%ABS from 38.75 to 47.98%. Number of rotatable bonds is
also crucial for the conformational flexibilities of molecules
for the receptor binding. It is considered that for passing oral
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Table 1. Physicochemical parameters of the title compounds (SS-1F to SS-19F).
Elemental analysisc)
(%)
Mol.
formula
Mol.
weight
%
Yield
Log
Compounds
R
m.p. (°C)
Rfa)
Pb)
C
H
N
SS-1F
Phenyl
C18H16N4O
304.13
318.14
322.33
334.37
338.79
332.41
332.41
332.41
332.41
360.45
354.40
373.24
394.46
396.45
346.44
380.44
348.41
383.24
310.37
210–212
141–143
114–117
184–186
138–140
156–158
201–203
131–133
126–128
163–165
296–298
159–161
267–269
242–244
178–180
147–149
193–195
151–153
270–272
0.78
0.48
0.67
0.59
0.42
0.64
0.72
0.68
0.49
0.57
0.72
0.64
0.45
0.78
0.59
0.48
0.63
0.76
0.51
52
72
63
48
78
40
56
38
77
61
32
70
58
44
60
47
76
63
59
2.40
2.89
2.56
2.28
2.96
3.31
3.38
3.38
3.38
4.05
3.40
3.52
4.49
3.94
3.72
4.08
2.61
3.23
2.38
71.06
71.70
67.09
68.28
63.84
72.29
72.30
72.28
72.31
73.33
74.58
57.94
76.15
72.74
72.83
75.80
68.97
56.43
61.94
5.32
5.73
4.71
5.47
4.49
6.08
6.09
6.10
6.08
6.74
5.15
3.81
5.64
5.10
6.42
5.33
5.82
3.97
4.57
18.43
17.62
17.40
16.79
16.57
16.88
16.89
16.88
16.89
15.57
15.83
15.03
14.22
14.17
16.20
14.75
16.11
14.64
18.07
SS-2F
p-Tolyl
C19H18N4O
SS-3F
p-Fluoro-phenyl
p-Anisyl
C18H15FN4O
19H18N4O2
C18 15ClN4O
C20H20N4O
20H20N4O
C20 20N4O
20H20N4O2
C22H24N4O
22H18N4O
18H14Cl2N4O
C25H22N4O
C24 20N4O2
21H22N4O
C24 20N4O
C20 20N4O2
C18 15BrN4O
16H14N4OS
SS-4F
C
SS-5F
p-Chloro-phenyl
p-Ethyl phenyl
3,4-Dimethylphenyl
2,5-Dimethylphenyl
2,4-Dimethylphenyl
p-Isobutyl phenyl
b-Naphthyl
H
SS-6F
SS-7F
C
SS-8F
H
SS-9F
C
SS-10F
SS-11F
SS-12F
SS-13F
SS-14F
SS-15F
SS-16F
SS-17F
SS-18F
SS-19F
C
C
3,4-Dichloro-phenyl
p-Benzyl phenyl
p-Phenoxyphenyl
p-Propyl phenyl
p-Biphenyl
p-Ethoxyphenyl
p-Bromo-phenyl
2-Thienyl
H
C
H
H
H
C
b)
a) Solvent system – ethyl acetate/hexane (6:4).
Log P was determined by octanol/phosphate buffer method. CLog P was
calculated using software Chem Draw Ultra 8.0. c) Elemental analysis for C, H, N were within ꢂ0.4% of the theoretical value.
bioavailability criteria, number of rotatable bonds should be
ꢁ10. All the compounds in general possess high number of
rotatable bonds (3–5) and therefore exhibit fair conforma-
tional flexibility. None of the compounds violated Lipinski
parameters (rule of 5) which make them potential
anticonvulsant.
As shown in Table 6, compound SS-4F was found to
significantly increase the GABA level as compared to control
animals for both i.p. and administration at 2 h and oral
treatment for 7 days. Compound SS-4F showed increase in
GABA level (71.4 mg/100 mg tissue 1.5-fold) in 2 h treatment
and (89.1 mg/100 mg 1.7-fold) in 7 days when compared with
control. The graphical representations of GABA level after 2 h
and 7 days are shown in Fig. 3.
with bigger phenoxy group led to drastic decrease in
activity. Among the aliphatic chain substitution on phenyl
ring, p-propyl ought to be most suitable, whereas p-
methyl, p-ethyl, isobutyl, 2,4-dimethyl showed less active.
Whereas lipophilic biphenyl (SS-16F), p-benzyl phenyl (SS-
13F), and thiophene derivative (SS-19F) are found to be
less potent. This may be attributed to the size of ring
structure which may have resulted in less binding affinity
with the receptor. In general, following interesting
pattern of anticonvulsant potencies of the phenyl ring
substituents of the benzimidazolyl-pyridazinone deriva-
tives was observed:
p-Anisyl > p-ethoxyphenyl > p-fluoro-phenyl > p-chloro-
phenyl, 3,4-dichloro-phenyl > 2-thienyl > p-propyl phenyl > p-
tolyl, p-ethyl phenyl > p-isobutyl phenyl, p-bromo-phenyl
> 3,4-dimethylphenyl, 2,4-dimethylphenyl, b-naphthyl > phe-
nyl, 2,5-dimethylphenyl, p-benzyl phenyl, p-phenoxyphenyl,
p-biphenyl.
Structure–activity relationship
The results of anticonvulsant studies of the synthesized
compounds revealed that the nature of substituents at the
sixth position of pyridazinone influences the anticonvul-
sant activity. Distal phenyl ring plays an important role to
the compound showing anticonvulsant activity. The result
indicated that electron rich groups such as p-methoxy and
p-ethoxy on the phenyl ring have a great influence on
anticonvulsant activity; however, replacement of alkoxy
Molecular docking studies
In this work, molecular docking computations were per-
formed using Maestro 10.5 program (Schrodinger 2013, Inc.,
USA) to unleash the possible mechanism of anticonvulsant
activity. The docking was performed on two main targets of
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Table 2. Anticonvulsant activity and minimal motor impairment of the synthesized compounds (SS-1F to SS-19F).
Intraperitoneal injection in mice
MES screen
scPTZ screen
Neurotoxicity screen
Compounds
0.5 h
4 h
0.5 h
4 h
0.5 h
4 h
SS-1F
SS-2F
SS-3F
SS-4F
SS-5F
SS-6F
SS-7F
SS-8F
300
100
100
30
–
300
100
30
100
300
–
–
–
300
100
100
300
300
–
X
–
X
–
300
100
100
100
300
300
–
–
–
–
–
100
100
300
300
300
300
300
100
300
300
100
300
30
–
–
–
–
–
–
–
–
X
X
–
X
X
–
SS-9F
–
300
300
300
100
–
–
SS-10F
SS-11F
SS-12F
SS-13F
SS-14F
SS-15F
SS-16F
SS-17F
SS-18F
SS-19F
Phenytoina)
Ethosuximidea)
300
–
–
–
X
X
X
X
–
X
X
–
100
–
300
–
–
–
–
X
–
300
–
100
–
300
–
300
–
300
–
100
300
100
30
100
300
300
–
–
300
100
30
300
300
–
–
–
–
–
100
100
100
300
The number of animals used ¼ 4; solvent used – polyethylene glycol; dose of 30, 100, and 300 mg/kg were administered i.p. The
figures in the table indicate the minimum dose whereby bioactivity was demonstrated in half or more of the mice. The animals
were examined at 0.5 and 4 h after injections were administered.
The dash (–) indicates an absence of activity at maximum dose administered (300 mg/kg). X denotes compound was not tested.
a)Data taken from Ref. [6] and dose in mg/kg.
commonly used anticonvulsant drugs: one is Naþ channel
and other is GABAA receptors. For this purpose, compound
SS-4F was selected because it displayed most potent
anticonvulsant action against both (MES and scPTZ) animal
screen models. Docking pose (Figs. 4 and 5) of compound
SS-4F with a homology model of the Naþ channel displayed
that SS-4F bound to the receptor in a pocket formed of
Asp7, Ala7C, Thr68D, Ser69D, Phe80, Phe84, Val87, Ile87,
Leu88, Val88, Phe91, and Lys7D. Among these amino acid
residues, SS-4F formed two hydrogen bonds, one hydrogen
bond formed between the amino group of benzimidazole
with Asp7 and the other between the keto groups of
pyridazinone with Lys7D. The docking and gliding scores
were found to be ꢀ5.580 and ꢀ5.593 within docking
interaction of SS-4F with a homology model of the Naþ
channel receptor. The docking study of compound SS-4F
with GABAA receptor was also carried out and docking pose
(Figs. 6 and 7) showed that SS-4F was surrounded by GABAA
receptors. Binding interaction of SS-4F with GABAA showed
that it was bound to a site formed of Val202, His101, Tyr159,
Table 3. Phase II quantitative anticonvulsant evaluation in mice.
a)
ED50
PIc)
b)
Compounds
MES
scPTZ
TD50
MES
scPTZ
SS-4F
25.10 (20.5–27.5)
9.5 (8.1–10.4)
>1000
85.33 (74.9–90.1)
>300
130
678.5 (565.6–890.7)
65.5 (52.5–72.9)
440.8
27.03
6.9
<0.44
7.95
<0.22
3.39
Phenytoind)
Ethosuximided)
Number of animals used ¼ 10; solvent used: polyethylene glycol (0.1 mL, i.p.).
a)Median effective dose eliciting anticonvulsant protection in 50% of animals. b) Median toxic dose eliciting minimal neurological
c)
d)
toxicity in 50% of animals. PI, protective index (TD50/ED50). Data taken from Ref. [4] and dose in mg/kg.
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Table 4. Liver enzyme and total protein estimation of the most potent compound SS-4F.
Alkaline
phosphatase ꢂ SEM
Total albumin
(g/100 mL) ꢂ SEM
Total protein
Comp.a)
SGOT ꢂ SEM
SGPT ꢂ SEM
(g/100 mL) ꢂ SEM
SS-4F
41.29 ꢂ 1.90
38.37 ꢂ 1.85
61.20 ꢂ 2.08
1.65 ꢂ 0.024
4.98 ꢂ 0.27ꢃ
Controlb)
37.18 ꢂ 1.71
34.23 ꢂ 1.27
57.29 ꢂ 2.01
1.57 ꢂ 0.021
5.23 ꢂ 0.21
a) Relative to control and data were analyzed by ANOVA followed by Student’s t-test for n ¼ 4 at ꢃp < 0.05. b) Control group were
treated with 0.5% methyl cellulose for 15 days.
Tyr209, Val190, Asn102, Arg132, Lys155, Asn60, Phe77,
His101, Met130, Leu140, Thr142, Arg144, Ser158, Ala160,
Glu189, and Ser204. The compounds SS-4F showed four
hydrogen bonds, one hydrogen bond is between serine 204
(shown in red color) and nitrogen of benzimidazole ring,
whereas the same amino acid is involved in another
hydrogen bonding with nitrogen of pyridazinone. The
amino acid arginine 144 shown in purple color displayed
hydrogen bond with keto groups of pyridazinone, whereas
NH of benzimidazole is involved in hydrogen bonding with
asparagine 60 (shown in green color). The docking and
gliding scores of compound SS-4F with GABAA docking
interaction were found to be ꢀ7.419 and ꢀ7.432. Lastly, the
result of docking interaction of the compound SS-4F
showed that it forms four hydrogen bonds with the GABAA
receptor with a high docking score as well as high gliding
score, whereas the binding interaction with Naþ channel
receptor gave less hydrogen bonding, less docking as well as
less gliding score. Hence, it may be concluded that our
compounds better act by GABAA receptors rather than Naþ
channel blocker.
postulated essential for the anticonvulsant. The results
of pharmacophore distance mapping of six standard
antiepileptic drugs were then compared with most active
compound SS-4F are shown in Table 7. It was found that the
distance between various groups essential for anticonvulsant
action was in good agreement with the clinically available
AEDs.
Conclusion
A series of hybrid benzimidazolyl-pyridazinone analogs were
synthesized and evaluated for their anticonvulsant activity
and neurotoxicity by MES, scPTZ, and minimal motor
impairment methods. Compound, 2-((1H-benzo[d]imidaz-
ole-2-yl)methyl)-6-(4-methoxyphenyl)-4,5-dihydropyridazin-
3(2H)-one (SS-4F) showed the most potent anticonvulsant
activity against both seizure models with no neurotoxicity.
An in silico pharmacophoric pattern and drug likeness
studies showed that compound fulfills all the requirements
crucial for the anticonvulsant drugs. Further, the most
potent compound SS-4F displayed in vivo increase in the
GABA level and better binding to GABAA receptor in
molecular docking studies, which indicates anticonvulsant
action through GABA-mediated mechanism rather than Naþ
channel inactivation. Thus hybrid benzimidazolyl-pyridazi-
nones could lead to the development of ideal scaffolds for
anticonvulsant agents acting through GABA-mediated
mechanism.
Pharmacophore distance mapping
The distance between various groups [one aryl(R) hydropho-
bic domain, one electron donor (D), and a hydrogen bond
acceptor/donor unit (HBD)] essential for anticonvulsant
activity were calculated using 3D optimized structures using
ACD freeware 3D viewer 12.0 version. Figure 8 shows the
distances between various groups of standard drugs
Figure 2. High power photomicrograph of
portal triad area of liver tissue from animals
treated with (a) control, (b) compound SS-4F
showing a normal histological appearance
(HE ꢄ 400). PT, portal triad; CV, central vein.
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Table 5. Important pharmacokinetics parameters for good oral bioavailability of title compounds (SS-1F to SS-19F).
%
ABS
TPSA
(A2)
Mol.
wt.
Molar
vol.
mi
LogP
n-OHNH
donor
n-ON
acceptor
Lipinski’s
violation
Compounds
R
n-ROTB
Rule
SS-1F
SS-2F
SS-3F
SS-4F
SS-5F
SS-6F
SS-7F
<500
304.35
<5
<5
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
<10
5
<1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Phenyl
p-Tolyl
p-Fluoro-phenyl
47.98 61.36
47.98 61.36
47.98 61.36
38.75 70.59
47.98 61.36
47.98 61.36
47.98 61.36
47.98 61.36
47.98 61.36
47.98 61.36
47.98 61.36
47.98 61.36
47.98 61.36
38.75 70.59
47.98 61.36
47.98 61.36
38.75 70.59
47.98 61.36
47.98 61.36
3
3
3
4
3
4
3
3
3
5
3
3
5
5
5
4
5
3
3
274.86
291.43
279.80
300.41
288.40
308.23
307.99
307.99
307.99
341.62
318.86
301.94
363.07
355.26
325.03
346.27
317.21
292.75
265.58
2.57
3.02
2.73
2.62
3.25
3.48
3.39
3.39
3.39
3.87
3.73
3.85
4.53
4.32
3.87
4.36
3.00
3.38
2.47
318.38
322.34
334.38
338.80
332.41
332.41
332.41
332.41
360.73
354.41
373.24
394.48
396.46
346.43
380.45
348.41
383.25
310.38
5
5
6
5
5
5
5
5
5
5
5
5
6
5
5
6
p-Anisyl
p-Chloro-phenyl
p-Ethylphenyl
3,4-Dimethylphenyl
2,5-Dimethylphenyl
2,4-Dimethylphenyl
p-Isobutyl phenyl
b-Nephthyl
3,4-Dichloro-phenyl
p-Benzyl phenyl
p-Phenoxyphenyl
p-Propyl phenyl
p-Biphenyl
SS-8F
SS-9F
SS-10F
SS-11F
SS-12F
SS-13F
SS-14F
SS-15F
SS-16F
SS-17F
SS-18F
SS-19F
p-Ethoxyphenyl
p-Bromo-phenyl
2-Thienyl
5
5
% ABS percentage of absorption, TPSA topological polar surface area, n-ROTB number of rotatable bonds, MW molecular
weight, MV molecular volume, n-OHNH number of hydrogen bond donors, n-ON number of hydrogen bond acceptors.
NewDelhi,India)andareuncorrected. Purityofthe compounds
was checked by thin-layer chromatography (TLC) plates (silica
Experimental
Chemistry
gelG)byusingethylacetate/hexane(4:6),whichwerevisualized
by exposing to iodine vapors and UV light. The FT-IR spectra
were recorded on (IR affinity SHIMADZU) FTIR spectrophotom-
eter usingKBrpellets;ymax valuesaregiven incmꢀ1 and1H NMR
spectra were recorded on Bruker model DRX-400MHz NMR
spectrometer (1H at 400 MHz, 13C at 100 MHz) in DMSO-d6.
Chemical shifts (d) are expressed in ppm relative to tetrame-
thylsilane (TMS) as an internal standard and coupling constants
(J values) are expressed in Hz. Mass spectra were recorded on
LCMS/MS (Perkin-Elmer and LABINDIA, Applied Biosystem)
model no. API 3000, presented as m/z. Elemental analysis (C, H,
and N) were undertaken with Perkin-Elmer model 240C
analyzer. Analyses for C, H, and N were within ꢂ0.4% of the
theoretical values.
General
All the chemicals used were of laboratory grade and procured
from E. Merck (Darmstadt, Germany) and S.D. Fine Chemicals
(Mumbai, India). Melting points were determined by open
capillary tubes in a Hicon melting point apparatus (Hicon,
Table 6. Effect of most potent compound in GABA
system.
GABA level in mice brain
(mg/100 mg of tissue)
2 h
7 days
post-treatment
Compounds
post-treatment
The InChI codes of the investigated compounds are
provided as Supporting Information.
SS-4F
Control
Clobazam
71.4 ꢂ 6.56ꢃ
45.5 þ 5.03
101 þ 6.77
89.1 ꢂ 4.23ꢃ
51.5 ꢂ 2.60
110 ꢂ 4.83
General procedure for the synthesis of ethyl 2-(6-oxo-3-
aryl-5,6-dihyropyidazin-1(4H)-yl acetate (SS-1c to SS-19c)
6-Substituted-phenyl-4,5-dihydropyridazin-3(2H)-one
(0.01 mol) and ethyl bromoacetate (0.03 mol) were dissolved
in 30 mL of dry acetone. In this mixture, potassium carbonate
(0.03 mol) was added, followed by heating under reflux for
12–14 h. The crude product was cooled at room temperature
and poured into ice-cold water. The obtained precipitates
were filtered off and recrystallized from appropriate solvent
to give the desired compounds [33].
The compounds were tested at a dose of 100 mg/kg (i.p.) and
clobazam (30 mg/ kg).
The data indicate the minimum concentration whereby at
least 50% inhibition was demonstrated in one or more time
points.
Each value represents the mean ꢂ SEM of six rats, significantly
different from the control at ꢃp< 0.005 (Student’s t-test). Animal
used ꢀ mice divided into three groups of six animals each.
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2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-phenyl-4,5-
dihydropyridazin-3(2H)-one (SS-1F)
IR (KBr) (cmꢀ1): 3347 (N–H str), 3085 (C–H, Ar–H str), 2960 (C–H
str), 1715 (C O str); 1H NMR (400 MHz, DMSO-d6) d (ppm):
–
–
12.09 (bs, 1H, NH, D2O exchangeable), 7.63–7.99 (m, 5H,
phenyl), 7.51 (d, 2H, J ¼ 7.6 Hz, H-4,7 benzimidazole), 7.10 (dd,
2H, J ¼ 4.8, 6.0 Hz, H-5,6 benzimidazole), 4.60 (s, 2H, –CH2),
2.69 (t, 2H, J ¼ 7.8 Hz, H5 pyridazinone), 3.58 (t, 2H, J ¼ 7.8 Hz,
H4 pyridazinone). 13C NMR (100 MHz, DMSO-d6) d (ppm):
165.56, 148.90, 140.32, 138.42, 136.86, 132.76, 128.80, 127.74,
123.41, 115.62, 51.31, 34.64, and 25.78. MS m/z: 305 [Mþ1]þ.
2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-p-tolyl-4,5-
dihydropyridazin-3(2H)-one (SS-2F)
Figure 3. Effects of compound SS-4F on GABA levels in mice
brain tissues (mg/100 mg tissue). Each value represents the
mean ꢂ SEM of six mice, significantly different from the control
at ꢃp < 0.005 (Student’s t-test).
IR (KBr) (cmꢀ1): 3452 (N–H str), 3030 (C–H, Ar–H str), 2881 (C–H
str), 1640 (C O str); 1H NMR (400 MHz, DMSO-d6) d (ppm):
–
–
11.87 (bs, 1H, NH, D2O exchangeable), 7.47–7.64 (m, 4H,
phenyl), 7.43 (d, 2H, J ¼ 8.0 Hz, H-4,7 benzimidazole), 7.34 (dd,
2H, J ¼ 2.4, 7.5 Hz, H-5,6 benzimidazole), 4.31 (s, 2H, –CH2),
2.67 (t, 2H, J ¼ 7.8 Hz, H5 pyridazone), 2.98 (t, 2H, J ¼ 7.8 Hz, H4
pyridazinone), 2.00 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6)
d (ppm): 162.78, 147.54, 141.32, 138.20, 136.80, 133.06,
128.90, 127.44, 123.40, 116.62, 53.30, 33.50, 24.78, and
22.42. MS m/z: 319 [Mþ1]þ.
General procedure for the synthesis of 2-((1H-benzo[d]-
imidazol-2-yl)methyl)-6-aryl-4,5-dihydropyridazin-3(2H)-
one (SS-1F to SS-19F)
A mixture of (0.01 mol) ester (SS-1c to SS-19c) and (0.01 mol)
o-phenylene diamine was refluxed with 4 N hydrochloric acid
at 100°C for 20 h. The resultant mixture was cooled and
poured in 10% ice cold aqueous sodium carbonate solution.
The solid product was obtained and washing was done with
hot water. The crude product was recrystallized with
appropriate solvent to obtain title compounds.
2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-(4-fluorophenyl)-
4,5-dihydropyridazin-3(2H)-one (SS-3F)
IR (KBr) (cmꢀ1): 3318 (N–H str), 3096 (C–H, Ar–H str), 2920 (C–H
str), 1649 (C O str); 1H NMR (400 MHz, DMSO-d6) d (ppm):
–
–
12.22 (bs, 1H, NH, D2O exchangeable), 7.63–7.98 (m, 4H,
phenyl), 7.55 (d, 2H, J ¼ 8.0 Hz, H-4,7 benzimidazole), 7.31 (dd,
2H, J ¼ 7.6, 10.4 Hz, H-5,6 benzimidazole), 4.39 (s, 2H, –CH2),
3.27 (t, 2H, J ¼ 7.8 Hz, H5 pyridazone), 2.60 (t, 2H, J ¼ 7.8 Hz, H4
pyridazinone). 13C NMR (100 MHz, DMSO-d6) d (ppm): 166.87,
163.89, 148.47, 142.54, 138.42, 136.76, 129.93, 128.83, 123.40,
115.26, 51.09, 33.67, and 25.88. MS m/z: 323 [Mþ1]þ.
2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-(4-
methoxyphenyl)-4,5-dihydropyridazin-3(2H)-one (SS-4F)
IR (KBr) (cmꢀ1): 3469 (N–H str), 3020 (C–H, Ar–H str), 2987 (C–H
str), 1692 (C O str); 1H NMR (400 MHz, DMSO-d6) d (ppm):
–
–
12.23 (bs, 1H, NH, D2O exchangeable), 7.83–7.97 (m, 4H,
phenyl), 7.71 (d, 2H, J ¼ 7.8 Hz, H-4,7 benzimidazole), 6.98 (dd,
2H, J ¼ 7.6, 12.0 Hz, H-5,6 benzimidazole), 4.62 (s, 2H, –CH2),
3.87 (t, 2H, J ¼ 7.8 Hz, H5 pyridazinone), 3.80 (s, 3H, –OCH3),
2.59 (t, 2H, J ¼ 7.8 Hz, H4 pyridazinone). 13C NMR (100 MHz,
DMSO-d6) d (ppm): 166.82, 159.35, 149.53, 141.78, 138.17,
135.93, 129.21, 128.45, 124.29, 115.91, 54.95, 51.05, 33.85, and
25.95. MS m/z: 335 [Mþ1]þ. Please see the Supporting
Information for the corresponding spectra.
Figure 4. Binding mode of compound SS-4F into Naþ channel
receptor pocket (homology model). Hydrogen bonds are shown
with yellow-dotted lines. One hydrogen bond is between NH of
benzimidazole and aspartate 7 shown in purple color and other
is between keto groups of pyridazinone and lysine 7D shown in
red color.
2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-(4-chlorophenyl)-
4,5-dihydropyridazin-3(2H)-one (SS-5F)
IR (KBr) (cmꢀ1): 3216 (N–H str), 3082 (C–H, Ar–H str), 2918 (C–H
str), 1683 (C O str); 1H NMR (400 MHz, DMSO-d6) d (ppm):
–
–
12.39 (bs, 1H, NH, D2O exchangeable), 7.78–7.89 (m, 4H,
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Figure 5. Lig plot of compound SS-4F show-
ing interaction into the binding site of Naþ
channel receptor (homology model).
phenyl), 7.62 (d, 2H, J ¼ 7.2 Hz, H-4,7 benzimidazole), 7.17 (dd,
2H, J ¼ 1.6, 6.9 Hz, H-5,6 benzimidazole), 4.38 (s, 2H, –CH2),
3.27 (t, 2H, J ¼ 6.4 Hz, H5 pyridazinone), 2.60 (t, 2H, J ¼ 6.0 Hz,
H4 pyridazinone). 13C NMR (100 MHz, DMSO-d6) d (ppm):
163.40, 145.50, 142.32, 139.72, 136.86, 132.76, 129.80, 127.74,
Figure 6. Binding mode of compound SS-4F into the GABAA
receptor pocket (homology model). Hydrogen bonds are shown
with yellow-dotted lines. The compounds showed four hydro-
gen bonds, one hydrogen bond is between serine 204 (shown in
red color) and nitrogen of benzimidazole ring, whereas the
same amino acid is involved in another hydrogen bonding with
nitrogen of pyridazinone. The amino acid arginine 144 shown in
purple color displayed hydrogen bond with keto groups of
pyridazinone, whereas NH of benzimidazole is involved in
hydrogen bonding with aspargine 60 (shown in green color).
Figure 7. Lig plot of compound SS-4F showing interaction into
the GABAA receptor pocket (homology model).
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Figure 8. Distance range between the essen-
tial structural elements R, D, and HBD.
123.84, 115.22, 51.76, 33.34, and 25.12. MS m/z: 339 [Mþ1]þ;
340 [Mþ2]þ.
2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-(2,5-
dimethylphenyl)-4,5-dihydropyridazin-3(2H)-one (SS-8F)
IR (KBr) (cmꢀ1): 3365 (N–H str), 3062 (C–H, Ar–H str), 2921 (C–H
str), 1754 (C O str); 1H NMR (400 MHz, DMSO-d6) d (ppm):
–
–
2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-(4-ethylphenyl)-
4,5-dihydropyridazin-3(2H)-one (SS-6F)
12.42 (bs, 1H, NH, D2O exchangeable), 7.97–7.85 (m, 3H,
phenyl), 7.79 (d, 2H, J ¼ 6.2, H-4,7 benzimidazole), 7.30 (dd,
2H, J ¼ 2.5, 7.9 Hz, H-5,6 benzimidazole), 4.13 (s, 2H, –CH2),
3.28 (t, 2H, J ¼ 7.8 Hz, H5 pyridazinone), 2.60 (t, 2H, J ¼ 7.8 Hz,
H4 pyridazinone), 2.18 (s, 6H, CH3). 13C NMR (100 MHz, DMSO-
d6) d (ppm): 166.21, 147.31, 139.61, 138.35, 136.32, 135.78,
131.43, 129.19, 127.69, 122.52, 113.29, 51.45, 34.60, 24.19,
20.76, 19.24. MS m/z: 333 [Mþ1]þ.
IR (KBr) (cmꢀ1): 3315 (N–H str), 3081 (C–H, Ar–H str), 2965
(C–H str), 1734 (C O str); 1H NMR (400 MHz, DMSO-d6) d
–
–
(ppm): 12.39 (bs, 1H, NH, D2O exchangeable), 7.63–7.99 (m,
4H, phenyl), 7.55 (d, 2H, J ¼ 7.4 Hz, H-4,7 benzimidazole),
7.20 (dd, 2H, J ¼ 3.2, 7.5 Hz, H-5,6 benzimidazole), 4.10 (s, 2H,
–CH2), 2.89 (t, 2H, J ¼ 7.6 Hz, H5 pyridazinone), 3.34 (t, 2H,
J ¼ 7.6 Hz, H4 pyridazinone), 2.55 (q, 2H, –CH2CH3), 1.30 (t,
3H, –CH2CH3). 13C NMR (100 MHz, DMSO-d6) d (ppm): 166.90,
147.82, 141.32, 138.70, 137.81, 131.72, 128.17, 127.67,
122.90, 115.80, 51.30, 33.70, 29.80, 26.09, and 20.47. MS
m/z: 333 [Mþ1]þ.
2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-(2,4-
dimethylphenyl)-4,5-dihydropyridazin-3(2H)-one (SS-9F)
IR (KBr) (cmꢀ1): 3390 (N–H str), 3110 (C–H, Ar–H str), 2934 (C–H
str), 1660 (C O str); 1H NMR (400 MHz, DMSO-d6) d (ppm):
–
–
2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-(3,4-
12.18 (bs, 1H, NH, D2O exchangeable), 7.87–7.72 (m, 3H,
phenyl), 7.69 (d, 2H, J ¼ 7.2 Hz, H-4,7 benzimidazole), 7.42 (dd,
2H, J ¼ 1.5, 6.9 Hz, H-5,6 benzimidazole), 4.17 (s, 2H, –CH2),
2.54 (t, 2H, J ¼ 7.8 Hz, H5 pyridazinone), 3.20 (t, 2H, J ¼ 7.8 Hz,
H4 pyridazinone), 2.15 (s, 6H, CH3). 13C NMR (100 MHz, DMSO-
d6) d (ppm): 168.20, 148.51, 139.60, 138.30, 137.31, 136.60,
131.40, 127.59, 126.52, 123.15, 122.61, 112.20, 52.42, 35.74,
24.18, 20.63, and 19.26. MS m/z: 333 [Mþ1]þ.
dimethylphenyl)-4,5-dihydropyridazin-3(2H)-one (SS-7F)
IR (KBr) (cmꢀ1): 3342 (N–H str), 3082 (C–H, Ar–H str), 2931
(C–H str), 1762 (C O str); 1H NMR (400 MHz, DMSO-d6) d
–
–
(ppm): 12.20 (bs, 1H, NH, D2O exchangeable), 7.98–7.82 (m,
3H, phenyl), 7.78 (d, 2H, J ¼ 7.6 Hz, H-4,7 benzimidazole),
7.31 (dd, 2H, J ¼ 3.2, 7.5 Hz, H-5,6 benzimidazole), 4.15 (s,
2H, –CH2), 2.64 (t, 2H, J ¼ 7.8 Hz, H5 pyridazinone), 3.80 (t,
2H, J ¼ 7.8 Hz, H4 pyridazinone), 2.15 (s, 6H, CH3). 13C NMR
(100 MHz, DMSO-d6)
d
(ppm): 165.45, 148.50, 139.30,
2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-(4-
139.40, 136.61, 135.41, 131.70, 127.56, 127.16, 124.54,
122.40, 114.60, 51.30, 34.61, 25.16, 21.56. MS m/z: 333
[Mþ1].
isobutylphenyl)-4,5-dihydropyridazin-3(2H)-one (SS-10F)
IR (KBr) (cmꢀ1): 3305 (N–H str), 3075 (C–H, Ar–H str), 2965 (C–H
str), 1721 (C O str); 1H NMR (400 MHz, DMSO-d6) d (ppm):
–
–
Table 7. Distance range between the essential structural elements R, D, and HBD.
Compounds
R-HBDa)
R-Da)
D-HBDa)
Lamotrigine
Phenytoin
Carbamazepine
Rufinamide
Diazepam
Zonisamide
SS-4F
5.807
3.042
6.517
2.407
4.793
4.058
5.48
3.301
3.868
3.931
7.474
4.827
5.651
7.42
4.598
2.497
5.554
5.209
1.497
6.729
5.46
a) Distances calculated for 3D optimized structures using MM3 and CHARMM parameterization (Argus Lab 4.0 and ACD/3D
viewer).
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12.10 (bs, 1H, NH, D2O exchangeable), 7.78–7.53 (m, 4H,
phenyl), 7.49 (d, 2H, J ¼ 8.0 Hz, H-4,7 benzimidazole), 7.23 (dd,
2H, J ¼ 1.7, 8.2 Hz, H-5,6 benzimidazole), 4.31 (s, 2H, –CH2),
2.53 (t, 2H, J ¼ 7.7 Hz, H5 pyridazinone), 2.99 (t, 2H, J ¼ 7.7 Hz,
H4 pyridazinone), 1.88 (d, 2H, –CH2), 1.64–1.72 (m, 1H, –CH),
0.90 (m, 6H, (CH3)2. 13C NMR (100 MHz, DMSO-d6) d (ppm):
166.16, 149.52, 141.31, 139.25, 136.75, 132.10, 128.71, 127.10,
123.19, 115.50, 50.17, 40.21, 35.87, 29.23, 24.17, and 22.81. MS
m/z: 361 [Mþ1]þ.
2.43 (t, 2H, J ¼ 7.6 Hz, H5 pyridazinone), 2.94 (t, 2H, J ¼ 7.6 Hz,
H4 pyridazinone). 13C NMR (100 MHz, DMSO-d6) d (ppm):
167.71, 158.80, 155.13, 148.51, 139.27, 135.40, 133.74, 130.19,
129.61, 128.43, 127.15, 125.83, 122.67, 115.30, 50.23, 34.72,
and 24.50. MS (m/z): 397 [Mþ1]þ.
2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-(4-propylphenyl)-
4,5-dihydropyridazin-3(2H)-one (SS-15F)
IR (KBr) (cmꢀ1): 3372 (N–H str), 3095 (C–H Ar str), 2964 (C–H
str), 1690 (C O str); 1H NMR (400 MHz, DMSO-d6) d (ppm):
–
–
2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-(naphthalene-1-
yl)-4,5-dihydropyridazin-3(2H)-one (SS-11F)
11.89 (bs, 1H, NH, D2O exchangeable), 7.75–7.69 (m, 4H,
phenyl), 7.56 (d, 2H, J ¼ 7.4 Hz, H-4,7 benzimidazole), 7.24 (dd,
2H, J ¼ 6.5, 7.9 Hz, H-5,6 benzimidazole), 4.24 (s, 2H, –CH2),
2.58 (t, 2H, J ¼ 7.8 Hz, H5 pyridazinone), 3.02 (t, 2H, J ¼ 7.8 Hz,
H4 pyridazinone), 2.42 (d, 2H, J ¼ 4.2 Hz, –CH2-phe), 1.64 (m,
2H, –CH2–CH3), 1.15 (t, 3H, J ¼ 3.3 Hz, –CH3). 13C NMR
IR (KBr) (cmꢀ1): 3373 (N–H str), 3115 (C–H, Ar–H str), 2965 (C–H
str), 1705 (C O str); 1H NMR (400 MHz, DMSO-d6) d (ppm):
–
–
12.19 (bs, 1H, NH, D2O exchangeable), 8.05–7.56 (m, 7H,
naphthyl), 7.50 (d, 2H, J ¼ 7.0 Hz, H-4,7 benzimidazole), 7.34
(dd, 2H, J ¼ 2.2, 8.6 Hz, H-5,6 benzimidazole), 4.41 (s, 2H,
–CH2), 2.92 (t, 2H, J ¼ 7.6 Hz, H5 pyridazinone), 3.24 (t, 2H,
J ¼ 7.6 Hz, H4 pyridazinone). 13C NMR (100 MHz, DMSO-d6) d
(ppm): 166.12, 147.90, 140.41, 138.30, 136.97, 134.73, 133.14,
132.52, 130.51, 130.46, 128.56, 127.41, 126.51, 126.40, 123.40,
115.61, 52.30, 34.51, and 25.75. MS m/z: 355 [Mþ1]þ.
(100 MHz, DMSO-d6)
d (ppm): 164.60, 148.95, 140.15,
138.42, 136.53, 132.60, 128.19, 127.42, 123.52, 115.23,
50.27, 37.89, 34.72, 23.65, 25.89, and 15.90. MS m/z: 347
[Mþ1]þ.
2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-(biphenyl-4-yl)-
4,5-dihydropyridazin-3(2H)-one (SS-16F)
2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-(3,4-
IR (KBr) (cmꢀ1): 3290 (N–H str), 3097 (C–H Ar str), 2998 (C–H
str), 1760 (C O str); 1H NMR (400 MHz, DMSO-d6) d (ppm):
–
–
dichlorophenyl)-4,5-dihydropyridazin-3(2H)-one (SS-12F)
IR (KBr) (cmꢀ1): 3345 (N–H str), 3114 (C–H Ar str), 2990 (C-H str),
12.38 (bs, 1H, NH, D2O exchangeable), 7.80–7.60 (m, 9H,
phenyl), 7.43 (d, 2H, J ¼ 7.2 Hz, H-4,7 benzimidazole), 7.26 (dd,
2H, J ¼ 3.5, 7.6 Hz, H-5,6 benzimidazole), 4.11 (s, 2H, –CH2),
2.86 (t, 2H, J ¼ 7.8 Hz, H5 pyridazinone), 3.24 (t, 2H, J ¼ 7.6 Hz,
H4 pyridazinone). 13C NMR (100 MHz, DMSO-d6) d (ppm):
161.20, 148.76, 143.14, 142.74, 139.67, 135.23, 133.97, 130.53,
129.35, 128.72, 127.14, 125.64, 122.80, 115.24, 50.90, 34.61,
and 25.60. MS (m/z): 381 [Mþ1]þ.
1
1760 (C ¼ O str); H NMR (400 MHz, DMSO-d6) d (ppm): 12.23
(bs, 1H, NH, D2O exchangeable), 7.89–7.83 (m, 3H, phenyl),
7.59 (d, 2H, J ¼ 7.1 Hz, H-4,7 benzimidazole), 7.21 (dd, 2H,
J ¼ 1.6, 8.5 Hz, H-5,6 benzimidazole), 4.41 (s, 2H, ꢀCH2), 2.57 (t,
2H, J ¼ 7.8 Hz, H5 pyridazinone), 3.28 (t, 2H, J ¼ 7.8 Hz, H4
pyridazinone). 13C NMR (100 MHz, DMSO-d6) d (ppm): 164.62,
149.19, 141.21, 138.40, 135.76, 136.62, 132.61, 130.43, 127.98,
127.70, 123.56, 115.75, 51.30, 34.56, and 25.64. MS (m/z): 373
[M þ 1]þ; 374 [M þ 2]þ.
2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-(4-ethoxyphenyl)-
4,5-dihydropyridazin-3(2H)-one (SS-17F)
2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-(4-benzylphenyl)-
4,5-dihydropyridazin-3(2H)-one (SS-13F)
IR (KBr) (cmꢀ1): 3345 (N–H str), 3082 (C–H Ar str), 2998 (C–H
str), 1735 (C O str); 1H NMR (400 MHz, DMSO-d6) d (ppm):
–
–
IR (KBr) (cmꢀ1): 3349 (N-H str), 3026 (C-H Ar str), 2973 (C–H str),
11.92 (bs, 1H, NH, D2O exchangeable), 7.91–7.75 (m, 4H,
phenyl), 7.59 (d, 2H, J ¼ 7.1 Hz, H-4,7 benzimidazole), 7.25 (dd,
2H, J ¼ 3.1, 7.6 Hz, H-5,6 benzimidazole), 4.45 (s, 2H, –CH2),
3.38 (q, 2H, –CH2–CH3), 2.50 (t, 2H, J ¼ 7.7 Hz, H5 pyridazi-
none), 2.96 (t, 2H, J ¼ 7.6 Hz, H4 pyridazinone), 1.32 (t, 3H,
J ¼ 3.2 Hz, –CH3). 13C NMR (100 MHz, DMSO-d6) d (ppm):
165.72, 161.70, 148.29, 141.20, 138.29, 132.10, 128.90, 127.54,
123.56, 115.70, 59.12, 50.37, 34.61, 24.42, and 15.36. MS m/z:
349 [Mþ1]þ.
1695 (C O str); 1H NMR (400 MHz, DMSO-d6) d (ppm): 12.51
–
–
(bs, 1H, NH, D2O exchangeable), 7.88–7.53 (m, 9H, phenyl),
7.45 (d, 2H, J ¼ 6.5 Hz, H-4,7 benzimidazole), 7.25 (dd, 2H,
J ¼ 2.1, 8.5 Hz, H-5,6 benzimidazole), 4.13 (s, 2H, N–CH2–C),
3.93 (s, 2H, –CH2), 2.56 (t, 2H, J ¼ 7.8 Hz, H5 pyridazinone), 2.96
(t, 2H, J ¼ 7.8 Hz, H4 pyridazinone). 13C NMR (100 MHz, DMSO-
d6) d (ppm): 166.90, 149.18, 142.34, 141.53, 139.42, 135.61,
133.56, 130.76, 129.23, 128.87, 127.30, 125.68, 122.49, 115.31,
50.29, 41.23, 34.52, and 25.91. MS (m/z): 395 [Mþ1]þ.
2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-(4-bromophenyl)-
4,5-dihydropyridazin-3(2H)-one (SS-18F)
2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-(4-
phenoxyphenyl)-4,5-dihydropyridazin-3(2H)-one (SS-14F)
IR (KBr) (cmꢀ1): 3340 (N–H str), 3091 (C–H Ar str), 2985 (C–H
IR (KBr) (cmꢀ1): 3314 (N–H str), 3092 (C–H Ar str), 2981 (C–H
str), 1740 (C O str); 1H NMR (400 MHz, DMSO-d6) d (ppm):
–
–
str), 1729 (C O str); 1H NMR (400 MHz, DMSO-d6) d (ppm):
11.95 (bs, 1H, NH, D2O exchangeable), 7.73–7.58 (m, 4H,
phenyl), 7.59 (d, 2H, J ¼ 8.0 Hz, H-4,7 benzimidazole), 7.21 (dd,
2H, J ¼ 3.3, 8.9 Hz, H-5,6 benzimidazole), 4.36 (s, 2H, –CH2),
2.62 (t, 2H, J ¼ 7.8 Hz, H5 pyridazinone), 3.29 (t, 2H, J ¼ 7.8 Hz,
–
–
11.72 (bs, 1H, NH, D2O exchangeable), 7.81–7.62 (m, 9H,
phenyl), 7.54 (d, 2H, J ¼ 7.5 Hz, H-4,7 benzimidazole), 7.23 (dd,
2H, J ¼ 2.1, 8.5 Hz, H-5,6 benzimidazole), 4.26 (s, 2H, –CH2),
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H4 pyridazinone). 13C NMR (100 MHz, DMSO-d6) d (ppm):
162.63, 146.25, 141.30, 138.21, 135.35, 131.28, 128.93, 127.24,
123.30, 115.25, 51.25, 34.74, and 25.80. MS m/z: 384 [Mþ1]þ;
385 [Mþ2].
scPTZ test
The scPTZ test was carried out according to the known
protocol [36]. In this method, a 0.5% solution of pentylene-
tetrazole (75 mg/kg) was utilized subcutaneously in the
posterior midline for inducing seizures. The animals were
observed for 0.5 and 4 h. Failure to observe even a threshold
seizure (a single episode of clonic spasms of at least 5 s
duration) was defined as protection.
2-((1H-Benzo[d]imidazol-2-yl)methyl)-6-(thiophen-2-yl)-
4,5-dihydropyridazin-3(2H)-one (SS-19F)
IR (KBr) (cmꢀ1): 3318 (N–H str), 3095 (C–H Ar str), 2995 (C–H
str), 1761 (C O str); 1H NMR (400 MHz, DMSO-d6) d (ppm):
–
–
11.96 (bs, 1H, NH, D2O exchangeable), 7.69 (dd, J ¼ 5.1,
7.5 Hz, 1H, H-5 thiophene), 7.52 (d, 2H, J ¼ 6.2 Hz, H-4,7
benzimidazole), 7.43 (dd, J ¼ 5.3 Hz, 1H, H-4 thiophene),
7.41 (d, J ¼ 6.9 Hz, 1H, H-3 thiophene), 7.22 (d, 2H, 6.9 Hz,
H-5,6 benzimidazole), 4.09 (s, 2H, –CH2), 2.54 (t, 2H,
J ¼ 7.8 Hz, H5 pyridazinone), 2.96 (t, 2H, J ¼ 7.8 Hz, H4
pyridazinone). 13C NMR (100 MHz, DMSO-d6) d (ppm):
164.16, 158.10, 141.55, 138.93, 128.67, 127.53, 125.10,
124.06, 123.0, 115.90, 51.0, 33.78, and 25.61. MS m/z: 311
[Mþ1]þ.
Neurotoxicity: Minimal motor impairment
The minimal motor impairment was measured in mice by the
rotarod test. The mice were trained to stay on an accelerating
rotarod of 3.2 cm diameter rotating at 10 rpm speed. Trained
animals were given i.p. injection of the test compounds at 30,
100, and 300 mg/kg. Neurotoxicity was observed as the
inability of the animal to maintain equilibrium on the rod
for at least 1 min in each of the trials done [37].
Phase II acute toxicity study
The phase I screening was quantified in phase II screening for
acute toxicity study (Table 3). Anticonvulsant activity was
expressed in terms of the median effective dose (ED50), and
neurotoxicity was expressed as the median toxic dose (TD50).
For the determination of ED50 and TD50 values, groups of 10
mice were given a range of intraperitoneal doses of the test
drug until at least three points were established in the range
of 10–90% seizure protection or minimal observed neurotox-
icity. ED50 and TD50 values at 95% confidence intervals were
calculated by using Probit Analysis program. The time to peak
effect of all compounds was determined at the interval of 0.5–
4 h range [38, 39].
Pharmacology
All the pharmacological experiments were performed
according to ethical principles after Institutional Animal
Ethics Committee (IAEC) approval. The investigations were
conducted on albino mice of either sex (25–30 g) obtained
from central animal house facility, Hamdard University, New
Delhi-62, Registration no. 173/Go/Re/S/2000 CPCSEA. The
animals were housed under optimal conditions and allowed
free access to standard pellet diet and water (ad libitum). The
preliminary anticonvulsant (phase I) screening of the test
compounds was assessed by following the standard proto-
cols of Antiepileptic Drug Development (ADD) program by
NINDS, US using two widely used standard models, namely
MES and scPTZ test, and minimal neurological toxicity was
evaluated by rotarod test. The most potent compound was
also subjected to phase II quantitative determination of
ED50, TD50, PI and probit calculations were done by means of
computer program BioStat 2009 using Finney’s method.
Hepatotoxicity studies of the compounds were also carried
out using procedures described elsewhere. Most active
anticonvulsant compounds in phase I screening were also
subjected to neurochemical estimation of GABAs levels in
the mice whole brain.
Serum enzyme activity and histopathology studies
For the liver enzyme and total protein estimation, the
compounds found active were administered to each animal
at a dose of 25 mg/kg/day in methylcellulose for a period of 2
weeks. After the stipulated period, each animal was
anesthetized using anesthetic ether, and blood was collected
from the liver to assess the biochemical parameters such as
SGOT, SGPT, alkaline phosphate, and total protein according
to the reported methods [40–42]. Further animal were
sacrificed and excised liver tissues were fixed in 10% neutral
buffered formalin and sections of maximum 5 mm thickness
were cut. The sections were stained with hematoxylin and
eosin for histopathology. The histomorphological evaluation
of the liver sections was done by a pathologist unacquainted
with the treatment groups.
MES test
MES test was performed as per the standard protocol [34,
35]. The test compounds were dissolved in polyethylene
glycol (PEG 400). In the preliminary screening, each
compound was administered at three dose levels (30, 100,
and 300 mg/kg body mass) and the activity was recorded
after 0.5 and 4.0 h intervals of administration. All the animals
were administered with the test drug via i.p. route. Albino
mice were stimulated by using corneal electrodes in which
50 mA current of 60 Hz frequency was applied for 0.25 s.
Abolition of hind limb tonic extension spasm was recorded as
the end point of test result.
GABA estimation
The GABA estimation was performed in brain tissue extract
spectrophotometrically after 2 h of drug administration
(100 mg/kg, i.p.). After dosing, the animals were decapitated
and brains were dropped into vials and rinsed with ice-cold
isotonic saline followed by homogenization with 0.1 mmol/L
phosphate buffer (pH 7.4). The homogenate (10% w/v) was
centrifuged at 20000ꢄg for 30 min and the supernatant so
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formed was applied on silica gel 60F254 aluminum sheets
(10 ꢄ 10 cm2) as stationary phase, using mobile phase com-
prising water-saturated phenol. TLC plates were prewashed
with methanol and activated in an oven at 50°C for 5 min prior
to chromatography. The 0.1 mL supernatant solutions were
applied on TLC plates in the form of bands. Ascending
development to 80 mm was performed in a glass chamber
saturated with the mobile phase for 30 min at room
temperature. The developed TLC plates were air dried and
sprayed with ninhydrin. Then developed spots were scraped
together, mixed with 3 mL distilled water and centrifuged.
The supernatant obtained was spectrophotometrically ana-
lyzed at 570 nm. The specificity of the method was ascertained
by taking standard GABA samples and confirmed by
comparing their Rf values. The calibration curve was prepared
by plotting OD versus concentration [43, 44].
and Naþ channel receptor by using Maestro 10.5 program
(Schrodinger, Inc., USA). These receptors are the most
important targets in the design and discovery of successful
antiepileptic drugs. The homology model of the diazepam
bound GABAA receptor developed by Richter [49] and
Lamotrigine bound Naþ channel by Lipkind and Fozzard [50]
was retrieved from the supplementary material of their
published paper.
One of author, Sangh Partap, is thankful to University Grants
Commission (UGC) New Delhi, India, for financial support.
Authors are also thankful to Jamia Hamdard, New Delhi, India
for providing facility for the research work.
The authors have declared no conflict of interest.
Distance mapping
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