Synthesis and anticonvulsant activity of new phenytoin derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.11.025

Hybrids between phenytoin and thiosemicarbazide, 1,3,4-oxadiazole, 1,3,4-thiadiazole or 1,2,4-triazole were synthesized and tested for anticonvulsant activity. Preliminary anticonvulsant screening was performed using standard maximal electroshock (MES) an

Full text of DOI:10.1016/j.ejmech.2012.11.025

Accepted Manuscript
Synthesis and Anticonvulsant Activity of New Phenytoin Derivatives
S. Botros, Nadia A. Khalil, Bassem H. Naguib, Yara El-Dash
PII:
S0223-5234(12)00694-0
10.1016/j.ejmech.2012.11.025
EJMECH 5851
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 May 2012
Revised Date: 14 November 2012
Accepted Date: 16 November 2012
Please cite this article as: S. Botros, N.A. Khalil, B.H. Naguib, Y. El-Dash, Synthesis and Anticonvulsant
Activity of New Phenytoin Derivatives, European Journal of Medicinal Chemistry (2012), doi: 10.1016/
j.ejmech.2012.11.025.
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ACCEPTED MANUSCRIPT
Graphical abstract
Synthesis and Anticonvulsant Activity of New Phenytoin Derivatives
Hybrid approach was used to design and synthesize hybrids between phenytoin and
thiosemicarbazide,
1,3,4-oxadiazole,
1,3,4-thiadiazole
or
1,2,4-triazole
pharmacophores to examine their anticonvulsant activity using MES and scPTZ
screens in mice.
N
SH
NHR
S
N
R
N
O
N
O
O
N
O
HN
NH
X
R
N
HN
N
N
HN
HN
O
O
O
X = O, S
7a-e
4, 6a-e
5a-e

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Research Highlights
•
•
•
•
•
Synthesis of phenytoin derivatives.
Anticonvulsant activity of all new synthesized compounds was determined.
Neurotoxicity of the most active compounds was estimated.
Compounds 4, 5b and 5d demonstrated marked anticonvulsant effect.
Elemental analysis and spectroscopic characterization of all new
compounds.

ACCEPTED MANUSCRIPT
Synthesis and Anticonvulsant Activity of New Phenytoin Derivatives
S. Botrosa, Nadia A. Khalila*, Bassem H. Naguiba, Yara El-Dasha
aPharmaceutical Organic Chemistry Department, Faculty of Pharmacy,
Cairo University, Cairo, Egypt
*Corresponding author: nadia_7394@live.com Tel: +0201006809136, P.O.Box 11562
Abstract
Hybrids between phenytoin and thiosemicarbazide, 1,3,4-oxadiazole, 1,3,4-
thiadiazole or 1,2,4-triazole were synthesized and tested for anticonvulsant activity.
Preliminary anticonvulsant screening was performed using standard maximal
electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) screens in mice.
The neurotoxicity was determined applying the rotarod test. Among these compounds,
4 and 5d showed the highest protection (80%) in the scPTZ test at a dose of 100
mg/kg, whereas the compound 5b displayed promising anticonvulsant effect in the
MES model.
Key words: Phenytoin/ Oxadiazole/ Thiadiazole/ Triazole/ Anticonvulsant activity
1. Introduction
Epilepsy is the most prevalent neurological disorder, affecting approximately 50
million people worldwide [1]. It is characterized by the periodic and unpredictable
occurrence of seizures that are caused by abnormal discharge of cerebral neurons [2].
Despite the significant advances have been made in epilepsy research, several of the
currently available antiepileptic drugs (AEDs) have been associated with severe side
effects, that range in harshness from minimal brain impairment and megaloblastic
anaemia, to death from aplastic anaemia or hepatic failure [3,4]. In addition, standard
drug therapy fails to control seizures in about 30% of epileptic patients [5-7]. These
limitations observed with conventional antiepileptic drugs, demand the need for the
development of more effective and safer candidates.
A rational drug design process for a new anticonvulsant could be achieved by the
traditional screening approach as well as the hybrid pharmacophore approach, which
is considered “the wave of drug research furnishing multitarget agents” [8].
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Hybrid pharmacophore approach relies on the use of two or more pharmacophores,
each with potential pharmacological activity to be combined in one molecule, aiming
that this hybridization could produce compounds with better pharmacological profile.
Phenytoin (Fig. 1), a well-established antiepileptic drug that act through inhibition
of brain sodium channels [9], has been chosen for its efficacy against partial and
generalized seizures [10].
Previous studies showed that compounds including thiosemicarbazide
pharmacophore in their structure, constitute a class of compounds with potential
anticonvulsant properties [11, 12]. Furthermore, many compounds bearing 1,3,4-
oxadiazole [13-15], 1,3,4-thiadiazole [16-18] or 1,2,4-triazole ring [19,20] in their
structure have been demonstrated anticonvulsant activity.
Taking in consideration the potential anticonvulsant properties observed by certain
thiosemicarbazides, oxadiazoles, thiadiazoles and triazoles, hybrids between
phenytoin and either of these pharmacophores were synthesized and evaluated as
anticonvulsant agents (Fig. 1).
Figure 1.
Despite the diversity of models that could potentially be used to screen their
anticonvulsant activity, the maximal electroshock model (MES) and the subcutaneous
pentylenetetrazole model (sc PTZ) remain the ‘‘gold standards’’ in the early stages of
testing. Most clinically significant AEDs have shown protection against seizures in at
least one of these two models.
The present work comprises the synthesis and anticonvulsant activity of 3-[(5-
sulphanyl-1,3,4-oxadiazol-2-yl)methyl]-5,5-diphenylimidazolidine-2,4-dione (4), 1-
[2-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)acetyl]-4-substituted thiosemicarbazides
(5a-e) 3-[(5-(alkyl/aryl)-1,3,4-thiadizol-2-yl)methyl]-5,5-diphenylimidazolidine-2,4-
diones (6a-e) and 3-[(4-alkyl/aryl-5-sulphanyl-4H-1,2,4-triazol-3-yl)methyl]-5,5-
diphenyl-imidazolidine-2,4-diones (7a-e). Their chemical structures were
1
characterized using IR, HNMR, 13CNMR, MS and elemental analysis techniques.
The anticonvulsant activity was evaluated by using MES and scPTZ experimental
epilepsy screens in mice. The rotarod assay was performed in mice to evaluate the
neurotoxicity of the most effective compounds.
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2. Results and discussion
2.1. Chemistry
The title compounds were prepared according to the synthetic strategy described in
Scheme 1. Ethyl 2-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)acetate was
2
synthesized by alkylation of phenytoin sodium 1 with ethyl chloroacetate in
anhydrous DMF as a solvent [21]. Reaction of 2 with hydrazine hydrate in absolute
ethanol, resulted in the formation of the corresponding hydrazide 3 [22]. The target 3-
[(5-sulphanyl-1,3,4-oxadiazol-2-yl)methyl]-5,5-diphenylimidazolidine-2,4-dione (4)
was obtained in 66 % yield by the cyclization of the hydrazide 3 with carbon
disulphide in ethanol in the presence of potassium hydroxide. It is interesting to note
that thiocarbonyl compounds are present in their thion-thiol tautomeric forms in
solution as indicated by their IR and 1H NMR spectra [23-25]. 1HNMR spectrum of 4
showed the presence of D2O exchangeable signals corresponding to NH and SH at δ
9.92 and 14.60 ppm respectively. In addition, 13CNMR spectrum showed the
appearance of two signals of the oxadiazole ring at δ 158.65 ppm and δ 177.70 ppm,
assignable to C-2 and C-5 respectively. Besides, the signal due to CH2 carbon was
observed at δ 33.25 ppm.
The intermediate thiosemicarbazides 5a-e were synthesized in 60-90 % yields
through the reaction of 3 with different isothiocyanates in absolute ethanol. A special
feature in the structure of thiosemicarbazides 5a-e is the presence of thiourea residue
(NH-CS-NH), which can be identified by IR spectral data. Compounds 5a-e exhibited
strong absorption in the range of 1188-1203 cm-1 attributable to C=S. The amidic
carbonyl absorption was observed as a broad band in the range of 1650-1680 cm-1.
1HNMR spectra for 5a-e showed the presence of a singlet in the range of δ 4.21-4.29
ppm assignable to CH2 protons flanked between the imidazolidine nitrogen and
carbonyl function. The four D2O exchangeable signals for the NH functions were
displayed in the range of δ 7.97-10.41 ppm.
On the other hand, the thiadiazole derivatives 6a-e were prepared through
cyclization of the thiosemicarbazides 5a-e, using concentrated sulphuric acid as a
dehydrating agent.
The structure of the target compounds 6a-e was confirmed by elemental analyses
1
and spectroscopic data (IR, HNMR, 13CNMR and MS). IR spectra showed broad
band in the range of 3471-3217 cm-1 corresponding to the two NH stretchrs.. 1HNMR
spectra of all compounds 6a-e, displayed a singlet signal in the range of δ 4.26-4.91
3

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ppm assignable to CH2 protons.13CNMR spectrum of the compound 6d showed the
presence of the C-2 and C-5 signals of the thiadiazole ring at δ 165.69 and 167.25
ppm respectively. The signal corresponding to OCH3 carbon was observed at 55.14
ppm.
Furthermore, the triazole derivatives 7a-e were obtained in 60-70 % yields via
cyclization of 5a-e in the presence of 2N NaOH solution. The characterization of the
isolated new compounds was based on elemental analyses and spectroscopic data (IR,
1HNMR, 13CNMR and MS).
Compounds 7a-e showed the presence of a possible thion-thiol tautomerism, as
1
indicated by their IR and H NMR spectra. A singlet signal in the range of δ 13.65-
13.90 ppm indicated the existence of SH. Besides, the characteristic singlet signal
corresponding to CH2 was observed in the range of δ 3.97-4.03 ppm. 13CNMR of the
compound 7c displayed the signal due to CH3 carbon at δ 20.75 ppm, in addition to
the C-3 and C-5 signals of the triazole ring at δ 150.56 and 167.84 ppm respectively.
2.2. Anticonvulsant activity
Phase I studies of the investigated compounds (4, 5a-e, 6a-e and 7a-e) involved
three tests: maximal electroshock seizure (MES), subcutaneous pentylenetetrazole
(scPTZ), and rotarod test for neurological toxicity. The MES test is a proven method
of generalized tonic-clonic seizures and identifies clinical candidates that prevent
seizure spread, whereas scPTZ test indicates their effectiveness against absence of
seizures and was used to identify compounds that elevate seizure threshold.
A valuable property of a candidate anticonvulsant is its ability to inhibit seizures upon
oral administration. The anticonvulsant activity of the test compounds was established
at a dose of 100 mg/Kg in the MES, scPTZ and neurotoxicity assays [23-26].
Compounds found to be effective in these seizure challenges are generally regarded to
be potentially useful candidates in treatment of partial, generalized and even absence
seizures. The results of the in vivo tests are summarized in Table 2, Figs. 2, 3.
Analyzing the activities of synthesized compounds 4, 5a-e, 6a-e and 7a-e, the
following results were obtained:
Preliminary anticonvulsant screening revealed that all new compounds showed
some degree of protection in MES screen, which was indicative of the good ability of
these compounds to prevent the seizure spread at a certain dose level. It was observed
that phenytoin-1,2,4-triazole conjugates 7a-e were generally more effective than the
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corresponding 1,3,4-oxadiazole derivative 4 and the 1,3,4-thiadiazole analogues 6a-e.
Moreover, compounds 7b-e, containing aromatic ring at the triazole N-3, displayed
higher protection against electrically induced seizures than the ethyl substituted
analogue 7a. On the other hand, among the thiosemicarbazide derivatives 5a-e, only
the phenyl substituted derivative, 5b showed protection from seizures superior to
phenytoin, indicating its possible efficacy in protection against generalized seizures of
the tonic–clonic (grandmal) type. Additionally, the anti-MES activity of the most
active compound, 5b, was further estimated at a lower dose (30 mg/ Kg). The results
revealed remarkable anticonvulsant activity of 5b at the tested dose, (Table 1). Other
test compounds displayed from good to moderate activity against electrically induced
seizures.
Concerning scPTZ screen, all test compounds showed varying degree (20-80 %) of
anticonvulsant activity except the thiosemicarbazide derivative 5e and the 1,2,4-
thiadiazole derivative 6e. Both compounds, bearing the 4-chlorophenyl substituent,
did not exhibit any anticonvulsant activity. On the other hand, the strongest anti-
scPTZ activity was observed by 4 and 5d, which were equipotent to the reference
drug, phenytoin (80 % protection of seizures).
Furthermore, the compounds showing significant activity in one or both of the
seizure models (4, 5b, 5d, 7b, 7d) were selected for evaluation of neurotoxicity using
rotarod test in mice. None of the compounds showed significant neurotoxic effect.
Compound 5b was found to be devoid of sedative effect, indicating that this
compound could possess a promising protective activity against electroshock-induced
convulsions at a given dose without affecting locomotor activity and coordination.
Table 2.
Figure 2.
Figure 3.
3. Conclusion
In summary, compounds 4 and 5d showed anticonvulsant activity comparable to the
reference drug, phenytoin in the scPTZ model. On the other hand, compound 5b
displayed anticonvulsant activity superior to phenytoin in the MES screen. In
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addition, it was devoid of neurotoxic properties in the neurotoxicity screen. These
preliminary results suggest further studies to really estimate their in vivo potency.
4. Experimental
4.1. Chemistry
All chemicals and reagents were obtained from Aldrich (Sigma–Aldrich, St.
Louis, MO, USA), and were used without further purification. Reactions were
monitored by TLC, performed on silica gel glass plates containing 60 GF-254, and
visualization on TLC was achieved by UV light or iodine indicator. IR spectra were
1
determined on Shimadzu IR 435 spectrophotometer (KBr, cm-1). HNMR spectra
were carried out using a Mercury 300-BB 300 MHz using TMS as an internal
standard. Chemical shifts (δ) were recorded in ppm on δ scale, Micro analytical
Center, Cairo University, Egypt. 13CNMR spectra were carried out using a Mercury
300-BB 300 MHz using TMS as an internal standard. Chemical shifts (δ) were
recorded in ppm on δ scale, Micro analytical Center, Cairo University, Egypt. Mass
spectra were recorded on Shimadzu Qp-2010 plus Spectrometer, Micro analytical
Center, Cairo University, Egypt. Elemental analyses were carried out at the Micro
analytical Center, Cairo University, Egypt. Their results corresponded to the
calculated values within experimental error. Melting points were determined with
Stuart apparatus and are uncorrected.
The starting materials, phenytoin sodium (5,5-diphenylimidazolidine-2,4-dione
sodium salt) (1) commercially available, ethyl 2-(2,5-dioxo-4,4-diphenylimidazolidin-
1-yl)acetate (2) [21] and 2-(2,5-dioxo-4,4-diphenylimidazolidin-1-yl)acetohydrazide
(3) [22] were prepared according to the reported procedures.
4.1. 3-[(5-Sulphanyl-1,3,4-oxadiazol-2-yl)methyl]-5,5-diphenylimidazolidine-2,4-
dione (4)
A mixture of the hydrazide 3 (0.32 g, 0.001 mol), carbon disulphide (2 mL) and
potassium hydroxide (0.5 g, 0.009 mol) in ethanol (5 mL, 95 %) was heated under
reflux for 12 h. The reaction mixture was filtered while hot, and the solvent was
distilled off under reduced pressure. The residue obtained was dissolved in water (10
mL) then neutralized with dil HCl. The resulting white precipitate was filtered,
washed with water, dried and crystallized from dioxane/ water.
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o
Yield 66 %; mp 225-226 C; IR (KBr) (cm-1): 3360 (N-H), 3032 (C-H aromatic),
2943, 2840 (C-H aliphatic), 1766, 1716 (C=O), 1639 (C=N), 1153 (C=S str.);
1HNMR (300 MHz, DMSO-d6) δ: 4.83 (s, 2H, CH2), 7.32-7.44 (m, 10H, Ar-H), 9.92
1
(s, 1H, NH, D2O exchangeable), 14.60 (s, 1H, SH, D2O exchangeable); 3CNMR
(DMSO-d6 ppm) δ: 33.25 (CH2), 69.52 (C-5 imidazolidine), 126.60, 128.29, 128.53,
139.01 (aromatic C's), 153.80 (C=O), 158.65 (oxadiazole C-2), 172.47 (C=O), 177.70
(oxadiazole C-5); EIMS (% rel. abundance): 367 (M+H , 15.42), 366 (M , 76.23),
180 (100). Anal. Calcd for C18H14N4O3S (366.39): C, 59.01; H, 3.85; N, 15.29,
Found, C, 58.96; H, 3.96; N, 15.43.
4.2. 1-[2-(2,5-Dioxo-4,4-diphenylimidazolidin-1-yl)acetyl]-4-substituted
thiosemicarbazides (5a-e)
To a solution of the hydrazide 3 (0.32 g, 0.001 mol) in absolute ethanol (10 mL),
the appropriate isothiocyanate (0.0015 mol) was added and the reaction mixture was
heated under reflux for 10 h. The solvent was concentrated under reduced pressure
then diluted with an ice-cold water (10 mL). The precipitated solid product was
filtered then crystallized from the appropriate solvent.
4.2.1.
1-[2-(2,5-Dioxo-4,4-diphenylimidazolidin-1-yl)acetyl]-4-
ethylthiosemicarbazide (5a)
o
Crystallized from benzene/ absolute ethanol mixture; yield 60 %; mp 189-190 C;
IR (KBr) (cm-1): 3433-3209 (N-H), 3040 (C-H aromatic), 2990, 2850 (C-H aliphatic),
1
1774, 1724-1680 (C=O), 1200 (C=S); HNMR (300 MHz, DMSO-d6) δ: 1.06 (t, 3H,
CH2-CH3), 3.48 (q, 2H, CH2-CH3), 4.21 (s, 2H, CH2-N), 7.34-7.43 (m, 10H, Ar-H),
7.97, 9.32, 9.67, 10.13 (each s, 4H, 4 NH, D2O exchangeable); EIMS (% rel.
abundance): 412 (M+H , 0.53), 411 (M , 1.80), 252 (100); Anal. Calcd for
C20H21N5O3S (411.48): C, 58.38; H, 5.14; N, 17.02, Found, C, 58.51; H, 5.23; N,
17.16.
4.2.2 . 1-[2-(2,5-Dioxo-4,4-diphenylimidazolidin-1-yl)acetyl]-4-
phenylthiosemicarbazide (5b)
o
Crystallized from benzene/ absolute ethanol mixture; yield 66 %; mp 186-187 C; IR
(KBr) (cm-1): 3406-3271 (N-H), 3062 (C-H aromatic), 2920, 2850 (C-H aliphatic),
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1766, 1716-1680 (C=O), 1597 (C=N), 1195 (C=S); 1HNMR (300 MHz, DMSO-d6) δ:
4.29 (s, 2H, CH2-N), 7.18-7.44 (m, 15H, Ar-H), 9.65, 9.69, 9.77, 10.40 (each s, 4H,
4N-H, D2O exchangeable); EIMS (% rel. abundance): 460 (M+H , 11.56), 459 (M ,
10.54), 366 (74), 104 (100); Anal. Calcd for C24H21N5O3S (459.52): C, 62.73; H,
4.61; N, 15.24, Found, C, 62.80; H, 4.72; N, 15.39.
4.2.3. 1-[2-(2,5-Dioxo-4,4-diphenylimidazolidin-1-yl)acetyl]-4-(4-methylphenyl)
thiosemicarbazide (5c)
o
Crystallized from benzene/ pet. ether mixture; yield 87 %; mp 110-111 C; IR (KBr)
(cm-1): 3400-3282 (N-H), 3059 (C-H aromatic), 2920, 2865 (C-H aliphatic), 1774,
1
1716-1665 (C=O), 1593 (C=N), 1192 (C=S); HNMR (300 MHz, DMSO-d6) δ: 2.28
(s, 3H, CH3), 4.28 (s, 2H, CH2-N), 7.13 (d, 2H, Ar-H), 7.24 (d, 2H, Ar-H), 7.35-7.44
(m, 10H, Ar-H), 9.60, 9.68, 9.70, 10.37 (each s, 4H, 4 NH, D2O exchangeable); EIMS
(% rel. abundance): 474 (M+H , 18.65), 473 (M+, 7.74), 308 (26.11), 251 (M-(
27.38); Anal. Calcd for C25H23N5O3S (473.55): C, 63.41; H, 4.90; N, 14.79, Found,
C, 63.48; H, 5.01; N, 14.88.
4.2.4 1-[2-(2,5-Dioxo-4,4-diphenylimidazolidin-1-yl)acetyl]-4-(4-methoxyphenyl)
thiosemicarbazide (5d)
o
Crystallized from benzene/ petroleum ether mixture; yield 90 %; mp 139-140 C; IR
(KBr) (cm-1): 3468-3302 (N-H), 3155 (C-H aromatic), 2927, 2839 (C-H aliphatic),
1770, 1724-1680 (C=O), 1593 (C=N), 1188 (C=S); 1HNMR (300 MHz, DMSO-d6) δ:
3.74 (s, 3H, OCH3), 4.28 (s, 2H, CH2-N), 6.92 (d, 2H, Ar-H), 7.25 (d, 2H, Ar-H),
7.39-7.44 (m, 10H, Ar-H), 9.42, 9.58, 9.68, 10.35 (each s, 4H, 4 NH, D2O
exchangeable); EIMS (% rel. abundance): 489 (M+, 1.55), 180 (100); Anal. Calcd for
C25H23N5O4S (489.55): C, 61.34; H, 4.74; N, 14.31, Found, C, 61.44; H, 4.79; N,
14.48.
4.2.5. 1-[2-(2,5-Dioxo-4,4-diphenylimidazolidin-1-yl)acetyl]-4-(4-chlorophenyl)
thiosemicarbazide (5e)
o
Crystallized from benzene/ petroleum ether mixture; yield 88 %; mp 99-100 C; IR
(KBr) (cm-1): 3464-3240 (N-H), 3055 (C-H aromatic), 2924, 2850 (C-H aliphatic),
1774, 1716-1675 (C=O), 1597 (C=N), 1203 (C=S); 1HNMR (300 MHz, DMSO-d6) δ:
4.28 (s, 2H, CH2-N), 7.33-7.43 (m, 14H, Ar-H), 9.32, 9.68, 9.87, 10.41 (each s, 4H, 4
8

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NH, D2O exchangeable); EIMS (% rel. abundance): 495 (M+2, 0.03), 493 (M ,
0.02); Anal. Calcd for C24H20ClN5O3S (493.97): C, 58.36; H, 4.08; N, 14.18, Found,
C, 58.39; H, 4.13; N, 14.27.
4.3. 3-{[5-(Alkyl/arylamino)-1,3,4-thiadiazol-2-yl]methyl}-5,5-diphenylimidazolidine-
2,4-diones (6a-e)
To an ice cooled and stirred solution of an appropriate thiosemicarbazide 5a-e
(0.0013 mol) in absolute ethanol (10 mL), concentrated sulphuric acid (5 mL) was
slowly added over a period of 15 min. The reaction mixture was stirred at room
temperature for 5 h then the mixture was poured onto equal volume of ice-cold water.
The white precipitate formed was filtered, washed with water, dried and crystallized
from DMF / water mixture.
4.3.1. 3-[(5-Ethylamino-1,3,4-thiadiazol-2-yl)methyl]-5,5-diphenylimidazolidine-2,4-
dione (6a)
o
Yield 60 %; mp 165-166 C; IR (KBr) (cm-1): 3450-3221 (N-H), 3105 (C-H
aromatic), 2980, 2852 (C-H aliphatic), 1774, 1716 (C=O), 1635 (C=N); 1HNMR (300
MHz, DMSO-d6) δ: 1.15 (t, 3H, CH2-CH3), 4.12 (q, 2H, CH2-CH3), 4.26 (s, 2H, CH2),
7.35-7.44 (m, 10H, Ar-H), 9.77 (s, 1H, NH, D2O exchangeable), 10.23 (s, 1H, NH,
D2O exchangeable); EIMS (% rel. abundance): 393 (M+, 0.01), 265 (18.38), 209
(100); Anal. Calcd for C20H19N5O2S (393.46): C, 61.05; H, 4.87; N, 17.80, Found, C,
61.13; H, 4.96; N, 18.11.
4.3.2. 3-[(5-Phenylamino-1,3,4-thiadiazol-2-yl)methyl]-5,5-diphenylimidazolidine-
2,4-dione (6b)
o
Yield 63 %; mp 255-256 C; IR (KBr) (cm-1): 3471-3290 (N-H), 3089 (C-H
aromatic), 2920, 2850 (C-H aliphatic), 1774, 1716 (C=O), 1595 (C=N); 1HNMR (300
MHz, DMSO-d6) δ: 4.88 (s, 2H, CH2), 6.90-7.52 (m, 15H, Ar-H), 9.85 (s, 1H, NH,
D2O exchangeable), 10.33 (s, 1H, NH, D2O exchangeable); EIMS (% rel. abundance):
442 (M+H, 31.17), 441 (M+, 100); Anal. Calcd for C24H19N5O2S (441.50): C, 65.29;
H, 4.34; N, 15.86, Found, C, 65.38; H, 4.51; N, 15. 98.
4.3.3. 3-{[5-(4-Methylphenylamino)-1,3,4-thiadiazol-2-yl]methyl}-5,5-
diphenylimidazolidine-2,4-dione (6c)
9

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o
Yield 66 %; mp 234-235 C; IR (KBr) (cm-1): 3394-3278 (N-H), 3062 (C-H
aromatic), 2920, 2854 (C-H aliphatic), 1774, 1716 (C=O), 1620 (C=N); 1HNMR (300
MHz, DMSO-d6) δ: 2.24 (s, 3H, CH3), 4.90 (s, 2H, CH2), 7.12-7.45 ( m, 14H, Ar-H),
9.85 (s, 1H, NH, D2O exchangeable), 10.25 (s, 1H, NH, D2O exchangeable); EIMS
(% rel. abundance): 456 (M+H, 31.20), 455 (M ,100); Anal. Calcd for C25H21N5O2S
(455.53): C, 65.92; H, 4.65; N, 15.37, Found, C, 66.14; H, 4.80; N, 15.44.
4.3.4. 3-{[5-(4-Methoxyphenylamino)-1,3,4-thiadiazol-2-yl]methyl}-5,5-
diphenylimidazolidine-2,4-dione (6d)
o
Yield 62 %; mp 220-221 C; IR (KBr) (cm-1): 3309-3217 (N-H), 3059 (C-H
aromatic), 2943, 2835 (C-H aliphatic), 1774, 1716 (C=O), 1624 (C=N); 1HNMR (300
MHz, DMSO-d6) δ: 3.73 (s, 3H, OCH3), 4.89 (s, 2H, CH2), 6.90-7.48 (m, 14H, Ar-H),
1
3
9.87 (s, 1H, NH, D2O exchangeable), 10.15 (s, 1H, NH, D2O exchangeable); CNMR
(DMSO-d6 ppm) δ: 36.97 (CH2), 55.14 (OCH3), 69.36 (C-5 imidazolidine), 114.18,
119.18, 126.59 , 128.21, 128.52, 133.81, 139.21, 154.23 )aromatic C's), 154.47
(C=O), 165.69 (thiadiazole C-2), 167.25 (thiadiazole C-5), 172.53 (C=O); EIMS (%
rel. abundance): 472 (M+2, 20.14), 471 (M , 100); Anal. Calcd for C25H21N5O3S
(471.53): C, 63.68; H, 4.49; N, 14.85, Found, C, 63.85; H, 4.58; N, 14.98.
4.3.5. 3-{[[5-(4-Chlorophenylamino)-1,3,4-thiadiazol-2-yl)methyl]-5,5-
diphenylimidazolidine-2,4-dione (6e)
o
Yield 60 %; mp 273-274 C; IR (KBr) (cm-1): 3468-3267 (N-H), 3062 (C-H
aromatic), 2927, 2850 (C-H aliphatic), 1774, 1716 (C=O), 1597 (C=N); 1HNMR (300
MHz, DMSO-d6) δ: 4.91 (s, 2H, CH2), 7.35-7.59 (m, 14H, Ar-H), 9.86 (s, 1H, NH,
D2O exchangeable), 10.48 (s, 1H, NH, D2O exchangeable); EIMS (% rel. abundance):
477 (M+2, 9.78), 475 (M , 8.09), 209 (100); Anal. Calcd for C24H18ClN5O2S
(475.95): C, 60.56; H, 3.81; N, 14.71, Found, C, 60.46; H, 3.90; N, 14.96.
4.4. 3-[(4-Alkyl/aryl-5-sulphanyl-4H-1,2,4-triazol-3-yl)methyl]-5,5-
diphenylimidazolidine-2,4-diones (7a-e)
The appropriate thiosemicarbazide 5a-e (0.005 mol) in sodium hydroxide solution
(2N, 10 mL) was heated under reflux for 4 h. After cooling, dil HCl was added to the
1

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reaction mixture till pH=2-3. The resulting precipitate was filtered, washed with
water, dried, and crystallized from benzene-absolute ethanol mixture.
4.4.1. 3-[(4-Ethyl-5-sulphanyl-4H-1,2,4-triazol-3-yl)methyl]-5,5-
diphenylimidazolidine-2,4-dione (7a)
o
Yield 60 %; mp 183-184 C; IR (KBr) (cm-1): 3375-3290 (N-H), 3089 (C-H
aromatic), 2924, 2854 (C-H aliphatic), 1774, 1724 (C=O), 1624 (C=N); 1HNMR (300
MHz, DMSO-d6) δ: 1.11 (t, 3H, CH2-CH3), 4.01 (s, 2H, CH2), 4.34 (q, 2H, CH2-CH3),
7.24-7.39 (m, 10H, Ar-H), 9.56 (s, 1H, NH, D2O exchangeable), 13.65 (s, 1H, SH,
D2O exchangeable); EIMS (% rel. abundance): 393 (M , 0.17), 265 (36.04); Anal.
Calcd for C20H19N5O2S (393.46): C, 61.05; H, 4.87; N, 17.80, Found, C, 61.15; H,
4.94; N, 18.07.
4.4.2. 3-[(4-Phenyl-5- sulphanyl-4H-1,2,4-triazol-3-yl)methyl]-5,5-
diphenylimidazolidine-2,4-dione (7b)
o
Yield 66 %; mp 162-163 C; IR (KBr) (cm-1): 3379 (N-H), 3059 (C-H aromatic),
1
2924, 2854 (C-H aliphatic), 1774, 1720 (C=O), 1595 (C=N); HNMR (300 MHz,
DMSO-d6) δ: 3.98 (s, 2H, CH2), 7.24-7.55 (m, 15H, Ar-H), 9.72 (s, 1H, NH, D2O
exchangeable), 13.87 (s, 1H, SH, D2O exchangeable); EIMS (% rel. abundance): 441
(M , 2.24); Anal. Calcd for C24H19N5O2S (441.50): C, 65.29; H, 4.34; N, 15.86,
Found, C, 65.32; H, 4.39; N, 16.12.
4.4.3. 3-{[4-(4-Methylphenyl)-5-sulphanyl-4H-1,2,4-triazol-3-yl]methyl}-5,5-
diphenylimidazolidine-2,4-dione (7c)
o
Yield 70 %; mp 151-152 C; IR (KBr) (cm-1): 3371 (N-H), 3059 (C-H aromatic),
1
2924, 2854 (C-H aliphatic), 1774, 1720 (C=O), 1624 (C=N); HNMR (300 MHz,
DMSO-d6) δ: 2.32 (s, 3H, CH3), 3.97 (s, 2H, CH2), 7.21-7.41 (m, 14H, Ar-H), 9.78 (s,
1
1H, NH, D2O exchangeable), 13.83 (s, 1H, SH, D2O exchangeable); 3CNMR
(DMSO-d6 ppm) δ: 20.75 (CH3), 35.34 (CH2), 68.48 (C-5 imidazolidine), 126.63,
126.75, 128.25, 128.37, 129.77, 130.52, 139.03, 141.69 (aromatic C's), 150.56
(triazole C-3), 156.43 (C=O), 167.84 (triazole C-5), 172.56 (C=O); EIMS (% rel.
1

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abundance): 456 (M+H, 1.59), 455 (M , 4.77); Anal. Calcd for C25H21N5O2S
(455.53): C, 65.92; H, 4.65; N, 15.37, Found, C, 66.04; H, 4.73; N, 15.49.
4.4.4. 3-{[4-(4-Methoxyphenyl)-5-sulphanyl-4H-1,2,4-triazol-3-yl]methyl}-5,5-
diphenyl imidazolidine-2,4-dione (7d)
o
Yield 68 %; mp 260 (dec.) C; IR (KBr) (cm-1): 3383 (N-H), 3059 (C-H aromatic),
1
2931, 2805 (C-H aliphatic), 1774, 1724 (C=O), 1624 (C=N); HNMR (300 MHz,
DMSO-d6) δ: 3.81 (s, 3H, OCH3), 3.98 (s, 2H, CH2), 7.01-7.43 (m, 14H, Ar-H), 9.80
(s, 1H, NH, D2O exchangeable), 13.81 (br, 1H, SH, D2O exchangeable); EIMS (% rel.
abundance): 472 (M+H, 7.48), 471 (M , 23.93); Anal. Calcd for C25H21N5O3S
(471.53): C, 63.68; H, 4.49; N, 14.85, Found, C, 63.77; H, 4.46; N, 15.03.
4.4.5. 3-{[4-(4-Chlorophenyl)-5-sulphanyl-4H-1,2,4-triazol-3-yl]methyl}-5,5-
diphenyl imidazolidine-2,4-dione (7e)
o
Yield 63 %; mp 149-150 C; IR (KBr) (cm-1): 3387 (N-H), 3093 (C-H aromatic),
1
2924, 2810 (C-H aliphatic), 1774, 1720 (C=O), 1597 (C=N); HNMR (300 MHz,
DMSO-d6) δ: 4.03 (s, 2H, CH2), 7.22-7.63 (m, 14H, Ar-H), 9.74 (s, 1H, NH, D2O
exchangeable), 13.90 (s, 1H, SH, D2O exchangeable); EIMS (% rel. abundance): 477
(M+2, 13.09), 475 (M , 31.66); Anal. Calcd for C24H18ClN5O2S (475.95): C, 60.56;
H, 3.81; N, 14.71, Found, C, 60.68; H, 3.92; N, 14.93.
4.5. Anticonvulsant screening
4.5.1. Materials and methods
All the compounds prepared herein were screened for their potential in vivo
anticonvulsant activity. The Phase I pharmacological screening involves MES, scPTZ
and neurotoxicity assays.
Adult albino mice weighing 20–25 g of both sexes (obtained from the animal
house colony in the National Research Center) were used throughout this study.
Animals were housed in groups of 4–5 and were allowed free access to food pellets
(vit mix 1 %, mineral mix 4 % corn oil 10 %, sucrose 20 %, cellulose 0.2 %, casein
(95% pure) 10.5 %, starch 54. 3%) and water except for the short time that animals
were removed from their cages for testing. All behavioural experiments were
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conducted during the period between 10:00 and 13:00 with normal room light (12 h
regular light/dark cycle) and temperature (22 ± 18°C). Animals’ housing and rearing
had been followed optimum standard rules. Unnecessary disturbance of animals was
avoided. Animals were treated gently; squeezing, pressure and tough manoeuvre is
avoided. Instruments used for drug preparation and animal injection were previously
sterilized. Anaesthesia had been used whenever applicable. The dose of each drug was
calculated accurately. Animals’ cadavers and parts of tissues were handled with care
by following the principles of healthy hygiene; dead bodies were incinerated in the
National Research Center incineration. All procedures were carried out in accordance
with the (National Research Center, Cairo, Egypt) institutional guidelines for animal
care and use as follows:
4.5.1.1. Pentylynetetrazole (PTZ) induced seizure test
The test compound or the reference drug (100 mg/Kg) was given orally to groups
of 6 mice. Another group of 6 mice have been taken as a control. Sixty min after oral
administration, pentylenetetrazole (PTZ) (85 mg/Kg) was injected subcutaneously.
Each animal was placed into an individual plastic cage for observation over a period
of 20 min. The incidence of tonic-clonic convulsions lasting for at least 5 sec was
recorded [27,28].
Animals devoid of generalized convulsions were considered to be protected and the
results were represented as percentage protection. Standard drug used was phenytoin
sodium at a dose of 100 mg/Kg (Table 2, Fig. 2).
4.5.1.2. MES-maximal electroshock seizure pattern test
The procedure was carried out as described by Krall, et al. [29] and Kitano et al.
[30]. A compound under investigation and the reference drug suspended in a vehicle
(Tween 80) was administered orally to a group of six mice (100 mg/Kg in 1% Tween
80) 60 min before the test. The control animals were administered the vehicle. An
increased electric current was applied via an ear electrode, initial current of 1 mA,
increment of 0.1 mA/0.2 s at 50 Hz (Ugo Basil, ECT Unit, 57800). Tonic extension of
hind limbs was taken as the end point. The mean convulsion threshold was considered
as the mean of the maximal electric current which could be tolerated by the animal at
the end point [31]. Results of the mean electrical stimulation (MES) test were
expressed in milli-Ampere of the mean convulsion threshold for each compound. The
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mean threshold current for electroshock-induced tonic hind limb extensor seizure was
calculated for each drug (Table 2, Fig. 3).
The mean convulsion threshold of compounds under investigation as well as the
standard error were calculated using chi-squared test with the aid of Graphpad Prism
software, version 5 (inc., San Diego, USA).
4.5.1.3. Neurotoxicity screening
Minimal motor impairment was measured in mice or rats by using standardized
rotarod test [32]. Prior to the experiment, male albino mice were placed on 3 cm rod
(Ugo-Basile Accele. ROTA-ROD for mice, 7650) rotating at 6 rpm, in two training
sessions that last 10 and 15 minutes respectively. The candidates under investigation
were injected i.p. (100 mg/Kg in 1% Tween 80) in four groups each of 6 mice. The
control group received 1% Tween 80 as a vehicle. One hour later, the animals were
again tested on the rotarod to assess the locomotor coordination and neurological
deficit (e.g., ataxia, sedation, hyper-excitability), which are reflected by the inability
of the animal to maintain equilibrium on the rod after the administration of a selected
candidate. The end point for minimal neurotoxicity assessment was reflected by the
inability of mice to maintain their equilibrium for at least 1 min in each of the four
trials.
ACKNOWLEDGEMENTS
The authors are grateful to Dr. Wafaa El-Eraky, Associate Professor of Pharmacology
and Toxicology Department, National Research Center, Cairo. Egypt for carrying out
the biological testing.
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Figure captions
Figure 1. Phenytoin and basic structures of the designed compounds.
Figure 2. Anticonvulsant activity of test compounds administrated orally to mice
(sc PTZ screen).
Figure 3. Anticonvulsant activity of test compounds administrated orally to mice
(MES screen).
Table 1. Anticonvulsant activity and neurotoxicity (NT) of compounds 4, 5a-e, 6a-e
and 7a-e administered orally to mice (100 mg/Kg).
Scheme 1. Reagents and conditions: a) ClCH2COOC2H5, DMF/ 2h b) N2H4, abs.
C2H5OH/ 25 oC/ 24 h c) CS2, KOH, C2H5OH/ 12 h d) RNCS, abs. C2H5OH/
10 h e) conc. H2SO4/ 5 h f) 2N NaOH/ 4h.
1

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Table 1. Anticonvulsant activity and neurotoxicity (NT) of compounds 4, 5a-e,
6a-e and 7a-e administered orally to mice (100 mg/Kg).
Compound PTZ
%protection
MES
NTa
mean convulsion
threshold
4
80*
40
50
60
80*
0
60
60
40
20
0
40
40
20
40
40
80*
5.5±0.2887
5.2±0.3742
8.4±0.5099*
5.8±0.3742
4.0±0.3162
5.4±0.5099
5.0±0.3162
5.0±0.4082
5.2±0.3742
4.0±0.3162
4.25±0.25
5.8±0.3742
6.6±0.400*
6.6±0.5099*
6.6±0.5099*
6.2±0.5831*
7.25±0.2500*
40
-
0
-
5a
5bb
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
16.67
-
-
-
-
-
-
-
33.33
-
50
-
Phenytoin
-
Values represent the mean ± S.E. of 5-6 animals for each group
Values with * are statistically significant (p<0.05) from the control group by using one way ANOVA
(followed by Tukey's as post tests).
aPercentage of animals that fail to maintain equilibrium on the accelerating rod for 300 seconds.
bThis compound was tested at a dose 30 mg/Kg in the MES screen, and the mean convulsion threshold
was found to be 6.143±0.1429*.

ACCEPTED MANUSCRIPT
NHR
S
O
O
O
HN
NH
NH
N
HN
HN
O
O
5a-e
Phenytoin
N
SH
N
R
N
O
N
O
N
X
R
N
N
HN
HN
O
O
7a-e
4, 6a-e
X = O, S
R= C2H5, C6H5, 4-CH3C6H4, 4-OCH3C6H4, 4-ClC6H4
Figure 1. Penytoin and basic structures of the designed compounds
90
80
70
60
50
40
30
20
10
0
4
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
phenytoin
compound No.
Figure 2. Anticonvulsant activity of test compounds administrated orally to mice
(sc PTZ screen).

ACCEPTED MANUSCRIPT
10
9
8
7
6
5
4
3
2
1
0
Figure 3. Anticonvulsant activity of test compounds administrated orally to mice
(MES screen).

ACCEPTED MANUSCRIPT
O
O
O
NH2
NH
O
O
b
a
O
NNa
N
N
NH
NH
NH
O
O
O
2
1
3
c
d
N
SH
NHR
S
O
N
O
N
O
O
NH
NH
O
N
NH
NH
O
4
5a-e
e
f
R
a
5, 6, 7
C2H5
N
b
c
NHR
N
C6H5
SH
N
O
N
O
N
S
4-CH3C6H4
4-OCH3C6H4
4-ClC6H4
N
R
N
d
e
NH
NH
O
O
6a-e
7a-e
Scheme 1. Reagents and conditions:a) ClCH2COOC2H5, DMF/ 2 h b) N2H4, abs.C2H5OH/ 25oC/ 24 h
c) CS2, KOH, C2H5OH/ 12 h d) RNCS, abs. C2H5OH/ 10 h e) conc H2SO4/ 5 h f) 2N NaOH/ 4 h