Design and synthesis of novel parabanic acid derivatives as anticonvulsants

Article abstract of DOI:10.1016/j.bioorg.2019.103473

In this work a set of novel derivatives of parabanic acid 9a-d, 12a-d and 13a-d was synthesized and their anticonvulsant potential was evaluated. All the compounds under investigation exhibited anticonvulsant activity in both scPTZ and MES tests. In phase II anticonvulsant study, the trimethoxy phenyl derivative 9a evoked the highest potency among the tested compounds in scPTZ test. It displayed 1.72- and 17.05-folds activity more than the standard drugs phenobarbital and ethosuximide, respectively. In addition, the margin of safety for compound 9a is better than that of the reference antiepileptic drug ethosuximide. Also, compound 9a was devoid of hepatotoxicity indicated by measurements of serum level of ALT, AST, ALP, albumin and total protein. Furthermore, treatment with compound 9a significantly increased the GABA brain level by 2.56-folds compared to the control value. Additionally, molecular docking was performed on the active site of GABA-AT to clarify the interactions of the most potent compound 9a with the enzyme. In MES test, compound 12a exhibited the most potent activity against electric stimuli-induced seizures with the lowest ED₅₀ = 13.7 mg/kg and protective index >36.5. Both candidates 9a and 12a could be a good starting point to develop new molecules as novel antiepileptic drugs.

Full text of DOI:10.1016/j.bioorg.2019.103473

BioorganicChemistryxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design and synthesis of novel parabanic acid derivatives as anticonvulsants
Mona Elsayed Aboutabla, Rasha Mohamed Hassanb, Aida Abdel-Sattar El-Azzounyb,
⁎
Mohamed Nabil Aboul-Eneinb, , Walaa Hamada Abd-Allahc
a Medicinal and Pharmaceutical Chemistry Department (Pharmacology Group), Pharmaceutical and Drug Industries Research Division, National Research Centre (ID:
60014618), P.O. 12622, Dokki, Giza, Egypt
b Medicinal and Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre (ID: 60014618), P.O. 12622,
Dokki, Giza, Egypt
c Pharmaceutical Chemistry Department, College of Pharmaceutical Science and Drug Manufacturing, Misr University for Science & Technology, P.O. 77, 6th of October
City, Giza, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this work a set of novel derivatives of parabanic acid 9a-d, 12a-d and 13a-d was synthesized and their an-
ticonvulsant potential was evaluated. All the compounds under investigation exhibited anticonvulsant activity in
both scPTZ and MES tests. In phase II anticonvulsant study, the trimethoxy phenyl derivative 9a evoked the highest
potency among the tested compounds in scPTZ test. It displayed 1.72- and 17.05-folds activity more than the
standard drugs phenobarbital and ethosuximide, respectively. In addition, the margin of safety for compound 9a is
better than that of the reference antiepileptic drug ethosuximide. Also, compound 9a was devoid of hepatotoxicity
indicated by measurements of serum level of ALT, AST, ALP, albumin and total protein. Furthermore, treatment
with compound 9a significantly increased the GABA brain level by 2.56-folds compared to the control value.
Additionally, molecular docking was performed on the active site of GABA-AT to clarify the interactions of the
most potent compound 9a with the enzyme. In MES test, compound 12a exhibited the most potent activity against
electric stimuli-induced seizures with the lowest ED50 = 13.7 mg/kg and protective index > 36.5. Both candidates
9a and 12a could be a good starting point to develop new molecules as novel antiepileptic drugs.
Synthesis
Anticonvulsants
Parabanic acids
GABA
1. Introduction
known antiepileptic drugs [6]. Accordingly, the development of novel
anticonvulsant agents could be achieved via screening investigations or
Epilepsy is a chronic neurological disorder affecting globally about
70 million people [1]. In spite of the discovery of new antiepileptic
drugs (AEDs) in recent years about 20–40% of epileptic patients suffer
from drug resistance to the currently used medications [2]. Moreover,
their use is accompanied by many untoward effects that affect quality of
life as ataxia, megaloblastic anemia as well as liver toxicity [3,4].
Therefore, a search for novel AEDs is importunate. The mechanism of
action of the marketed AEDs could be classified into: (i) enhancement of
action of the inhibitory neurotransmitter γ-aminobutyric acid (GABA)
by allosteric modulation of GABAA receptor or inhibition of GABA
aminotransferase (GABA-AT) which is responsible for GABA degrada-
tion, (ii) modulation of sodium and calcium gated channels and/or (iii)
inhibition of synaptic excitation activated by glutamate receptors [5].
The use of target-based studies in discovery of novel antiepileptics is
uncommon due to the insufficiency in information of molecular
pathway of epilepsy as well as complex mode of action for most of
use of hybridization approach [7]. The latter depends on using two or
more pharmacophoric scaffolds, each with remarkable pharmacological
effect to be gathered in one single structure to obtain new components
with better biological effect. Amide and imide linkages are important
fragments of anticonvulsants. They are found in chain form or as a part
of heterocyclic ring (Fig. 1A) [8]. According to literature survey, there
is a paucity of studying parabanic acids which are considered as cyclic
imides as anticonvulsants [9,10]. Moreover, parabanic acid is con-
sidered as a lower homologue of barbituric acid which is the basic
nucleus in the antiepileptic drug phenobarbital that acts by enhance-
ment of GABA action (Fig. 1B).
On the other hand, the 1,3-benzodioxole ring system is the core
moiety of the antiepileptic drug stiripentol which has a GABA enhance-
ment effect [11] as well as several synthetic anticonvulsant candidates as
compounds I [12] and II [13]. Also, the higher homologue of 1,3-ben-
zodioxole, 1,4-benzodioxine, is found in the structure of some
⁎ Corresponding author at: Medicinal and Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre,
12622 Dokki, Giza, Egypt.
E-mail address: mnaboulenein@yahoo.com (M.N. Aboul-Enein).
https://doi.org/10.1016/j.bioorg.2019.103473
Received 2 August 2019; Received in revised form 21 October 2019; Accepted 23 November 2019
0045-2068/©2019ElsevierInc.Allrightsreserved.
Pleasecitethisarticleas:MonaElsayedAboutabl,etal.,BioorganicChemistry,https://doi.org/10.1016/j.bioorg.2019.103473

M.E. Aboutabl, et al.
BioorganicChemistryxxx(xxxx)xxxx
Fig. 1. (A) Structures of some AEDs containing amide and imide bonds, (B) Structure similarity between parabanic acid and barbituric acid, (C) Structures of some
anticonvulsants having 1,3-benzodioxoles and 1,4-benzodioxines moieties.
anticonvulsant agents as JNJ-26489112 [14] (Fig. 1C). With this back-
ground in mind and as a continuation of our work in the field of dis-
covery new anticonvulsant agents [15–20] parabanic acid conjugates
with trimethoxy phenyl moiety, 1,3-benzodioxoles or 1,4-benzodioxines
having the general structural formulae 9a-d, 12a-d and 13a-d (Fig. 2)
were synthesized for the development of new effective anticonvulsant
agents. These new hydrids fulfill the essential pharmacophoric features
which are generally needed for the anticonvulsant activity [21] as hy-
drophobic domain (R), electron donor atom (D) as well as hydrogen
donor/acceptor unit (HAD) (Fig. 2). The screening of the anticonvulsant
activity of the new candidates 9a-d, 12a-d and 13a-d was performed
using subcutaneous pentylenetetrazole (scPTZ) and maximal electric
shock seizure (MES) models. Moreover, the most active compounds were
evaluated for their median effective dose (ED50), median toxic dose
(TD50), protective index (PI) as well as neurotoxic and hepatotoxic side
effects. Furthermore, the most active compound in scPTZ test was further
investigated to measure its effect on GABA brain level. In addition,
molecular docking study was performed for the most active compound
on the active site of GABA-AT enzyme to study its GABAergic activity.
prepared from the corresponding acid 1 using PTSA as a catalyst [22].
This ester is subjected to either complete methoxylation with methyl
iodide to obtain methyl 3,4,5-trimethoxybenzoate (3) [23] or selective
methoxylation using dimethyl sulphate and sodium borate to achieve
methyl 5-methoxygallate (5) [24]. Cyclization of the latter using either
dichloromethane (DCM) or 1,2-dichloroethane (DCE) yielded the 1,3-
benzodioxole and 1,4-benzodioxine congeners 6a [25] and 6b [26], re-
spectively. The hydrazides 4 [27], 7a [28] and 7b were obtained by
hydrazinolysis of the corresponding esters 3, 6a and 6b, respectively. The
new semicarbazides 8a-d, 10a-d and 11a-d were prepared by reaction of
hydrazides 4, 7a or 7b, respectively, with the appropriate isocyanate in
1,2-dichloroethane. Subsequent cyclization of 8a-d, 10a-d and 11a-d
using oxalyl chloride in benzene afforded the parabanic acids 9a-d, 12a-
d and 13a-d, respectively. The structures of the new parabanic acids 9a-
d, 12a-d and 13a-d were confirmed in 1H NMR by presence of only one
signal of NH at 11.3 ppm as well as in 13C NMR by existence of three
carbonyl carbons characteristic for parabanic acid ring at 150–155 ppm.
In the current study, the structures of the novel derivatives were further
substantiated through elemental analyses and other spectral data.
2. Results and discussion
2.2. Pharmacology
2.1. Chemistry
2.2.1. Anticonvulsant activity
2.2.1.1. Phase I anticonvulsant evaluation. The phase I screening was
performed according to the standard procedure of Antiepileptic Drug
Development (ADD) program of the National Institute of Neurological
The designed parabanic acids 9a-d, 12a-d and 13a-d were prepared
as illustrated in scheme 1. The key intermediate methyl gallate (2) was
2

M.E. Aboutabl, et al.
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Fig. 2. Structure of some antiepileptics and the target compounds 9a-d, 12a-d and 13a-d, with essential pharmacophoric structural features required for antic-
onvulsant activity.
Disorders and Stroke (NINDS), Epilepsy section [29]. It consists of using
the “gold standards” screen tests; scPTZ and MES tests. The
anticonvulsant activity of the synthesized parabanic acid derivatives
9a-d, 12a-d and 13a-d were evaluated for scPTZ and MES screen tests.
These screen tests were used for phase I evaluations in order to identify
the best candidates for phase II anticonvulsant evaluation. The
compounds were administered intraperitoneally at doses of 30, 100
and 300 mg/kg at 0.5 h and 4 h interval. The data recorded in Table 1
represents the minimum dose demonstrated protection in half or more
of the mice. Neurotoxicity was evaluated by rotarod test (c.f. Table 1).
In phase I preliminary anticonvulsant screening, scPTZ screen test is
used to identify compounds elevating the seizure threshold [30]. In the
current study all compounds displayed activity in scPTZ which in-
dicated their positive efficacy against generalized myoclonic seizures.
The tested compounds 9a-d, 12a, 12b and 13a-d exhibited protection
at 0.5 h and 4 h at the dose level of 30 mg/kg revealing their rapid onset
and longer duration of action (c.f. Table 1).
MES screen test is used to identify compounds having potential ef-
fect against generalized tonic-clonic seizures [30]. All compounds 9a-d,
12a-d and 13a-d demonstrated protection in MES test. Compounds 9d,
12a, 12c, 13b and 13d showed protection from seizures at the lowest
dose 30 mg/kg after 0.5 h and 4 h revealing their rapid onset and longer
duration of action at dose 30 mg/kg. Compounds 9c and 12b at dose
level of 30 mg/kg displayed long duration of anticonvulsant activity in
MES test at 4 h, while at higher dose level (100 mg/kg) they exert
anticonvulsant effect at 0.5 h. Compounds 12d, 13a and 13c were ac-
tive after 0.5 and 4 h of compound administration at 100 mg/kg. Both
compounds 9a and 9b evoked quick onset of action at 0.5 h at higher
doses of 300 and 100 mg/kg, respectively and long duration of action at
4 h at doses of 100 and 300 mg/kg, respectively. All the tested com-
pounds were devoid of neurotoxicity at the maximum administered
dose (300 mg/kg) (c.f. Table 1).
2.2.1.2. Phase II anticonvulsant evaluation. In this study, the selected
compounds 9a, 13a and 13c (scPTZ) and 12a, 13b and 13d (MES) were
found to have significant anticonvulsant potential as they demonstrated
the highest percentage of protection at dose 30 mg/kg at both 0.5 h and
4 h. The encouraging results of phase I study urged the study of phase II
anticonvulsant evaluation which focused on the quantitative
pharmacological parameters.
Compounds 12d and 12c exhibited rapid onset of action (0.5 h) at
dose level of 30 mg/kg and 100 mg/kg, respectively. Surprisingly,
compound 12c exhibited long duration of action (4 h) at dose 30 mg/
kg. Compound 12d demonstrated a protective effect at low dose in the
short time interval while needed a higher dose in order to exert an
effect at the longer time interval demonstrating its shorter duration of
action and that its effect fades in short time because the low dose was
not effective in the 4 h interval and when increased to the next dose
level the effect was improved. Such effect was found in the reference
drug ethosuximide. Compounds 9a-d, 12a, 12b, 12dand 13a-d showed
greater potency than the standard drugs phenobarbital and ethosux-
imide at 0.5 h, in addition they exhibited similar potency compared to
phenobarbital at 4 h, and higher anticonvulsant activity than etho-
suximide at 4 h (c.f. Table 1).
The median effective dose (ED50) as well as the protective index (as
a ratio of TD50 and ED50) values for the above mentioned compounds
were calculated (c.f. Tables 2 and 3). Compound 9a displayed the po-
tent anticonvulsant activity against scPTZ test with the lowest
ED50 = 7.656 mg/kg followed by compound 13c (ED50 = 8.884 mg/
kg) then compound 13a (ED50 = 10.37 mg/kg). In MES test, compound
12a exhibited the most potent activity against electric stimuli-induced
seizures with the lowest ED50 = 13.7 mg/kg followed by compound
13b and 13d which showed an equipotent activity ED50 = 26.59 mg/
kg and 26.79 mg/kg, respectively (c.f. Tables 2 and 3).
In the scPTZ model, the seizure protection of the tested candidates is
possibly due to GABA-mediated mechanisms, where seizures were
mostly blocked by AEDs acting at the GABAA receptor such as barbi-
turates, valproate and benzodiazepines [31].
The protective index (PI), which is the ratio of TD50 to ED50
(PI = TD50/ED50), of the most active compounds 9a, 13a and 13c in
3

M.E. Aboutabl, et al.
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Scheme 1. Synthesis of target compounds 9a-d, 12a-d and 13a-d. Reagents and conditions: (i) PTSA, Methanol, 65 °C, 18 h. (ii) CH3I, K2CO3, DMF, 85 °C, 2 days. (iii)
98% Hydrazine hydrate, MeOH, 65 °C, 6 h. iv) Sodium borate, NaOH, (CH3O)2SO4, r.t., overnight. (v) Dichloromethane or 1,2-dichloroethane, K2CO3, DMF, 90 °C,
12 h. (vi) Isocynate derivative, 1,2-dichloroethane, reflux, 3 h. (vii) (COCl)2, benzene, reflux, 3 h.
scPTZ test demonstrated values in the range of > 48.22 to > 65.31
which were much higher than that of the reference drug ethosuximide
having PI value = 2.68 (Table 2). Moreover, the most active com-
pounds 12a, 13b and 13d in MES test showed PI values in the range
of > 18.66 to > 36.50 which were much higher than phenytoin as
reference drug having PI value = 6.59 (Table 3).
This indicated no signs of hepatotoxicity.
Moreover, in acute toxicity study, all animals were devoid of acute
toxicity signs till up to 500 mg/kg except 12d.
2.2.2. γ-Aminobutyric acid estimation
It has become increasingly evident that the significant AED design
and development can be reached only by observing the mode of drug
action and cellular targets of epilepsy. Therefore, to demonstrate the
mechanism of action, the effect of the promising compound 9a (it was
selected due to having the lowest ED50 in sc-PTZ test) on GABA neu-
rotransmitter level in the whole mice brain was investigated. GABA
estimation data showed that compound 9a increased the GABA level
(54.07 ug/g brain tissue) significantly as compared to standard drug
bromazepam (30.21 ug/g brain tissue) and control (21.08 ug/g brain
tissue) (Fig. 3). Thus, it could be speculated that the anticonvulsant
potential of these parabanic derivatives might be related to the in-
creased GABA level in mice brain which is the main inhibitory
2.2.1.3. Hepatotoxicity. Additionally, the most active compounds 9a,
13a, 13c (scPTZ), compounds 12a, 13b and 13d (MES) were tested for
the possible hepatotoxic effect through assessment of serum enzyme
activity assays. Elevation of liver enzymes is considered as specific
markers of liver dysfunction. The liver enzymes analysis data including:
alanine aminotransferase (ALT), aspartate aminotransferase (AST), and
alkaline phosphatase (ALP), as well as albumin and total protein serum
levels were represented as mean
SEM (c.f. Table 4). Accordingly, the
above mentioned compounds did not show any significant change in
serum enzymes level, albumin and total protein compared to control.
4

M.E. Aboutabl, et al.
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Table 1
neurotransmitter in the brain. Enhancement of the GABA system can be
attributed to the following: (1) direct binding to the GABA-A receptors
which results in increasing the influx of chloride ion which leads to
more negative resting membrane potential and thus decreasing the
excitability of the postsynaptic membrane, (2) blocking of presynaptic
GABA re-uptake through blocking the GABA transporter 1 by GABA re-
uptake inhibitors, and consequently inhibition of GABA re-uptake at the
synapse, (3) inhibiting the metabolism of GABA via inhibition of GABA
transaminase enzyme, (4) increasing GABA synthesis via modulation of
glutamic acid decarboxylase enzyme which enhance the production of
GABA through the decarboxylation of glutamate [32].
Anticonvulsant activity and neurotoxicity of the synthesized compounds 9a-d,
12a-d and 13a-d.
Compound
scPTZ (mg/kg)
MES (mg/kg)
Neurotoxicity
0.5 h
4 h
0.5 h
4 h
0.5 h
4 h
9a
30
30
30
30
30
30
100
30
30
30
30
30
100
30
30
30
30
30
30
30
100
30
30
30
30
30
300
100
100
30
100
300
30
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
9b
9c
9d
30
12a
30
30
12b
100
30
30
12c
30
12d
100
100
30
100
100
30
2.3. Structure activity relationship (SAR)
13a
13b
13c
100
30
100
30
In the scPTZ test according to the above mentioned results of phase I
anticonvulsant evaluation, the substitution in the 1,3-benzodioxoles
parabanic acids hybrids 12c and 12d has an impact on the dose re-
quired to achieve the rapid onset and long duration of action. In com-
pound 12c which is substituted with long aliphatic side chain the dose
needed to reach rapid onset of action (0.5 h) is 100 mg/kg but the
anticonvulsant effect was maintained for long time (4 h) at 30 mg/kg
only. However, the opposite occurred in compound 12d which is sub-
stituted with aralkyl side chain. Meanwhile, the substitution pattern on
the side chain of the parabanic acid does not change the onset or
duration of action of the anticonvulsant effects of both trimethoxy
phenyl and 1,4-benzodioxines parabanic acids hybrids as shown in
compounds 9a-d and 13a-d, respectively. By analysis of the quantita-
tive results obtained from phase II anticonvulsant evaluation, it is clear
that the trimethoxy phenyl derivative 9a has more potency than the
corresponding 1,4-benzodioxine analogue 13a. Also, substitution with
longer aliphatic side chain (n-butyl) in the 1,4-benzodioxines accen-
tuated the activity than substitution with shorter aliphatic side chain (n-
propyl) as shown in compounds 13c and 13a, respectively.
13d
Phenobarbital
Phenytoin
Ethosuximide
30
30
100
300
The mice received dose of 30, 100 and/or 300 via intraperitoneal administra-
tion.
The data represented in the table indicates the minimum dose demonstrated
protection in half or more of the mice (n = 4). The animals were examined at
0.5 h and 4 h after compounds administration. The dash (–) indicates absence of
activity at the maximum dose administered (300 mg/kg).
Table 2
The quantitative pharmacological parameters ED50, TD50 and PI in scPTZ test.
Compound
ED50 (mg/kg)
TD50 (mg/kg)
PI (TD50
/
ED50
)
9a
7.656 (9.56–5.94)
10.37 (12.17–8.70)
8.884 (11.79–6.40)
130.55
> 500
> 65.31
> 48.22
> 56.28
2.68
13a
> 500
13c
> 500
Ethosuximide*
350.10
Regarding the MES test, in the trimethoxy phenyl series 9a-d
compounds substituted with aliphatic side chains 9a-c have rapid onset
of action at doses greater than that of the benzyl derivative 9d.
However, compounds that bear long aliphatic (9c) or aralkyl side chain
(9d) have long duration of action at a dose level lower than that needed
for the shorter aliphatic side chain (9a and 9b). Concerning the 1,3-
benzodioxoles 12a-d, the dose needed to achieve rapid onset is only
30 mg/kg in the derivatives substituted with normal aliphatic side
chains (12a and 12c). While, compounds substituted with branched
aliphatic (12b) or aralkyl (12d) side chain needed a higher dose
(100 mg/kg) to give a rapid onset. It was observed that 1,3-benzo-
dioxoles derivatives having an aliphatic side chain (12a-c) could
maintain a long duration of action (4 h) at a dose level lower than that
required for the benzyl analogue 12d. On the other hand, in the series
of 1,4-benzodioxines 13a-d compounds that have branched aliphatic
(13b) or aralkyl (13d) side chain required a lower dose to achieve rapid
onset and long duration of action than those substituted with normal
aliphatic side chains (13a and 13c). From the quantitative results in the
(177.87–98.78)
13.2 (15.90–6.80)
(346.20–268.90)
nd
Phenobarbital*
nd
ED50: median effective dose in scPTZ test.
TD50: median toxic dose in neurotoxicity test (rotarod test).
PI: Protective index. *Data from Reference [1]. nd: not determined.
Table 3
The quantitative pharmacological parameters ED50, TD50 and PI in MES test.
Compound
ED50 (mg/kg)
TD50 (mg/kg)
PI (TD50/ED50)
12a
13.7 (15.26–12.25)
26.59 (27.52–25.69)
26.79 (28.3–23.78)
9.50 (10.96–7.24)
> 500
> 36.50
> 18.80
> 18.66
6.59
13b
> 500
13d
> 500
Phenytoin*
62.64 (69.82–50.52)
ED50: median effective dose in MES test.
TD50: median toxic dose in neurotoxicity test (rotarod test).
PI: Protective index. *Data from Ref. [1].
Table 4
Liver enzyme, albumin and total protein determination of the most active compounds 9a, 12a and 13a-d.
Compd. No.
ALT (U/L)
AST (U/L)
ALP (U/L)
Albumin (g/dL)
Total protein (g/dL)
Control
9a
22.80
23.80
23.30
23.25
21.55
23.93
21.55
2.29
0.25
4.13
1.10
0.89
1.09
2.51
74.31
71.68
70.04
70.04
68.95
76.67
66.13
0.66
9.37
8.60
4.55
6.27
11.61
6.53
12.35
12.28
12.13
11.89
12.33
12.47
12.59
0.53
1.47
0.89
2.11
0.58
0.63
0.56
3.31
3.47
3.14
3.39
3.30
3.43
3.43
0.22
0.12
0.16
0.11
0.16
0.14
0.10
7.64
7.49
7.71
7.60
7.31
7.44
7.88
0.54
0.30
0.18
0.23
0.12
0.19
0.49
12a
13a
13b
13c
13d
Data are represented as mean
SEM and were analyzed by ANOVA followed by Student Newman Keul post hoc comparison, n = 6. ALT, alanine aminotransferase;
AST, aspartate aminotransferase; ALP, alkaline phosphatase.
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For this study, the coordinates corresponding to chain A were ex-
tracted. The active site residues of chain A consist of His44A, Tyr69A,
Ile72A, Gly136A, Ser137A, Phe189A, Arg192A, Lys203A, Ile205A,
His206A, Glu265A, Ser269A, Glu270A, Glu298A, Val300A, Lys329A,
Arg422A, Asn423A, Ile426A, Gly438A, Gly440A [5]. Concerning the
GABA-AT inhibitor, it showed three hydrogen bond interactions with
Arg 192, Lys 329 and Glu 270. Additionally, the GABA-AT inhibitor
showed Van der Waals interaction with Ile 72, Phe 189, Val 300, Hist
206, Arg445 and Gln301 residues. Compound 9a formed five hydrogen
bond interactions, one with Arg192 residue through carbonyl moiety,
the second one with Ser 137 residue via methoxy group, two hydrogen
bond with Lys 329 through carbonyl moiety and one hydrogen bond
with Arg 445 through carbonyl moiety. Lipophilicity plays a crucial role
in the inhibitory effect of compound 9a, it showed Van der Waals in-
teraction with Phe 189 and Gly 191 residues. Furthermore, compound
9a showed pi-alkyl interaction with Ile 72 residue and pi-anion inter-
action with Glu 270 residue. The results revealed good interaction of
compound 9a with GABA-AT enzyme which might be the cause of the
observed increase in GABA brain level.
Fig. 3. Effect of compound 9a on GABA brain level. Results are presented as
mean
SEM. *P < 0.05 compared with control value. # P < 0.05 compared
with bromazepam value. $P < 0.05 compared with compound 9a value.
3. Conclusion
phase II anticonvulsant evaluation it is clear that substitution pattern
doesn’t affect the anticonvulsant activity in the 1,4-benzodioxines
where both the isopropyl (13b) and benzyl (13d) derivatives have
equipotent activity. It is worth to mention that in both scPTZ and MES
tests the most active compounds is n-propyl derivative as represented in
9a and 12a, respectively.
The anticonvulsant activity of the novel parabanic acid derivatives
9a-d, 12a-d and 13a-d was evaluated. All compounds exhibited an-
ticonvulsant effect in both scPTZ and MES tests. The trimethoxy phenyl
derivative 9a demonstrated the highest potency among the tested
compounds in scPTZ test as well as exhibiting high safety margin
compared with ethosuximide as reference drug. Compound 9a was
devoid of neurotoxicity and hepatotoxicity. Furthermore, it sig-
nificantly increased the GABA brain level by 2.56- and 1.79-folds
compared to control and bromazepam, respectively. Additionally, mo-
lecular docking study of compound 9a illustrated good binding mode
and interaction on the active site of GABA-AT. All results revealed that
this compound could be a promising antiepileptic agent. In MES test,
compound 12a exhibited the most potent activity against electric sti-
muli-induced seizures with the lowest ED50 = 13.7 mg/kg and pro-
tective index > 36.5. Both candidates 9a and 12a could be a good
starting point to develop new molecules as novel antiepileptic drugs
2.4. Molecular modeling
To obtain more proof regarding the GABAergic effect of compound
9a, a molecular docking study was performed using Discovery Studio
2016 program on γ-amino butyric acid aminotransferase (GABA-AT)
enzyme. The latter is responsible for GABA metabolism and conse-
quently its inhibition increase GABA level in the brain [33]. GABA-AT
used in this study was obtained from PDB, code: 1OHW [5]. The
binding mode and interaction of GABA-AT inhibitor and compound 9a
are illustrated in Fig. 4.
Fig. 4. 2D Interaction diagram (A) for co-crystallized ligand, (B) for compound 9a.
6

M.E. Aboutabl, et al.
BioorganicChemistryxxx(xxxx)xxxx
4. Experimental
3H, J = 8 Hz, CH3), 1.24–1.30 (m, 2H, CH2eCH3),1.34–1.40 (m, 2H,
CH2eCH2eCH3), 3.03 (q, 2H, J = 8 Hz, CH2eCH2eCH2eCH3), 3.71 (s,
3H, OCH3), 3.83 (s, 6H, 2OCH3), 6.45 (s, 1H, NH), 7.23 (s, 2H, Har.),
7.79 (s, 1H, NH), 10.07 (s, 1H, NH).13C NMR: 13.75 (CH3), 19.46
(CH2eCH3), 32.04 (CH2eCH2eCH3), 40.21 (CH2eCH2eCH2eCH3),
56.02 (2OCH3), 60.11 (OCH3), 105.08, 127.82, 140.24, 152.57
(aromatic carbons), 158.42, 164.70 (2C]O), MS (EI) m/z (%):
325.12 (M+, 22.3%), Anal. calcd. for C15H23N3O5: C, 55.37; H, 7.13;
N, 12.92. Found: C, 55.31; H, 7.22; N, 13.33.
4.1. Chemistry
Melting points were measured by the Electrothermal Capillary ap-
paratus and were uncorrected. Infrared spectra were recorded as KBr
pellets with [JASCO FT/IR-6100 spectrometer] and the values are
characterized in cm−1
.
1H NMR (400 MHz) and 13C NMR (100 MHz)
spectra were performed on [Jeol ECA 500 MHz spectrometer] and
chemical shift values were recorded in ppm. Mass spectral data were
measured with electron impact ionization technique at 70 eV.
Elemental microanalyses were performed at Microanalytical Units in
the National Research Centre as well as Cairo University.
4.1.2.4. N-Benzyl-2-(3,4,5-trimethoxybenzoyl)hydrazine-1-carboxamide
(8d). White solid, m.p. 179 °C, yield 88%, IR (KBr, cm−1) 3444, 3326,
3132 (3NH), 1752, 1683 (2C]O); 1H NMR: 3.71 (s, 3H, OCH3), 3.84 (s,
6H, OCH3), 4.24 (s, 2H, CH2eC6H5), 6.95 (s, 1H, NH), 7.22–7.76 (m,
7H, Har.), 8.00 (s, 1H, NH), 10.14 (s, 1H, NH). 13C NMR: 43.12
(CH2eC6H5), 56.50 (2OCH3), 60.57 (OCH3), 105.62, 126.93, 126.98,
127.41, 128.26, 128.53, 128.57, 140.75, 153.02 (aromatic carbons),
159.01, 166.27 (2C]O), MS (EI) m/z (%): 359.4 (M+, 17%), Anal.
calcd. for C18H21N3O5: C, 60.16; H, 5.89; N, 11.96. Found: C, 60.22; H,
6.13; N, 12.20.
Preparation of the intermediates 2, 3, 4, 5, 6 and 7a was performed
according to reported procedures [18–24].
4.1.1. Synthesis
of
8-methoxy-2,3-dihydrobenzo[b][1,4]dioxine-6-
carbohydrazide (7b)
A mixture of 5 mmol of ester 6b and 25 mmol of hydrazine hydrate
in methanol was refluxed for five days. After cooling a white precipitate
of the hydrazide 7b was formed and filtered off. Yield 80%; m.p.
178–180 °C, IR (KBr, cm−1) 3436, 3298 (NH, NH2), 1611 (C]O); 1H
NMR (DMSOd6, δ, ppm): 3.89 (s, 3H, OCH3), 4.20 (s, 4H, 2CH2-benz-
dioxine), 4.39 (s, 2H, NH2), 6.98–7.03 (m, 2H, Har.), 9.58 (s, 1H, NH);
13CNMR (DMSOd6, δ, ppm): 56.32 (OCH3), 64.39, 64.511 (2CH2-
benzdioxine), 103.87, 109.27, 125.37, 136.05, 143.79, 148.88 (aro-
matic carbons), 165.84 (C]O); MS (EI) m/z (%): 224 (M+, 14); Anal.
calcd. for C10H12N2O4: C, 53. 57; H, 5.39; N, 12.49. Found: C, 53.71; H,
5.50; N, 12.32.
4.1.2.5. 2-(7-Methoxybenzo[d][1,3]dioxole-5-carbonyl)-N-
propylhydrazine-1-carboxamide (10a). White solid, m.p. 181 °C, yield
85%, IR (KBr, cm−1) 3430, 3097, 2929 (3NH), 1633, 1565 (2C]O); 1H
NMR (DMSOd6, δ, ppm): 0.84 (t, 3H, J = 8 Hz, CH3), 1.36–1.45 (m, 2H,
CH2eCH3), 3.00 (t, 2H, J = 8 Hz, CH2eCH2eCH3), 3.88 (s, 3H, OCH3),
6.09 (s, 2H, CH2-benzodioxole), 6.48 (s, 1H, NH), 7.14–7.27 (m, 2H,
H
ar.), 7.76 (s, 1H, NH), 9.97 (s, 1H, NH). 13C NMR (DMSOd6, δ, ppm):
11.70 (CH3), 23.54 (CH2eCH3), 41.45 (CH2eCH2eCH3), 56.80 (OCH3),
102.05 (CH2-benzodioxole), 102.48, 108.55, 127.34, 138.12, 143.26,
148.71 (aromatic carbons), 158.89, 165.82 (2C]O), MS (EI) m/z (%):
295 (M+, 13.27), Anal. calcd. for C13H17N3O5: C, 52.88; H, 5.80; N,
14.23. Found: C, 52.61; H, 5.82; N, 14.21.
4.1.2. General procedures for synthesis of N-alkyl/aralkyl-2-(3,4,5-
trimethoxybenzoyl)hydrazine-1-carboxamide (8a-d), 2-(7-methoxybenzo
[d][1,3]dioxole-5-carbonyl)-N-alkyl/aralkylhydrazine-1-carboxamide
(10a-d) and 2-(8-methoxy-2,3-dihydrobenzo[b][1,4]dioxine-6-carbonyl)-
N-propylhydrazine-1-carboxamide (11a-d)
4.1.2.6. N-Isopropyl-2-(7-methoxybenzo[d][1,3]dioxole-5-carbonyl)
hydrazine-1-carboxamide (10b). White solid, m.p.189 °C, yield 90%, IR
(KBr, cm−1) 3448, 3333, 3082 (3NH), 1691, 1631 (2C]O); 1H NMR
(DMSOd6, δ, ppm): 1.07 (d, J = 6.4 Hz, 6H, CHe(CH3)2), 3.40 (q, 1H,
J = 7.5 Hz, CHe(CH3)2), 3.88 (s, 3H, OCH3), 6.09 (s, 2H, CH2-
benzodioxole), 6.25 (s, 1H, NH), 7.14–7.27 (s, 2H, Har.), 7.72 (s, 1H,
NH), 10.00 (s, 1H, NH).13C NMR (DMSOd6, δ, ppm): 23.34 (2CH3),
41.62 (CHe(CH3)2), 56.9 (OCH3), 101.98 (CH2-benzodioxole), 102.47,
108.08, 127.26, 138.18, 143.27, 148.73 (aromatic carbons), 158.14,
165.77 (2C]O), MS (EI) m/z (%): 295 (M+, 1.38), Anal. calcd. for
A mixture of 0.01 mol of hydrazide 4, 7a or 7b and 0.01 mol of the
appropriate isocyante derivative in 30 ml dichloroethane was refluxed
for 3 h. The formed white precipitate was filtered off then washed with
diethyl ether to afford 8a-d, 10a-d or 11a-d, respectively.
4.1.2.1. N-Propyl-2-(3,4,5-trimethoxybenzoyl)hydrazine-1-carboxamide
(8a). White solid, m.p. 179 °C, yield 85%, IR (KBr, cm−1) 3770, 3442,
2927 (3NH), 1639, 1594 (2C]O); 1H NMR (DMSOd6, δ, ppm): 0.83 (t,
3H, J = 8 Hz, CH3), 1.36–1.42 (m, 2H, CH2eCH3), 3.00 (t, 2H,
J = 8 Hz, CH2eCH2eCH3), 3.71 (s, 6H, 2OCH3), 3.83 (s, 3H, OCH3),
6.48 (s, 1H, NH), 7.23 (s, 2H, Har.), 7.81 (s, 1H, NH), 10.08 (s, 1H,
NH).13C NMR (DMSOd6, δ, ppm): 11.71 (CH3), 23.55 (CH2eCH3),
41.46 (CH2eCH2eCH3), 56.48 (2OCH3), 58.48 (OCH3), 105.52,
128.25, 140.68, 153.90 (aromatic carbons), 158.90, 166.14 (2C]O),
MS (EI) m/z (%): 311.5 (M+, 10%), Anal. calcd. for C14H21N3O5: C,
54.01; H, 6.80; N, 13.50. Found: C, 54.12; H, 6.85; N, 13.52.
C
13H17N3O5: C, 52.88; H, 5.80; N, 14.23. Found: C, 52.86; H, 5.83; N,
14.25.
4.1.2.7. N-Butyl-2-(7-methoxybenzo[d][1,3]dioxole-5-carbonyl)
hydrazine-1-carboxamide (10c). White solid, m.p. 164 °C, yield 82%, IR
(KBr, cm−1) 3446, 3271, 3086 (3NH), 1687, 1626 (2C]O). 1H NMR
(DMSOd6, δ, ppm): 0.87 (t, 3H, J = 7.2 Hz, CH3), 1.24–1.30 (m, 2H,
CH2eCH3), 1.36–1.40 (m, 2H, CH2eCH2eCH3), 3.03 (q, 2H,
J = 6.6 Hz, CH2eCH2eCH2eCH3), 3.87 (s, 3H, OCH3), 6.08 (s, 2H,
CH2-benzodioxole), 6.44 (s, 1H, NH), 7.14 (s, 1H, Har.), 7.26 (s, 1H,
4.1.2.2. N-Isopropyl-2-(3,4,5-trimethoxybenzoyl)hydrazine-1-
carboxamide (8b). White solid, , m.p. 181 °C, yield 90%, IR (KBr, cm−1
)
3777, 3440, 2930 (3NH), 1645, 1590 (2C]O); 1H NMR (DMSOd6, δ,
ppm): 1.04 (d, 6H, J = 8 Hz, CHe(CH3)2), 3.36 (q, 1H, J = 7.5 Hz,
CHe(CH3)2), 3.71 (s, 3H, OCH3), 3.82 (s, 6H, 2OCH3), 6.25 (s, 1H, NH),
7.23 (s, 2H, Har.), 7.74 (s, 1H, NH), 10.08 (s, 1H, NH). 13C NMR: 22.96
(2CH3), 41.22 (CHe(CH3)2), 56.03 (2OCH3), 60.12 (OCH3), 105.04,
127.79, 140.27, 152.61 (aromatic carbons.), 157.70, 165.65 (2C]O),
MS (EI) m/z (%): 311.3 (M+, 64.2%), Anal. calcd. for C14H21N3O5: C,
54.01; H, 6.80; N, 13.50. Found: C, 54.05; H, 6.75; N, 13.51.
H
ar.), 7.74 (s, 1H, NH), 9.97 (s, 1H, NH); 13C NMR (DMSOd6, δ, ppm):
14.19 (CH3eCH2), 19.89 (CH2eCH3), 32.46 (CH2eCH2eCH3), 40.60
(CH2eCH2eCH2eCH3), 56.82 (OCH3), 102.05 (CH2-benzodioxole),
102.48, 108.57, 127.38, 138.13, 143.27, 148.73 (aromatic carbons.),
158.85, 165.83 (2C]O), MS(EI) m/z (%): 309 (M+, 8%), Anal. calcd.
for C14H19N3O5: C, 54.36; H, 6.19; N, 13.58. Found: C, 54.32; H, 6.21;
N, 13.63.
4.1.2.8. N-Benzyl-2-(7-methoxybenzo[d][1,3]dioxole-5-carbonyl)
hydrazine-1-carboxamide (10d). White solid, m.p. 172 °C, yield 95%, IR
(KBr, cm−1) 3375, 3259, 3071 (3NH), 1691, 1615 (2C]O); 1H NMR
(DMSOd6, δ, ppm): 3.82 (s, 3H, OCH3), 4.19 (s, 2H, CH2eC6H5), 6.04 (s,
4.1.2.3. N-Butyl-2-(3,4,5-trimethoxybenzoyl)hydrazine-1-carboxamide
(8c). White solid, m.p. 185 °C, yield 80%, IR (KBr, cm−1) 3446, 3271,
3086 (3NH), 1685, 1644 (2C]O); 1H NMR (DMSOd6, δ, ppm): 0.87 (t,
7

M.E. Aboutabl, et al.
BioorganicChemistryxxx(xxxx)xxxx
2H, CH2-benzodioxole), 7.04–7.27 (m, 7H, Har.), 7.93 (s, 1H, NH),
10.03 (s, 1H, NH); 13C NMR (DMSOd6, δ, ppm): 40.18 (CH2eC6H5),
56.89 (OCH3), 102.20 (CH2-benzodioxole), 102.56, 108.71 127.04,
127.47, 128.63, 132.31, 138.22, 141.15, 143.34, 148.79 95 (aromatic
carbons), 159.10, 166.04 (2C]O), MS (EI) m/z (%):434 (M+, 23%),
Anal. calcd. for C17H17N3O5: C, 59.47; H, 4.99; N, 12.24. Found: C,
59.44; H, 5.11; N, 12.27.
4.1.3. General method for synthesis of 3,4,5-trimethoxy-N-(2,4,5-trioxo-3-
alkyl/aralkylimidazolidin-1-yl)benzamide (9a-d), 7-methoxy-N-(2,4,5-
trioxo-3-alkyl/aralkylimidazolidin-1-yl)benzo[d][1,3]dioxole-5-
carboxamide
(12a-d)
and
8-methoxy-N-(2,4,5-trioxo-3-alkyl/
aralkylimidazolidin-1-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide
(13a-d)
A mixture of 0.001 mol of 8a-d, 10a-d or 11a-d and 0.002 mol of
oxalyl chloride in 5 ml benzene was refluxed for 3 h. The solvent was
evaporated and the residue was purified by column chromatography
using silica gel as stationary phase and a mixture of chloroform and
methanol as mobile phase by gradient elution to achieve 9a-d, 12a-d or
13a-d, respectively.
4.1.2.9. 2-(8-Methoxy-2,3-dihydrobenzo[b][1,4]dioxine-6-carbonyl)-N-
propylhydrazine-1-carboxamide (11a). White solid, m.p. 218 °C, yield
90%, IR (KBr, cm−1) 3382, 3297, 3087 (3NH), 1684, 1646 (2C]O); 1H
NMR (DMSOd6, δ, ppm): 0.83 (t, 3H, J = 8 Hz, CH3), 1.39 (m, 2H,
CH2eCH3), 2.97 (t, 2H, J = 8 Hz, CH2eCH2eCH3), 3.80 (s, 3H, OCH3),
4.27 (m, 4H, CH2-benzodioxine), 6.45 (s, 1H, NH), 7.10–7.13 (m, 2H,
4.1.3.1. 3,4,5-Trimethoxy-N-(2,4,5-trioxo-3-propylimidazolidin-1-yl)
benzamide (9a). White solid, m.p. 190 °C, yield 75%, IR (KBr, cm−1
)
H
ar.), 7.73 (s, 1H, NH), 9.95 (s, 1H, NH); 13C NMR (DMSOd6, δ, ppm):
3444, (NH), 1752, 1683, 1670, 1592 (4C]O); 1H NMR (CDCl3, δ,
ppm): 0.85 (t, 3H, J = 7.4 Hz, CH3), 1.57–1.64 (m, 2H, CH2eCH3),
3.56 (t, 2H, J = 7.2 Hz, CH2eCH2eCH3), 3.74 (s, 6H, 2OCH3), 3.80 (s,
3H, OCH3), 7.03 (s, 2H, Har.), 9.08 (s, 1H, NH); 13C NMR (CDCl3, δ,
ppm): 11.02 (CH3), 21.24 (CH2eCH3), 41.57 (CH2eCH2eCH3), 56.23
(2OCH3), 60.96 (OCH3), 105.17, 124.55, 142.22, 151.57 (aromatic
carbons), 153.19, 155.22, 155.47 (3C]O, imidazolidine), 165.42 (C]
O), MS (EI) m/z (%): 365 (M+, 4%), Anal. calcd. for C16H19N3O7: C,
52.60; H, 5.24; N, 11.50. Found: C, 52.63; H, 5.28; N, 11.51.
11.29 (CH3), 23.11 (CH2eCH3), 41.03 (CH2eCH2eCH3), 55.88 (OCH3),
63.90, 64.11 (2CH2-benzodioxine), 103.93, 109.54, 124.25, 136.02,
143.26, 148.36 (aromatic carbons), 158.50, 165.68 (2C]O), MS(EI) m/
z (%): 309 (M+, 60%), Anal. calcd. for C14H19N3O5: C, 54.36; H, 6.19;
N, 13.58. Found: C, 54.33; H, 6.22; N, 13.62.
4.1.2.10. N-Isopropyl-2-(8-methoxy-2,3-dihydrobenzo[b][1,4]dioxine-6-
carbonyl)hydrazine-1-carboxamide (11b). Yellowish solid, m.p. 120 °C,
yield 85%, IR (KBr, cm−1) 3382, 3297, 3087 (3NH), 1684, 1646 (2C]
O); 1H NMR (DMSOd6, δ, ppm): 1.06 (d, 6H, J = 6.5 Hz, CHe(CH3)2),
3.88 (s, 3H, OCH3), 3.72 (q, 1H, J = 6.5 Hz, CHe(CH3)2), 3.88 (s, 3H,
OCH3), 4.25–4.28 (m, 4H, CH2-benzodioxine), 6.18 (s, 1H, NH),
7.10–7.13 (m, 2H, Har.), 7.66 (s, 1H, NH), 9.93 (s, 1H, NH); 13C NMR
(DMSOd6, δ, ppm): 23.39 (2CH3), 41.59 (CHe(CH3)2), 56.32 (OCH3),
64.32, 64.53 (2CH2-benzodioxine), 104.32, 109.91, 124.65, 136.46,
143.71, 148.09 (aromatic carbons), 158.09, 165.96 (2C]O), MS (EI)
m/z (%): 323 (M+, 1.2%), Anal. calcd. for C14H19N3O5: C, 54.36; H,
6.19; N, 13.58. Found: C, 54.40; H, 6.16; N, 13.44.
4.1.3.2. N-(3-Isopropyl-2,4,5-trioxoimidazolidin-1-yl)-2-(3,4,5-
trimethoxybenzoyl)hydrazine-1-carboxamide (9b). White solid, m.p.
198 °C yield 72%, IR (KBr, cm−1) 3341 (NH), 1746, 1682, 1677,
1592 (4C]O); 1H NMR (CDCl3, δ, ppm): 1.38 (d, 6H, J = 6.88 Hz,
2CH3), 3.76 (s, 6H, 2OCH3), 3.82 (s, 3H, OCH3), 4.38 (q, 1H,
J = 6.9 Hz, CHe(CH3)2), 7.03 (s, 2H, Har.), 9.06 (s, 1H, NH); 13C
NMR (CDCl3, δ, ppm): 19.69 (2CH3), 45.93 (CHe(CH3)2), 56.23
(2OCH3), 60.95 (OCH3), 105.17, 124.81, 142.20, 151.06 (aromatic
carbons), 153.18, 155.12, 155.29 (3C]O, imidazolidine), 165.43 (C]
O), MS (EI) m/z (%): 365 (M+, 4.4%), C16H19N3O7: C, 52.60; H, 5.24;
N, 11.50. Found: C, 52.61; H, 5.22; N, 11.62.
4.1.2.11. N-Butyl-2-(8-methoxy-2,3-dihydrobenzo[b][1,4]dioxine-6-
carbonyl)hydrazine-1-carboxamide (11c). White solid, m.p. 190 °C,
yield 88%, IR (KBr, cm−1) 3446, 3271, 3086 (3NH), 1685, 1644
(2C]O); 1H NMR (DMSOd6, δ, ppm): 0.87 (t, 3H, J = 7.2 Hz, CH3),
1.24–1.30 (m, 3H, CH2eCH3), 1.34–1.39 (m, 2H, CH2eCH2eCH3), 3.03
(t, 2H, J = 6.6, CH2eCH2eCH2eCH3), 3.80 (s, 3H, OCH3), 4.25–4.26
(m, 2H, CH2-benzodioxine), 6.40 (s, 1H, NH), 7.10–7.13 (m, 2H, Har.),
7.72 (s, 1H, NH), 9.94 (s, 1H, NH); 13C NMR (DMSOd6, δ, ppm): 14.19
(CH3eCH2), 19.89 (CH2eCH3), 32.46 (CH2eCH2eCH3), 40.59
(CH2eCH2eCH2eCH3), 56.82 (OCH3), 64.32, 64.53 (2CH2-
benzodioxine) 104.37, 109.97, 124.69, 136.45, 143.69, 148.79
(aromatic carbons), 158.88, 166.05 (2C]O), MS (EI) m/z (%): 323
(M+, 3.7%), Anal. calcd. for C15H21N3O5: C, 55.72; H, 5.55; N, 13.00.
Found: C, 55.61; H, 5.62; N, 13.31.
4.1.3.3. N-(3-Butyl-2,4,5-trioxoimidazolidin-1-yl)-2-(3,4,5-
trimethoxybenzoyl)hydrazine-1-carboxamide (9c). White solid, m.p.
188 °C yield 75%, IR (KBr, cm−1) 3446 (NH), 1685, 1644, 1590,
1570 (4C]O); 1H NMR (DMSOd6, δ, ppm): 0.89 (t, 3H, J = 8 Hz, CH3),
1.30–13.7 (m, 2H, CH2eCH3), 1.57–1.64 (m, 2H, CH2eCH2eCH3), 3.62
(t, 2H, J = 8 Hz, CH2eCH2eCH2eCH3), 3.75 (s, 3H, OCH3), 3.86 (s,
6H, 2OCH3), 7.31 (s, 2H, Har.), 11.51 (s, 1H, NH);13C NMR (DMSOd6, δ,
ppm): 13.41 (CH3eCH2), 19.21 (CH2eCH3), 29.38 (CH2eCH2eCH3),
40.15 (CH2eCH2eCH2eCH3), 56.12 (2OCH3), 60.12 (OCH3), 105.5,
124.92, 141.38, 152.29 (aromatic carbons), 152.85, 155.49, 155.85
(3C]O, imidazolidine), 164.61 (C]O), MS (EI) m/z (%): 379 (M+
,
1%), C17H21N3O7: C, 53.82; H, 5.58; N, 11.08. Found: C, 53.78; H, 5.61;
N, 11.11.
4.1.3.4. N-(3-Benzyl-2,4,5-trioxoimidazolidin-1-yl)-2-(3,4,5-
trimethoxybenzoyl)hydrazine-1-carboxamide (9d). White solid, m.p.
200 °C yield 77%, IR (KBr, cm−1) 3450 (NH), 1720, 1670, 1644,
1590 (4C]O); 1H NMR (DMSOd6, δ, ppm): 3.75 (s, 3H, OCH3), 3.85 (s,
6H, 2OCH3), 4.83 (s, 2H, CH2eC6H5), 7.31 (s, 2H, Har.), 7.37–7.40 (m,
5H, Har.), 11.50 (s, 1H, NH), 13C NMR (DMSOd6, δ, ppm): 42.23
(CH2eC6H5), 56.09 (2OCH3), 60.20 (OCH3), 105.49, 128.58, 127.57,
127.89, 128.58, 128.70, 135.04, 141.37 (aromatic carbons.), 152.13,
152.83, 155.38 (3C]O, imidazolidine), 164.66 (C]O), MS (EI) m/z
(%): 413 (M+, 1.4%), Anal. calcd. for C20H19N3O7: C, 58.11; H, 4.63; N,
10.17. Found: C, 58.22; H, 4.65; N, 10.20.
4.1.2.12. N-Benzyl-2-(8-methoxy-2,3-dihydrobenzo[b][1,4]dioxine-6-
carbonyl)hydrazine-1-carboxamide (11d). White solid, m.p. 180 °C,
yield 95%, IR (KBr, cm−1) 3349, 3085, 3001(3NH), 1691, 1640
(2C]O); 1H NMR (DMSOd6, δ, ppm): 3.79 (s, 3H, OCH3), 4.23–4.24
(m, 6H, CH2eC6H5 and CH2-benzodioxine), 7.12–7.27 (m, 7H, Har.),
7.93 (s, 1H, NH), 10.04 (s, 1H, NH);13C NMR (DMSOd6, δ, ppm): 43.06
(CH2eC6H5), 56.89 (OCH3), 64.32, 64.54 (2CH2-benzodioxine), 104.3,
105.5, 124.55, 127.04, 127.37, 128.60, 136.49, 140.67, 143.67, 148.78
(aromatic carbons), 159.12, 166.42 (2C]O), MS (EI) m/z (%): 357
(M+, 4%), Anal. calcd. for C18H19N3O5: C, 60.50; H, 5.36; N, 11.76.
Found: C, 60.61; H, 5.42; N, 11.85.
4.1.3.5. 7-Methoxy-N-(2,4,5-trioxo-3-propylimidazolidin-1-yl)benzo[d]
[1,3]dioxole-5-carboxamide (12a). White solid, m.p. 175 °C, yield 95%,
IR (KBr, cm−1) 3434 (NH), 1749, 1675, 1631, 1590 (4C]O); 1H NMR
8

M.E. Aboutabl, et al.
BioorganicChemistryxxx(xxxx)xxxx
(DMSOd6, δ, ppm): 0.90 (t, 3H, J = 7.4 Hz, CH3), 1.61–1.67 (m, 2H,
CH2eCH3), 3.59 (t, 2H, J = 7 Hz, CH2eCH2eCH3), 3.88 (s, 3H, OCH3),
6.14 (s, 2H, CH2-benzodioxole), 7.21 (s, 1H, Har.), 7.35 (s, 1H, Har.),
11.44 (s, 1H, NH); 13C NMR (DMSOd6, δ, ppm): 11.40 (CH3), 21.23
(CH2eCH3), 41.08 (CH2eCH2eCH3), 56.89 (OCH3), 102.37 (CH2-
benzodioxole), 102.92, 109.19, 124.39, 139.34, 143.53, 149.09
(aromatic carbons), 152.70, 155.96, 156.30 (3C]O, imidazolidine),
164.74 (C]O), MS (EI) m/z (%): 349 (M+, 16.7%), Anal. calcd. for
dihydrobenzo[b][1,4]dioxine-6-carboxamide (13b). White solid, m.p.
262 °C, yield 80%, IR (KBr, cm−1) 3341, (NH), 1746, 1682, 1592,
1493 (4C]O), 1H NMR (DMSOd6, δ, ppm): 1.40 (d, 6H, J = 8 Hz,
2CH3), 3.82 (s, 3H, OCH3), 4.26–4.39 (m, 5H, 2CH2-benzodioxine and
CHe(CH3)2), 7.15–7.36 (m, 2H, Har.), 11.36 (s, 1H, NH); 13C NMR
(DMSOd6, δ, ppm): 19.02 (2CH3), 44.91 (CHe(CH3)2), 56.37 (OCH3),
64.32, 64.64 (2CH2-benzodioxine), 104.61, 110.49 (2CHar.), 121.18,
128.76, 137.64, 143.94 (aromatic carbons.), 149.11, 152.17, 155.89
C
15H15N3O7: C, 51.58; H, 4.33; N, 12.03. Found: C, 51.47; H, 4.51; N,
(3C]O, imidazolidine), 164.94 (C]O), MS (EI) m/z (%): 363 (M+
,
12.33.
49%), Anal. calcd. for C16H17N3O7: C, 52.89; H, 4.72; N, 11.57. Found:
C, 52.88; H, 4.80; N, 11.61.
4.1.3.6. N-(3-Isopropyl-2,4,5-trioxoimidazolidin-1-yl)-7-methoxybenzo[d]
[1,3]dioxole-5-carboxamide (12b). White solid, m.p. 264 °C, yield 90%,
IR (KBr, cm−1) 3434 (NH), 1747, 1680, 1631, 1595 (4C]O); 1H NMR
(DMSOd6, δ, ppm): 1.40 (d, 6H, J = 8 Hz, 2CH3), 3.90 (s, 3H, OCH3),
4.36 (q, 1H, J = 8 Hz, CHe(CH3)2), 6.14 (s, 2H, CH2-benzodioxole),
7.20 (s, 1H, Har.), 7.34 (s, 1H, Har.), 11.38 (s, 1H, NH); 13C NMR
(DMSOd6, δ, ppm): 19.59 (2CH3), 44.48 (CHe(CH3)2), 56.43 (OCH3),
101.91 (CH2-benzodioxole), 102.46, 108.7, 123.96, 128.33, 138.05,
143.06 (aromatic carbons), 148.62, 151.71, 155.45 (3C]O,
imidazolidine), 164.28 (C]O), MS (EI) m/z (%): 349 (M+, 11%),
Anal. calcd. for C15H15N3O7: C, 51.58; H, 4.33; N, 12.03. Found: C,
51.55; H, 4.32; N, 12.11.
4.1.3.11. N-(3-Butyl-2,4,5-trioxoimidazolidin-1-yl)-8-methoxy-2,3-
dihydrobenzo[b][1,4]dioxine-6-carboxamide (13c). White solid, m.p.
198 °C, yield 90%, IR (KBr, cm−1) 3271, 3086 (3NH), 1748, 1686,
1670 1593 (4C]O); 1H NMR (DMSOd6, δ, ppm): 0.90 (t, 3H, J = 8 Hz,
CH3), 1.23–1.36 (m, 2H, CH2eCH3), 1.56–1.63 (m, 2H,
CH2eCH2eCH3), 3.60 (t, 2H, J = 8 Hz, CH2eCH2eCH2eCH3), 3.82
(s, 3H, OCH3), 4.27–4.32 (m, 4H, CH2-benzodioxine), 7.18–7.36 (m,
2H, Har.), 11.39 (s, 1H, NH); 13C NMR (DMSOd6, δ, ppm): 13.61 (CH3),
19.42
(CH2eCH3),
29.58
(CH2eCH2eCH3),
38.92
(CH2eCH2eCH2eCH3), 56.15 (OCH3), 64.09, 64.42 (2CH2-
benzodioxine), 106.3, 105.5, 121.59, 128.54, 137.43, 143.72
(aromatic carbons), 152.52, 155.74, 156.02 (3C]O, imidazolidine),
164.71 (C]O), MS (EI) m/z (%): 377 (M+, 23%), Anal. calcd. for
C17H19N3O7: C, 54.11; H, 5.08; N, 11.14. Found: C, 54.28; H, 5.11; N,
11.20.
4.1.3.7. N-(3-Butyl-2,4,5-trioxoimidazolidin-1-yl)-7-methoxybenzo[d]
[1,3]dioxole-5-carboxamide (12c). White solid, m.p. 200 °C, yield 85%,
IR (KBr, cm−1) 3422 (NH), 1751, 1683, 1631, 1589 (4C]O); 1H NMR
(DMSOd6, δ, ppm): 0.89 (t, 3H, J = 8 Hz, CH3), 1.30–1.35 (m, 2H,
CH2eCH3), 1.57–1.64 (m, 2H, CH2eCH2eCH3), 3.60 (t, 2H, J = 8 Hz,
CH2eCH2eCH2eCH3), 3.89 (s, 3H, OCH3), 6.13 (s, 2H, CH2-
benzodioxole), 7.19–7.20 (m, 1H, Har.), 7.31–7.35 (m, 1H, Har.),
11.42 (s, 1H, NH); 13C NMR (DMSOd6, δ, ppm): 13.20 (CH3), 19.0
(CH2eCH3), 29.16 (CH2eCH2eCH3), 38.52 (CH2eCH2eCH2eCH),
56.82 (OCH3), 101.71 (CH2-benzodioxole), 102.26, 108.49, 123.71,
138.76, 142.86, 148.42 (aromatic carbons.), 152.08, 155.28, 155.59
4.1.3.12. N-(3-Benzyl-2,4,5-trioxoimidazolidin-1-yl)-8-methoxy-2,3-
dihydrobenzo[b][1,4]dioxine-6-carboxamide (13d). White solid, m.p.
202 °C, yield 88%,IR (KBr, cm−1) 3341, (NH), 1744, 1680, 1664,
1596 (4C]O); 1H NMR (DMSOd6, δ, ppm): 3.82 (s, 3H, OCH3),
4.29–4.31 (m, 4H, 2CH2-benzodioxine), 4.83 (s, 2H, CH2eC6H5),
7.19–7.40 (m, 7H,
H
ar.), 13C NMR (DMSOd6, δ, ppm): 42.68
(CH2eC6H5), 56.3 (OCH3), 64.33, 64.66 (2CH2-benzodioxine),
104.05, 110.52, 121.81, 128.08, 128.35, 129.15, 135.50, 137.71,
143.97, 149.15 (aromatic carbons.), 152.63, 155.89, 156.09 (3C]O,
imidazolidine), 164.99 (C]O), MS (EI) m/z (%): 411 (M+, 18%), Anal.
calcd. for C20H17N3O7: C, 58.40; H, 4.17; N, 10.21. Found: C, 58.43; H,
4.23; N, 10.33.
(3C]O, imidazolidine), 164.07(C]O), MS (EI) m/z (%): 363 (M+
,
32%), Anal. calcd. for C16H17N3O7: C, 52.89; H, 4.72; N, 11.57 Found:
C, 52.73; H, 4.68; N, 11.62.
4.1.3.8. N-(3-Benzyl-2,4,5-trioxoimidazolidin-1-yl)-7-methoxybenzo[d]
[1,3]dioxole-5-carboxamide (12d). White solid, m.p. 167 °C, yield 92%,
IR (KBr, cm−1) 3442 (NH), 1748, 1683, 1631, 1594 (4C]O); 1H NMR
(DMSOd6, δ, ppm): 3.37 (s, 3H, OCH3), 4.77 (s, 2H, CH2eC6H5), 6.10 (s,
4.2. Pharmacology
2H, CH2-benzodioxole), 7.16–7.35 (m, 7H, Har.), 11.42 (s, 1H, NH); 13
C
4.2.1. Anticonvulsant activity
NMR (DMSOd6, δ, ppm): 40.20 (CH2eC6H5), 56.94 (OCH3), 102.44
(CH2-benzodioxole), 109.25, 124.41, 128.15, 128.42, 128.86, 129.22,
135.55, 139.52, 143.59 (aromatic carbons), 149.16, 152.68, 156.17
4.2.1.1. Experimental animals. The anticonvulsant potential of
compounds 9a-d, 12a-d and 13a-d was performed on adult male
albino mice weighing 20–25 g. Mice were supplied from the Animals
House Colony of National Research Centre, Giza, Egypt. Mice were
housed in polypropylene cages under standardized conditions of light
(3C]O, imidazolidine), 164.85 (C]O), MS (EI) m/z (%): 397 (M+
,
7%), Anal. calcd. for C19H15N3O7: C, 57.43; H, 3.81; N, 10.58. Found: C,
57.35; H, 3.84; N, 10.63.
(12 h light/dark cycle), temperature (room temperature 23
2 °C)
and humidity (relative humidity 55
5%). Mice were allowed free
4.1.3.9. 8-Methoxy-N-(2,4,5-trioxo-3-propylimidazolidin-1-yl)-2,3-
dihydrobenzo[b][1,4]dioxine-6-carboxamide (13a). White solid, m.p.
178 °C, yield 95%, IR (KBr, cm−1) 3432, (NH), 1749, 1675, 1637,
1598 (4C]O); 1H NMR (DMSOd6, δ, ppm): 0.87 (t, 3H, J = 8 Hz, CH3),
1.61–1.66 (m, 2H, CH2eCH3), 3.59 (t, 2H, J = 8 Hz, CH2eCH2eCH3),
3.82 (s, 3H, OCH3), 4.27–4.32 (m, 4H, CH2-benzodioxine), 7.18–7.21
(m, 2H, Har.), 11.41 (s, 1H, NH); 13C NMR (DMSOd6, δ, ppm): 10.96
(CH3), 20.79 (CH2eCH3), 41.22 (CH2eCH2eCH3), 55.95 (OCH3),
63.88, 64.21 (2CH2-benzodioxine), 104.17, 110.07, 121.38, 128.33,
137.22, 143.52 (aromatic carbons), 152.30, 155.55, 155.85 (3C]O,
imidazolidine), 164.50 (C]O), MS (EI) m/z (%): 363 (M+, 2%), Anal.
calcd. for C16H17N3O7: C, 52.89; H, 4.72; N, 11.57. Found: C, 52.65; H,
4.61; N, 11.63.
access to water and standard rat chow. All experimental procedures
involving animals were performed according to the guidelines of the
ethical committee of National Research Centre for experimental animal
use. Mice were acclimatized to laboratory conditions before the
beginning of the experiment. All the target compounds were
suspended in 7% Tween 80 as a vehicle.
4.2.1.2. Phase I study anticonvulsant evaluation
4.2.1.2.1. scPTZ-induced seizures test. PTZ was administered
subcutaneously at a dose of 85 mg/kg in the loose fold of the skin on
the back of the mice neck [34]. This dose is known to induce clonic
seizures that persists for at least five seconds in 97 percent of the test
animals. Compounds 9a-d, 12a-d, 13a-d and reference drugs were
injected intraperitoneally (i.p.) at doses of 30, 100, and 300 mg/kg, and
after 0.5 h and 4 h interval, the test was carried out by injecting PTZ
4.1.3.10. N-(3-Isopropyl-2,4,5-trioxoimidazolidin-1-yl)-8-methoxy-2,3-
9

M.E. Aboutabl, et al.
BioorganicChemistryxxx(xxxx)xxxx
followed by 30 min observation for seizure appearance. A threshold
convulsion is defined as one episode of clonic convulsions which persist
for at least a 5 s. If such threshold seizure is not observed, this indicates
the potency of the test compound to protect against PTZ-induced
seizure threshold [35,36].
homogenization, sample extraction, and GABA analysis were
performed according to Wojnicz et al. [40].
4.2.3.2. Mass spectrometry conditions. ESI-MS positive ion acquisition
mode was performed on a XEVO TQD triple quadruple instrument
Waters Corporation, Milford, MA01757 U.S.A, mass spectrometer. This
system was operated in multiple reaction monitoring (MRM) mode.
Samples (10 μL) were injected into the ultra-performance liquid
chromatography (UPLC) instrument equipped with reverse phase C-18
4.2.1.2.2. MES test. Mice were injected with the test compound and
reference drug at doses of 30, 100, and 300 mg/kg, i.p. After 0.5 h and
4 h, an electric current of fixed intensity of 25 mA and stimulus
duration of 0.2 s was produced via Rodent Shocker generator (constant-
current stimulator Type 221, Hugo Sachs Elektronik, Freiburg,
Germany). This electric current was delivered via ear-clip electrode
producing electroconvulsions in mice. The ability of the test compound
to prevent the tonic hind limb extension indicates its ability to inhibit
MES-induced seizure spread [37].
column (ACQUITY UPLC
- BEH C18 1.7 µm particle size −
2.1 × 50 mm Column). The mobile phase of the sample was filtered
using 0.2 μm filter membrane disc and degassed by sonication before
injection. Mobile phase elution conditions was performed with the flow
rate of 0.2 ml/min using gradient mobile phase comprising two eluents:
eluent A is H2O acidified with 0.1% formic acid and eluent B is MeOH
acidified with 0.1% formic acid. Elution was carried out under the
above gradient conditions.
4.2.1.2.3. Neurotoxicity. Neurotoxicity is determined by using
rotarod test. It is well
a documented test to detect minimal
neurological deficit. Mice were trained to maintain equilibrium on a
revolving 1-inch-diameter knurled plastic rod at a speed of 10 rpm for
1 min in each of three trials using a rotarod device (UGO Basile, 47600,
Varese, Italy). Only animals that fulfill this criterion were used in the
experiment. Trained mice were given i.p. injection of the compounds
9a-d, 12a-d and 13a-d at doses of 30, 100 and 300 mg/kg.
Neurotoxicity was determined by the inability of the animal to keep
equilibrium on the rotating rod for at least 1 min [38].
After separation by high performance liquid chromatography, the
peak corresponding to the transition m/z for the quantifier ion was
measured relative to that of the transition m/z for its IS (caffeine). In
the identification of GABA the m/z reaction for the qualifier ion was
also monitored to add specificity. The analysis of the parameters were
carried out using negative ion mode: source temperature 150 °C, cone
voltage 30 eV, capillary voltage 3 kV, desolvation temperature 440 °C,
cone gas flow 50 L/h, and desolvation gas flow 900 L/h. Mass spectra
were detected in the ESI negative ion mode between m/z 100–1000.
The integration of the peaks and spectra of the MRM transitions of each
analyte were processed using the Maslynx 4.1 software.
4.2.1.3. Phase II study anticonvulsant evaluation. The median effective
dose (ED50) is defined as the dose of a drug protecting 50% of animals
against scPTZ and MES seizures. For the determination of ED50, the test
drug was administered to different groups of mice (n = 6) at various
doses until at least three points were established in the range of 16–83%
seizure protection. The neurotoxicity was expressed as the median toxic
dose (TD50).The protective index (PI) value was calculated as the ratio
of TD50 to ED50 (PI = TD50/ED50) [1,17].
4.2.4. Statistical analysis
Data were presented as mean
standard error of the mean (SEM).
The experimental groups were analyzed using one-way analysis of
variance followed by Student–Newman–Keuls post hoc comparison. A
result was considered statistically significant when P < 0.05.
4.2.1.4. Hepatotoxicity. Mice were divided into 7 groups (n = 6).
Group 1 served as control group and received the vehicle (7%
aqueous suspension of Tween 80); Groups 2–7 received the test
compounds 9a-d, 12a-d and 13a-d, respectively at a dose of (30 mg/
kg, i.p.) dissolved in the vehicle. The treatments were carried out for
7 days. 24 h after the last injection of the test compounds, the mice
were anaesthetized and blood samples were collected by cardiac
puncture then centrifuged at 3000 rpm for 10 min for serum
separation. Analyses of the liver enzymes ALT, AST and ALP, in
addition to total protein and albumin were determined in serum via
commercially available kits (Biodiagnostic, Egypt) [1,18].
4.3. Molecular modeling
All docking studies were performed using Discovery Studio 2016.
The structure of γ-aminobutyric acid aminotransferase GABA-AT) was
retrieved from RCSB protein data bank (PDB: 1OWH). Generally, the
protein structure was prepared then adding of hydrogen bonds. The
prepared molecules were minimized and docked into the protein
structure using CDOCKER protocol.
Declaration of Competing Interest
4.2.2. Acute toxicity test
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Different doses of compounds 9a-d, 12a-d and 13a-d (10, 100, 300
and 500 mg/kg suspended in 7% tween 80) were injected via i.p. to
adult male albino mice (n = 6). After the administration of these
compounds, mice were observed for the first 2 h continuously in order
to detect any gross behavioral changes and deaths, then at hourly in-
tervals for the next 4 h. The observation was continued every 2 h until
hour 12 and then at hour 22 and 24 in order to detect any delayed
effects. The animal’s behavior and other toxic symptoms were observed
daily for 72 h, and the mice were kept under observation for 14 days
[18,39].
Acknowledgement
The authors acknowledge the National Research Centre for sup-
porting this work through project AR 110211.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103473.
4.2.3. Determination of γ-aminobutyric acid in brain homogenate
4.2.3.1. Sample preparation. The γ-aminobutyric acid (GABA)
estimation was performed in whole brain tissue homogenate using
liquid chromatography–spectrometry (LC–MS). Mice were treated
either with compound 9a (30 mg/kg, i.p.) or the standard drug
bromazepam (30 mg/kg, i.p.). Then 2 h later, the control and treated
animals were sacrified by decapitation and brains were extracted and
weighed then immediately frozen in −80 freezer. Brain
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