Design, synthesis and docking studies of novel benzopyrone derivatives as anticonvulsants

Article abstract of DOI:10.1016/j.bioorg.2020.103738

A series of coumarin derivatives 6–8, 9a-h, 11 and 13a, b -16a, b was synthesized and screened for their anticonvulsant profile. Screening of these analogues using the 'gold standard methods' revealed variable anticonvulsant potential with remarkable effects observed particularly in chemically-induced seizure test. Compounds 6, 7, 13b disclosed the highest potency among the series with 100% protection against scPTZ. Quantification study confirmed that compound 6 (ED₅₀ 0.238 mmol/kg) was the most active congener in the scPTZ model and was approximately 1.5 folds more potent than ethosuximide as reference drug Meanwhile, in the MES test, candidate drugs exhibited mild to moderate anticonvulsant efficacy, the highest of which was compound 14a, imparting 50% protection at 2.1 mmol/kg, followed by other compounds with activity ranging from 14 to 33%, as compared to diphenylhydantoin. Additionally, all candidate compounds were screened for acute neurotoxicity using the rotarod method to identify motor impairment, where almost all compounds passed the test. Further neurochemical investigation was performed to unravel the effect of the most active compound (6) on GABA level in mouse brain, where a significant elevation was evident by 4 and 1.4 folds with respect to that of the control and reference groups at p < 0.05. Molecular modeling study using Discovery Studio program was performed, where compound 6 exhibited good binding interaction with γ-aminobutyric acid aminotransferase (GABA-AT) enzyme and this was consistent with the attained experimental results.

Full text of DOI:10.1016/j.bioorg.2020.103738

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Design, Synthesis and docking studies of novel benzopyrone derivatives as
anticonvulsants
Walaa Hamada Abd-Allah, Essam Eldin A Osman, Mostafa Abd-El-Mohsen
Anwar, Hanan Naeim Attia, Samir M El Moghazy
PII:
S0045-2068(20)30098-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.103738
YBIOO 103738
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
14 January 2020
9 February 2020
7 March 2020
Please cite this article as: W. Hamada Abd-Allah, E. Eldin A Osman, M. Abd-El-Mohsen Anwar, H. Naeim
Attia, S.M. El Moghazy, Design, Synthesis and docking studies of novel benzopyrone derivatives as
anticonvulsants, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103738
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Design, Synthesis and docking studies of novel benzopyrone derivatives as anticonvulsants
a,*
b
a
Walaa Hamada Abd-Allah , Essam Eldin A Osman , Mostafa Abd-El-Mohsen Anwar , Hanan
c
b
Naeim Attia , Samir M El Moghazy
aPharmaceutical Chemistry Department, College of Pharmaceutical Science and Drug
Manufacturing, Misr University for Science & Technology, P.O. 77, 6th of October
City, Giza, Egypt
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, 11562
Cairo, Egypt
cMedicinal and Pharmaceutical Chemistry Department (Pharmacology Group) Pharmaceutical
and Drug Industries Research Division, National Research Centre (ID: 60014618), P.O. 12622,
Dokki, Giza, Egypt.
*
Corresponding author: Walaa Hamada Abd-Allah, Pharmaceutical Chemistry Department,
College of Pharmaceutical Science and Drug Manufacturing, Misr University for Science
&
Technology, P.O. 77, 6th of October City, Giza, Egypt. Email: walaa.abdalla@must.edu.eg.
Abstract
A series of coumarin derivatives 6-8, 9a-h, 11 and 13a, b -16a, b was synthesized and screened
for their anticonvulsant profile. Screening of these analogues using the 'gold standard methods'
revealed variable anticonvulsant potential with remarkable effects observed particularly in
chemically-induced seizure test. Compounds 6, 7, 13b disclosed the highest potency among the
series with 100% protection against scPTZ. Quantification study confirmed that compound 6
(
ED 0.238 mmol/kg) was the most active congener in the scPTZ model and was approximately
50
1
.5 folds more potent than ethosuximide as reference drug Meanwhile, in the MES test,
candidate drugs exhibited mild to moderate anticonvulsant efficacy, the highest of which was
compound 14a, imparting 50% protection at 2.1 mmol/kg, followed by other compounds with
activity ranging from 14-33%, as compared to diphenylhydantoin. Additionally, all candidate
compounds were screened for acute neurotoxicity using the rotarod method to identify motor
impairment, where almost all compounds passed the test. Further neurochemical investigation
was performed to unravel the effect of the most active compound (6) on GABA level in mouse
brain, where a significant elevation was evident by 4 and 1.4 folds with respect to that of the
1

control and reference groups at p<0.05. Molecular modeling study using Discovery Studio
program was performed, where compound 6 exhibited good binding interaction with γ-
aminobutyric acid aminotransferase (GABA-AT) enzyme and this was consistent with the
attained experimental results.
Key words:
Synthesis; coumarin; anticonvulsants; GABA; Molecular Modeling
1
. Introduction
Epilepsy is defined as disorders of neuronal excitability characterized by periodic and
unpredictable occurrences of seizures and transient changes of behavior as a result of
synchronous disordered rhythmic firing of neural populations of the central nervous system [1-
3
]. It is known to be one of the most common serious neurological disorders that affect 1-2%
people worldwide. Seizures could be attributed to an abnormality in the major neurotransmitters
of the brain; such as over-activity of the excitatory transmitters or impaired activity of the
inhibitory transmitters or possibly through a combination of both. However, it is quite difficult to
pinpoint a singular biochemical disorder that results in epileptogenesis due to the marked
heterogeneity of epilepsy syndromes and emergence of therapy-resistant epilepsy as well [4].
Despite the presence of several novel antiepileptic drugs (AEDs), the control of seizures is
attained in only 50% of patients and the decrease in incidence is found in only 75% of patients
[2, 5]. Additionally, they have side effects such as ataxia, hepatotoxicity, and megaloblastic
anemia [6, 7] .Consequently, search for new antiepileptics with lower toxicity and better efficacy
is still a paramount demand. Antiepileptic drugs (AEDs) comprise a diverse range of molecules
acting mostly through: i) modulation of voltage-gated ion channels (sodium or calcium), ii)
reduction of excitatory, particularly glutamate-mediated neurotransmitter or iii) promotion of
GABA receptors directly through positive allosteric modulation of GABA receptors or
A
indirectly through enhancing GABA level via GABA transaminase inhibition [8]. GABA is
considered the main inhibitory neurotransmitter in the brain of mammals and plays a
considerable role in the pathogenesis of epilepsy [9]. GABAergic inhibition is considered the
main mechanism balancing glutamatergic excitation and hypersynchronous neuronal discharges
inhibition.
2

Coumarins are the privileged structure in medicinal chemistry for the discovery of therapeutic
agents [10-12]. They were reported to exhibit various therapeutic profiles among which is
antiepileptic activity as represented in compounds A [13], B [13], C [14], D [15] and E [16].
Interestingly, incorporation of dioxopyrrole, thiazoline, imino thiazolidinone and
imidazolidinetrione moieties to the scaffold of coumarin entity could improve their anti-
convulsant potential [17].
Using target-based studies in the discovery of new antiepileptic agents is uncommon due to lack
of information regarding the molecular pathway of epilepsy and complex mode of action for
most of the known antiepileptic drugs. Therefore, the development of novel anticonvulsants may
be achieved through screening investigations or using hybrid approach [18, 19]. The latter
depends on the use of two or more pharmacophoric scaffolds, each having a remarkable
pharmacological effect, to be joined in one single structure to attain new components having
better biological profile [20].
The designed hybrid of the target compounds 6-8, 9a-h, 11, 13a, b -16a, b possesses all
pharmacophoric elements essential anticonvulsant activity such as lipophilic aryl ring (A),
hydrogen bonding domain (HBD) and electron donor moiety (D) [5, 8, 21, 22] (fig 2). The
anticonvulsant evaluation for these benzopyran derivatives was performed through two standard
animal seizure models; subcutaneous pentylenetetrazole (scPTZ) and a maximal electric shock
(MES) seizure tests. Additionally, the effect of the synthesized entities on motor function was
assessed via rotarod test. The most potent compounds which had remarkable efficacy in scPTZ
were further investigated for its effect on GABA brain level. To calculate physicochemical
properties of these benzopyran derivatives, computational studies were carried out using
swissADME a free online web tool. In addition, docking study was accomplished on the active
site of GABA-AT enzyme to confirm GABAergic activity.
Insert Fig.1
Insert Fig.2
3

2
. Results and discussions
2
.1. Chemistry
The synthetic route of the target compounds 6-8, 9a-h, 11, 13a, b -16a, b is illustrated in
scheme 1-3. The compounds were produced in a reasonable yield and their structure was
confirmed with spectral and elemental analysis. The key intermediate 5 [23]was prepared
through hydrazinolysis of the corresponding ester 4.
Reacting of the acid hydrazide 5 with phthalic anhydride in presence of glacial acetic acid
producing pyrrolidin-2, 5-dione 6 (Scheme 2). The IR spectra illustrated bands at 1626-1720 cm-
1
1
for 4C=O groups. H NMR spectra displayed the disappearance of NH and NH signals of
2
1
3
parent acid hydrazide 5 and presence of one signal at 10.98 ppm for NH. C NMR spectrum
showed presence of 4C=O groups.
Reacting of acid hydrazide 5 with acetylacetone in ethanol afforded compound 7 [23]. The IR
-1
1
spectra illustrated bands at 1712 and 1720 cm for 2C=O groups. H NMR spectrum showed
1
3
presence of three peaks at 1.76, 2.00 and 2.39 ppm corresponding to protons of 3CH . C NMR
3
spectrum showed 3 peaks at 16.3, 18.6 and 26.2 for carbons of 3CH3.
Condensation of acid hydrazide 5 with ethyl acetoacetate in presence of absolute ethanol
achieved the corresponding pyrazol-5-one derivative 8 (Scheme 2). IR spectra showed a band for
-1
-1 1
NH at 3446 cm and 3C=O bands at 1680-1730 cm . H NMR spectra showed a broad singlet at
.09 ppm corresponding to NH and exchangeable with D O and another singlet signals at 2.40
2
2
ppm assigned to CH at C3 pyrazolone, 4.97 ppm regarding CH pyrazolone.
3
Refluxing of the hydrazide 5 with appropriate aromatic aldehyde in presence of glacial acetic
acid yielded the benzylidene acetohydrazide derivatives 9a-h (Scheme 2). IR spectra showed
-1
1
bands at 3275–3446 cm regarding NH group. H NMR showed one signal at 8.04–8.38 ppm
regarding benzylidene proton and one signal at 11.67–11.96 ppm regarding NH. Additionally,
13CNMR showed peak at 142.8-147.4 ppm corresponding to (N=C).
4
-Benzyl-1-acylsemicarbazide 10 was attained through refluxing of the corresponding hydrazide
5
with benzyl isocyanate in presence of dichloroethane (Scheme 2). IR spectra displayed bands
-1
1
at 3305– 3385 cm for 3NH. The structure was confirmed in H NMR by presence of one single
4

signal at 4.25 ppm and a multiplet signal at 7.27–7.36 ppm for CH and phenyl protons of the
2
benzyl moiety, respectively. Furthermore, there are three a singlet signals at 8.04, 9.28 and 9.82
ppm conforming 3NH protons.
Cyclization of acylsemicarbazide 10 using oxalyl chloride achieved imidazolidin-2,4,5-trione 11
-1
-1
(
Scheme 2). IR spectra illustrated a band at 3329 cm for NH and bands at 1643–1720cm for
1
13
5
C=O groups. H NMR exposed a singlet signal at 11.20 ppm regarding one NH as well as C
NMR showed peaks at 155.9, 158.4 and 158.8 corresponding to 3C=O imidazolidin-2,4,5-trione.
Thiosemicarbazides 12a[23], b were attained through the reaction of the respective hydrazide 5
with the appropriate isothiocyanate as illustrated in scheme 3. IR spectra showed three bands at
-1
1
3
169–3458 cm corresponding to 3NH. H NMR spectrum revealed a triplet signal at 1.25 ppm
as well as quartet signal at 4.05 ppm related to the ethyl protons in 12a spectrum. Furthermore,
compound 12b was confirmed via presence of singlet signal at 4.77 and a multiplet at 7.22-7.27
ppm corresponding to the phenyl protons. 13C NMR exhibited a single peak at 183.2
corresponding to C=S. MS spectrum illustrated their molecular ion peaks.
1
3a, b congeners were achieved through cyclization of thiosemicarbazides 12a, b with a 2 N
-1
NaOH yielded (Scheme 3). IR spectra revealed bands at 3124 and 3419 cm corresponding to
1
NH. H NMR of 13a [23] congener revealed a broad singlet signal at 13.90 ppm referring NH
proton while compound 13b exhibited a singlet signal at 10.63 ppm regarding one NH proton.
Also, 13CNMR for compound 13b showed peak at 180.1ppm corresponding to C=S. MS
spectrum illustrated their molecular ion peaks.
Compounds 14a,b were elucidated through their IR spectra showing disappearance of a band at
1
282–1251 cm-1 corresponding to C=S and only appearance one band at 3419 and 3477 cm-1
1
corresponding to one NH. Additionally, their H NMR showed a singlet signal at 6.97 and 6.99
1
3
ppm assigned to CH thiazoline. C NMR for compound 14a showed peak at 108.3 regarding
CH-thiazoline and another peak at 158.0 for C=N.
1
,3,4-thiadiazole derivatives 15a, b were achieved through cyclization of thiosemicarbazides
1
2a,b with concentrated sulfuric acid on cold (Scheme 3). Their structures were confirmed
-1
through IR spectra via disappearance of C=S band at 1282- 1251 cm and appearance of band at
5

-1
1
3
157 and 3277 cm corresponding to NH. H NMR revealed one singlet signal at 7.82 and 8.36
ppm referred to NH proton.
Iminothiazolidin-4-ones 16a,b were produced through reaction of the respective
thiosemicarbazides 12a,b with chloroacetic acid and anhydrous sodium acetate in presence of
glacial acetic acid (Scheme 3). Their structures were confirmed through IR spectra showing band
-1
-1
at 3348 and 3500 cm related NH and three bands at 1732–1699 cm assigned to three C=O of
1
respective congener. H NMR shown two singlet signals at 4.15 ppm, 4.07 ppm corresponding to
CH group of the thiazolidinone as well as 10.56 and 10.57 ppm exchanged with D O assigned to
2
2
NH as well as 13C NMR for compound 16a showed peak at 33.2 corresponding to CH2-
thiazolidinone.
Insert Sheme1
Insert Scheme 2
Insert Scheme 3
2
2
.2. Pharmacology
.2.1. Anticonvulsant evaluation
The anticonvulsant evaluation was performed according to an anticonvulsant drug
development (ADD) protocol [24], where submitted potential anticonvulsants undergo a list of
screening tests in various animal seizure models in the discovery phase of drug development.
The preliminary screening in phase I for all the compounds in the series was adopted to detect
the most potent candidate drug using the gold standards scPTZ and MES models in normal male
albino mice, in addition to evaluation of the potential acute neurotoxic effects by the rotarod test.
Phase II followed phase I for the quantitative determination of the median effective dose ED50
values of the compounds that conferred 100 % protection against one or both tests. Finally,
neurochemical estimation of GABA inhibitory neurotransmitter level for the most active
compound was carried out in whole mouse brain, isolated post-administration of the most potent
congener possessing the least ED value, using gabapentin as reference standard drug, in order
5
0
to predict the mechanism of action.
6

2
.2.2. Phase I: Preliminary screening for anticonvulsant activity:
Compounds 6-8, 9a-h, 11 and 13a, b -16a, b were pharmacologically screened by
intraperitoneal administration of a fixed dose; 100 mg/kg to male albino mice in order to
evaluate anticonvulsant activity after 45 min post-drug treatment. This acute dose was selected
according to a pilot study performed prior to the main study. The results of the primary (phase-I)
screening are summarized in Table 1. Candidate drugs showed variable activity against scPTZ–
induced absence epilepsy ranging from 17–100% protection at the tested dose level equivalent to
1
00 mg/kg, where compounds 6, 7 and 13b exhibited the highest protection against PTZ screen
(100%) at 0.238, 0.239 and 0.283 mmol/kg which is almost 1.49, 1.48, 1.25 folds more potent
than reference drug ethosuximide, respectively. The remaining candidates revealed
anticonvulsant activity in the following descending order; 9h (83%, 0.243mmol) =15a (83%,
0
.279mmol) >16a (67%, 0.24mmol) = 8 (67%, 0.282mmol) >14a (40%, 0.218mmol) > 11
(33%, 0.210mmol)=9b (33%, 0.237mmol) = 9d (33%, 0.246) = 9g (33%, 0.253mmol) = 9a
(33%, 0.256mmol) = 9c (33%, 0.256mmol) =13a (33%, 0.279) >9f (29%, 0.214mmol)>14b
(17%, 0.186)=16b (17%, 0.209mmol), > 9e (13%, 0.224mmol). On the other hand, all
compounds failed to fully protect animals against MES-induced grand mal seizures, which imply
that they lack the ability to completely abolish generalized tonic-clonic seizures and are unable to
prevent seizure spreading in all animal groups [25, 26]. In this test, candidate drugs exhibited
mild to moderate anticonvulsant efficacy, the highest of which was compound 14a imparting
5
3
0% protection at 2.1 mmol/kg, followed by weaker compounds with activity ranging from 14-
3% in the following descending order; 14b (33%, 0.186mmol) = 9d (33%, 0.246 mmol) >9b
(
20%, 0.237mmol) = 9a (20%, 0.256mmol) = 8 (20%, 0.282mmol)> 6 (17%, 0.25mmol) = 9f
17%, 0.214mmol)= 9c (17%, 0.256mmol) =9g (17%, 0.253mmol)=13a (17%,
(
0
1
.279mmol)=15a (17%, 0.279mmol)> 9e (14%, 0.224mmol) whereas compounds, 7, 9h, 11,
3b,16a, and 16b at doses ranging from 2.09 to 0.283 mmol/kg had absolutely no effect on the
electrically-induced seizures. Noteworthy, compound 15b showed no anticonvulsant potential in
both chemical and electro-convulsive tests. Additionally, all candidate compounds underwent an
acute neurotoxicological study using the rotarod apparatus to identify motor impairment. All
compounds passed the test as the motor coordination in animals was not affected by any
7

unexpected toxicities, except for compound 9a, where 2 out of 6 animals were unable to
maintain equilibrium during 60 s. (c.f. Table 1).
2
.2.3. Phase II: Quantitative estimation of median effective dose ED :
50
Target compounds 6, 7 and 13b that raised seizure threshold and conferred 100%
protection in the scPTZ screening test, were further analyzed in order to quantitate their median
effective doses (ED ) at 95% confidence interval. The candidate with the least ED will be
5
0
50
selected for the final step to predict mechanism of action c.f Table 2. Compound 6 (ED 54.86
5
0
mg/kg, 0.131 mmol/kg) possessed the highest potency and the least ED with respect to 13b
5
0
(77.11 mg/kg, 0.183 mmol/kg), and 7 (69.93 mg/kg, 0.198 mmol/kg), with confidence limits of
(57.51-65.34), (97.20-61.18) and (55.83-73.20), respectively. Regarding reference standards in
the scPTZ test for median effective dose, compound 6 protected 50 % of animals at almost half
the dose of ethosuximide; 130.55 mg/kg (177.87–98.78), which confirmed its superior activity.
2
.2.4. GABA estimation in whole mouse brain
Further neurochemical assessment was carried out to predict the possible mechanism of
anticonvulsant action for compound 6, which possessed highest potency with the least median
effective dose as previously shown in phases I and II (c.f. tables 1, 2). The target candidate and
gabapentin were given to a new set of animal groups (n=6) at dose of 100 mg/kg, i.p., which
were then sacrificed 60 min post-treatment. Quantitative estimation of GABA concentration in
whole brain homogenate of the target compound was compared against control and gabapentin
as reference standard in the current study. As graphically represented in figure (3), GABA level
in whole brain of control animals recorded 2.09±0.07µg/g tissue, whereas compound 6
significantly elevated brain GABA level to 4 folds more than that of the normal control group at
p<0.05. Interestingly, the candidate drug further exhibited 36% higher GABA level than that of
gabapentin (6.28±0.25 µg/ g tissue) at p<0.05. Hence, it could be proposed that compound 6
exerted its anticonvulsant activity via a GABA-mediated mechanism, the principal inhibitory
neurotransmitter that exerts a hyperpolarizing action in all forebrain neurons. One possible
mechanism may be through non-vesicular release of GABA, which may be mediated in part by
the GABA transporters; hence increases inhibitory tone according to During et al. (1995) [27].
Other mechanisms may involve GABA receptor activation or GABA receptor inhibition, or
A
B
8

modulation of enzymes involved in metabolic pathways, thus promoting GABA synthesis or
release. In the current investigation, we proposed the possible elevation in GABA brain level to
be mediated via the latter mechanism, thus we carried out docking studies using the γ-
aminobutyric acid aminotransferase enzyme as will be explained below (fig 4).
9

Table 1
Phase I anticonvulsant activity and minimal motor impairment of the synthesized compounds 6-
8
, 9a-h, 10-11, 13a, b -16a, b
Compound
Dose
% Protection
Neurotoxicity
mg/kg mmol/kg scPTZ MES
6
7
8
9
9
9
9
100
100
100
100
100
100
100
0. 239
0.283
0.282
0. 256
0. 237
0. 256
0.246
100
100
67
33
33
17
0
0/6
0/6
0/6
2/6
0/6
0/6
0/6
20
20
20
17
33
a
b
c
33
33
d
9
9
e
f
100
100
0. 224
0.214
13
29
14
17
0/6
0/6
9
9
1
1
1
1
1
1
1
1
1
g
h
1
100
100
100
100
100
100
100
100
100
100
100
0.253
0. 243
0.210
0.279
0. 238
0. 218
0. 186
0. 279
0. 238
0. 24
33
83
33
33
100
40
17
83
0
17
0
0
17
0
50
33
17
0
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
3a
3b
4a
4b
5a
5b
6a
6b
67
17
0
0
0. 209
Phenobarbital
Ethosuximide
Diphenyl Hydantoin
30
50
45
0.129
0.354
0.178
100
100
-
-
-
-
-
-
100
Table 2
The quantification study for determination of ED of the most active compounds in scPTZ screening test
5
0
Compound
ED (mg/kg)
ED (mmol/kg)
5
0
50
6
7
3b
54.86 (57.51-65.34)
69.93 (55.83-73.20)
77.11 (97.20-61.18)
130.55 (177.87–98.78)
13.2 (15.90–6.80)
0.131(0.137-0.156)
0.166 (0.133-0.174)
0.183 (0.231-0.145)
0.92 (1.26-0.699)
1
Ethosuximide
Phenobarbital
0.056 (0.068-0.029)
ED : median effective dose
5
0
1
0

Insert Fig.3
2
.3. Structure activity relationship
The synthesized benzopyran-2-one hybrids 6-8, 9a-h, 11 and 13a, b -16a, b were found as a
lead for anticonvulsant drug development. According to the above mentioned results we can
deduce a conclusion illustrating the relation between the structure and their activity. The
protection obtained by these compounds was indicating their ability as anticonvulsants and this
property is due to incorporation of various heterocyclic moieties and acylhydrazone scaffold to
the coumarin ring.
Compounds 6, 7 and 13b hybrid containing pyrrolidin-2, 5-dione, pyrazoles and triazole
moieties, respectively produced the most potent activity in the preliminary in vivo screening
against sc-PTZ seizure.
Incorporation of pyrroilidin- 2, 5-dione and pyrazoles entities (compounds 6 and 7) to the
coumarin ring increase the activity against sc-PTZ seizure. Meanwhile, incorporation of
pyrazole-5-one scaffold (compound 8) decreased the activity.
Regarding acylhydrazone derivatives 9a-h, compound 9h bearing 4-Cl substitution exhibited the
most potent activity against sc-PTZ seizure.
Concerning 14a, b -16a, b hybrid, substitution of iminothiazole, thiadiazole and oxathiazolidine
with ethyl moiety increased the activity more than the substitution with benzyl moiety.
Meanwhile, in the triazole scaffold 13a,b hybrid the substitution with ethyl moiety deceased the
activity.
2
.4. Molecular modeling study
Computer-aided drug design is considered an important tool in designing selective and
potent inhibitors [5, 8, 28]. Docking study can be used to illustrate the molecular interaction of
the novel candidates at protein– ligand interface. GABA-AT (γ-aminobutyric acid
aminotransferase) is a validated target for AEDs and being a catabolic in nature, its selective
inhibition raises GABA concentrations in brain [8, 21]. The enzyme was obtained from PDB,
code: 1OHW [29]. The binding interaction for the inhibitor (viagabatrin) and compound 6 were
clarified in Figure 4.
1
1

In this work, the amino acids coordinates corresponding to chain A were extracted. The residues
containing in the active site of chain A consisting of His44A, Tyr69A, Ile72A, Gly136A,
Ser137A, Phe189A, Arg192A, Lys203A, Ile205A, His206A, Glu265A, Ser269A, Glu270A,
Glu298A, Val300A, Lys329A, Arg422A, Asn423A, Ile426A, Gly438A, Gly440A [8, 22].
Concerning the viagabatrin, it showed two hydrogen bond interactions with Arg 192 and Lys
3
1
29. Additionally, the GABA-AT inhibitor showed Van der Waals interaction with Ile 72, Phe
89, Val 300, Hist 206, Arg445 and Gln301 residues. Furthermore, compound 6 showed good
interaction with GABA-AT enzyme, it formed seven hydrogen bond interactions, two hydrogen
bonds with Arg 192 which is a gate-keeper residue in the GABA active site, two hydrogen
A
bonds with Gly136, one hydrogen bond with His206, one hydrogen bond with Ser 328 and one
hydrogen bond with Lys329 and shown Van der Waals interaction with Arg 445, Gln 301 and
Val 300. The results illustrated the good interaction of the tested compounds with GABA-AT
binding site which may justify the increased GABA level in the brain.
Insert Fig.4
2
.5. Pharmacophore distance mapping
The distance between the essential features [presence of one aryl (R) hydrophobic domain,
one electron donor (D), and a hydrogen bond acceptor/donor unit (HBD)] for anticonvulsant
activity was calculated using Discovery Studio. The result of pharmacophore distance mapping
of the most potent compound 6 was illustrated on the generated hypothetical model, which was
in good agreement with the clinically available AEDs [5, 8].
Insert Fig.5
2
.6. In silico studies
CNS bioavailability of the drug is greatly affected by physicochemical parameters such as
log P (partition coefficient), molecular weight (MW), hydrogen bond acceptors and donor. The
synthesized molecules were analyzed for physicochemical parameters using swissadme online
tool ref (Table 3).
1
2

The lipophilicity of all target candidates were l enough (>2) and having the ability to cross the
BBB and being potent anticonvulsants. TPSA (topological polar surface area) define molecules
ability to penetrate blood brain barrier and absorption through the intestine. TPSA value < 140 is
essential for absorption via intestine and <90 for the blood brain barrier penetration [8, 30].
Conformational flexibility and binding of the molecules to the receptors are affected by number
of rotatable bonds. Generally, all the compounds possess high number of rotatable bonds (7–11).
The other physiochemical properties such as hydrogen bond donors and hydrogen bond
acceptors were complemented with Lipniski rule of five ref and these parameters strongly show
the suitability of the new compounds as drug like candidates. None of the synthesized entities
violated Lipinski parameters (rule of 5) which may induce their potential anticonvulsant activity.
1
3

Table 3
Physicochemical parameters of the title compounds (6-8, 9a-h, 10-11, 13a, b -16a, b).
a
LogPd
s
Compounds TPSA
n-
ROTB
Mol.
Wt.
n-HB
donor
n-HB
acceptor violation
Lipinski’
2
b
c
e
f
h
(A )
6
7
8
9
9
9
9
9
9
9
9
1
1
1
1
1
1
1
1
1
105.92
74.33
94.30
80.90
126.72
80.90
90.13
84.14
108.59 11
80.90
80.90
126.23
105.14
105.14
114.07
7
6
6
8
9
8
9
10
418.40 2.41
352.38 3.54
354.36 2.24
376.41 2.59
421.40 2.26
390.43 2.86
406.43 2.92
433.50 3.03
466.48 3.52
394.40 2.64
410.85 2.85
475.45 2.64
357.43 3.10
419.50 3.10
475.56 3.60
551.66 3.74
357.43 3.26
419.50 3.64
1
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
5
5
5
7
5
6
5
8
6
5
7
4
4
5
5
5
5
6
6
a
b
c
d
e
f
g
h
8
8
9
6
7
9
1
3a
3b
4a
4b
5a
5b
6a
6b
114.07 11
105.49
105.49
126.51
126.51
7
8
8
9
415.46
3
477.53 3.40
All values were calculated using online software swissadme.com, [a] Topological polar surface area, [b] Total number of free rotated bounds, [c]
Molecular weight [d] Theoretical log
p
, , [e] Number of hydrogen bond donors, [f] Number of
hydrogen bond acceptors
1
4

3
. Conclusion
A series of benzopyrone derivatives 6-8, 9a-h, 10-11, 13a, b -16a, b were synthesized and
evaluated for their anticonvulsant activity and possible side effects (motor dysfunction) through
MES, scPTZ, rotarod methods, respectively. Compound 6 was the most potent congener among
the tested compounds in scPTZ test and exhibited the highest safety margin as compared to
ethosuximide as reference drug. Additionally, compound 6 was devoid of neurotoxicity. In silico
pharmacophoric pattern as well as drug likeness study illustrated that compound 6 fulfilled all
the requirements which were crucial for anticonvulsant drugs. Compound 6 significantly
elevated brain GABA level to 4 folds that of the control group and showed good binding
interaction in molecular modeling studies, which indicate that the anticonvulsant action might be
through GABA-mediated.
4
. Experimental
4
.1. Chemistry
All chemicals were commercially available. Infrared (IR) spectra were tested as KBr pellets (for
1
13
solids) using JASCO FT/IR-6100 spectrometer. H NMR (400 MHz) and C NMR (125 MHz)
spectra were performed through Jeol ECA 500 MHz spectrometer and the values of chemical
shift were represented as ppm on d scale. Mass spectral data were attained using electron impact
(EI) ionization technique at 70 eV. Elemental analyses were carried out in Microanalytical Units
at National Research Centre and Cairo University. Melting points were measured on a Walden
Precision Electro thermal 9300 apparatus (Stone, Staffordshire, England) and are uncorrected.
Synthesis of 7-hydroxy-4-methyl-2H-chromen-2-one (1)
Yield: 95%, m.p. 185 °C as reported [31].
Synthesis of 7-(allyloxy)-4-methyl-2H-chromen-2-one (2)
Yield: 90%, m.p. 105 °C as reported[32].
Synthesis of 8-allyl-7-hydroxy-4-methyl-2H-chromen-2-one (3)
Yield: 65%, m.p. 197 °C as reported[32].
Synthesis of ethyl 2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetate (4)
1
5

Yield: 85%, m.p. 103 °C as reported [33].
Synthesis of 2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetohydrazide (5)
-1
Yield: 80%, m.p. 165 °C as reported [33]; IR (KBr, cm ) exhibited bands at 3356, 3317 (NH ),
2
3
215 (NH), 3007, 3059, 3082, 3107 (CH Ar), 2856, 2924, 2968 (CH aliphatic), 1668, 1712
1
(
2C=O); HNMR (DMSO d , δ, ppm): 2.40 (s, 3H, CH at C4), 3.58 (d, 2H, J=8 Hz, CH -CH),
6
3
2
4
.75 (s, 2H, OCH ), 4.77 (d, 1H, J=8 Hz, CH=CH), 5.02 (d, 1H, J=2 Hz, CH=CH,), 5.89-5.99
2
(
m, 1H, CH-CH ), 6.24 (s, 1H, CH at C3), 7.03 (d, 1H, J=8 Hz, CH at C6), 7.59 (d, 1H, J=8 Hz,
2
CH at C5), 8.56 (br.s, 2H, NH ), 10.09 (br.s, 1H, NH, D O-exchangeable).
2
2
Synthesis of 2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-N-(1,3 dioxoisoindolin-2-
yl)acetamide (6)
A mixture of compound 5 (10 mmol, 2.7gm) and phthalic anhydride (10 mmol, 1.48gm,) were
dissolved in dioxane (20 mL) containing glacial acetic acid (0.14 mL) and heated under reflux
for 7h. Subsequently, the reaction mixture was concentrated under reduced pressure then the
deposited solid was filtered, dried and recrystallized from ethanol.
-1
Yield: 96%, m.p. 260 °C [23]; IR (KBr, cm ) exhibited bands at 3379 (NH), 3001, 3076, 3116
1
(
CH Ar), 2922, 2954, 2976 (CH aliphatic), 1626, 1672, 1712, 1720 (4C=O), 1608 (NH); HNMR
(
DMSO d , δ, ppm): 2.43 (s, 3H, CH at C4), 3.58 (d, J=4 Hz, 2H, CH -CH), 4.81 (s, 2H.
6
3
2
OCH ), 4.98 (d, 1H, J=8 Hz, CH=CH), 5.04 (d, 1H, J=12 Hz, CH=CH), 5.93-5.97 (m, 1H, CH-
2
CH ) , 6.26 (s, 1H, CH at C3) , 7.07(d, 1H, J=12 Hz, H6-Ar,) , 7.67 (d, 1H, J=4 Hz, H5-Ar) ,
2
1
3
7
1
.94 (s, 4H, H ), 10.35 (br.s, 1H, NH, exchanged with D O ); CNMR (DMSO d , δ, ppm):
ar. 2 6
9.7 (CH at C4), 27.1 (CH -CH), 66.8 (O-CH ), 109.4 (C6), 111.3 (C10), 112.08 (C3), 114.6
3
2
2
(CH =CH), 115.9 (C8), 116.0 (C5), 124.3 (2CH ), 129.3 (2CH Ar), 135.7 (2C ), 135.8
2 ar ar.
(
CH=CH ), 152.2 (C9), 154.04 (C4), 158.5 (C2, coumarin), 160.5, 165.2, 167.8 (3C=O); MS (EI)
2
m/z (%):418.3 ([M+, 58.4); Anal. Calcd. for C H N O : C, 66.03; H, 4.34; N, 6.70, Found: C,
2
3
18
2
6
6
6.32; H, 4.65; N, 6.96.
Synthesis of 8-allyl-7-(2-(3,5-dimethyl-1H-pyrazol-1-yl)-2-oxoethoxy)-4-methyl-2H-chromen-2-
one (7)
Acetylacetone (10 mmol, 1mL) was added to a solution of compound 5 (10 mmol, 2.7 gm) in
absolute ethanol (20 ml) containing few drops of triethylamine and was refluxed for 12h. The
1
6

reaction mixture was concentrated and poured into ice-HCL and the resultant solid was filtered,
washed with diluted ethanol, dried and recrystallized from ethanol to give 7 as white solid [23].
-1
Yield: 98%, m.p. 178 °C; IR (KBr, cm ) exhibited bands at 3001, 3076, 3116 (CH Ar), 2922,
1
2
954, 2976 (CH aliphatic), 1712, 1720 (2C=O), 1625 (C=N), HNMR (DMSO d , δ, ppm): 1.76
6
(
s, 3H, N=C-CH ) , 2.00 (s, 3H, CH at C4), 2.39 (s, 3H, N=C-CH ), 3.55 (d, 2H, J=6 Hz, CH -
3 3 3 2
CH),4.96 (d, 1H, J=1.6 Hz, CH=CH), 4.99 (d, 1H, J=1.6 Hz, CH=CH), 5.09 (s, 2H, OCH ),
2
5
.93-6.03 (m, 1H, CH -CH), 6.22 (s, 1H, CH at C3) , 6.50 (s, 1H, CH-pyrazole), 6.91 (d, 1H,
2
1
3
J=8.8 Hz, H6-Ar) , 7.58 (d, 1H, J=8.8 Hz, H5-Ar); CNMR (DMSO d , δ, ppm): 16.3, 18.6 (
6
2
1
1
CH - pyrazole), 26.2 (CH -at C4), 27.1 (CH -CH), 66.9 (OCH ), 109.1 (C6), 111.6 (C10),
3 3 2 2
13.9 (C3), 115.2 (CH =CH), 118.8 (C8), 124.8 (C5), 135.7 (CH=CH ), 152.3 (C9), 154.0 (C4),
2
2
+
59.3 (C7), 160.5 (C2-coumarin), 164.7 (N-C=O); (EI) m/z (%):, 419 ([M +1, 39.66), 84.3 (100),
Anal.calcd. for C H N O : C, 68.17; H, 5.72; N, 7.95, Found: C, 68.25; H, 5.61; N, 7.85.
2
3
18
2
6
Synthesis of 2-(2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetyl)-5-methyl-1,2-dihydro-
3
H-pyrazol-3-one (8)
A mixture of compound 5 (10 mmol, 2.7 gm) and ethyl acetoacetate (10 mmol, 1.3 gm) in
absolute ethanol (20 mL) were refluxed for 4h. The reaction mixture was concentrated; the
formed precipitate was filtered, washed with water and recrystallized from ethanol to give 8 as
yellowish white solid.
-1
Yield: 90%, m.p. 200 °C; IR (KBr, cm ) exhibited bands at 3446 (NH), 1680, 1708, 1730
1
(
3C=O), 1604 (NH); HNMR (DMSO d , δ, ppm): 2.09 (br.s, 1H, NH), 2.40 (s, 3H, CH -
6
3
pyrazolone) , 2.50 (s , 3H, CH -at C4 ), 3.53 (d, 2H, J=5.8 Hz, CH -CH) , 4.15-4.20 (m, 2H,
3
2
OCH ) , 4.97 (s, 2H, CH=CH, CH-pyrazolone ), 5.01 (s, 1H, CH=CH) , 5.91-5.93 (m, 1H, CH -
2
2
CH), 6.24 (s, 1H, CH at C3), 6.96 (d, 1H, J=8 Hz, H6-Ar), 7.63 (d, J=8 Hz, 1H, H5-Ar); MS
+
(
EI) m/z (%): , 354.36 ([M +1, 70); Anal.calcd. for C H N O : C, 64.40; H, 5.12; N, 7.91,
6
19 18
2
4
Found: C, 64.63; H, 5.33; N, 8.20.
General method for synthesis of (E)-2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl) oxy)-N'-
benzylideneacetohydrazide (9a-h)
A solution of the acid hydrazide 5 (0.01 mol, 2.88 gm) and the appropriate aromatic aldehyde
(0.01mol) in glacial acetic acid (20 mL) was refluxed for 6h. The crude mixture was evaporated
under pressure and recrystallized from ethanol.
1
7

(E)-2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-N'-benzylideneacetohydrazide (9a)
-1
Yield: 77%, m.p. 245 °C [23]; IR (KBr, cm ) exhibited bands at 3336 (NH), 3030, 3064, 3078
1
(
CH Ar), 2862, 2922, 2978 (CH aliphatic), 1685, 1716 (2C=O), 1604 (C=N); HNMR (DMSO
d , δ, ppm): 2.39 (s, 3H, CH at C4) , 3.35 (d, 2H, J=4 Hz, CH -CH) , 4.85 (s, 2H, OCH ) , 4.96
6
3
2
2
(
d, 1H, J=1.6 Hz, CH=CH) , 5.01 (d, 1H, J=1.6 Hz, CH=CH), 5.94-5.99 (m, 1H, CH -CH,) 6.24
2
(s, 1H, CH at C3) , 7.02 (d, 1H, J=8 Hz, H6-Ar), 7.61 (d, 1H, J=8 Hz, H5-Ar) , 7.72-7.73 (m,
1
3
5
H, H ), 8.28 (s, 1H, N=CH), 11.67 (br.s, 1H, NH, exchanged with D2O); CNMR (DMSO d ,
ar 6
δ, ppm): 19.5 (CH at C4), 27.1 (CH -CH), 66.1 (OCH ), 109.1 (C6), 111.6 (C10), 112.5 (C3),
3
2
2
1
15.4 (CH=CH ), 121.6 (C8), 112.4 (C5), 127.5, 129.2, 130.4 (3CH ), 134.4 (C ), 135.7
2 ar ar
(
CH=CH ), 144.4 (N=CH), 152.3 (C9), 154.01 (C4), 159.2 (C7), 160.7 (C2), 169.07 (CO-NH);
2
+
MS (EI) m/z (%): , 376.41 ([M +1, 39.66), 84.3 (100); Anal.calcd. for C H N O C, 70.21; H,
6
22 20
2
4
5
.36; N, 7.44, Found: C, 70.45; H, 5.61; N, 7.53.
(E)-2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-N'-(4-nitrobenzylidene)acetohydrazide
(9b)
-1
Yield: 77%, m.p. 250 °C; IR (KBr, cm ) exhibited bands at 3275 (NH), 3080, 3196 (CH Ar),
1
2
922, 2943, 2970 (CH aliphatic), 1685, 1705 (2C=O), 1600 (NH), 1521, 1344 (NO ). HNMR
2
(
DMSO d , δ, ppm): 2.56 (s, 3H, CH at C4) , 3.56 (d, 2H, J=6 Hz, CH -CH) , 4.90 (s, 2H,
6 3 2
OCH ), 4.97 (d, 1H, J=10.4 Hz, CH=CH), 5.04 (s, 1H, CH=CH), 5.93-5.95 (m, 1H, CH -CH),
2
2
6
.24 (s, 1H, H3-Ar) 7.04 (d , 1H, J=8 Hz, H6-Ar), 7.61 (d, 2H, J=12 Hz, H5-Ar) ,7.97 (d, 2H,
J=8 Hz, H ) , 8.27 (d, 2H, J=8 Hz, H ), 8.38 (s, 1H, N=CH), 11.96 (br.s,1H, NH, exchanged
ar
ar
1
3
with D O); CNMR (DMSO d , δ, ppm): 18.6 (CH at C4), 27.28 (CH -CH), 68.2 (O-CH ),
2
6
3
2
2
1
1
1
4
09.2 (C6), 115.9 (CH=CH ), 120.3 (C9), 124.4 (CH ), 124.5 (C5), 128.43 (CH ), 130.0 (C ),
2 ar ar ar
35.6 (CH=CH ), 140.7 (N=CH-C), 142.8 (N=CH), 150.9 (C=NO ), 152.3 (C4), 159.2 (C=O),
2
2
+
69.5 (CO-NH); MS (EI) m/z (%): , 421.41 ([M , 25), Anal.calcd. for C H N O C, 62.70; H,
6
22 19
3
6
.54; N, 9.97, Found: C, 62.95; H, 4.67; N, 710.16.
(E)-2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-N'-(4-methylbenzylidene)acetohydrazide
(9c)
-1
Yield: 78%, m.p. 250 °C; IR (KBr, cm ) exhibited bands at 3336 (NH), 3001, 3089, 3194 (CH
1
Ar), 2872, 2933, 2976 (CH aliphatic), 1685, 1720 (2C=O), 1639 (C=N), 1604 (NH), HNMR
1
8

(
DMSO d , δ, ppm): 2.39 (s, 3H, CH -C H ), 2.51 (s, 3H, CH at C4) , 3.57 (d , 2H, J=8 Hz,
6 3 6 5 3
CH -CH) , 4.83 (s, 2H, OCH ) , 4.97 (t, 1H, J=8 Hz, CH=CH), 5.03 (t, 2H, J=8 Hz, CH=CH),
2
2
5
.92-6.00 (m, 1H, CH-CH ) , 6.24 (s ,1H, CH at C3), 6.95 (d, 2H, J=8 Hz, H ), 7.01 (d, 1H, J=
2 ar
8
Hz, H6- Ar) , 7.61 (d, 2H, J=8 Hz, H ), 7.65 (d, 1H, J=8 Hz, H5- Ar), 7.9 (s, 2H, H ), 8.21
ar. ar.
1
3
(
s, 1H, N=CH) , 11.5 (br.s, 1H, NH, exchanged with D2O); CNMR (DMSO d , δ, ppm): 19.9
6
(
CH -C H ), 20.1 (CH at C4), 27.1 (CH -CH), 66.1 (OCH ), 109.2 (C6), 111.6 (C10), 112.9
3
6
5
3
2
2
(
C3), 115.9 (CH=CH ), 119.9 (C8), 124.5 (C5), 127.04, 129.04 (CH ), 129.7 (C ), 134.5
2 ar ar.
(
CH=CH ), 144.5 (N=CH), 150.9 (C-NO ), 152.3 (C9), 154.06 (C4), 159.3 (C7), 160.5 (C2),
2
2
+
1
5
68.8 (CO-NH); MS (EI) m/z (%): , 390.44 ([M , 40),; Anal.calcd. for C H N O : C, 70.75; H,
6
23 22
2
4
.68; N, 7.18, Found: C, 70.92; H, 5.77; N, 7.26.
(E)-2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-N'-(4-methoxybenzylidene)acetohydrazide
(9d)
-1
Yield: 77%, m.p. 250 °C;IR (KBr, cm ) exhibited bands at 3446 (NH), 3082, 3032, 3001 (CH
1
Ar), 2978, 2966, 2933 (CH aliphatic), 1722, 1685 (2C=O), 1604 (C=N); HNMR (DMSO d , δ,
6
ppm): 2.39 (s, 3H, CH at C4) , 3.59 (d, 2H, J=6 Hz, CH -CH), 3.80 (s, 3H, OCH ) , 4.83 (s, 2H,
3
2
3
OCH ) , 4.98 (d, 1H, J=1.6 Hz, CH=CH), 5.01 (d, 1H, J=1.6 Hz, CH=CH), 5.92-6.02 (m, 1H,
2
CH -CH), 6.24 (s, 1H, CH at C3), 7.00 (d, 1H, J=6 Hz, H6-Ar) , 7.03 (d, 2H, J=9 Hz, H ), 7.63
2
ar.
(
d, 2H, J=8.8 Hz, H ), 7.65(d, 1H, J=8.8 Hz, H5-Ar) , 8.21 (s, 1H, N=CH), 11.4 (br.s,1H, NH,
ar
1
3
exchanged with D2O); CNMR (DMSO d , δ, ppm): 19.5 (CH at C4), 27.1 (CH -CH), 55.7
6
3
2
(
OCH ), 66.1 (OCH ), 109.1 (C6), 111.6 (C10), 112.2 (C3), 115.9 (CH=CH), 120.2 (C8), 122.4
3 2
(
C5), 127.1 (C ), 129.1 (2CH ), 135.7 (CH=CH ), 144.5 (N=CH), 152.2 (C9), 154.05 (C4),
ar. ar 2
+
1
59.3 (C7), 160.4 (C2), 161.2 (C ), 168.2 (CO-NH); MS (EI) m/z (%): , 406.44 ([M , 30);
ar. 6
Anal.calcd. for C H N O : C, 67.97; H, 5.46; N, 6.89, Found: C, 67.83; H, 5.09; N, 6.56.
2
3
22
2
5
2
-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-N'-((1E)-3-(4-
(dimethylamino)phenyl)allylidene)acetohydrazide (9e)
-1
Yield: 70%, m.p. 260 °C; IR (KBr, cm ) exhibited bands 3446 (NH), 3062, 3035, 3012 (CH Ar),
1
2
976, 2960, 2926 (CH aliphatic), 1714, 1627 (2C=O); HNMR (DMSO d , δ, ppm): 2.41 (s, 3H,
6
CH at C4) , 2.86 (s, 6H, N(CH ) ) , 3.57 ( d, 2H, J=6 Hz, CH -CH), 4.81 ( s, 2H, OCH ), 4.97
3
3 2
2
2
(
d, 1H, J=8.6 Hz, CH=CH) , 5.02 (s, 1H, CH=CH), 5.90-6.00 (m, 1H, CH -CH), 5.23 ( br.s, 1H,
2
NH, exchangeable with D O), 6.25 (s, 1H, H3-Ar), 6.65 (d, 2H, J=8.8 Hz, H ) 6.99 (d, 2H,
2
ar.
1
9

J=8.5 Hz, H6-Ar) , 7.05 (d, 2H, J=8.8 Hz, H ), 8.04 (s, 1H, N=CH), 7.64 (d, 1H, J=8.8 Hz, H5-
ar.
+
Ar), 10.26 (br.s, 1H, NH); MS (EI) m/z (%): , 445.52 ([M , 23); Anal.calcd. for C H N O : C,
6
26 27
3
4
6
8.72; H, 6.01; N, 10.02, Found: C, 68.88; H, 6.22; N, 10.30.
(E)-2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-N'-(3,4,5-
trimethoxybenzylidene)acetohydrazide (9f)
-1
Yield: 76%, m.p. 250 °C;IR (KBr, cm ) exhibited bands at 3213 (NH), 3076 (CH Ar), 2935,
1
2
954, 2974 (CH aliphatic), 1712,1720 (2C=O), 1624 (C=N), 1604 (NH); HNMR (DMSO d , δ,
6
ppm): 2.40 (s, 3H, CH at C4), 3.55 (d, 2H, J=4 Hz, CH -CH), 3.71 (s, 3H, OCH ), 3.81 (m, 6H,
3
2
3
OCH ), 4.85 (s, 2H, O-CH ), 4.99 (d, 1H, J=6.8 Hz, CH=CH), 5.03 (d, 1H, J=6 Hz, CH=CH),
3
2
5
.91-5.95 (m, 1H, CH=CH ), 6.23 (s, 1H, CH at C3), 7.00 (d, 1H, J=7.4 Hz, H6-Ar), 7.22 (s, 2H,
2
CH-Ar), 7.64 (d, 1H, J=8 Hz, H5-Ar), 7.93 (s, 1H, N=CH), 11.56 (br.s, 1H, NH, exchanged with
1
3
D O); CNMR (DMSO d , δ, ppm): 18.22 (CH at C4), 26.71 (CH -CH), 55.94 (OCH ), 60.12
2
6
3
2
3
(
2 OCH ), 65.5 (O-CH ), 104 (CH ), 108.6 (C6), 111.1 (C10), 113.4 (C3), 115.4 (CH=CH ),
3 2 ar 2
1
1
24.09 (C8), 124.3 (C5), 129.4 (C ), 135.2 (CH=CH ), 140.2 (C ), 147.7 (N=CH), 151.8 (C9),
ar 2 ar
+
53.16 (C ), 158.8 (C-O), 160 (C2), 168.6 (CO-NH); MS (EI) m/z (%):, 466.49 ([M , 5.04);
ar
Anal.calcd. for C H N O : C, 64.37; H, 5.36; N, 6.01, Found: C, 64.53; H5.39; N, 6.24.
2
5
26
2
7
(E)-2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-N'-(4-fluorobenzylidene)acetohydrazide
(9g)
-1
Yield: 77%, m.p. 240 °C;IR (KBr, cm ) exhibited bands at 3446 (NH), 3086, 3176 (CH Ar),
1
2
924, 2943, 2976 (CH aliphatic), 1687, 1716 (2C=O), 1639 (C=N), 1602 (NH); HNMR (DMSO
d , δ, ppm): 2.39 (s, 3H, CH at C4), 3.56 (d, 2H, J=8 Hz, CH -CH), 4.85 (s, 2H, OCH ), 4.97 (d,
6
3
2
2
1
H, J=8 Hz, CH=CH), 5.03 (s, 1H, J=8 Hz, CH=CH), 5.92-6.04 (m, 1H, CH -CH), 6.23 (s, 1H,
2
H3-Ar), 7.00 (d, 1H, J=8 Hz, H6 Ar), 7.27 (d, 2H, J=8 Hz, H ), 7.29 (d, 2H, J=8 Hz ,H ), 7.66
ar.
ar.
(
d, 1H, J=8 Hz, H5- Ar), 8.28 (s, 1H, N=CH), 11.6 (br.s, 1H, NH, exchanged with D O);
2
1
3
CNMR (DMSO d , δ, ppm): 18.22 (CH at C4), 26.71 (CH -CH), 66.93 (OCH ), 108.7 (C6),
6
3
2
2
1
11.45 (C9), 113.53 (C3), 115.7 (CH ), 115.93 (CH=CH ), 124.1 (C8), 124.3 (C5), 129.3, 130.5
ar. 2
(
2 CH ), 135.2 (CH=CH ), 146.6 (N=CH), 151.8 (C9), 153.5 (C4), 158.8 (C7), 160 (C2), 164.2
ar 2
+
(
C ), 168.6 (CO-NH); MS (EI) m/z (%):, 394.40 ([M , 15); Anal.calcd. for C H FN O : C,
ar
22 19
2
4
6
7.00; H, 4.86; N, 7.10, Found: C, 67.27; H, 5.04; N, 7.32.
2
0

(E)-2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-N'-(4-chlorobenzylidene)acetohydrazide
(9h)
-1
Yield: 77%, m.p. 250 °C;IR (KBr, cm ) exhibited bands at 3481 (NH), 3066, 3082, 3130 (CH
1
Ar), 2945, 2962, 2978 (CH aliphatic), 1685, 1716 (2C=O), 1602 (C=N), 1570 (NH); HNMR
(
DMSO d , δ, ppm): 2.51 (s, 3H, CH3 at C4), 3.55 (d, 2H, J=6 Hz, CH -CH), 4.78 (s, 2H,
6
2
OCH ), 4.97 (t, 1H, J =8 Hz, CH=CH), 5.03 (d, 1H, J=7 Hz, CH=CH), 5.91-6.02 (m, 1H, CH -
2
2
CH), 6.23 (s, 1H, CH at C3), 7.01 (d, 1H, J=4 Hz, H6-Ar), 7.5 (d, 2H, J=8 Hz, H ), 7.73 (d, 1H,
ar.
J=4 Hz, H5 Ar), 7.91 (d, 2H, J=8 Hz, H ), 8.27 (s, 1H, N=CH), 11.67 (br.s, 1H, NH, exchanged
ar.
with D2O); MS (EI) m/z (%):, 410.85 ([M+, 45.11); Anal.calcd. for C H ClN O : C,64.32; H,
2
2
19
2
4
4
.66; N, 6.82, Found: C, 64.35; H, 4.92; N, 6.96.
Synthesis of 2-(2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetyl)-N-benzylhydrazine-1-
carboxamide (10)
A mixture of compound 5 (0.01 mol, 2.88 gm) and benzyl isocyanate (0.01 mol, 1.59 gm) in
dichloroethane was stirred and refluxed for 3h. The formed precipitate was filtered, washed with
diethyl ether and dried. The crude mixture was crystallized from isopropanol to give 10 as a
white solid.
-1
Yield: 95%, m.p. 278 °C; IR (KBr, cm ) exhibited bands at 3305, 3331, 3385 (3NH) 3089,
1
3
064, 3034 (CH Ar), 2956, 2926 (CH aliphatic), 1747, 1710, 1693 (3C=O); HNMR (DMSO d ,
6
δ, ppm): 2.39 (s, 3H, CH at C4), 3.57 (d, 2H, J= 4Hz, CH -CH), 4.25 (s, 2H, N-CH ), 4.76 (s,
3
2
2
2
H, OCH ), 4.97 (d, 1H, J=4 Hz, CH=CH), 5.02 (d, 1H, J=4 Hz, CH=CH), 5.90-6.00 (m, 1H,
2
CH -CH), 6.23 (s, 1H, CH at C3), 7.01 (d, 1H, J=12 Hz, CH at C6), 7.27-7.36 (m, 5H, CH-Ar),
2
7
.58 (d, 1H, J=8 Hz, CH at C5), 8.04 (br.s, 1H, O=NH, exchanged with D O), 9.28 (br.s, 1H,
2
1
3
O=C-NH, exchanged with D O), 9.82 (br.s, 1H, NH, exchanged with D O); CNMR (DMSO d ,
2
2
6
δ, ppm): 18.2 (CH at C4), 26.6 (CH -CH), 45.11 (NH-CH ), 66.6 (O-CH ), 108.7 (C6), 111.4
3
2
2
2
(
C10), 113.8 (C3), 115.4 (CH=CH ), 123.9 (C8), 124.1 (C5), 126.8, 128.1 (CH ), 135.3
2
a
r
.
(
CH=CH ), 140.4 (NH-CH -C), 151.7 (C9), 153.5 (C4), 158.7 (NH-C=O), 160.01 (C7), 160.08
2 2
+
(
C2), 167.2 (NH-CO); MS (EI) m/z (%):, 421.45 ([M , 42); Anal.calcd. for C H N O : C,65.55;
23 23 3 5
H, 5.55; N, 9.97, Found: C, 65.82; H, 5.75; N, 10.08.
Synthesis
of
2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)
oxy)-N-(3-benzyl-2,4,5-
trioxoimidazolidin-1-yl) acetamide (11)
2
1

To a solution of compound 10 (0.001 mol, 0.47 gm) in dry benzene (5 mL), oxalyl chloride
0.002 mol, 0.25 gm) was added dropwise while stirring, then refluxed at 60-65℃ for 2h. The
(
solvent was distilled under reduced pressure and the obtained residue was dried. The crude
product was crystallized from ethanol to obtain 11 as a white solid.
General procedures for the synthesis of 2-(2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-
yl)oxy)acetyl)-N-aralkylhydrazine-1-carbothioamide (12a, b)
To a solution of compound 5 (0.01 mol, 2.88 gm) in ethanol (30 mL), an appropriate
substituted isothiocyanate (0.01 mol) was added, and then the reaction mixture was refluxed
under stirring for 12h. The separated solid was filtered, washed with diethyl ether and dried. The
crude product was crystallized from isopropanol.
2
-(2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetyl)-N-ethylhydrazine-1-carbothioamide
(12a)
-1
Yield: 76%, m.p. 225 °C ; IR (KBr, cm ) exhibited bands at 3169, 3329, 3379 (3NH), 3078 (CH
1
Ar), 2976, 2931 (CH aliphatic), 1720, 1710 (2C=O), 1122 (C=S); HNMR (DMSO d , δ, ppm):
6
1
.25 (t, 3H, J=6.8 Hz, CH -CH ), 2.41 (s, 3H, CH at C4), 3.47 (d, 2H, J=8 Hz, CH -CH), 4.05
2 3 3 2
(
q, 2H, J=6.8 Hz, CH -CH ), 4.77 (s, 2H, OCH ), 4.94 (t, 1H, J=9.6 Hz, CH=CH), 5.02 (t, 1H,
2 3 2
J=1.2 Hz, CH=CH), 5.87-5.91 (m, 1H, CH-CH ), 6.26 (s, 1H, CH at C3), 7.04 (d, 1H, J=8 Hz,
2
H6-Ar), 7.28 (s, 1H, -CH ), 7.71 (d, 1H, J=8 Hz, H5-Ar), 8,0 (br.s, 1H, NH=O, exchanged with
2
1
3
D O), 9.3 (br.s, 1H, NH=S, exchanged with D O); CNMR (DMSO d , δ, ppm): 15.32 (CH -
2
2
6
2
CH ), 19.8 (CH at C4), 27.1 (CH -CH), 47.4 (NH-CH ), 67.8 (OCH ), 109.2 (C6), 111.9 (C10),
3
3
2
2
2
1
1
14.3 (C3), 115.4 (CH=CH ), 124.6 (C8), 125.4 (C5), 128.5 (CH ), 152.2 (C9 ), 152.9 (C4),
2 ar .
+
58.7 (C7 ), 160.4 (C2), 166.5(C=O), 183.2 (C=S); MS (EI) m/z (%):, 375.44 ([M , 34);
.
Anal.calcd. for C H N O S: C, 57.58, H, 5.64, N, 11.19, Found: C, 57.81, H, 5.70, N, 11.33.
1
8
21
3
4
2
-(2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetyl)-N-benzylhydrazine-1-
carbothioamide (12b)
-1
Yield: 80%, m.p. 230 °C; IR (KBr, cm ) exhibited bands at 3344, 3444, 3458 (3NH), 3005,
1
3
034, 3070, 3099 (CH Ar), 2916, 2937, 2947, 2980 (CH aliphatic), 1716, 1705 (2C=O); HNMR
(
DMSO d , δ, ppm): 2.50 (s, 3H, CH at C4) , 3.58 (d, 2H, J=6 Hz, CH -CH), 4.75(s, 2H,
6 3 2
OCH ), 4.77 (t, 1H, J=6 Hz, CH=CH), 4.78 (s, 2H, CH -C H ), 4.98 (t, 1H, J=10.4 Hz,
2
2
6
5
2
2

CH=CH), 5.0 (d, 2H, J=2 Hz, CH=CH) , 5.90-5.98 (m , 1H, CH -CH,) , 6.24 (s, 1H, CH at C3) ,
2
7
1
.03 (d, 1H , J=8 Hz, H6-Ar), 7.22-7.27 (m, 5H, H ), 7.29 (d, 1H, J=4 Hz, H5-Ar), 8.5 (br.s,
ar.
H, NH-CH, exchanged with D O), 10.09 (br.s,1H, NH-C=S, exchanged with D O); MS (EI)
2
2
+
m/z (%):, 437.51 ([M , 14); Anal.calcd. for C H N O S: C,63.14; H, 5.30; N, 9.60, Found: C,
2
5
21
3
7
6
3.35; H, 5.39; N, 9.71.
General procedures for the synthesis 8-allyl-7-((4- aralkyl-5-thioxo-4,5-dihydro-1H-1,2,4-
triazol-3-yl) methoxy)-4-methyl-2H-chromen-2-one (13a,b)
A solution of 2 N sodium hydroxide (12 mL) was added to a solution of compounds 12a,b
(0.04 mol) then refluxed with stirring for 12h. The reaction mixture was cooled and acidified
with 10% hydrochloric acid to PH 5. The produced precipitate was filtered, washed and
crystallized from methanol.
8
2
-allyl-7-((4-ethyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methoxy)-4-methyl-2H-chromen-
-one (13a)[23]
Yield: 70%, m.p. 200 °C 3419 (NH), 3003, 3043, 3157 (CH Ar), 2939, 2953, 2976 (CH
1
aliphatic), 1705 (C=O), 1608 (C=N), 1568 (NH); HNMR (DMSO d , δ, ppm): 1.25 (t, 3H, J=4
6
Hz, CH -CH ), 2.43 (s, 3H, CH at C4), 3.44 (d, 2H, J=4 Hz, CH -CH), 4.05 (d, 2H, J=4 Hz,
2
3
3
2
CH -CH ), 4.83 (d, 1H, J=16 Hz, CH=CH), 4.92 (d, 1H, J=12 Hz, CH=CH), 5.40 (s, 2H,
2
3
OCH ), 5.82-5.91 (m, 1H, CH-CH ), 6.22 (s, 1H, CH at C3), 7.28 (d, 1H, J=8 Hz, CH at C6),
2
2
7
.68 (d, 1H, J=8 Hz, CH at C5), 13.90 (br.s, 1H, NH, exchanged with D O); MS (EI) m/z (%):,
2
+
3
57.43 ([M , 100); Anal.calcd. for C H N O S: C,60.49; H, 5.36; N, 11.76, Found: C, 60.69;
18
19
3
3
H, 5.531; N, 11.95.
8
2
-allyl-7-((4-benzyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methoxy)-4-methyl-2H-chromen-
-one (13b)
Yield: 70%, m.p. 200 °C 3124 (NH), 3043, 3103 (CH Ar), 2904, 2935, 2983 (CH aliphatic),
1
1
705 (C=O), 1602 (C=N); HNMR (DMSO d , δ, ppm): 2.5 (s, 3H, CH at C4), 3.07 (d, 2H, J=8
6
3
Hz, CH -CH), 4.73 (s, 2H, N-CH ), 4.75 (d, 1H, J=8 Hz, CH=CH ), 4.87 (d, 1H, J=8 Hz ,
2
2
2
CH=CH ), 5.24 (s, 2H, CH -C H ), 5.53 (s, 2H, OCH ), 5.91-5.98 (m, 1H, CH-CH ), 6.23 (s,
2
2
6
5
2
2
1
H, CH at C3), 7.14 (d, 1H, J=8 Hz, H6-Ar), 7.18-7.28 (m, 5H, H ), 7.64 (d, 1H, J=8.8 Hz, H5-
ar.
1
3
Ar), 10.63 (br.s, 1H, NH, exchanged with D O); CNMR (DMSO d , δ, ppm): 18.4 (CH at
2
6
3
2
3

C4), 26.5 (CH -CH), 48.6 (CH -C H ), 61.6 (O-CH ), 109.06 (C6), 112.09 (C10 ), 114.5 (C3),
2
2
6
5
2
.
1
1
15.6 (CH=CH ), 124.7 (C8), 125.3 (C5), 126.1, 128.07, 129.23 (3CH ), 135.6 (CH=CH ),
2 ar 2
35.8 (C ), 148.2 (C9), 152.1 (C4), 153.9 (N=C), 157.9 (C7), 160.3 (C=O), 180.1 (C=S); MS
ar
+
(
EI) m/z (%):, 419.50 ([M , 45.11); Anal.calcd. for C H N O S: C,65.85; H5.05; N, 10.02,
2
3
21
3
3
Found: C, 66.03; H, 5.15; N, 10.32.
General procedures for the synthesis of (Z)-2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-N-
(2-(ethylimino)-4-phenylthiazol- or N-(4-phenyl-2-(phenylimino)thiazol- 3(2H)-yl)acetamide
(14a, b)
A mixture of compound 12a,b (0.002 mol) and the appropriate phenacyl bromide (0.002 mol,
0
.39gm) in ethanol/chloroform 1:3 mixture (100 mL) were refluxed for 3h, concentrated, left to
cool, filtered and dried.
(Z)-2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-N-(2-(ethylimino)-4-phenylthiazol-3(2H)-
yl)acetamide (14a)
Yield: 75%, m.p. 200 °C 3477 (NH), 3078,3061 (CH Ar), 2974, 2933 (CH aliphatic), 1712, 1600
1
(
2C=O), 702 (C-S); HNMR (DMSO d , δ, ppm): 0.94 (s, 3H, CH -CH ), 1.11 (q, 2H, J=8 Hz,
6
2
3
CH -CH ), 2.42 (s, 3H, CH at C4), 3.61 (d, 2H, J=8 Hz, CH -CH), 3.91 (q, 2H, J=12 Hz, CH -
2
3
3
2
2
CH ), 4.97 (s, 2H, O-CH ), 4.99 (t, 1H, J=8 Hz, CH=CH), 5.02 (t, 1H, J=4 Hz, CH=CH), 5.91-
3
2
5
.98 (m, 1H, CH -CH), 6.26 (s, 1H, CH at C3), 6.97 (s, 1H, CH-thiazoline), 7.09 (d, 1H, J=12
2
1
3
Hz,H6-Ar), 7.49-7.58 (m, 5H, H ), 7.7 (d, 1H, J=8 Hz, H5-Ar); CNMR (DMSO d , δ,
ar.
6
ppm):12.6 (CH -CH ), 18.2 (CH at C4), 26.6 (CH -CH), 42.2 (CH -CH ), 66.6 (OCH ), 108.3
2
3
3
2
2
3
2
(
CH-thiazoline), 111.5 (C6), 114.1 (C3), 115.0 (C3), 115.49 (CH=CH ), 124.3 (C8), 128.7 (C5),
2
1
29.01, 129.46, 130.26 (3 CH ), 135.2 (C ), 141.66 (CH =CH), 151.83 (C9), 153.5 (C4), 158.0
ar ar. 2
(
C=N-), 159.98 (C7), 160.7 (C2), 161.5 (C-C H , thiazoline), 170.4 (CO-NH); MS (EI) m/z (%):,
6 5
+
4
5
75.56 ([M , 50.2); Anal.calcd. for C H N O S: C,65.67; H,5.30; N, 8.84, Found: C, 65.81; H,
26 25 3 4
.39; N, 8.91.
(Z)-2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-N-(2-(benzylimino)-4-phenylthiazol-
3
(2H)-yl)acetamide (14b)
Yield: 70%, m.p. 236 °C 3419 (NH), 3030, 3061, 3107 (CH Ar), 2926, 2978 (CH aliphatic),
1
1
724, 1618 (2C=O), 1602 (C=N); HNMR (DMSO d , δ, ppm): 2.40 ( s, 3H, CH ), 2.42 (s, 2H,
6
3
2
4

CH -C H ), 3.53 (d, 2H, J=4 Hz, CH -CH), 4.87 (s, 2H, OCH ), 4.98 (s, 1H, CH=CH), 5.14 (s,
2
6
5
2
2
1
7
H, CH=CH), 5.94-5.97 (m, 1H, CH-CH ), 6.25 (s, 1H, H3-Ar.), 6.99 (s, 1H, CH-thiazoline),
2
.08 (d, 1H, J=8 Hz,H6-Ar.), 7.25-7.44 (m, 10H, H ), 7.63 (d, 1H, J=8.8 Hz, H5-Ar.); MS (EI)
ar.
+
m/z (%):, 537.63 ([M , 42.5); Anal.calcd. for C H N O S: C,69.26; H,5.06; N, 7.82, Found: C,
3
1
27
3
4
6
9.36; H, 5.25; N, 8.05.
General procedures for the synthesis of 8-allyl-7-((5-(aralkyl amino)-1,3,4-thiadiazol-2-yl)
methoxy)-4-methyl-2H-chromen-2-one (15a,b)
Conc.H SO (10 mL) was added to compound 12a,b. and the mixture allowed to stand overnight.
2
4
The reaction mixture was cooled then poured onto ice water and neutralized with ammonium
hydroxide solution to PH 7. The produced precipitate was filtered off, washed and crystallized
from glacial acetic acid.
8
-allyl-7-((5-(ethylamino)-1,3,4-thiadiazol-2-yl)methoxy)-4-methyl-2H-chromen-2-one (15a)
Yield: 90%, m.p. 248 °C 3277 (NH), 3061,3041 (CH Ar), 2964, 2931 (CH aliphatic), 1735
1
(
C=O), 617 (C-S); HNMR (DMSO d , δ, ppm): 1.16 (t, 3H, J=8 Hz, CH -CH ), 2.39 (s, 3H,
6
2
3
CH at C4), 3.29 (d, 2H, J=8 Hz, CH -CH), 3.40 (s, 2H, CH -CH ), 4.92 (s, 1H, CH=CH), 5.03
3
2
2
3
(
s, 1H, CH=CH), 5.83-5.90 (m, 1H, CH -CH), 5.45 (s, 2H, OCH ), 6.22 (s, 1H, CH at C3), 7.20
2 2
(
d, 1H, 8 Hz, H6-Ar), 7.61 (d, 1H, 8 Hz, H6-Ar), 7.82 (br.s, 1H, NH, exchanged with D O); MS
2
+
(
EI) m/z (%):, 357.34 ([M , 45.11); Anal.calcd. for C H N O S: C,60.49; H,5.36; N, 11.76,
1
8
19
3
3
Found: C, 60.2; H, 5.31; N, 11.65.
8
-allyl-7-((5-(benzylamino)-1,3,4-thiadiazol-2-yl)methoxy)-4-methyl-2H-chromen-2-one (15b)
1
Yield: 90%, m.p. 250 °C 3157 (NH), 1705 (C=O), 1600 (C=N), 1566 (NH).; HNMR (DMSO
d , δ, ppm): 2.40 (s, 3H, CH at C4), 2.92 (d, 2H, J=8 Hz, CH -CH), 4.49 (s, 2H, NH-CH ), 4.55
6
3
2
2
(
d, 1H, J=4 Hz, CH=CH), 5.18 (s, 1H, CH=CH), 5.37 (s, 2H, OCH ), 5.89-5.92 (m, 1H, CH -
2 2
CH), 6.24 (s, 1H, CH at C3), 7.09 (d, 1H, J=8 Hz, CH at C6), 7.64 (d, 1H, J=12 Hz, CH at C5),
+
8
.36 (br.s, 1H, NH, exchanged with D O); MS (EI) m/z (%):, 419.50 ([M , 45.11); Anal.calcd.
2
for C H N O S: C, 65.85; H,5.05; N, 10.02, Found: C, 65.81; H, 5.39; N, 8.91.
2
3
21
3
3
General procedures for the synthesis of 2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl) oxy)-N'-(3-
aralkyl -4-oxothiazolidin-2-ylidene) acetohydrazide (16a, b)
2
5

To a suspension of substituted thiosemicarbazide derivatives 12a,b (0.01 mol) in glacial acetic
acid (5 mL), anhydrous sodium acetate (0.02 mol, 1.64gm) and chloroacetic acid (0.02mol,
1
.89gm) were added. The reaction mixture was refluxed for 4h then cooled, diluted with water
and allowed to stand overnight. The product was filtered off, washed and dried.
(Z)-2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-N'-(3-ethyl-4-oxothiazolidin-2-
ylidene)acetohydrazide (16a)
Yield: 65%, m.p. 166 °C 3500 (NH), 3016, 3188 (CH Ar), 2877, 2935, 2980 (CH aliphatic),
1
1
699, 1710, 1728 (3C=O), 1602 (C=N); HNMR (DMSO d , δ, ppm): 0.87 (t, 3H, J=4Hz, CH -
6
2
CH ), 1.10 (q, 2H, J=4Hz, CH -CH ), 2.40 (s, 3H, CH at C4), 3.46 (d, 2H, J= 8 Hz, CH -CH),
3
2
3
3
2
4
2
.07 (s, 2H, CH -thiazolidinone), 4.83 (s, 2H, OCH ), 4.86 (t, 1H, J=4 Hz, CH=CH), 4.95 (q,
2 2
H, J=8 Hz, CH -CH ), 5.02 (t, 1H, J=4 Hz, CH=CH), 5.91-5.98 (m, 1H, CH -CH), 6.25 (s, 1H,
2
3
2
H3-Ar.), 7.06 (d, 1H, J=8 Hz, H6-Ar.), 7.67 (d, 1H, J=8 Hz, CH at C5), 10.57 (br.s, 1H, NH,
1
3
exchanged with D O); CNMR (DMSO d , δ, ppm): 12.5 (CH -CH ), 18.7 (CH at C4), 27.1
2
6
2
3
3
(
CH -CH), 33.2 (CH -thiazolidinone), 37.9 (CH -CH ), 67.2 (OCH ), 109.5 (C6), 111.8 (C10),
2 2 2 3 2
1
1
14.5 (C3), 115.9 (CH=CH ), 124.7 (C8), 124.9 (C5), 135.7 (CH=CH ), 151.4 (C9), 154 (C4),
2 2
58.8 (C-O), 160.5 (C2), 169.7 (CO-NH), 171.6 (CO-thiazolidinone); MS (EI) m/z (%):, 415.46
+
(
[M , 25); Anal.calcd. for C H N O S: C, 57.82; H, 5.1; N, 10.11; Found: C, 57.62; H, 5.22;
2
0
21
3
5
N, 10.31.
(Z)-2-((8-allyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-N'-(3-benzyl-4-oxothiazolidin-2-
ylidene)acetohydrazide (16b)
Yield: 65%, m.p. 170 °C 3348 (NH), 3008, 3034, 3064 (CH Ar), 2931, 2958, 2985 (CH
1
aliphatic), 1732, 1708, 1604 (2C=O), 1604 (C=N), 1570 (NH); HNMR (DMSO d , δ, ppm):
6
2
.40 (s, 3H, CH at C4), 3.87 (d, 2H, J=12 Hz, CH -CH), 4.15 (s, 2H, CH -thiazolidinone), 4.81
3 2 2
(
s, 2H, OCH ), 4.96 (t, 1H, J=8 Hz, CH=CH), 5.01 (s, 1H, CH=CH), 5.40 (s, 2H, CH -C H ),
2
2
6
5
5
5
.93-5.97 (m, 1H, CH-CH ), 6.23 (s, 1H, CH at C3), 7.03 (d, 1H, J=8 Hz, H6-Ar), 7.29-7.35 (m,
2
H, H ), 7.64 (d, 1H, J=4 Hz, H5-Ar), 10.56 (br.s, 1H, NH, exchanged with D O); MS (EI) m/z
ar
2
+
(
%):, 477.54 ([M , 45.11); Anal.calcd. for C H N O S: C, 62.88; H,4.85; N, 8.80, Found: C,
25 23 3 5
6
3.01; H, 4.95; N, 9.02.
4
.2. Pharmacology
2
6

4
.2.1. Materials and Methods
Materials
4
.2.1.1. Animals
In the current study, 200 adult male Swiss albino mice (18–25 g) were purchased from
European Reef Animal House Colony (Egypt). They were kept under standardized conditions;
0
1
5
2 h-light/dark cycle with room temperature adjusted to 23 + 2 C and relative humidity at 55 +
%. Free access to tap water and standard rat chow was provided daily during the entire
experimental period. Animal care and handling were performed after receiving approval of the
ethical committee of Misr University for Science & Technology (approval no ID MC3).
Procedures followed herein were in accordance with the recommendations for the proper care
and use of laboratory animals “Canadian Council on Animal Care Guidelines, 1984” and
"ARRIVE guidelines, 2010". To minimize animal suffering, extensive efforts were made with
the least number of animals (n=6) in order to obtain reliable data.
4
.2.1.2. Drugs and Chemicals
Ethosuximide (Pfizer Co., Giza, Egypt), diphenylhydantoin (Nasr Co., Giza, Egypt) and
gabapentin (Pfizer Co., Egypt) were used as standard drugs for the study. Tween-80 and
pentylenetetrazole were purchased from Sigma (St. Louis, MO, USA). Animals were given
candidate drugs or standards (0.1 mL/10 g body weight, i.p) as a suspension in tween-80 (7%).
Methods
One week following adaptation to laboratory conditions, animals were randomly assigned to
series and reference standard groups consisting of six mice each in preparation for phase I study.
Another set of animals were allocated for phases II and neurochemical assay to determine ED50
as well as mechanistic action of the most active congener, respectively.
4
4
.2.1.3. Phase I: Preliminary screening for anticonvulsant activity
.2.1.3.1. Subcutaneous Pentylenetetrazole (scPTZ) Screen
This test, which simulates human absence/petit mal epilepsy or myoclonic seizures,
produces threshold or minimal (clonic) seizures [34, 35]. An aqueous solution of PTZ (85
mg/kg) was administered subcutaneously (sc) in the loose fold of skin of the mouse nape. This
2
7

dose induces seizures in more than 97% of animals, which is known as the convulsive dose 97
CD97) according to Löscher et al., 1991 [36]. Compounds of the series (at a dose level
(
equivalent to 100 mg/kg) were injected intraperitoneal (i.p), 45 min prior to scPTZ. Animals
were closely monitored for seizure episodes for 30 min. An episode of clonic convulsions that
persisted for at least 5 s, with loss of righting reflex was considered as a threshold convulsion.
Absence of a single 5 s episode of clonic spasms during the 30 min period of observation is taken
as the end point in this test and considered positive [34, 37].
4
.2.1.3.2. Maximal Electroshock Seizure (MES) Screen
The test was performed according to standard protocol followed by the National Institute of
Health NIH [38] and Krall et al. [34]. Electro-convulsions were induced using a Rodent Shocker
generator (constant-current stimulator Type 221, Hugo Sachs Elektronik, Freiburg, Germany)
adjusted to a fixed 25 mA current intensity and 0.2 s stimulus duration, delivered via ear clip
electrodes, forty-five min post-injection of the test compound (i.p). Occurrence of tonic hind
limb extension, i.e. the hind limbs of animals outstretched 180 degree to the plane of the body
axis, was considered as the criterion for positive seizure activity [39].
4
.2.1.3.3. Neurotoxicity
Neurotoxicity of test compounds was assessed by adopting the rotarod method to determine
any signs of motor impairment [40]. In this test, the animals were pre-trained to maintain balance
on a rotating 1 inch diameter knurled plastic rod (rotarod, UGO Basile, 47600, Varese, Italy) at a
fixed speed of 10 rpm for 60 s. Animals that fulfill this criterion and pass the test after three trials
were included in the experiment. This test was carried out forty-five min post- injection (i.p) of
each of the candidate drugs of the series. The mice were re-located on the rotating rod and the
motor performance was closely monitored for up to 60 s. Neurotoxicity was indicated by the loss
of motor coordination and inability of the animal to maintain equilibrium on the rod for at least
one minute.
4
.2.1.4. Phase II: Quantitative estimation of median effective dose (ED )
:
50
For the determination of median effective dose ED , the most potent surrogates were
5
0
administered to 5 groups of 6 mice at variable selected doses that cover 10-90% protection range
against PTZ-induced seizure. The median effective dose (at 95% confidence interval) was
2
8

calculated as the dose of the candidate drug that protects 50% of animals in the group against
scPTZ [41].
4
.2.1.5. Quantitative Estimation of GABA in whole mouse Brain
Free γ-aminobutyric acid (GABA) content in the brain tissue homogenate was determined
by High Performance Liquid Chromatography (HPLC) according to the method described by
Heinrikson and Meredith [42]. The HPLC system of Agilent (Germany) consisted of
quaternary pump; a column oven, Rheodine injector, 20μl loop and UV detector.
The technique involved a two-step process; sample preparation then derivatization. The first step
was sample preparation, where whole mouse brain was weighed and homogenized in 1/10
weight/volume of 75% aqueous HPLC grade methanol. The second step involved derivatization
procedure which involved a series of processing, whereby derivatizing agent consisted of 7:1:1:1
mixture (by volume) of methanol:, triethylamine (TEA): double distilled deionized water:
phenylisothiocyanate (PITC). Derivatized samples were re-dried by vacuum (70millitore),
diluted by a sample diluent then mixed with acetoniltrile. 20μl derivatized amino acid standards
and derivatized samples were injected into the column at flow rate 2ml/min for separation by
PICO- TAG column (Waters, Milford, USA) for free-amino acid analysis (3.9 x 30 cm). The
assay conditions were adjusted at 46 °C, and the resulting chromatogram (at 250 nm) identified
each amino acid position and concentration from the sample as compared to that of the amino
acids standard. The report and chromatogram were obtained from chemstation program. The
content of GABA in brain homogenate was calculated as μg per gram tissue.
4
.2.2. Statistical Analysis
Statistical analysis of the obtained data regarding GABA level in mouse brain was
accomplished by means of one-way analysis of variance (one-way ANOVA), followed by
Tukey's post hoc test. Results were expressed as mean ± SEM. The level of statistical
significance was set at p < 0.05.
4
.3. Docking study
Molecular modeling study was accomplished using Discovery Studio 2016. The crystal
structure of γ-aminobutyric acid aminotransferase GABA-AT) was retrieved from RCSB protein
2
9