Synthesis and antiseizure activity of (E)-1,2-diarylethylidenehydrazine carboximidamides against tonic-clonic seizures: an intracerebroventricular and electrophysiological study

Article abstract of DOI:10.1007/s00044-020-02576-7

A series of (E)-1,2-diarylethylidenehydrazine carboximidamides 2a–j were synthesized and characterized by NOESY experiment as anticonvulsant agents and their antiseizure activity was evaluated by intracerebroventricular administration of compounds. Most of the compounds had significant protection against tonic-clonic seizures and 2a was found to be as equipotent as carbamazepine in seizures control. In order to find their anticonvulsant mechanism of action, 2a was subjected to further electrophysiological studies using patch-clamp technique. The results confirmed that this compound is neither a voltage-gated sodium channel blocker nor a NMDA/AMPA antagonist. Although 2a did not show any direct GABA agonistic activity, it could decrease EPSP and increase IPSP frequency without any change in amplitude. Finally, the results indicated most likely a presynaptic GABA-mediated mechanism of 2a for its antiseizure activity such as inhibition of the GABA-T which was validated by molecular docking.

Full text of DOI:10.1007/s00044-020-02576-7

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02576-7
ORIGINAL RESEARCH
Synthesis and antiseizure activity of (E)-1,2-diarylethylidenehydrazine
carboximidamides against tonic-clonic seizures: an
intracerebroventricular and electrophysiological study
Fariba Abedi Firouzjaei1 Elmira Heidarli2 Shabnam Ravan2 Sayed Masoud Hosseini2 Nima Naderi2
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Kiarash Almasyan3 Afshin Sarvary1 Hamid Irannejad
3,4
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Received: 5 January 2020 / Accepted: 27 May 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
A series of (E)-1,2-diarylethylidenehydrazine carboximidamides 2a–j were synthesized and characterized by NOESY experiment
as anticonvulsant agents and their antiseizure activity was evaluated by intracerebroventricular administration of compounds. Most
of the compounds had significant protection against tonic-clonic seizures and 2a was found to be as equipotent as carbamazepine in
seizures control. In order to find their anticonvulsant mechanism of action, 2a was subjected to further electrophysiological studies
using patch-clamp technique. The results confirmed that this compound is neither a voltage-gated sodium channel blocker nor a
NMDA/AMPA antagonist. Although 2a did not show any direct GABA agonistic activity, it could decrease EPSP and increase
IPSP frequency without any change in amplitude. Finally, the results indicated most likely a presynaptic GABA-mediated
mechanism of 2a for its antiseizure activity such as inhibition of the GABA-T which was validated by molecular docking.
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Keywords Epilepsy Hydrazinecarboximidamide Intracerebroventricular Patch-clamp Tonic-clonic seizures GABA-T
Introduction
in the world live with epilepsy and 70% of epileptic patients
respond to antiepileptic drugs (AED). AEDs may cause
adverse effects which are common among patients receiving
medicines and can have a considerable impact on quality of
life, contributing to failure of treatment. The most common
side effects of AEDs include dizziness, nausea, headache,
vomiting, fatigue, vertigo, ataxia and blurred vision, and so on
which are a motivation for drug discontinuation in 40% of
cases. Therefore, the emerge of further research to develop
novel AEDs with fewer side effects and higher effectiveness
is of great importance (Perucca and Meador 2005).
Epilepsy is a chronic cerebral disorder characterized by partial
or generalized seizures of the body. Seizures are due to suc-
cessive electrical discharges of neurons which tend to unin-
tentional body movements. Approximately, 50 million people
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02576-7) contains supplementary
material, which is available to authorized users.
Owing to the complex mechanisms involved in seizures,
structurally various AEDs with different mechanisms have been
discovered and introduced. Among compounds classes, carba-
zides and carbazones are an interesting scaffold for consideration
in AED structures due to their hydrogen bonding potential,
because hydrogen bonding domain is a main part of pharma-
cophoric elements of AEDs. More specifically, hydrazine-
carboxamides and hydrazinecarbothioamides have prominent
and various biological and pharmacological activities like try-
panocidal, cathepsin L (Compound I in Fig. 1) and tyrosinase
inhibition (Compound II) (Fatondji et al. 2013; Haldys et al.
2018; Kumar et al. 2010; Song et al. 2017). Moreover, anti-
retroviral activity of thiosemicarbazone metal complexes against
* Nima Naderi
naderi@sbmu.ac.ir
* Hamid Irannejad
irannejadhamid@gmail.com
1
Department of Chemistry, Faculty of Science, Babol Noshirvani
University of Technology, Babol, Iran
2
Department of Pharmacology & Toxicology, School of Pharmacy,
Shahid Beheshti University of Medical Sciences, Tehran, Iran
3
Department of Medicinal Chemistry, Faculty of Pharmacy,
Mazandaran University of Medical Sciences, Sari, Iran
4
Pharmaceutical Sciences Research Center, Mazandaran University
of Medical Sciences, Sari, Iran

Medicinal Chemistry Research
Fig. 1 Structures of some semicarbazone/thiosemicarbazone derivatives reported in literature as active pharmacological agents and the structure of
newly designed compounds
HIV-1 and HTLV-1 has been studied and reported by Pelosi
et al. (2010). Recently, Urease inhibition activity has been
reported for some benzophenone semicarbazone/thiosemicarba-
zone derivatives (Compound III) and also thiosemicarbazide
derivatives have displayed antibacterial and antiproliferative
effects (Arshia et al. 2016; Pitucha et al. 2016). Above all,
hydrazinecarbothioamides IV and V have been reported several
times in literature as anticonvulsant agents (Nevagi et al. 2014;
Shafiee et al. 2009; Tripathi and Kumar 2013).
In this study, some hydrazinecarboximidamides (Fig. 1)
were designed and synthesized by conventional methods and
their correct structures were confirmed by NMR and Mass
spectra. Designing was based on our previously reported
anticonvulsant agents with 5,6-diaryl-1,2,4-triazine structure
(Irannejad et al. 2014; Irannejad et al. 2015). In order to have a
highly potential hydrogen bonding domain which is a requisite
pharmacophoric element in anticonvulsant agents, 1,2,4-tria-
zine ring was considered to be in the form of an open chain to
confer more flexibility and hydrogen bonding capability (Fig.
2). In this case, hydrazone moiety can exist in two geometrical
isomeric forms E/Z which were characterized by NOESY
experiment and compared with the previously reported similar
compounds. Subsequently, their antiseizure activity was eval-
uated by intracerebroventricular (i.c.v.) injection of compounds
against seizures induced by subcutaneous (s.c) injection of
pentylenetetrazole (PTZ) and their mechanism of antiseizure
activity was studied by voltage and current-clamp method on
DG hippocampal neuron cells.

Medicinal Chemistry Research
Fig. 2 The strategy of
designing open chain
hydrazinecarboximidamides by
ring opening of our previously
reported 5,6-diaryl-1,2,4-triazine
anticonvulsant agents
Methods and materials
catalytic amounts of hydrochloric acid (37%) and the
mixture was refluxed for 24 h to complete the reaction
monitored by TLC. After completion of reaction, it was
cooled to room temperature to precipitate the product,
then filtered off and washed by water. If necessary,
recrystallization was performed in 2-propanol to give the
pure product.
All chemical reagents and solvents were purchased from
Sigma–Aldrich or Merck suppliers and used without further
purification. The progress of reactions was checked by thin-
layer chromatography (TLC) by using TLC Silica gel 60 F254
(Merck, Darmstadt, Germany). UV cabinet with wavelengths
254 and 360 nm was used for visualizing spots on TLC. Col-
umn chromatography was performed by Silica gel 60
(0.040–0.063 mm), Merck, Germany. Melting points were
measured in open capillaries on a Stuart Scientific apparatus
and are uncorrected. IR spectra were recorded on a FT-IR
Perkin Elmer spectrometer. NMR spectra were recorded on
Bruker 400 MHz (Germany) and tetramethylsilane was used as
standard and chemical shifts are expressed in ppm units.
Coupling constants are reported in Hertz (Hz). Low resolution
mass spectra were measured using a HP 5975 Mass Selective
Detector (Agilent technologies, USA). Elemental analyses for
C, H, and N were performed by using a CHN elemental ana-
lyzer (GmbH-Germany), and the results are within 0.4% of
the theoretical values.
(E)-2-(2-(4-methoxyphenyl)-1-(4-(methylthio)phenyl)
ethylidene) hydrazinecarboximidamide (2a)
White powder (0.21 g, yield 35%); mp 231–233 °C. IR
(KBr) (νmax /cm−1): 3455, 3061, 2989, 1684, 1622, 1513,
1
1439, 1246, 1030, 830. H-NMR (400 MHz, DMSO-d6):
δH (ppm) 2.46 (3H, s, SCH3), 3.67 (3H, s, OCH3), 4.24
3
(2H, s, CH2), 6.83 (2H, d, JHH = 8.6 Hz, H–Ar), 7.10
3
3
(2H, d, JHH = 8.6 Hz, H–Ar), 7.21 (2H, d, JHH
=
8.5 Hz, H–Ar), 7.80 (3H, brs, NH and NH2), 7.91 (2H, d,
3JHH = 8.5 Hz, H–Ar), 11.64 (1H, brs, NH). 13C-NMR
(100 MHz, DMSO-d6) δC (ppm) 14.2 (SCH3), 31.3
(CH2), 55.0 (OCH3), 114.1, 125.1, 127.2, 127.7, 129.3,
132.2, 140.5, 152.5, 156.1, 157.9. EI-MS, m/z (%): 328
(M+, 15), 243(20), 151(50), 109(100), 80(30), 45(60).
Anal. Calcd for C17H20N4OS: C, 62.17; H, 6.14; N,
17.06. Found: C, 62.21; H, 4.16; N, 17.11.
General procedure
Synthesis of 1,2-Diarylethanones (1a–j)
(E)-2-(1-(4-(methylthio)phenyl)-2-(p-tolyl)ethylidene)
To a mixture of arylacetic acid derivatives (1 eq), anisole or
thioanisole (1.2 eq) and phosphoric acid (1.2 eq), was added
trifluoroacetic anhydride (4 eq) and the resulting mixture was
stirred at room temperature for 10 min. Crushed ice was added
to the mixture to precipitate the product and then it was filtered
and washed with water and petroleum ether. It was used in the
next step without further purification. Compounds character-
ization was previously reported by us in our recent publication
(Kucukkilinc et al. 2017; Valipour et al. 2019).
hydrazinecarboximidamide (2b)
White powder (0.32 g, yield 53%); mp 236–238 °C. IR
(KBr) (νmax /cm−1): 3465, 3269, 1682, 1621, 1495, 1318,
1140, 988, 761. 1H-NMR (400 MHz, DMSO-d6): δH
(ppm) 2.20 (3H, s, CH3), 2.46 (3H, s, SCH3), 4.27 (2H, s,
3
CH2), 7.06 (4H, s, H–Ar), 7.20 (2H, d, JHH = 8.5 Hz,
H–Ar), 7.85 (3H, brs, NH and NH2), 7.90 (2H, d, 3JHH
=
8.5 Hz, H–Ar), 11.69 (1H, brs, NH). 13C-NMR
(100 MHz, DMSO-d6) δC (ppm) 14.1 (SCH3), 20.5
(CH3), 31.8 (CH2), 125.0, 127.7, 128.1, 129.2, 132.2,
132.4, 135.6, 140.5, 152.3, 156.1. EI-MS, m/z (%): 312
(M+,10), 207 (21), 151(50), 109(72), 81(20), 44(100).
Anal. Calcd for C17H20N4S: C, 65.35; H, 6.45; N, 17.93.
Found: C, 65.42; H, 6.43; N, 17.98.
Synthesis of (E)-1,2-
Diarylethylidenecarboximidamides (2a–j)
To a suspension of 1,2-diarylethanones (1a–j) (1 eq) in
2-propanol, was added aminoguanidine (1.5 eq) and

Medicinal Chemistry Research
(E)-2-(1-(4-(methylthio)phenyl)-2-phenylethylidene)
hydrazinecarboximidamide (2c)
1260, 1026, 810. 1H-NMR (400 MHz, DMSO-d6): δH
(ppm) 3.67 (3H, s, OCH3), 3.76 (3H, s, OCH3), 4.23 (2H, s,
CH2), 6.83 (2H, d, 3JHH = 8.6 Hz, H–Ar), 6.90 (2H, d, 3JHH
3
White powder (0.46 g, yield 75%); mp 218–220 °C. IR (KBr)
(νmax /cm−1): 3465, 3152, 2989, 1689, 1623, 1495, 1322, 1095,
= 8.9 Hz, H–Ar), 7.10 (2H, d, JHH = 8.6 Hz, H–Ar), 7.85
(3H, brs, NH and NH2), 7.92 (2H, d, 3JHH = 8.9 Hz, H–Ar),
11.55 (1H, brs, NH). 13C-NMR (100 MHz, DMSO-d6) δC
(ppm) 31.4 (CH2), 55.0 (OCH3), 55.2 (OCH3), 113.6,
114.1, 127.3, 128.3, 128.9, 129.3, 152.7, 156.0, 157.9,
160.4. EI-MS, m/z (%): 312 (M+, 100), 238 (88), 229 (95),
191 (91), 149 (75), 121 (80), 104 (65), 77 (79). Anal. Calcd
for C17H20N4O2: C, 65.37; H, 6.45; N, 17.94. Found: C,
65.46; H, 6.49; N, 17.88.
1
739. H-NMR (400 MHz, DMSO-d6): δH (ppm) 2.46 (3H, s,
SCH3), 4.33 (2H, s, CH2), 7.18–7.29 (7H, m, H–Ar), 7.85 (3H,
brs, NH and NH2), 7.91 (2H, d, 3JHH = 8.50 Hz, H–Ar), 11.66
(1H, brs, NH). 13C-NMR (100 MHz, DMSO-d6) δC (ppm) 14.1
(SCH3), 32.2 (CH2), 125.1, 126.5, 127.6, 128.2, 128.7, 132.2,
135.5, 140.5, 152.1, 156.1. EI-MS, m/z (%): 298(M+,95), 224
(60), 207 (58), 151(60),109(90), 91 (30), 45 (100). Anal. Calcd
for C16H218N4S: C, 64.40; H, 6.08; N, 18.78. Found: C, 64.32;
H, 6.11; N, 18.84.
(E)-2-(1-(4-methoxyphenyl)-2-(p-tolyl)ethylidene)
hydrazinecarboximidamide (2g)
(E)-2-(2-(4-chlorophenyl)-1-(4-(methylthio)phenyl)
ethylidene)hydrazinecarboximidamide (2d)
White powder (0.52 g, yield 85%); mp 218–220 °C. IR
(KBr) (νmax /cm−1): 3425, 3255, 2933, 1595, 1517, 1513,
1463, 1260, 1027, 846. 1H-NMR (400 MHz, DMSO-d6): δH
(ppm) 2.21 (3H, s, CH3), 3.75 (3H, s, OCH3), 4.25 (2H, s,
White powder (0.09 g, yield 77%); mp 250–252 °C. IR (KBr)
(νmax /cm−1): 3466, 3265, 2920, 1685, 1623, 1493, 1435, 1094,
1
3
1014, 821. H-NMR (300 MHz, DMSO-d6): δH (ppm) 2.47
CH2), 6.90 (2H, d, JHH = 8.9 Hz, H–Ar), 7.06 (4H, s,
(3H, s, SCH3), 4.34 (2H, s, CH2), 7.20 (2H, d, 3JHH = 8.4 Hz,
H–Ar), 7.84 (3H, brs, NH and NH2), 7.91 (2H, d, JHH
=
3
3
3
H–Ar), 7.22 (2H, d, JHH = 8.4 Hz, H–Ar), 7.34 (2H, d, JHH
= 8.40 Hz, H–Ar), 7.80 (3H, brs, NH and NH2), 7.91 (2H, d,
3JHH = 8.40 Hz), 11.70 (1H, brs, NH). 13C-NMR (75 MHz,
DMSO-d6) δC (ppm) 14.5 (SCH3), 31.3 (CH2), 125.5, 128.1,
129.1, 130.5, 131.7, 132.3, 135.1, 141.2, 152.1, 156.5. EI-MS,
m/z (%): 332 (M+, 9), 275 (9), 207 (16), 151 (43), 109 (81), 44
(100). Anal. Calcd for C16H17ClN4S: C, 57.74; H, 5.15; N,
16.83. Found: C, 57.81; H, 5.18; N, 16.75.
8.9 Hz, H–Ar), 11.55 (1H, brs, NH). 13C-NMR (100 MHz,
DMSO-d6) δC (ppm) 20.5 (CH3), 31.9 (CH2), 55.2 (OCH3),
113.6, 128.1, 128.3, 128.8, 129.2, 132.5, 135.5, 152.5,
156.0, 160.4. EI-MS, m/z (%): 296 (M+, 96), 237 (34), 222
(87), 191 (100), 149 (74), 134 (74), 105 (46). Anal. Calcd
for C17H20N4O: C, 68.90; H, 6.80; N, 18.90. Found: C,
68.81; H, 6.88; N, 18.98.
(E)-2-(1-(4-methoxyphenyl)-2-phenylethylidene)
(E)-2-(2-(4-fluorophenyl)-1-(4-(methylthio)phenyl)
hydrazinecarboximidamide (2h)
ethylidene)hydrazinecarboximidamide (2e)
White powder (0.35 g, yield 57%); mp 188–190 °C. IR (KBr)
(νmax /cm−1): 3464, 3262, 2836, 1684, 1619, 1513, 1253,
White powder (0.19 g, yield 70%); mp 253–255 °C. IR (KBr)
(νmax /cm−1): 3469, 30264, 1686, 1622, 1600, 1508, 1324,
1
1028, 838. H-NMR (400 MHz, DMSO-d6): δH (ppm) 3.75
1
1221, 1096, 822. H-NMR (400 MHz, DMSO-d6): δH (ppm)
(3H, s, OCH3), 4.31 (2H, s, CH2), 6.90 (2H, d, 3JHH = 8.9 Hz,
H–Ar), 7.16–7.29 (5H, m, H–Ar), 7.84 (3H, brs, NH and
3
2.47 (3H, s, SCH3), 4.33 (2H, s, CH2), 7.10 (2H, t, JHH
=
3
8.7 Hz, H–Ar), 7.20–7.24 (4H, m, H–Ar), 7.89 (3H, brs, NH
and NH2), 7.91 (2H, d, 3JHH = 8.5 Hz, H–Ar), 11.76 (1H, brs,
NH). 13C-NMR (100 MHz, DMSO-d6) δC (ppm) 14.1 (SCH3),
31.3 (CH2), 115.5 (d, 2JCF = 21.10 Hz), 125.1, 127.7, 130.1 (d,
NH2), 7.92 (2H, d, JHH = 8.9 Hz, H–Ar), 11.55 (1H, brs,
NH). 13C-NMR (100 MHz, DMSO-d6) δC (ppm) 32.3 (CH2),
55.2 (OCH3), 113.7, 126.5, 128.2, 128.3, 128.7, 128.8, 135.7,
152.3, 156.0, 160.5. EI-MS, m/z (%): 282 (M+, 100), 223
(24), 208 (64), 191 (88), 165 (24), 149 (65), 134 (29), 91 (31).
Anal. Calcd for C16H18N4O: C, 68.06; H, 6.43; N, 19.84.
Found: C, 68.19; H, 6.40; N, 19.79.
4
3JCF = 7.9 Hz), 131.7 (d, JCF = 3.0 Hz), 132.0, 140.7, 152.0,
1
156.1, 161.0 (d, JCF = 241.1 Hz). EI-MS, m/z (%): 303 (20),
272(40), 224 (24), 183(20), 151 (22), 134 (29), 44(100). Anal.
Calcd for C16H17FN4S: C, 60.74; H, 5.42; N, 17.71. Found: C,
60.64; H, 5.49; N, 17.77.
(E)-2-(2-(4-chlorophenyl)-1-4-(methoxyphenyl)ethylidene)
hydrazinecarboximidamide (2i)
(E)-2-(1,2-bis(4-methoxyphenyl)ethylidene)
hydrazinecarboximidamide (2f)
White powder (0.54 g, yield 89%); mp 222–224 °C. IR
(KBr) (νmax /cm−1): 3461, 2952, 2559, 1688, 1569, 1492,
1
White powder (0.55 g, yield 91%); mp 233–240 °C. IR
(KBr) (νmax /cm−1): 3421, 3150, 2936, 1660, 1511, 1464,
1305, 845. H-NMR (400 MHz, DMSO-d6): δH (ppm) 3.76
3
(3H, s, OCH3), 4.32 (2H, s, CH2), 6.91 (2H, d, JHH
=

Medicinal Chemistry Research
3
8.8 Hz, H–Ar), 7.21 (2H, d, JHH = 8.3 Hz, H–Ar), 7.33
(2H, d, 3JHH = 8.3 Hz, H–Ar), 7.80 (3H, brs, NH and NH2),
experimental groups. Drugs were dissolved in DMSO, then
using a 5-μl Hamilton syringe connected to a PE-10 tube,
100 µg (dissolved in 5 µL DMSO) of drug was injected over
2 min and the needle remained in place for an additional 1 min
before it was slowly retracted. Ten minutes after drug admin-
istration, seizure was induced by s.c. injection of freshly pre-
pared PTZ (95 mg/kg) and the animal was observed in a
transparent Plexiglas box for 30 min. Seizure severity was
measured according to the methods described by Pohl and
Mares (1987). These stages are as follow: 1—isolated myo-
clonic jerks; 2—unilateral and incomplete seizures; 3—clonic
seizures; 4—tonic-clonic seizures with a suppression of tonic
phase; 5—generalized tonic-clonic seizures. In our study,
latency to occurrence for stage 1 and stage 5 of seizure were
recorded. These seizure types describe in part absence and
generalized tonic-clonic seizure and contribute to different
mechanisms of actions of antiseizure drugs. The antiseizure
effect of the test compounds was compared with vehicle-treated
(control) group and with a standard treatment (carbamazepine
100 µg).
3
7.91 (2H, d, JHH = 8.8 Hz, H–Ar), 11.63 (1H, brs, NH).
13C-NMR (100 MHz, DMSO-d6) δC (ppm) 31.6 (CH2), 55.2
(OCH3), 113.7, 128.1, 128.6, 128.8, 130.1, 131.1, 134.8,
151.8, 156.1, 160.5. EI-MS, m/z (%): 314 (17), 281 (11),
238 (9), 218 (24), 135 (100), 103 (34), 77 (45). Anal. Calcd
for C16H17ClN4O: C, 60.66; H, 5.41; N, 17.69. Found: C,
60.75; H, 5.44; N, 17.76.
(E)-2-(2-(4-fluorophenyl)-1-(4-methoxyphenyl)ethylidene)
hydrazinecarboximidamide (2j)
White powder (0.21 g, yield 34%); mp 190–192 °C. IR
(KBr) (νmax /cm−1): 3466, 3274, 2837, 1689, 1570, 1510,
1181, 1028, 1030, 831. 1H-NMR (400 MHz, DMSO-d6): δH
(ppm) 3.75 (3H, s, OCH3), 4.30 (2H, s, CH2), 6.90 (2H, d,
3JHH = 8.8 Hz, H–Ar), 7.09 (2H, t, JHH = 8.7 Hz, H–Ar),
3
7.21 (2H, dd, 3JHH = 8.5, 5.5 Hz, H–Ar), 7.72 (3H, brs, NH
3
and NH2), 7.91 (2H, d, JHH = 8.8 Hz, H–Ar), 11.60 (1H,
brs, NH). 13C-NMR (100 MHz, DMSO-d6) δC (ppm) 31.4
2
(CH2), 55.3 (OCH3), 113.7, 115.4 (d, JCF = 21.1 Hz),
128.3, 128.8, 130.2 (d, JCF = 7.90 Hz), 132.0, 152.2,
Whole-cell patch-clamp recording in hippocampal
brain slices
3
1
156.4, 160.5, 161.0 (d, JCF = 240.9 Hz). EI-MS, m/z (%):
300 (M+, 100), 241 (28), 226 (68), 191 (93), 149 (65), 134
(71), 109 (61). Anal. Calcd for C16H17FN4O: C, 63.99; H,
5.71; N, 18.66. Found: C, 63.87; H, 5.76; N, 18.74.
Since the hippocampus is an important brain region in seizure
development, the hippocampal dentate gyrus (DG) cells were
considered for evaluation of the antiseizure mechanism of the
effective compound 2a at 10 µM concentration.
Pharmacology
Brain slice preparation
The antiseizure effects of the test compounds were eval-
uated using PTZ-induced seizure in rats. Male wistar rats
(150–200 g; Pasteur Institute, Tehran, Iran) were used in
this study. They were kept in animal facility with free
access to food and water and were housed on a 12/12 h
light/dark cycle (lights on at 7:00 a.m.), and temperature of
23 2 °C. Efforts were made to minimize the number of
animals in each group. Animal care and experimental pro-
cedures were in accordance with Iranian Ministry of Health
and Medical Education Guidelines and were approved by
University Ethics Committee (approval No. IR.SBMU.
PHNM.1395.543).
Rats were deeply anesthetized with i.p. injection of ketamine
(100 mg/kg) and xylazine (10 mg/kg) and transcardially per-
fused with cold (4 °C) modified artificial cerebrospinal fluid
(ACSF) containing high sucrose and low sodium (in mM):
206 sucrose, 2.8 KCl, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose,
1 CaCl2, 2 MgSO4, 1 MgCl2, pH = 7.4, oxygenated with 95%
O2 and 5% CO2. Rats were then decapitated and the brain
rapidly removed and placed in the cold sucrose-ACSF solution
with the procedure mentioned above. Transverse hippocampal
slices (300-μm thick) were prepared using a vibrating slicer
(Campden Instruments Ltd, UK) and transferred to a holding
chamber continuously perfused at room temperature with
ACSF containing (in mM): 124 NaCl, 3 KCl, 1.24 NaH2PO4,
26 NaHCO3, 10 glucose, 2 CaCl2, 2 MgSO4, pH 7.4, oxyge-
nated with 95% O2 and 5% CO2.
Stereotaxic surgery and drug administration
Rats were anesthetized by intraperitoneal injection of ketamine
(100 mg/kg) and xylazine (10 mg/kg) and were placed in a
stereotaxic surgery device (Stoelting, IL, USA). A 23-gauge
stainless-steel cannula was implanted 1 mm above the left lat-
eral ventricle (at the following coordinates with reference to
bregma: AP = −1.2 mm, L = 2 mm, DV = 2.8 mm) according
to the atlas of Paxinos and Watson (2005). One week after
surgery, rats were randomly assigned for one of the
Whole-cell patch-clamp recording
Whole-cell recordings were done under visual control using
infrared difference interference contrast optics. DG granule
cells (DG-GCs) were visualized by infrared video imaging
(Hamamatsu, ORSA, Japan) with a ×40 water immersion

Medicinal Chemistry Research
objective. Whole-cell recordings were made using Multiclamp
700B amplifier (Axon Instruments, CA, USA) equipped with
Digidata 1320 A/D converter (Axon Instruments, CA, USA).
For the recordings, the patch pipettes were pulled with a PC10
vertical puller (Narishige, Japan) from borosilicate glass
capillary (1.2 mm O.D., 0.9 mm I.D. with inner filament).
Pipettes had a 3–5 MΩ tip resistance when they were filled with
intracellular solution. Recordings were accepted only if the
series resistance was <25 MΩ, and if it did not vary by 20%
during the experiment. In voltage-clamp recording, the pipettes
were filled with a solution containing (in mM); 100 Cs2MeSO3,
50 CsCl, 10 HEPES, 1.1 EGTA, 2 Mg-ATP, 0.3 Na2GTP, 5
QX-314, 5 TEA-Cl, 5 4-AP, and 10 Na2-Phosphocreatine at
pH 7.3 adjusted with CsOH, and the osmolarity of the pipette
solution was 290 mOsm. For current-clamp recording, pipettes
were filled with intracellular solution containing (in mM) 140
K-Gluconate, 10 HEPES, 2 MgCl2, 2 Na2-ATP, 1.1 EGTA,
0.1 CaCl2, and 0.4 Mg-ATP. The pH of the internal solution
was set to 7.3 by KOH, and the osmolarity was adjusted to
290 mOsm. The recordings were filtered by low-pass Bessel at
10 kHz, sampled at 20 kHz and stored on a personal computer
for offline analysis. Spontaneous excitatory postsynaptic cur-
rents (EPSCs) were recorded in the presence of GABAA
receptor antagonist bicuculline (30 μM) and were blocked by
the ionotropic glutamate receptor antagonists AP-5 (50 μM)
and CNQX (20 μM). Spontaneous inhibitory postsynaptic
currents (IPSCs) were recorded with cesium-containing elec-
trodes in the presence of AP-5 (50 μM) and CNQX (20 μM)
and were blocked by the GABAA receptor antagonist bicu-
culline (30 μM).
test was used to compare seizure latencies between groups.
Also, chi-squared test was used to compare the ratio of
seizure occurrence between control and drug-treated groups.
Statistical analysis was performed by GraphPad Prism 6.0
(San Diego, CA, USA). P values <0.05 were considered as
significant difference.
Results and discussion
Chemistry
Preparation of the target compounds 2a–j, is outlined in
Scheme 1. As presented, synthetic route was started by an
acylation-like reaction between phenylacetic acid deriva-
tives and anisole or thioanisole in the presence of tri-
fluoroacetic anhydride and phosphoric acid which has been
reported by us in our previous works (Kucukkilinc et al.
2017; Valipour et al. 2019). in the next step, to the 1,2-
diarylethanones 1a–j, was added aminoguanidine in acidic
solution of 2-propanol and the mixture was refluxed to give
the final products 2a–j. Target compounds 2a–j were
obtained in pure form by crystallization in 2-propanol.
Subsequently, they were accurately characterized by 1H and
13C-NMR as well as mass spectroscopy (spectra in Sup-
plementary material) and the data confirmed their correct
1
structures. In the H-NMR spectra of compounds 2a–j, a
singlet was observed in the range of 4.23–4.34 ppm with
integral of two protons which is attributed to the aliphatic
methylene (Ar–CH2–C=N) protons. Methylthio protons in
compounds 2a–e, appeared as a singlet in the range of
2.46–2.47 ppm and protons of methoxy groups (CH3X in
Scheme 1) in compounds 2f–j are located in the range of
3.75–3.76 ppm. Notably, protons of methoxy groups in the
R position of compounds 2a and 2f were found in 3.67 ppm.
Exchangeable protons attached to nitrogen atoms in
hydrazine carboximidamide moiety can be seen as a singlet
at a range of 11.54–11.76 and a broad singlet at
7.7–7.8 ppm. Existence of exchangeable protons was con-
firmed by adding D2O to the deuterated-DMSO in NMR
solution of compound 2i and observing disappearance of the
corresponding signals at their own locations (Fig. 3).13C-
NMR spectra of compounds 2a–j showed that the methy-
lene carbon atoms (Ar–CH2–C=N) are in the range of
31.3–32.3 ppm, the methylthio carbon atoms at
14.1–14.6 ppm and methoxy carbon atoms (CH3X–) are in
the range of 55.2–55.3 ppm. Imine carbon atoms in the
ethylidene moiety of compounds display a signal in the
range of 151.8–152.8 ppm and carbon atoms of the guani-
dine moiety are at the downfield part of the spectrum at the
range of 156.0–156.6 ppm.
Molecular docking
PDB code 1OHW was used for molecular docking of vig-
abatrin and 2a into the active site of GABA-T. Validation of
the method was performed by docking of vigabatrin and
valproic acid into the active site and comparing the results
by a recently reported study on GABA-T inhibitors (Sahu
et al. 2018). Docking was performed by AutoDock 4.2 and
AutoDock Tools 1.5.6 was used to prepare the protein and
the required parameter files. Protonation of the protein and
the ligands was performed at pH = 7.4 and the ligands were
energy minimized at PM3 level prior to docking calcula-
tions. Center of the grid was X = 9.49, Y = −2.656,
Z = 17.132, grid spacing was 0.375 and the number of
points in the grid was 50, 46, and 50 in each Cartesian
coordinates. Lamarckian genetic algorithm was used to
search the space and the number of GA runs were set to 20.
Statistical analysis
Data of seizure latency were shown as mean SEM. One-
way analysis of variances (ANOVA) followed by Donnette
Due to the presence of the imine bond in the structure of
2a–j, stereoisomeric E/Z forms could be formed during the

Medicinal Chemistry Research
Scheme 1 Synthetic route to prepare (E)-1,2-diarylethylidenehydrazine carboximidamides 2a–j and representation of their hydrazone-azine
tautomerism
Fig. 3 1H-NMR spectrum of compound 2i. a 1H-NMR spectrum of compound 2i in DMSO-d6. b 1H-NMR spectrum of compound 2i in a mixture
of DMSO-d6 and D2O
reaction or after product formation by inversion of the nitrogen
atom. Many reports exist in the literature debating and identi-
fying the type and percentage of isomers by 1H-NMR chemical
shifts or by NOE relationships (Dehestani et al. 2018; Silva
et al. 2016). The 1H-NMR spectra of 2a–j shows that all
compounds are in pure isomeric forms because only one peak
exists for each proton in the structure. To determine the type of
E/Z isomer of compounds, NOESY assignment was performed
for compound 2i and is shown in Fig. 4. As shown, there is a
correlation between methylene protons at 4.36 ppm and gua-
nidine protons at 11.73 ppm and hence confirms the absolute
E-isomeric form of all compounds.
Hydrazone forms of compounds such as semi-
carbazone, thiosemicarbazone, and guanylhydrazone

Medicinal Chemistry Research
Fig. 4 NOESY spectrum of
compound 2i. Arrows represent
the correlation between
methylene (CH2) and guanidine
protons
have been used extensively in the literature for the
design of new drug-like compounds. But according to
quantum and structural studies on these type of com-
pounds, hydrazone-azine tautomerism exists which pre-
dominates to the azine formation (Chourasiya et al.
2016). Accordingly, hydrazine carboximidamides 2a–j
are predominantly in the form of azine in which the
double bonds are conjugated in the guanylhydrazone
moiety as presented in Scheme 1.
However, neither the test compounds 2a–j nor carba-
mazepine could protect rats against PTZ-induced myoclonic
seizures. One-way ANOVA and post tests revealed no
significant difference in latency to myoclonic seizure
occurrence between groups suggesting no protection against
myoclonic seizure with any of the test compounds.
Considering the chemical structure of compounds, the
results suggested that the presence of lipophilic atoms or
groups in both R and X positions such as 2b (R = Me, X = S)
and 2d (R = Cl, X = S) diminishes antiseizure activity of
compounds. On the other hand, presence of polar groups in
the same positions as in 2f (R = MeO, X = O) also inactivates
compounds against seizures control. Therefore, a combination
of a polar and a nonpolar group on the two aryl rings confers
optimum antiseizure activity. According to the results, lipo-
philicity seems to play a key role in the antiseizure activity of
the compounds. As shown by the calculated lipophilicity
values in Table 1, the most lipophilic compounds 2b and 2d
with logP value of 3.7 and the most polar compound 2f with
logP value of 2.5 are inactive against tonic-clonic seizures.
Reasoning of this finding is described in the molecular
docking results.
The protective effects of 2a–j against PTZ-induced
tonic-clonic and myoclonic seizures
In this study, all tested compounds and carbamazepine as a
standard drug were used at a dose of 100 µg/rat and the results
were compared with the control group received DMSO as
solvent. As shown in Table 1, compounds 2a, 2c, 2e, 2g, 2h,
2i, and 2j significantly increased latency time to the tonic-
clonic seizures compared with the control group (p < 0.05) and
compounds 2b, 2d, and 2f did not show any protection against
tonic-clonic seizures. Accordingly, 2j had the lowest activity
among compounds while compounds 2e and 2g showed
latency to tonic-clonic seizures with a p value < 0.01. Inter-
estingly, compounds 2c, 2h, and 2i increased the latency to
tonic-clonic seizures with a higher magnitude and a p value <
0.001. More interestingly, compound 2a had the highest pro-
tection against tonic-clonic seizures with the longest latency
time and a p value < 0.0001 which was equipotent as the
standard drug carbamazepine.
Effects of 2a in in vivo studies on active and passive
properties of hippocampal neurons
In order to explore the possible mechanism of anticonvulsant
activity of 2a, which was very active in behavioral study, it was
subjected to electrophysiological investigation to study its

Medicinal Chemistry Research
Table 1 Protective effect of pretreatment with carbamazepine and compounds against PTZ-induced tonic-clonic and myoclonic seizures
Compound
Substitution
logP Tonic-clonic seizure latency (s)
p valuea
Myoclonic seizure latency (s)
p value
(mean SEM)
(mean SEM)
Control (DMSO)
–
–
458 63
–
161 19
–
2a
R = MeO, X = S 3.1 1800
0
p < 0.0001 360 90
NS 228 51
p < 0.001 216 40
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
2b
R = Me, X = S
R = H, X = S
R = Cl, X = S
R = F, X = S
3.7 1092 297
3.2 1548 209
3.7 1170 134
3.3 1428 209
2c
2d
NS
384 52
315 66
564 236
791 319
2e
p < 0.01
NS
2f
R = MeO, X = O 2.5
941 244
2g
R = Me, X = O
R = H, X = O
R = Cl, X = O
R = F, X = O
–
3.1 1450 245
2.6 1620 180
3.2 1574 226
2.8 1249 278
p < 0.01
2h
p < 0.001 642 253
p < 0.001 535 316
2i
2j
p < 0.05
521 320
Carbamazepine
–
1800 0
p < 0.0001 302 49
The antiseizure effect of drugs (100 µg/5 µL vehicle; i.c.v. administration) in PTZ-induced seizure in rats
Drugs (100 µg/rat) or DMSO (in control group) were microinjected (5 µl) into left lateral ventricle 15 min before s.c. administration of PTZ (95 mg/
kg). After PTZ injection, rats were monitored for 30 min for seizure occurrence (cutoff point 1800s). N = 6–8 per group
NS not significant
aStatistically significant compared with the control (DMSO) group
effects on electrophysiological properties of DG neurons in rat
hippocampus.
changed from 77.56 12.8 pA at the baseline to 76.12
13.37 pA after bath application of 2a. Similarly, the
utilization time (latency to the first action potential in
Ramp test) changed from 381.6 63.47 ms at the base-
line to 377.75 63.9 ms after bath application of 2a.
A change in AHP (after hyperpolarization) could suggest
the effect of 2a on calcium-activated potassium channels,
which is important in seizure treatment. As shown in Fig. 8,
no significant effect of treatment on AHP amplitude was
shown in different steps of depolarizing currents [F(1,33) =
4.114; p = 0.05], although a slight increase in AHP was
detected after bath application of 2a.
Current-clamp studies: effect of bath application of
2a on active and passive properties of membrane
ion channels in DG hippocampal neurons
Basically, DG-GCs had an initial membrane conductance
of 0.27 0.05 pS and bath application of 2a did not
significantly change the membrane conductance (0.39
0.13 pS; Fig. 5). Depolarizing currents-induced neuronal
excitability as shown in Fig. 6 by increased number of
action potential and eventually inactivation of sodium
channels. Two-way ANOVA showed no significant
effect of 2a on the number of action potentials before and
after bath application of 2a [F(1,39) = 0.3764; p =
0.543] suggesting no blocking effect of 2a on voltage-
gated sodium channels. Moreover, bath application of 2a
did not change rheobase current and utilization time
compared with the baseline (Fig. 7). The rheobase cur-
rent (minimal required current to induce action potential)
Voltage-clamp studies: effect of bath application of
2a on excitatory and inhibitory synaptic
transmission in DG hippocampal neurons
In order to evaluate the effects of 2a on synaptic transmission
of DG hippocampal neurons, the inter-event intervals (IEI)
and amplitude of IPSCs and EPSCs were determined at
baseline values and after bath application of 2a. As shown in

Medicinal Chemistry Research
A
B
0.8
0.6
0.4
0.2
0.0
after
before
Fig. 5 a Representative traces and relative current–voltage (I–V) curve
obtained by applying hyperpolarizing currents to DG cells. The slope
of the fitted line represents total resistance of the cell membrane.
b Average of membrane resistance before and after bath application of
2a shows no significant effect of the drug on membrane resistance.
Data were shown as mean SEM (n = 5)
Fig. 9a, for IPSCs, a left shift in cumulative histogram of IEI
in a DG neuron suggested a decrease in IEI or an increase in
the number of IPSCs. Paired t-test revealed a significant
decrease in IEI (p = 0.027) but no change in IPSCs amplitude
compared with the baseline values, suggesting a possible
presynaptic augmentation of inhibitory neurotransmission by
2a. Moreover, 2a caused a right shift in cumulative histogram
of IEI for EPSCs with no change in amplitude (Fig. 10a).
Also, paired t-test revealed a significant increase in IEI of
EPSCs (p < 0.05) but no change in EPSCs amplitude com-
pared with the baseline values (Fig. 10b), suggesting a pos-
sible presynaptic inhibition of EPSCs by 2a in DG neurons.
Accordingly, it is believed that 2a might act through the
inhibition of GABA-transaminase (GABA-T) to exert its
antiseizure activity. Since this enzyme is one of the main
targets for some well-known anticonvulsant drugs like
vigabatrin and valproic acid, 2a was further considered for
docking into the active site of GABA-T as a potential target
for (E)-1,2-diarylethylidenehydrazine carboximidamides.
Molecular docking study
Fig. 6 2a could not alter depolarizing currents-induced action potential
properties. a Representative traces and b average of the number of
action potentials at different depolarizing currents show no change
after bath application of 2a compared with the baseline values. Data
were shown as mean SEM (n = 5)
According to the results obtained by the electrophysiological
patch-clamp study, the possible mechanism of action of (E)-
1,2-diarylethylidenehydrazine carboximidamide 2a was sus-
pected to be through the inhibition of GABA-T. This enzyme
degrades the inhibitory neurotransmitter GABA to end its
physiological action and its inhibition increases the synaptic
concentration of GABA.
To model the binding strength of 2a and its hypothetical
interactions with the key residues in the active site of
GABA-T, molecular docking is a reliable and useful tool for
medicinal chemists. Since the PDB structure of the X-ray
crystallized Homo sapiens GABA-T is not available yet, the
PDB code 1OHW which is the Sus scrofa GABA-T can be
used for molecular docking. According to a recent study on
GABA-T inhibitors, human GABA-T (UniProt id-P80404)
has 95.2% sequence identity with the Sus scrofa (UniProt
id-P80147) and the active site residues are highly conserved

Medicinal Chemistry Research
Fig. 7 Effect of 2a on membrane excitability of DG cells in Ramp test.
a Representative trace showing the minimum current of electrical
stimulus to excite the membrane (rheobase), the time taken by a sti-
mulus of rheobase strength to excite the membrane (utilization time),
and the threshold voltage. b No change in rheobase, utilization time, or
threshold voltage were shown after bath application of 2a compared
with the baseline values. Data were shown as mean SEM (N = 5)
(>96%), therefore PDB code 1OHW can be used with
confidence (Sahu et al. 2018).
methoxyphenyl, has pi-donor or arene-H interactions with
amide NH of Gly136A (2.92 Å) and Thr353B (3.22 Å) as
well as a pi-alkyl interaction with Cys135A (4.06 Å). Cor-
respondingly, amidine NH of 2a has conventional hydrogen
bonds with carbonyl oxygen of Phe189A (2.05 Å) and
carboxylate group of Asp298A (1.78 and 2.92 Å) (Fig. 11).
Similarly, hydrazone form of 2a produced very similar
results to the azine form in binding energy and conforma-
tion in the docking calculations.
Likewise, carboxylate oxygen atom of vigabatrin has
hydrogen bonding to the NH of Gln301A (1.96 Å) and amine
group of Lys329A (2.12 Å). Correspondingly, ammonium
group of vigabatrin has two attractive ionic interactions with
Glu265A and Glu299A (3.84 Å) and conventional hydrogen
bonding to the carboxylate group of Asp298A (1.74 Å).
Similarly, the binding conformation of vigabatrin has located in
the same space as predicted by a previously reported study on
GABA-T inhibitors and have Lys329A and Gln301A as
common interacting residues (Sahu et al. 2018).
Basically, the enzyme exists in a tetrameric form and its
active site is located at the interface of the two monomer
including the residues surrounding the pyridoxal phosphate
(PLP). Correspondingly, 2a in its azine form and vigabatrin
as a standard GABA-T inhibitor were subjected to mole-
cular docking into the active site of the enzyme.
The calculated binding energy was found to be
−3.08 kcal/mol for vigabatrin while 2a showed better
binding energy (−5.56 kcal/mol) and higher affinity than
vigabatrin into the active site of the enzyme. The con-
formational binding mode and interactions of 2a and viga-
batrin in the active site of GABA-T are illustrated in Fig.
11. In the case of 2a, phenyl ring of the 4-methylthiophenyl,
has pi–pi T-shaped interactions with Phe189A (4.77 Å) and
its methylthio group has pi-alkyl and pi-sulfur interactions
with the phenyl ring of Phe351B (5.13 and 4.52 Å,
respectively). Likewise, the other phenyl ring in the 4-

Medicinal Chemistry Research
Fig. 8 Effect of bath application
of 2a on latency to first action
potential (left) and AHP
amplitude (right) in DG cells.
Representative traces (a) and
average of data (b) show no
significant change in either
latency to first action potential or
AHP amplitude after drug
application compared with the
baseline values. Data were
shown as mean SEM (N = 5)
As noted earlier in the in vivo results, for the optimum
antiseizure activity of compounds, one of the aromatic
rings has to bear a lipophilic moiety while the other ring
must contain a polar group. This fact was further con-
firmed by presenting the surface hydrophobicity of
GABA-T active site residues in Fig. 12. As illustrated,
the methylthio group of 2a is located into a more
hydrophobic space (brown color) due to the presence of
Phe351 and Phe189 than the methoxy group. This find-
ing confirms that compounds which have hydrophobic
groups on the both of the two aryl rings (2b and 2d) or
the ones which have polar groups on the both of them
(2f) cannot accommodate well into the GABA-T active
site and hence will be inactive.
Interestingly, 2a has acceptable Human Intestinal Absorption,
Caco-2 permeability and blood–brain barrier penetration.
However, it has poor oral bioavailability predicted by this web-
server probably due to its high hepatic clearance. On the other
hand, 2a is neither a P-glycoprotein (P-gp) substrate nor a P-gp
inhibitor and hence does not have drug resistance due to P-gp
efflux. Correspondingly, 2a was predicted to be an inhibitor of
CYP3A4 and substrate of CYP2D6 which are the factors
affecting its low oral bioavailability.
Conclusion
(E)-1,2-Diarylethylidenehydrazine carboximidamides as
new anticonvulsant agents were synthesized and char-
acterized by NOESY experiment to confirm their (E)-
stereoisomeric form. Evaluation of their antiseizure
activity by i.c.v. administration showed their prominent
activity against tonic-clonic seizures. Correspondingly,
2a was found to be the most potent one as equipotent as
carbamazepine in controlling the seizures. Patch-clamp
results confirmed that 2a is neither a voltage-gated
Prediction of ADME parameters
Prediction of the druglikeness and ADME properties of com-
pound 2a were calculated by admetSAR 2.0 web-service
(Yang et al. 2018) and is summarized in Table 2. As shown in
the Table, 2a has no violation of Lipinski’s Rule of Five and all
the parameters of druglikeness are in the normal range.

Medicinal Chemistry Research
A
B
30
20
10
15000
10000
5000
0
*
0
er
er
t
t
ore
ore
f
f
af
af
e
e
b
b
Fig. 9 2a increased IPSCs frequency with no change in IPSCs
amplitude. a Representative IPSCs and the cumulative histograms of
inter-event interval (IEI) and amplitude of the same cell. Bath appli-
cation of 2a could shift the histogram to the left (i.e. a decrease in IEIs)
with no change in IPSCs amplitude. b Bath application of 2a sig-
nificantly increased the number of IPSCs during 10 min of recording
compared with the baseline values. Data were shown as mean SEM
(N = 5). *p < 0.05 (paired t-test)
Fig. 10 2a decreased EPSCs frequency with no change in EPSCs
amplitude. a Representative EPSCs and the cumulative histograms of
inter-event interval (IEI) and amplitude of the same cell. Bath appli-
cation of 2a could shift the histogram to the right (i.e. an increase in
IEIs) with no change in amplitude. b Bath application of 2a
significantly increased inter-event intervals of EPSCs during 10 min of
recording compared with the baseline values while no change in
EPSCs amplitude. Data were shown as mean SEM (N = 5). *p <
0.05 (paired t-test)

Medicinal Chemistry Research
Fig. 11 2D map of the binding
conformation and interactions of
(a) 2a and (b) vigabatrin in the
active site of GABA-T
sodium channel blocker, nor an AMPA or NMDA
antagonist. Besides, 2a did not induce any direct GABA
current but could decrease EPSP and increase IPSP fre-
quency without any change in amplitude. Totally, the

Medicinal Chemistry Research
Fig. 12 Surface hydrophobicity
presentation of the GABA-T
active site residues
Table 2 Physicochemical
Parameter
Value
Parameter
Value
Parameter
Value
parameters and ADME profile of
compound 2a predicted by
admetSAR 2.0 web-service
Molecular weight
AlogP
328.44
Human intestinal absorption
Caco-2
Yes
Yes
Yes
No
CYP3A4 substrate
CYP2C9 substrate
CYP2D6 substrate
CYP3A4 inhibitor
CYP2C9 inhibitor
CYP2D6 inhibitor
No
No
Yes
Yes
No
No
2.85
4
H-bond acceptor
H-bond donor
Rotatable bonds
Applicability domain
Blood–brain barrier
3
Human oral bioavailability
P-glycoprotein inhibitor
P-glycoprotein substrate
6
No
Yes
No
results guided us to conclude a presynaptic mechanism
of 2a for its antiseizure activity and prompted us to
consider GABA-T as a probable target for (E)-1,2-Dia-
rylethylidenehydrazine carboximidamides.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
References
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Mazandaran University of Medical Sciences (grant No. 1796) and also
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Conflict of interest The authors declare that they have no conflict of
interest.

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