Synthesis, anti-convulsant activity and molecular docking study of novel thiazole pyridazinone hybrid analogues

Article abstract of DOI:10.1016/j.bioorg.2020.103584

Pyridazinone analogues have been known to be potential candidates for anticonvulsant agents. We have identified several pyridazinone-based anticonvulsant agents. As a continuation to our previous research, a series of hybrid pyridazinone-thiazole connected through amide linkage were designed and synthesized. Among these, compound SP-5F demonstrated significant anticonvulsant activity with median effective dose of 24.38 mg/kg (MES) and 88.23 mg/kg (scPTz). Results of GABA estimation showed a marked increase in the GABA level when compared with control. Molecular docking studies at the active site of GABA receptor, further confirmed the GABA modulatory effects of SP-5F.

Full text of DOI:10.1016/j.bioorg.2020.103584

BioorganicChemistry99(2020)103584
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, anti-convulsant activity and molecular docking study of novel
thiazole pyridazinone hybrid analogues
⁎
Aness Ahmad Siddiquia, , Sangh Partapa, Subuhi Khisala, Mohammad Shahar Yara,
Ravinesh Mishrab
a Department of Pharmaceutical Chemistry, School of Pharmaceutical Education and Research, Jamia Hamdard University, New Delhi, India
b School of Pharmacy and Emerging Sciences, Baddi University of Emerging Sciences & Technology, Baddi (Solan), Himachal Pradesh 173205, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Pyridazinone analogues have been known to be potential candidates for anticonvulsant agents. We have iden-
tified several pyridazinone-based anticonvulsant agents. As a continuation to our previous research, a series of
hybrid pyridazinone-thiazole connected through amide linkage were designed and synthesized. Among these,
compound SP-5F demonstrated significant anticonvulsant activity with median effective dose of 24.38 mg/kg
(MES) and 88.23 mg/kg (scPTz). Results of GABA estimation showed a marked increase in the GABA level when
compared with control. Molecular docking studies at the active site of GABA receptor, further confirmed the
GABA modulatory effects of SP-5F.
Thiazole
Pyridazinone
Anticonvulsant activity
Pharmacophore based drug design
GABA
1. Introduction
receptor-mediated seizure protective activity [9–11]. In previous
manuscript, we detailed our efforts to identify hybrid pyridazinone
Epilepsy is very dangerous CNS diseases due to hyper synchronous
electrical discharge in neurons. Globally, around fifty million people
are suffering from this destructive neurological condition and out of
these, ninety percent patients are from developing countries [1,2].
Moreover, the risk of seizure, trauma, hospitalization and mortality is
very high in epileptic patients which have very strong negative impact
on patient's physical, mental and social health [3]. Although, several
newer classes of antiepileptic drugs (AEDs) have emerged in the past
15 years, but complete treatment of epilepsy is still a long way [4,5].
Moreover, these AEDs are ineffective against the 30% refractory epi-
leptic patients and possess severe side effects such as, neurotoxicity,
anemia, hepatic failure etc. [6]. Thus, there is always a constant need to
develop newer effective anticonvulsant agent with minimal side-effects
(see Fig. 1).
analogues as anticonvulsant agents. These compounds were found to be
safer and effective anticonvulsant agents having GABA modulatory ef-
fects [12]. Subsequently, an attempt has also been made for hit-to-lead
optimization of pyridazinoneanalogues by introducing amide linkage as
hydrogen bonding domain between the two lipophilic nuclei. Results
revealed that some of these derivatives demonstrated admirable in vi-
troGABA-ATinhibition. However, the partition coefficient of these de-
rivatives was found to be higher (logP > 5) which conceivably con-
tributed to their high neurotoxicity [13]. Encouraged by these
observations and in continuation of our research program [14], it was
of interest to further optimize pyridazinone analogues by the mod-
ification the lipophilic domain which resulted into optimal lipophilic
derivatives.
Pyridazinone is an attractive lead for anticonvulsants, is a cyclized
derivative of β-aroylpropionic acid which contain GABA like pharma-
cophore [7]. Several pyridazinone derivatives have been reported to
possess seizure protective activity in number of animal models [8].
Moreover, designing of anticonvulsant agents based on pharmacophoric
pattern has received much attention in the AED discovery. Based on
these, several newer anticonvulsant agents have been described pre-
viously that demonstrated significant in vivo and in vitro binding and
2. Result and discussion
2.1. Chemistry
In the first step, β-aroyl propionic acids (SP-1a to SP-19a) were
synthesized by Lewis acid-catalyzed Friedel-Crafts acylation of appro-
priate hydrocarbon with succinic anhydride [15]. Further, condensa-
tion of the same (SP1a-SP19a) with hydrazine hydrate afforded
⁎ Corresponding author at: Department of Pharmaceutical Chemistry, School of Pharmaceutical, Education and Research, Jamia Hamdard, New Delhi 110062,
India.
E-mail address: anees_asiddiqui@rediffmail.com (A.A. Siddiqui).
https://doi.org/10.1016/j.bioorg.2020.103584
Received 20 October 2019; Received in revised form 6 December 2019; Accepted 12 January 2020
Availableonline27January2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

A.A. Siddiqui, et al.
BioorganicChemistry99(2020)103584
Fig. 1. Lead optimization of anticonvulsant pyridazinones.
different 6-substitued-4,5-dihydropyridazine-3(2H)-one (SP1b-SP18b)
in the second step [16]. The synthetic route of synthesized derivatives is
outlined in Scheme 1. Subsequently, 6-substitued-4,5-dihydropyr-
idazine-3(2H)-one on treatment with 2-chloro-N-(thiazol-2-yl)acet-
amide in the presence of potassium carbonate in dry DMF produce
5F with 13C NMR spectra as a prototype revealed two up-field peaks at δ
54.12 ppm of methylene carbons and the carbonyl carbon appeared
downfield at δ 168.29 ppm.
2.2. Pharmacology
substituted
phenyl-5,6-dihydropyridazin-1(4H)-yl)-N-(thiazol-2-yl)
acetamide(SP1F-SP18F). The synthetic route of synthesized derivatives
is outlined in Scheme 1.
All the synthesized compounds were evaluated for maximal elec-
troshock seizure (MES) and subcutaneous pentylenetetrazole (scPTZ)
tests. The compounds were administered intraperitoneally (ip) in-
different doses of 30,100 and 300 mg/kg and end points were recorded
at two different time intervals of 0.5 h and 4 h, respectively.
Neurotoxicity of the synthesized compounds was evaluated by rotarod
method. Results of anticonvulsant screenings along with neurotoxicity
studies were summarized in Table 2.
The completion of reactions and purity of synthesized compounds
was confirmed by TLC using ethyl acetate: hexane (4:6) as solvent
system and melting points were recorded. The structures were char-
acterized on the basis of elemental and spectral data analyses. The SP-
5F recorded IR spectra in the range of 1640 and 3410 cm−1 which
shows the appearance of C]O and NeH absorption bands respectively.
A broad singlet proton signal (-NHD2O exchangeable) was shown by
H1-NMR spectra at δ 9.32 ppm. SP-5F having methylene proton dis-
played a characteristic singlet signal at δ 3.92 ppm and the triplet peaks
at δ 3.62& 2.42 ppm confirmed the pyridazinone proton. Compound SP-
In the preliminary MES screening, all the compounds showed some
degree of protection which is clear indicative that these pyridazinone
analogues have potential to control seizure effective. Among the eigh-
teen compounds tested in MES, compound SP-5Fhaving p-chlorophenyl
2

A.A. Siddiqui, et al.
BioorganicChemistry99(2020)103584
Scheme 1. Synthesis route to the synthesized title compound SP (1a-18a) to SP (1F- 18F).
substitution prevented the hind limb tonic extension (HLTE) after 0.5
and 4 h at the lower dose of 30 mg/kg against the 50 mA electroshock.
Thus, compound SP-5F (LogP 2.03) emerged as the most effective an-
ticonvulsant having rapid onset and longer duration of action. At the
same dose of 30 mg/kg, compound SP-17F (LogP 2.31) having p-bro-
mophenyl substitution also provided protection against HLTE with
rapid onset and short duration of action however, at higher dose of
100 mg/kg showed protection at longer duration. Compound SP-12F
(LogP 2.59) having dicholorophenyl group showed protection at a dose
of 100 mg/kg after 0.5 and 4 h. Rest of the compounds showed pro-
tection only at highest dose (> 300 mg/kg).
The compounds which were found active during MES test were
further screened for scPTZ test. The ScPTZ is a confirmatory screening
test for the compounds to be active against the absence seizure. In
Table 1
Physicochemical parameters of the title compounds (SP-1F to SP-18F).
Compound
R
(SP-1F to SP-18F)
Mol. Formula
C15H14N4O2S
Mol. Weight
M.P. (°C)
Rfa
% Yield
Log Pb
SP-1F
Phenyl
314.36
332.35
328.39
344.39
348.81
342.42
342.42
342.42
342.42
370.47
364.42
383.25
404.48
406.46
356.44
390.46
393.26
320.39
189–191
238–242
270–272
218–220
262–264
298–300
258–260
248–250
254–256
290–292
268–270
178–180
201–203
229–231
287–289
244–246
169–171
195–197
0.48
0.64
0.76
0.55
0.61
0.42
0.68
0.51
0.48
0.72
0.57
0.62
0.48
0.71
0.58
0.76
0.68
0.56
46
57
73
38
68
60
44
59
58
47
63
70
40
56
38
32
61
49
1.48
1.63
1.96
1.35
2.03
2.45
2.45
2.45
2.45
3.13
2.47
2.59
3.57
3.01
2.80
3.15
2.31
1.46
SP-2F
p-Fluro-phenyl
p-Tolyl
C
15H13N4O2S
C16H16N4O2S
16H16N4O3S
ClN4O2S
17H18N4O2S
17H18N4O2S
SP-3F
SP-4F
p-Anisyl
C
SP-5F
p-Chloro-phenyl
p-Ethyl phenyl
3,4-Dimethylphenyl
2,5-Dimethylphenyl
2,4-Dimethylphenyl
p-Isobutyl phenyl
Naphthyl
C15 13
H
SP-6F
C
C
SP-7F
SP-8F
C17H18N4O2S
SP-9F
C
17H18N4O2S
C19H22N4O2S
19H16N4O2S
Cl2N4O2S
22H20N4O2S
21H18N4O3S
SP-10F
SP-11F
SP-12F
SP-13F
SP-14F
SP-15F
SP-16F
SP-17F
SP-18F
C
3,4-Dichloro-phenyl
p-Benzyl phenyl
p-Phenoxy phenyl
p- Propyl phenyl
p-Biphenyl
C15 12
H
C
C
C18H20N4O2S
C
C
C
21H18N4O2S
p-Bromo-phenyl
2-Thienyl
15H13 BrN4O2S
13H12N4O2S2
a
b
Solvent system- Ethyl acetate: Hexane (4:6).
Log P was determined by octanol: phosphate buffer method.
3

A.A. Siddiqui, et al.
BioorganicChemistry99(2020)103584
Table 2
Table 4
Anticonvulsant activity and minimal motor impairment of the synthesized
compounds (SP-1F to SP-18F).
Effect of most potent compound in GABA system.
Compound
GABA level in mice brain (μg/100 mg of tissue) 2 h post treatment
7 days post treatment
Compound
Intraperitoneal injection in mice
SP-5F
72.4
45.5
101
6.56*
5.03
90.1
51.5
110
4.23*
2.60
Control
Clobazam
MES screen scPTZ screen
Neurotoxicity screen
6.77
4.83
0.5 h
4 h
0.5 h
4 h
0.5 h
4 h
a) The compounds were tested at a dose of 100 mg/kg (i.p.) and clobazam
(30 mg/ kg).
SP-1F
300
100
300
300
30
–
–
–
X
–
X
–
SP-2F
300
–
100
300
300
100
300
300
X
300
–
b) The data indicate the minimum concentration whereby at least 50% in-
hibition was demonstrated in one or more time points.
c) Each value represents the mean (SEM of six rats, significantly different from
the control at.
SP-3F
X
X
–
X
X
–
SP-4F
–
–
SP-5F
30
300
X
100
X
SP-6F
100
300
300
300
300
100
100
–
–
–
* p < 0.005 (Student’s t-test). Animal used – adult Wistar rats divided into
three groups of six animals each.
SP-7F
X
–
–
SP-8F
–
X
X
X
X
–
X
X
X
–
SP-9F
300
300
300
100
–
X
X
SP-10F
SP-11F
SP-12F
SP-13F
SP-14F
SP-15F
SP-16F
SP-17F
SP-18F
Phenytoina
Ethosuximide
300
100
100
–
X
analogue, whole brain GABA estimation of rats was carried out.
Compound SP-5F, significantly increased the GABA level after both i.p.
administration after 2 h and with an oral treatment for 7 days. After
seven days of chronic oral administration, there was 1.74 folds increase
in GABA level in rat brain as compared to control. Thus, the pyr-
idazinone analogues might be acting through GABA modulatory me-
chanism (Table 4, Fig. 3).
300
300
–
–
–
X
X
–
X
X
–
300
100
–
X
X
X
300
–
100
–
300
–
X
–
X
–
30
100
–
100
–
–
–
–
X
100
–
X
100
–
It is well known that almost all AEDs like Carbamazepine and val-
poric acid are associated with hepatotoxicity [17]. Since most ther-
apeutic agents limit the hepatotoxic reactions, the liver function test for
all the compounds was performed. Result of the same has been shown
in Table 5. The most active compound SP-5F was administered
chronically to animals for 15 days and their biochemical parameters
(SGOT, SGPT, alkaline phosphatase, total albumin and total protein)
were evaluated. Compound SP-5F did not show any significant increase
or decrease in the said parameters as shown in Table 4. By using he-
matoxylin and eosin staining technique, the histopathological evalua-
tion of liver tissue of the groups which were treated with compound SP-
5F showed histological features which were normal (Fig. 2) (see
Table 6).
30
30
–
–
a
100
300
The Number of animals used = 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 where by bioactivity was demonstrated in half or more of
the mice. The animals were examined at 0.5 h 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.
scPTZ model only compound SP-5F showed protection at both time
intervals at a dose level of 100 mg/kg, showing active nature of com-
pound against absence seizure. Rest of the compounds was found to be
active at a higher dose level of 300 mg/kg after 4 h time interval. Thus,
optimal lipophilicity for anticonvulsant activity of pyridazinone ana-
logues is around 2.03.
CNS bioavailability of a drug or molecule is greatly affected by
various physicochemical descriptors such as LogP (partition coeffi-
cient), molecular weight (MW), hydrogen bond acceptors and donor
counts. By using octanol-phosphate buffer method, the partition coef-
ficient of all the compounds was determined experimentally (Table 1).
The compounds were found to be lipophilic enough (> 2) with po-
tential to cross the blood brain barrier which proves their antic-
onvulsant action potential. Due to high lipophilicity, rapid onset and
shorter duration of action of the said compound may be reasoned. The
log P values were found to be in optimal range of 1.38–3.56(< 5),
otherwise it may lead to CNS toxicities like motor impairement etc. the
topological polar surface area (TPSA) was calculated using Mol in-
spiration and the results have been presented in Table 5. The com-
pounds showed %ABS from 80.24 to 83.42%. Number of rotatable
bonds for the receptor binding is also crucial for conformational flex-
ibilities. The number of rotatable bond should be < 10 for passing the
oral bioavailability criteria. All the compounds in general possessed
None of the tested compounds showed any sign of neurological
deficit at the highest dose of 300 mg/kg.
Results of preliminary phase I anticonvulsant screening prompted us
to further assess the most promising compound in phase II screening.
The selected compound SP-5Fwas found to have significant antic-
onvulsant potential during Phase II study. As compared to standard
drugs phenytoin and ethosuximide, compounds SP-5F demonstrated
effective median dose (ED50) 24.38 and 88.23 and higher protective
index (PI) of 27.30 and 7.54 (Table 3) in MES and scPTZ screening
respectively. The p-chlorophenyl substitution at pyridazinone was ob-
served essential for better anticonvulsant activity.
To evaluate the GABA modulatory mechanism of pyridazinone
Table 3
Phase II quantitative anticonvulsant evaluation in mice.
PIc MES
scPTZ
a
b
Comp.
ED50 MES
scPTZ
TD50
SP-5F
24.38 (20.5–27.5)
9.5 (8.1–10.4)
> 1000
88.23 (80.7–95.4)
665.7 (532.6–740.5)
65.5 (52.5–72.9)
440.8
27.30
6.9
7.54
d
Phenytoin
> 300
130
< 0.22
3.39
Ethosuximided
< 0.44
Number of animals used = 10; Solvent used: polyethylene glycol (0.1 mL, i.p.).
a
b
c
d
Median effective dose eliciting anticonvulsant protection in 50% animals.
Median toxic dose eliciting minimal neurological toxicity in 50% animals.
PI = Protective index (TD50/ED50).
Data taken from Ref. [4] and dose in mg/kg.
4

A.A. Siddiqui, et al.
BioorganicChemistry99(2020)103584
Fig. 2. High power photomicrograph of portal triad area of liver tissue from animals treated with (a) control (b) compound SP-5Fshowing a normal histological
appearance (HE × 400). PT, portal triad; CV, central vein.
high number of rotatable bonds (3–5) and therefore provided fair
conformational flexibility. No compounds violated Lipinski parameters
(rule of 5), which makes them potential anticonvulsant agent.
2.3. Structure activity relationship
The results of anticonvulsant studies of the synthesized compounds
(Table 1) indicated that electron withdrawing groups like (Cl, Br and F)
on the phenyl attached at the 6th position of pyridazinone ring resulted
into higher seizure protection. Electron donating group such as methyl,
methoxy and dimethyl substituted phenyl did not show promising an-
ticonvulsant activity. However, compounds with disubstituted methyl
retains the anticonvulsant activity but at higher dose. Among the ali-
phatic chain substitution on phenyl ring, n-propyl was found to be more
active than the isobutyl substitution. An introduction of groups which
increase the length of molecules (biphenyl, phenoxyphenyl and benzyl
phenyl), unsubstituted phenyl and heteroaromatic ring (thienyl) does
not showed remarkable anticonvulsant activity except compound with
Fig. 3. Effect of compound SP-5F on GABA levels in mice brain tissues (µg/
napthyl ring. In general, following interesting pattern of anticonvulsant
100 mg wet tissue).
potencies of the phenyl ring substituents of the thiazole amide-pyr-
idazinone derivatives were observed
anticonvulsant action against both (MES and scPTZ) animal screen
models. Docking pose (Figs. 4 and 5) of compound SP-5F with a
homology model of the Na+ channel displayed that SP-5F bound to the
receptor in a pocket formed of Asp7, Ala7C, Thr68D, Ser69D, Phe80,
Phe84, Val87, Leu88, Phe91, Ser83, Thr87, Phe84 and Lys7D. Among
these amino acid residues, SP-5F formed two hydrogen bonds, one
hydrogen bond formed between the amino group of thiazole with Asp7
and the other between the keto groups of pyridazinone with Lys7D. The
docking and gliding scores were found to be −5.291 and −4.160
within docking interaction of SP-5F with a homology model of the
voltage-gated sodium channel receptor. The docking study of com-
pound SP-5F with GABAA receptor was also carried out and docking
pose (Figs. 6 and 7) showed that SP-5F was surrounded by GABAA re-
ceptors. Binding interaction of SP-5F with GABAA showed that it was
p-chlorophenyl > p-bromophenyl > 3,
4-dichloro-phenyl >
phenyl > p-ethylben-
phenyl > 2,4-Dimethyl-
Naphthyl > p-fluorophenyl > p-
Propyl
zene > 3,4-Dimethylphenyl > p-Isobutyl
phenyl > p-tolyl > p-Anisyl > 2,5-Dimethylphenyl > Phenyl > p-
Benzyl phenyl > thienyl > p-Biphenyl > p-Phenoxy phenyl.
2.4. Molecular docking studies
In this work, molecular docking computations were performed using
Maestro 10.5 program (Schrodinger 2013, Inc., USA) to unleash the
possible mechanism of anticonvulsant activity. The docking was per-
formed on two main targets of commonly used anticonvulsant drugs:
one is Na+ channel and other is GABAA receptors. For this purpose,
compound SP-5F was selected because it displayed most potent
Table 5
Liver enzyme and total protein estimation of the most potent compound SP-5F.
Comp.a
SGOT
SEM
SGPT
SEM
Alkaline phosphatase
SEM
Total albumin(g/100 mL)
SEM
Total protein (g/100 mL)
SEM
SP-5F
40.12
37.18
1.90
1.71
37.58
34.23
1.85
1.27
58.13
56.11
2.08
2.01
1.63
1.56
0.024
0.021
5.28
5.23
0.24*
0.21
controlb
a
Relative to control and data were analyzed by ANOVA followed by Student’s t test for n = 4 at.
* p < 0.05.
b
Control group were treated with 0.5% methyl cellulose for 15 days.
5

A.A. Siddiqui, et al.
BioorganicChemistry99(2020)103584
Table 6
Important pharmacokinetics parameters for good oral bioavailability of title compounds (SP-1F to SP-18F).
Comp.
R
% ABS
TPSA (A2)
n-rotb
Mol.Wt.
Molar Vol.
mi LogP
n-OHNH donor
n- ON acceptor
Lipinski’s violation
Rule
< 500
316.39
334.38
330.41
346.41
350.83
344.44
344.44
344.44
344.44
386.52
366.45
385.28
406.51
408.48
358.47
392.48
395.28
322.42
< 5
1.48
1.64
1.93
1.53
2.15
2.39
2.30
2.30
2.30
3.56
2.64
2.76
3.44
3.23
2.78
3.27
2.29
1.38
< 5
1
< 10
< 1
0
SP-1F
SP-2F
SP-3F
SP-4F
SP-5F
SP-6F
SP-7F
SP-8F
SP-9F
SP-10F
SP-11F
SP-12F
SP-13F
SP-14F
SP-15F
SP-16F
SP-17F
SP-18F
Phenyl
83.42
83.42
83.42
80.24
83.42
83.42
83.42
83.42
83.42
83.42
83.42
83.42
83.42
80.24
83.42
83.42
83.42
83.42
74.14
74.14
74.14
83.37
74.14
74.14
74.14
74.14
74.14
74.14
74.14
74.14
74.14
83.37
74.14
74.14
74.14
74.14
4
4
4
5
4
5
4
4
4
7
4
4
6
6
6
5
4
4
274.17
279.10
290.73
299.72
287.71
307.53
307.29
307.29
307.29
357.73
318.16
301.24
362.38
354.56
324.34
345.58
292.06
264.88
6
6
6
7
6
6
6
6
6
6
6
6
6
7
6
6
6
6
p-Fluro-phenyl
p-Tolyl
1
0
1
0
p-Anisyl
1
0
p-Chloro-phenyl
p-Ethylphenyl
3,4-Dimethylphenyl
2,5-Dimethylphenyl
2,4-Dimethylphenyl
p- Isobutyl phenyl
β- Nephthyl
1
0
1
0
1
0
1
0
1
0
1
0
1
0
3,4-Dichloro-phenyl
p- Benzyl phenyl
p- Phenoxy phenyl
p-Propyl phenyl
p-Biphenyl
1
0
1
0
1
0
1
0
1
0
p-Bromo-phenyl
2-Thienyl
1
0
1
0
% 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.
Fig. 4. Binding mode of compound SP-5F
into voltage-gated sodium channel receptor
pocket (homology model). Hydrogen bonds
are shown with yellow dotted lines. One
hydrogen bond is between NH of thiazole
and aspartate7 shown in brown color and
other is between keto groups of pyr-
idazinone and lysine 7D shown in purple
color.
bound to a site formed of Val202, His101, Tyr159, Tyr209, Val211,
Arg132, Lue131, Phe77, Met130, Leu140, Thr142, Ser204, Glu189,
Thr206, Thr141, Phe99, Gly157, Ser158, Val210 and Ser204. Hydrogen
bonds are shown with yellow dotted lines and pi-pi interaction by blue
dotted lines. The compounds (SP-5F) showed four hydrogen bonds;
among them two of the hydrogen bond is with common amino acid Thr
206 and the C]O group of the pyridazinone and the aliphatic amide
chain. The NH of the amide group showed key interaction with Tyr 159.
This underscores the importance of C]O and NH group present in the
compound.
our compounds better act by GABAA receptors rather than Na+ channel
blocker.
2.5. Pharmacophore distance mapping
The distance between various groups [one aryl (R) hydrophobic
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.
Fig. 8 shows the distances between various groups of standard drugs
postulated essential for the anticonvulsant. The results of pharmaco-
phore distance mapping of six standard antiepileptic drugs were then
compared with most active compound (SP-5F) are shown in Table 7. It
was found that the distance between various groups essential for an-
ticonvulsant action was in good agreement with the clinically available
AEDs.
The docking and gliding scores of compound (SP-5F) with GABAA
docking interaction were found to be −7.357 and −6.714. Lastly, the
result of docking interaction of the compound SP-5F 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 in-
teraction with Na+ channel receptor gave less hydrogen bonding, less
docking as well as less gliding score. Hence, it may be concluded that
6

A.A. Siddiqui, et al.
BioorganicChemistry99(2020)103584
Fig. 5. Lig plot of compound SP-5F showing interaction into the binding site of Na+ channel receptor (homology model).
3. Conclusion
were synthesized as anticonvulsants. Among all eighteen synthesized
compounds, 2-(3-(4-chlorophenyl)-6-oxo-5,6-dihydropyridazin-1(4H)-
yl)-N-(thiazol-2-yl)acetamide (SP-5F) emerged as the most potent and
safer anticonvulsant agent acting through GABA mediated mechanism.
In continuation of our previous studies on pyridazinone analogues
as anticonvulsant, a series of hybrid thiazole-pyridazinone analogues
Fig. 6. Binding mode of compound SP-5F
into the GABAA receptor pocket (homology
model). Hydrogen bonds are shown with
yellow dotted lines and pi-pi interaction by
blue dotted lines. The compounds showed
four hydrogen bonds; among them two of
the hydrogen bond is with common amino
acid Thr 206 and the C]O group of the
pyridazinoneand the aliphaticamide chain.
The NH of the amide group showed key in-
teraction with Tyr 159. This underscores the
importance of C]O and NH group present
in the compound.
7

A.A. Siddiqui, et al.
BioorganicChemistry99(2020)103584
Fig. 7. Lig plot of compound SP-5F showing interaction into the GABAA receptor pocket (homology model).
In silico pharmacophoric pattern and drug likeness studies showed that
all the compounds fulfill the requirements which are crucial to be a
potent anticonvulsant drug. Further, molecular docking studies on the
validated target protein substantiate the binding pattern of the ligand as
hypothesized by the pharmacophore pattern. Thus, hybrid thiazole-
pyridazinones may lead to the development of ideal scaffolds for an-
ticonvulsant agents acting through GABA mediated mechanism.
Table 7
Distance range between the essential structural elements R, D and HBD.
Compound
R-HBDa
R-Da
D-HBDa
Gabapentin
Phenytoin
Carbamazepine
Remacemide
Progabide
SP-5F
4.02
4.97
6.51
5.06
6.65
6.38
4.14
5.22
4.61
6.74
4.49
6.03
4.45
4.63
3.44
4.37
5.12
3.95
4. Experimental
a
Distances calculated for 3D optimized structures using MM3 and CHARMM
parameterization (Argus Lab 4.0 and ACD/3D viewer).
4.1. General
Hicon melting point apparatus (Hicon, New Delhi, India) and are un-
corrected. Purity of the compounds was checked by thin-layer chro-
matography (TLC) plates (silica gel G) by using ethylacetate: Hexane
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
Fig. 8. Distance range between the essential structural elements R, D and HBD.
8

A.A. Siddiqui, et al.
BioorganicChemistry99(2020)103584
(4:6), which were visualized by exposing to iodine vapours and UV
light. The FT-IR spectra were recorded on (IR affinity SHIMADZU) FTIR
spectrophotometer using KBrpellets; υmax values are given in cm−1 and
1H NMR spectra were recorded on Bruker model DRX-300 and 400 MHz
NMR spectrometer (1H at 300 and 400 MHz, 13C at 100 MHz) in
DMSO‑d6. Chemical shifts (δ) are expressed in ppm relative to tetra-
methylsilane (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 0.4% of the theoretical values.
N, 16.89.
4.2.5. 2-(3-(4-methoxyphenyl)-6-oxo-5,6-dihydropyridazin-1(4H)-yl)-N-
(thiazol-2-yl)acetamide(SP-4F)
Yield: 38%; mp 218–220 °C; Rf = 0.55; IR (KBr) (cm−1): 3446 (NeH
str), 1697 (C]O str), 1644 (C]N str); 1H NMR (400 MHz, DMSO‑d6)
δ(ppm): 9.47 (s, 1H, NH), 7.92–7.72 (m, 4H, phenyl), 7.53 (d, J = 7.4,
H4-thiazole), 7.29 (d, J = 4.1, H3-thiazole), 3.84 (s, 3H, eOCH3), 3.56
(s, 2H, eCH2), 2.92 (t, J = 7.8, 2H, CeCH2), 2.54 (t, J = 7.8, 2H, CH2-
CO Pyridazinone). 13C NMR (100 MHz, DMSO‑d6) δ (ppm): 168.37,
162.96, 162.54, 162.01, 146.27, 132.81, 128.76, 114.98, 112.08,
55.81, 54.28, 32.87 and 24.65. ESI MS (m/z): 345[M+1]; Anal. Calcd.
for C16H16N4O3S: C, 55.80; H, 4.68; N, 16.27; Found: C, 55.82; H, 4.70;
N, 16.29.
4.2. General procedure for the synthesis of 6-substituted-phenyl- 4, 5-
dihydropyridazin-3(2H)-one (SP-1b to SP-18b)
4.2.6. 2-(3-(4-chlorophenyl)-6-oxo-5,6-dihydropyridazin-1(4H)-yl)-N-
(thiazol-2-yl)acetamide(SP-5F)
The appropriate substituted β-aroyl propionic acids were reacted
with hydrazine hydrate to get corresponding pyridazinone and char-
acterized on the basis of spectral data as per earlier reported procedure
[16].
Yield: 68%; mp 262–264 °C; Rf = 0.61; IR (KBr) (cm−1): 3410 (NeH
str), 1640 (C]O str), 1617 (C]N str); 1H NMR (400 MHz, DMSO‑d6)
δ(ppm): 9.32 (s, 1H, NH), 7.80–8.12 (m, 4H, phenyl), 7.41 (d, J = 7.8,
H4-thiazole), 7.15 (d, J = 4.5, H3-thiazole), 3.92 (s, 2H, eCH2), 3.62 (t,
J = 7.8, 2H, CeCH2), 2.42 (t, J = 7.8, 2H, CH2-CO Pyridazinone). 13C
NMR (100 MHz, DMSO‑d6) δ (ppm): 168.29, 165.16, 162.90, 162.44,
146.36, 136.67, 132.46, 128.83, 128.64, 112.28, 56.25, 54.12, 32.12
and 24.28. ESI MS (m/z): [M+1], 350[M+2]; Anal. Calcd. for
4.2.1. General procedure for the synthesis of 2-(6-oxo-3-substitutedaryl-
5,6-dihydropyridazin-1(4H)-yl)-N-(thiazol-2-yl)acetamide(SP-1F to SP-
18F)
To a mixture of 6-substituted-phenyl- 4, 5-dihydropyridazin-3(2H)-
one (0.01 mol) (SP-2b to SP-18b) and 2-chloro-N-(thiazol-2-yl) acet-
amide (0.02 mol) in dry DMF (10 mL) was added potassium carbonate.
The reaction mixture was reflux for 5 h until TLC showed the single
spot. It was cooled and poured into crushed ice. The solid obtained was
filtered and recrystallized with methanol.
C
15H13ClN4O2S: C, 51.65; H, 3.76; N, 16.06; Found: C, 51.68; H, 3.77;
N, 16.08.
4.2.7. 2-(3-(4-ethylphenyl)-6-oxo-5,6-dihydropyridazin-1(4H)-yl)-N-
(thiazol-2-yl)acetamide(SP-6F)
Yield: 60%; mp 298–300 °C; Rf = 0.42; IR (KBr) (cm−1): 3360 (NeH
str), 1685 (C]O str), 1635 (C]N str); 1H NMR (400 MHz, DMSO‑d6)
δ(ppm): 9.46 (s, 1H, NH), 7.72–8.06 (m, 4H, phenyl), 7.41 (d, J = 7.6,
H4-thiazole), 7.23 (d, J = 4.6, H3-thiazole), 3.87 (s, 2H, eCH2), 2.96 (t,
J = 7.8, 2H, CeCH2), 2.52 (q, 2H, eCH2CH3), 2.52 (t, J = 7.8, 2H, CH2-
CO Pyridazinone), 1.23 (t, 3H, eCH3). 13C NMR (100 MHz, DMSO‑d6) δ
(ppm): 167.98, 165.46, 162.79, 147.08, 146.35, 133.48, 132.24,
127.99, 127.24, 112.46, 54.78, 32.42, 30.15, 24.12 and 16.52. ESI MS
(m/z): 343[M+1]; Anal. Calcd. for C17H18N4O2S: C, 59.63; H, 5.30; N,
16.36; Found: C, 59.66; H, 5.32; N, 16.38.
4.2.2. 2-(6-oxo-3phenyl-5, 6-dihydropyridazin-1(4H)-yl)-N-(thiazol-2-yl)
acetamide (SP-1F)
Yield: 46%; mp 189–191 °C; Rf = 0.48; IR (KBr) (cm−1): 3350 (NeH
str), 1695 (C]O str), 1562 (C]N str); 1H NMR (400 MHz, DMSO‑d6)
δ(ppm): 9.37 (s, 1H, NH), 7.82–7.67(m, 5H, phenyl), 7.53 (d, J = 7.8,
H4-thiazole), 7.29 (d, J = 4.3, H3-thiazole), 3.89 (s, 2H, eCH2), 2.98 (t,
J = 7.8, 2H, CeCH2), 2.53 (t, J = 7.8, 2H, CH2-CO Pyridazinone). 13C
NMR (100 MHz, DMSO‑d6) δ (ppm): 168.40, 162.12, 159.50, 145.87,
136.25, 132.73, 130.94, 128.30, 126.59, 112.37, 54.80, 32.48 and
24.64. Mass (m/z): 315[M+1]; Anal. Calcd. for C15H14N4O2S: C, 57.31;
H, 4.49; N, 17.82; Found: C, 57.34; H, 4.51; N, 17.85.
4.2.8. 2-(3-(3,4-dimethylphenyl)-6-oxo-5,6-dihydropyridazin-1(4H)-yl)-
N-(thiazol-2-yl)acetamide(SP-7F)
4.2.3. 2-(3-(4-fluorophenyl)-6-oxo-5,6-dihydropyridazin-1(4H)-yl)-N-
(thiazol-2-yl)acetamide (SP-2F)
Yield: 44%; mp 258–260 °C; Rf = 0.68; IR (KBr) (cm−1): 3450 (NeH
str), 1710 (C]O str), 1599 (C]N str); 1H NMR (400 MHz, DMSO‑d6)
δ(ppm): 9.26 (s, 1H, NH), 7.54–7.78 (m, 3H, phenyl), 7.45 (d, J = 7.8,
H4-thiazole), 7.30 (d, J = 4.4, H3-thiazole), 3.58 (s, 2H, eCH2), 2.96 (t,
J = 7.8, 2H, CeCH2), 2.46 (t, J = 7.8, 2H, CH2-CO Pyridazinone), 2.32
(s, 6H, CH3). 13C NMR (100 MHz, DMSO‑d6) δ (ppm): 168.44, 162.88,
162.08, 146.50, 139.76, 136.57, 132.61, 132.29, 130.82, 129.15,
124.82, 112.25, 54.79, 34.23, 32.46, 25.03. ESI MS (m/z): 343[M+1];
Anal. Calcd. for C17H18N4O2S: C, 59.63; H, 5.30; N, 17.82; Found: C,
57.35; H, 4.52; N, 17.85.
Yield: 57%; mp 238–242 °C; Rf = 0.64; IR (KBr) (cm−1): 3431 (NeH
str), 1705 (C]O str), 1615 (C]N str); 1H NMR (400 MHz, DMSO‑d6)
δ(ppm): 9.36 (s, 1H, NH), 7.98–7.64 (m, 4H, phenyl), 7.52 (d, J = 7.6,
H4-thiazole), 7.38 (d, J = 4.2, H3-thiazole), 3.87 (s, 2H, eCH2), 2.96 (t,
J = 7.8, 2H, CeCH2), 2.54 (t, J = 7.8, 2H, CH2-CO Pyridazinone). 13C
NMR (100 MHz, DMSO‑d6) δ (ppm): 168.21, 162.10, 165.32, 159.76,
146.23, 132.49, 131.92, 129.32, 115.85, 112.78, 54.67, 32.56 and
24.79. ESI MS (m/z): 333 [M+1]; Anal. Calcd. for C15H13 FN4O2S: C,
54.21; H, 3.94; N, 16.86; Found: C, 54.24; H, 3.97; N, 16.89;
4.2.9. 2-(3-(2,5-dimethylphenyl)-6-oxo-5,6-dihydropyridazin-1(4H)-yl)-
N-(thiazol-2-yl)acetamide(SP-8F)
4.2.4. 2-(6-oxo-3-(p-tolyl)-5,6-dihydropyridazin-1(4H)-yl)-N-(thiazol-2-
yl)acetamide (SP-3F)
Yield: 59%; mp 248–250 °C; Rf = 0.51; IR (KBr) (cm−1): 3360 (NeH
str), 1690 (C]O str), 1609 (C]N str); 1H NMR (400 MHz, DMSO‑d6)
δ(ppm): 9.41 (s, 1H, NH), 7.71–7.54 (m, 3H, phenyl), 7.41 (d, J = 7.6,
H4-thiazole), 7.33 (d, J = 4.6, H3-thiazole), 3.74 (s, 2H, eCH2), 2.94 (t,
J = 7.8, 2H, CeCH2), 2.53 (t, J = 7.8, 2H, CH2-CO Pyridazinone), 2.46
(m, 6H, CH3). 13C NMR (100 MHz, DMSO‑d6) δ (ppm): 168.51, 162.98,
162.17, 146.94, 135.63, 132.97, 132.01, 131.19, 130.37, 129.52,
128.80, 112.75, 54.62, 32.19, 24.67, 21.20, and 19.53. ESI MS (m/z):
343[M+1]; Anal. Calcd. for C17H18N4O2S: C, 59.63; H, 5.30; N, 17.82;
Found: C, 57.35; H, 4.52; N, 17.85.
Yield: 76%; mp 270–272 °C; Rf = 0.73; IR (KBr) (cm−1): 3346 (NeH
str), 1690 (C]O str), 1612 (C]N str); 1H NMR (400 MHz, DMSO‑d6)
δ(ppm): 9.33 (s, 1H, NH), 8.59–8.35 (m, 4H, phenyl), 7.54 (d, J = 8.8,
H4-thiazole), 7.04 (d, J = 4.8, H3-thiazole), 4.67 (s, 2H, eCH2), 2.80 (t,
J = 8.0, 2H, CeCH2), 2.45 (t, J = 8.0, 2H, CH2-CO Pyridazinone), 2.32
(s, 3H, CH3). 13C NMR (100 MHz, DMSO‑d6) δ (ppm): 167.96, 162.71,
162.42, 146.59, 140.86, 133.20, 132.09, 129.62, 127.56, 112.76,
54.82, 32.66, 24.10, and 21.89. ESI MS (m/z): 329[M+1]; Anal. Calcd.
for C16H16N4O2S: C, 58.52; H, 3.94; N, 16.86; Found: C, 58.54; H, 3.97;
9

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BioorganicChemistry99(2020)103584
4.2.10. 2-(3-(2,4-dimethylphenyl)-6-oxo-5,6-dihydropyridazin-1(4H)-yl)-
N-(thiazol-2-yl)acetamide(SP-9F)
4.2.15. 2-(6-oxo 3-(4-phenoxyphenyl)-5,6-dihydropyridazin-1(4H)-yl)-N-
(thiazol-2-yl)acetamide (SP-14F)
Yield: 58%; mp 254–256 °C; Rf = 0.48; IR (KBr) (cm−1): 3420 (NeH
str), 1710 (C]O str), 1660 (C]N str); 1H NMR (400 MHz, DMSO‑d6)
δ(ppm): 9.51 (s, 1H, NH), 7.70–7.59 (m, 3H, phenyl), 7.48 (d, J = 7.2,
H4-thiazole), 7.28 (d, J = 4.1, H3-thiazole), 3.92 (s, 2H, eCH2), 2.96 (t,
J = 7.8 Hz, 2H, CeCH2), 2.43 (t, J = 7.8, 2H, CH2-CO Pyridazinone),
2.51 (m, 6H, CH3). 13C NMR (100 MHz, DMSO‑d6) δ (ppm): 167.93,
162.87, 162.04, 146.34, 138.61, 136.90, 131.09, 132.45, 129.18,
126.88, 126.03, 112.44, 54.63, 32.57, 31.26, 25.72 and 24.38. ESI MS
(m/z): 343[M+1]; Anal. Calcd. for C17H18N4O2S: C, 59.63; H, 5.30; N,
17.82; Found: C, 57.35; H, 4.52; N, 17.85.
Yield: 56%; mp 229–231 °C; Rf = 0.71; IR (KBr) (cm−1): 3487 (NeH
str), 1720 (C]O str), 1585 (C]N str); 1H NMR (400 MHz, DMSO‑d6)
δ(ppm): 9.34 (s, 1H, NH), 7.89–7.64 (m, 9H, phenyl), 7.52 (d, J = 7.2,
H4-thiazole), 7.27 (d, J = 4.6, H3-thiazole), 3.76 (s, 2H, eCH2), 2.91 (t,
J = 7.8, 2H, CeCH2), 2.44 (t, J = 7.8, 2H, CH2-CO Pyridazinone). 13C
NMR (100 MHz, DMSO‑d6) δ (ppm): 168.21, 162.91, 159.58, 157.43,
145.38, 136.12, 132.47, 130.59, 128.33, 126.59, 121.11, 118.23,
117.52, 112.53, 54.03, 32.84 and 24.83. ESI MS (m/z): 407[M+1];
Anal. Calcd. for C21H18N4O3S: C, 62.05; H, 4.46; N, 13.78; Found: C,
62.07; H, 4.48; N, 13.79.
4.2.11. 2-(3-(4-isobutylphenyl)-6-oxo-5,6-dihydropyridazin-1(4H)-yl)-N-
(thiazol-2-yl)acetamide(SP-10F)
4.2.16. 2-(6-oxo 3-(4-propylphenyl)-5,6-dihydropyridazin-1(4H)-yl)-N-
(thiazol-2-yl)acetamide (SP-15F)
Yield: 47%; mp 290–292 °C; Rf = 0.72; IR (KBr) (cm−1): 3345 (NeH
str), 1725 (C]O str), 1592 (C]N str); 1H NMR (400 MHz, DMSO‑d6)
δ(ppm): 9.43 (s, 1H, NH), 7.54–7.78 (m, 4H, phenyl), 7.41 (d, J = 7.4,
H4-thiazole), 7.25 (d, J = 4.8, H3-thiazole), 3.84 (s, 2H, eCH2), 2.98 (t,
J = 7.8, 2H, CeCH2), 2.43 (t, J = 7.8, 2H, CH2-CO Pyridazinone), 0.91
(d, 6H, (CH3)2, 1.63–1.71 (m, 1H, eCH), 1.98 (d, 2H, eCH2). 13C NMR
(100 MHz, DMSO‑d6) δ (ppm): 168.76, 162.80, 162.01, 146.38, 143.76,
133.59, 132.06, 128.46, 126.65, 112.50, 54.78, 44.61, 32.19, 29.48,
24.15 and 22.71. ESI MS (m/z): 371[M+1]; Anal. Calcd. for
Yield: 38%; mp 287–289 °C; Rf = 0.58; IR (KBr) (cm−1): 3325 (NeH
str), 1640 (C]O str), 1620 (C]N str); 1H NMR (400 MHz, DMSO‑d6)
δ(ppm): 9.29 (s, 1H, NH), 7.81–7.58 (m, 4H, phenyl), 7.43 (d, J = 7.2,
H4-thiazole), 7.31 (d, J = 4.6, H3-thiazole), 3.76 (s, 2H, eCH2), 2.90 (t,
J = 7.8, 2H, CeCH2), 2.60 (t, J = 3.6, 2H, eCH2CH2), 2.47 (t, J = 7.8,
2H, CH2-CO Pyridazinone), 1.62 (m, 2H, eCH2CH3), 1.10 (t, J = 3.2,
3H, eCH2CH3) . 13C NMR (100 MHz, DMSO‑d6) δ (ppm): 167.96,
162.71, 159.55, 145.78, 136.12, 132.57, 130.89, 128.63, 126.59,
112.43, 54.18, 37.40, 32.04, 24.64, 24.10, and 15.76. ESI MS (m/z):
357[M+1]; Anal. Calcd. for C18H20N4O2S: C, 60.65; H, 5.66; N, 15.72;
Found: C, 60.68; H, 5.69; N, 15.74.
C
19H22N4O2S: C, 61.60; H, 5.99; N, 15.12; Found: C, 61.62; H, 6.01; N,
15.15.
4.2.12. 2-(3-(naphthalen-1-yl)-6-oxo-5,6-dihydropyridazin-1(4H)-yl)-N-
(thiazol-2-yl)acetamide (SP-11F)
4.2.17. 2-(3-([1,1′-biphenyl]-4-yl)-6-oxo-5,6-dihydropyridazin-1(4H)-
yl)-N-(thiazol-2-yl)acetamide(SP-16F)
Yield: 63%; mp 268–270 °C; Rf = 0.57; IR (KBr) (cm−1): 3355 (NeH
str), 1700 (C]O str), 1602 (C]N str); 1H NMR (400 MHz, DMSO‑d6)
δ(ppm): 9.27 (s, 1H, NH), 7.96–7.75 (m, 7H, phenyl), 7.54 (d, J = 7.6,
H4-thiazole), 7.35 (d, J = 4.6, H3-thiazole), 3.86 (s, 2H, eCH2), 2.96 (t,
J = 7.8, 2H, CeCH2), 2.43 (t, J = 7.8, 2H, CH2-CO Pyridazinone). 13C
NMR (100 MHz, DMSO‑d6) δ (ppm): 168.45, 162.97, 162.10, 146.67,
134.52, 133.18, 132.85, 130.27, 129.04, 127.25, 126.12, 125.39,
112.24, 54.68, 32.81 and 24.86. ESI MS (m/z): 365[M+1]; Anal. Calcd.
for C19H16N4O2S: C, 62.62; H, 4.49; N, 17.82; Found: C, 62.66; H, 4.51;
N, 17.86.
Yield: 32%; mp 244–246 °C; Rf = 0.76; IR (KBr) (cm−1): 3368 (NeH
str), 1699 (C]O str), 1578 (C]N str); 1H NMR (400 MHz, DMSO‑d6)
δ(ppm): 9.33 (s, 1H, NH), 7.96–7.54 (m, 9H, phenyl), 7.45 (d, J = 7.8,
H4-thiazole), 7.33 (d, J = 4.4, H3-thiazole), 3.80 (s, 2H, eCH2), 2.96 (t,
J = 7.8, 2H, CeCH2), 2.48 (t, J = 7.8, 2H, CH2-CO Pyridazinone). 13C
NMR (100 MHz, DMSO‑d6) δ (ppm): 168.04, 162.74, 159.63, 145.48,
143.59, 140.75, 136.52, 132.97, 130.09, 129.51, 128.73, 1127.49,
126.49, 112.33, 54.13, 32.74 and 24.53. ESI MS (m/z): 391[M+1];
Anal. Calcd. for C21H18N4O2S: C, 64.60; H, 4.65; N, 14.35; Found: C,
64.62; H, 4.67; N, 14.38.
4.2.13. 2-(3-(3,4-dichlorophenyl)-6-oxo-5,6-dihydropyridazin-1(4H)-yl)-
N-(thiazol-2-yl)acetamide (SP-12F)
4.2.18. 2-(3-(4-bromophenyl)-6-oxo-5,6-dihydropyridazin-1(4H)-yl)-N-
(thiazol-2-yl)acetamide (SP-17F)
Yield: 70%; mp 178–180 °C; Rf = 0.62; IR (KBr) (cm−1): 3360 (NeH
str), 1685 (C]O str), 1610 (C]N str); 1H NMR (400 MHz, DMSO‑d6)
δ(ppm): 9.48 (s, 1H, NH), 7.67–7.86 (m, 3H, phenyl), 7.50 (d, J = 7.8,
H4-thiazole), 7.34 (d, J = 4.2, H3-thiazole), 3.87 (s, 2H, eCH2), 2.94 (t,
J = 7.8, 2H, CeCH2), 2.48 (t, J = 7.8, 2H, CH2-CO Pyridazinone). 13C
NMR (100 MHz, DMSO‑d6) δ (ppm): 168.44, 162.95, 162.14, 146.58,
135.77, 133.95, 132.96, 130.49, 129.22, 126.82, 112.44, 54.92, 32.61
and 24.74. ESI MS (m/z): 383[M+1], 384[M+2]; Anal. Calcd. for
Yield: 61%; mp 169–171 °C; Rf = 0.68; IR (KBr) (cm−1): 3358 (NeH
str), 1685 (C]O str), 1598 (C]N str); 1H NMR (400 MHz, DMSO‑d6)
δ(ppm): 9.30 (s, 1H, NH), 7.87–7.55 (m, 4H, phenyl), 7.39 (d, J = 7.8,
H4-thiazole), 7.24 (d, J = 4.1, H3-thiazole), 3.78 (s, 2H, eCH2), 2.96 (t,
J = 7.8, 2H, CeCH2), 2.42 (t, J = 7.8, 2H, CH2-CO Pyridazinone). 13C
NMR (100 MHz, DMSO‑d6) δ (ppm): 168.35, 162.51, 159.68, 145.49,
136.21, 132.27, 130.03, 128.43, 126.02, 112.80, 54.41, 32.30 and
24.38. ESI MS (m/z): 393[M+1], 394[M+2]; Anal. Calcd. for C15H13
BrN4O2S: C, 45.81; H, 3.33; N, 14.25; Found: C, 45.83; H, 3.35; N,
14.27.
C
15H12Cl2N4O2S: C, 47.01; H, 3.16; N, 14.62; Found: C, 47.04; H, 3.18;
N, 14.65.
4.2.14. 2-(3-(4-benzylphenyl)-6-oxo-5,6-dihydropyridazin-1(4H)-yl)-N-
(thiazol-2-yl)acetamide (SP-13F)
4.2.19. 2-(6-oxo
3-(thiophen-2-yl)-5,6-dihydropyridazin-1(4H)-yl)-N-
Yield: 40%; mp 201–203 °C; Rf = 0.48; IR (KBr) (cm−1): 3450 (NeH
str), 1699 (C]O str), 1600 (C]N str); 1H NMR (400 MHz, DMSO‑d6)
δ(ppm): 9.51 (s, 1H, NH), 7.99–7.74 (m, 9H, phenyl), 7.46 (d, J = 7.1,
H4-thiazole), 7.39 (d, J = 7.6, H3-thiazole), 3.86 (s, 2H, eCH2), 3.42
(s, 2H, eCH2), 2.98 (t, J = 7.8, 2H, CeCH2), 2.53 (t, J = 7.8, 2H, CH2-
CO Pyridazinone). 13C NMR (100 MHz, DMSO‑d6) δ (ppm): 168.74,
162.79, 162.02, 146.65, 143.85, 141.25, 136.13, 133.78, 132.56,
129.44, 128.62, 127.17, 126.02, 112.42, 54.28, 41.34, 32.14 and
24.64. ESI MS (m/z): 405[M+1]; Anal. Calcd. for C22H20N4O2S: C,
65.33; H, 4.98; N, 13.85; Found: C, 65.35; H, 4.99; N, 13.87.
(thiazol-2-yl)acetamide (SP-18F)
Yield: 49%; mp 195–197 °C; Rf = 0.56; IR (KBr) (cm−1): 3350 (NeH
str), 1680 (C]O str), 1608 (C]N str); 1H NMR (400 MHz, DMSO‑d6)
δ(ppm): 9.49 (s, 1H, NH), 7.83–7.51 (m, 3H, thiazole), 7.41 (d, J = 7.2,
H4-thiazole), 7.21 (d, J = 4.8, H3-thiazole), 3.82 (s, 2H, eCH2), 2.94 (t,
J = 7.8, 2H, CeCH2), 2.48 (t, J = 7.8, 2H, CH2-CO Pyridazinone). 13C
NMR (100 MHz, DMSO‑d6) δ (ppm): 168.41, 162.18, 159.30, 145.71,
136.22, 132.49, 130.19, 128.50, 126.91, 112.52, 54.30, 32.26 and
24.88. ESI MS (m/z): 321[M+1]; Anal. Calcd. for C13H12N4O2S2: C,
48.73; H, 3.78; N, 14.25; Found: C, 48.75; H, 3.79; N, 14.27.
10

A.A. Siddiqui, et al.
BioorganicChemistry99(2020)103584
4.3. Pharmacology
4.8. Serum enzyme activity
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/2000CPCSEA.
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 protocols of Antiepileptic Drug Development
(ADD) program by NINDS, US using two widely used standard models
namely, maximal electroshock seizure (MES), subcutaneous pentyle-
netetrazole (scPTZ) test and minimal neurological toxicity was eval-
uated by rotarod test. The most potent compound was also subjected to
phase II quantitative determination of ED50, TD50, PI and probit cal-
culations were done by means of computer program Bio Stat 2009 using
Finney’s method. Hepatotoxicity studies of the compounds were also
carried out using procedures described elsewhere. Most active antic-
onvulsant compounds in phase I screening were also subjected to neuro
chemical estimation of GABAs levels in the mice whole brain.
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 anaesthetized 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 [24–26].
4.9. Gamma-aminobutyric acid estimation
The GABA estimation was performed in brain tissue extract spec-
trophotometrically 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 homo-
genization with 0.1 mmol/L phosphate buffer (pH 7.4). The homo-
genate (10% w/v) was centrifuged at 20000g for 30 min and the su-
pernatant so formed was applied on silica gel 60F254 aluminum sheets
(10 cm × 10 cm) as stationary phase, using mobile phase comprising
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 tem-
perature. The developed TLC plates were air dried and sprayed with
ninhydrin. Ten developed spots were scraped together, mixed with
3 mL distilled water and centrifuged. The supernatant obtained was
spectrophotometrically analyzed at 570 nm. The specificity of the
method was ascertained by taking standard GABA samples and con-
firmed by comparing their Rf values. The calibration curve was pre-
pared by plotting OD versus concentration [27,28].
4.4. Maximal electroshock (MES) test
MES test was performed as per the standard protocol [18,19]. 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 re-
corded 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 sec. Abolition of hind limb tonic ex-
tension spasm was recorded as the end point of test result.
5. Distance mapping
4.5. Subcutaneous pentylenetetrazole (scPTZ) test
The pharmacophore pattern studies, in which the distance between
various groups postulated as essential for anticonvulsant activity was
determined from the 3D optimized structures using ACD/3D viewer
version 12.01 and Argus Lab 4.0 Mark A. Thompson Planaria Software,
LLC. A molecular model was suggested, through conformational ana-
lysis of clinically effective, well-known and structurally different an-
ticonvulsant drugs such as a Carbamazepine, Gabapentin, Lamotrigine,
Phenytoin, Rufinamide, Diazepam, and Zonisamideon the basis of
molecular dynamics distance estimations [29,30].
The scPTZ test was carried out according to the known protocol
[20]. In this method, a 0.5% solution of pentylenetetrazole (75 mg/kg)
was utilized subcutaneously in the posterior midline for inducing sei-
zures. The animals were observed for 0.5 h 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.
4.6. Neurotoxicity-minimal motor impairment
6. Prediction of ADME properties
The minimal motor impairment was measured in mice by the ro-
torod test. The mice were trained to stay on an accelerating rotorod 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 trial done [21].
A computational study of the title compounds was performed for
prediction of ADME properties. Polar surface area (TPSA), miLog P,
number of rotatable bonds, molecular volume, and number of hydrogen
donor and acceptor atoms and violations of Lipinski’s rule of five were
calculated by the Molinspiration online property calculation toolkit.
The percentage absorption (% ABS) was also calculated by the formula:
% ABS = 109-(0.345 - TPSA) [31].
4.7. Phase II acute toxicity study
7. Log P determination
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
TD50values, groups of 10 mice were given a range of intrapeitoneal
doses of the test drug until at least three points were established in the
range of 10–90% seizure protection or minimal observed neurotoxicity.
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 [22,23].
The partition coefficient was determined at room temperature using
octanol and phosphate buffer mixture. 10 mL of octanol and 10 mL
phosphate buffer were mixed and placed in a glass stopper graduated
tube. In this mixture, 5 mg of accurately weighed compound was added,
followed by shaking with mechanical shaker for 24 h at room tem-
perature. After that, the mixture was transferred to a separating funnel
and allowed to dynamically equilibrate for 6 h. The aqueous and oc-
tanol phases were separated and filtered through membrane filter. The
drug content in aqueous phase was analyzed by UV spectroscopy [32].
11

A.A. Siddiqui, et al.
BioorganicChemistry99(2020)103584
8. Docking study
and muscle relaxant activities of new 2-((5-amino-1,3,4-thiadiazol2-yl)methyl)-6-
phenyl-4,5-dihydropyridazin-3(2H)-one derivatives, Med. Chem. Res. 23 (2014)
146–157.
Compound SP-5F was selected as ligand for docking studies with
two well-established epilepsy receptors namely GABAA and Voltage-
gated sodium channel receptor by using Maestro 10.5 program
(Schrodinger, Inc., USA). These receptors are the most important tar-
gets in the design and discovery of successful antiepileptic drugs. The
homology model of the diazepam bound GABAA receptor developed by
Richter [33] and Lamotrigine bound Voltage-gated sodium channel by
Lipkind and Fozzard [34] was retrieved from the supplementary ma-
terial of their published paper.
[9] B. Malawska, A. Scatturin, Application of pharmacophore models for the design and
synthesis of new anticonvulsant drugs, Mini. Rev. Med. Chem. 3 (4) (2003 Jun)
341–348.
[10] Mohd. Amir, S. AsifIsrar Ali, Mohd. Zaheen Hassan, Synthesis of benzothiazole
derivatives having acetamido and carbothioamidopharmacophore as antic-
onvulsant agents, Med. Chem. Res. 21 (9) (2012) 2661–2670.
[11] Mohamed F. Zayed, Saleh K. Ihmaid, Hany E.A. Ahmed, Khaled El-Adl, Ahmed
M. Asiri, Abdelsattar M. Omar, Synthesis, Modelling, and Anticonvulsant Studies of
New Quinazolines Showing Three Highly Active Compounds with Low Toxicity and
High Affinity to the GABA-A Receptor, Molecules 22 (2017) 188.
[12] S. Partap, M.S. Yar, M.Z. Hassan, M.J. Akhtar, A.A. Siddiqui, Design, Synthesis, and
Pharmacological Screening of Pyridazinone Hybrids as Anticonvulsant Agents,
Arch. Pharm. (Weinheim) 350 (10) (2017), https://doi.org/10.1002/ardp.
201700135 Epub 2017 Sep 1.
Declaration of Competing Interest
[13] Md. SanghPartap, Jawaid Akhtar, Mohammed Shahar Yar, Mohd Zaheen Hassan,
Anees Ahmad Siddiqui, Pyridazinone hybrids: Design, synthesis and evaluation as
potential anticonvulsant agents, Bioorg. Chem. 77 (2018) 74–83.
[14] A.A. Siddiqui, R. Mishra, M.S. Yar, Eur. J. Med. Chem. 45 (2010) 2283–2290.
[15] M.S.Y. Khan, A.A. Siddiqui, Indian J. Hetrocyclic Chem. 39B (2000) 614–619.
[16] A.A. Siddiqui, M. Islam, M. Asif, C. Asthana, J. Ultra. Chem. 3 (2007) 176–178.
[17] Raghda R.S. Hussein, Rasha H. Soliman, Ahmed M. Abdelhaleem, Mona H. Ali,
Mohamed E.A. Tawfeik, Abdelrahim, Effect of antiepileptic drugs on liver enzymes,
Beni-Suef Univ. J. Basic Appl. Sci. 2 (1) (2013) 14–19.
The authors declare no conflict of interest.
Acknowledgement
One of authors, 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.
[18] Anticonvulsant Screening Project, Antiepileptic Drug Development Program,
National Institute of Health, DHEW Publ (NIH), US, 1978, pp. 78–1093.
[19] L. Krall, J.K. Penry, B.G. White, H.J.E. Kupferberg, A. Swinyard, Epilepsia 19 (1978)
409–428.
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
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12