Design, synthesis and anticonvulsant activity of new imidazolidindione and imidazole derivatives

Article abstract of DOI:10.1016/j.bioorg.2020.104020

New imidazolidindiones and tetra-substituted imidazole derivatives were designed, synthesized, and evaluated for the anticonvulsant activity through pentylenetetrazole (PTZ)-induced seizures and maximal electroshock (MES) tests using valproate sodium and

Full text of DOI:10.1016/j.bioorg.2020.104020

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Design, synthesis and anticonvulsant activity of new imidazolidindione and
imidazole derivatives
Adel A. Marzouk, Amr K.A. Bass, Montaser Sh. Ahmed, Antar A.
Abdelhamid, Yaseen A.M.M. Elshaier, Asmaa M.M. Salman, Omar M. Aly
PII:
S0045-2068(20)31317-1
DOI:
https://doi.org/10.1016/j.bioorg.2020.104020
YBIOO 104020
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
22 November 2019
6 June 2020
12 June 2020
Please cite this article as: A.A. Marzouk, A.K.A. Bass, M.S. Ahmed, A.A. Abdelhamid, Y.A.M. Elshaier,
A.M.M. Salman, O.M. Aly, Design, synthesis and anticonvulsant activity of new imidazolidindione and
imidazole derivatives, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104020
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Design, synthesis and anticonvulsant activity of new imidazolidindione and
imidazole derivatives
Adel A. Marzouka, Amr K. A. Bassb, Montaser Sh. Ahmeda, Antar A. Abdelhamidc,d, Yaseen A.
M. M. Elshaiere, Asmaa MM Salmanf, and Omar M. Alyg
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Assiut
Branch, Egypt.
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Menoufia University, Egypt.
c Department of Chemistry, Faculty of Science, Sohag University, Egypt.
d Chemistry Department, Faculty of Science, Albaha University, Albaha (P.O. Box 1988), Saudi
Arabia.
e Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Sadat City,
Sadat City, Menoufia, Egypt.
f Medicinal and Pharmaceutical Chemistry Department, National Research Centre, Dokki, Cairo,
Egypt.
g Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, Egypt.
*Corresponding author: e-mail; adelmarzouk@azhar.edu.eg, Tel.: +201021213733 address:
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Assiut
Branch, Egypt.
Abstract
New imidazolidindiones and tetra-substituted imidazole derivatives were designed, synthesized,
and evaluated for the anticonvulsant activity through pentylenetetrazole (PTZ)-induced seizures
and maximal electroshock (MES) tests using valproate sodium and phenytoin sodium as reference
drugs, respectively. Most of the target compounds showed excellent activity against
pentylenetetrazole (PTZ)-induced seizures with fair to no-activity against MES. Compounds 3d,
4e, 11b, and 11e showed higher activity (120 %) than that of valproate sodium in PTZ model.
Almost all compounds showed no neurotoxicity, as indicated by the rotarod test. Estimation of
physicochemical properties and pharmacokinetic profiles of the target compounds were studied.
The chemical structures of the target compounds were characterized by different spectrometric
methods and elemental analysis.
Keywords: Spiro[fluorene-9,4ʹ-imidazolidine]; Imidazolidinediones, Imidazoles; Anticonvulsant
Activity; Neurotoxicity; shape similarity.

1. Introduction
Epilepsy is a common neurological disease that affects about 50 million people of all ages in the
world. Noteworthily, about 80% of them live in low- and middle-income countries. Epilepsy is
diagnosed by recurrent seizure attacks which involve a group of symptoms that is spontaneous and
intermittent with abnormal electrical activity in the brain accompanied by episodes of the motor,
sensory or autonomic phenomenon. The attacks occur with or without loss of consciousness[1,2].
Currently, clinically available drugs produce acceptable seizure control in about 60 –70% of
patients. Unfortunately, still, about 30% of patients suffer uncontrolled seizure attacks and they
are pharmaco-resistant to the available therapy[3]. In addition, the available drugs have major side-
effects, narrow therapeutic indices and difficulty to be formulated. The anticonvulsant drug must
reach its receptors in the central nervous system to exert its therapeutic effect [4].
It is noticed that most of the antiepileptic drugs don’t have any site of action or known mechanism
of action[5]. The shortage of understanding and complexity of the mechanism of action certainly
affects the development of new candidates as possible Anti-Epileptic Drugs (AEDs) through
mechanism-driven designs. So, the design of a new antiepileptic drug depends on shape similarities
as basic descriptors of computational drug discovery to model and the correct understanding of the
protein-ligand interactions. Shape exhibits good neighboring behaviors, thus, similarities in the
shape reflects similarities in biological activities most notably in 2D structures.
Also, calculations of molecular properties are very important in filtering new drug candidates as
important tools to achieve good bioavailability[6]. Consideration of Lipinski rule known as Role
of Five ((RO5)[7-10] and estimation of molecular polar surface area (PSA) or topological polar
surface area (TPSA) are very important parameters for the prediction of drug transport
properties[7]. Molecular polar surface area (PSA) is used to calculate the percentage of ABS using
the expression: %ABS = 109 - 0.345 PSA [11].
Anti-epileptic research mainly focuses on the study of new anticonvulsant agents through
conventional screening and structural modifications rather than mechanism-based drug design. So,
drug identification is usually conducted via in vivo screening tests based on the seizure type rather
than the etiology.
Evidently, the anticonvulsant activity is mainly due to the presence of the aryl binding site with
aryl/alkyl hydrophobic group, hydrogen bonding domain, and electron donor group. These groups
are the crucial requirements for the molecules to show potential action [12-15].

Encouraged by the promising pharmaceutical profiles of imidazoles and hydantoins as potent
anticonvulsant entities [16-18], and in the continuation of our efforts [19, 20], new anticonvulsant
compounds were prepared to look for safer, more selective and more effective candidates. In this
study, two series of compounds as a hybrid structure from phenytoin and valproic acid were
designed. The side chain was selected to mimic the length and lipophilic nature of valproic acid.
Two series of imidazolidinediones and imidazoles were designed, synthesized, structurally
characterized, and evaluated for the anticonvulsant and neurotoxic activities. Also, similarity to
known drugs (phenytoin and valproic acid) was studied using ROCS (Rapid Overlay of Chemical
Structure) technique. Moreover, physicochemical properties for final compounds were estimated
to assess if these compounds possess drug-likeness properties or not.
Figure1. The design strategy of novel phenytoin-valproic acid analogs
2. Results and Discussion
2.1 Synthesis

Sequential processes were done for the synthesis of the new compounds of imidazolidinedione and
tetra-substituted imidazole derivatives (Schemes 1 and 2).
Scheme 1: Synthesis of imidazolidinedione derivatives (3a-e) and (4a-e).

Scheme 2: Synthesis of multi-substituted imidazoles derivatives (9a-e), (10a-e) and (11a-e).
Imidazolidinediones (hydantoins) (3a-e) were prepared in good yield by alkylation of the parent
spiro hydantoin (spiro[fluorene-9,4'-imidazolidine]-2',5'-dione) 2 by heating of 9-fluorenone 1
with potassium cyanide and anhydrous ammonium carbonate as guided by Bucherer-Bergs
reaction while following the method described by Nagasawa et al [21, 22]. This alkylation was
done using methyl 4-bromomethyl benzoate, ethyl bromoacetate, ethyl 3-bromopropanoate, ethyl
4-bromobutyrate, or ethyl 6-bromohexanoate, in DMF and anhydrous K2CO3 to form the
corresponding compounds (3a-e). The alkylation step was followed by a hydrolysis step which
resulted in compounds (4a-e) with good yield (Scheme 1).
IR spectra of compounds 3a-e showed significant stretching broad bands due to imidic NH groups
at 3399-3174 cm-1, three sharp stretching bands due to three carbonyl groups; two imidic and one
1
ester moiety at 1778-1620 cm-1. H-NMR spectra of compounds 3a–d showed the ethyl ester
protons as a triplet integrated signal for methyl group (CH2CH3) from δ 1.20-1.16 ppm and a
quartet at δ 4.05 ppm (CH2CH3). For compound 3e, a singlet integrated signal for methyl group
protons at δ 3.87 ppm and a singlet signal for 2 protons of CH2 between N and the phenyl moiety
at δ 4.79 ppm appeared. The amidic NH proton appeared as a singlet at δ 9.03-8.90 ppm, while
both the aromatic moieties and aliphatic chains exhibited their signals at the expected δ values.
13C-NMR spectra of compounds 3a-d showed signals for the ethyl ester carbons at approximately
δ 14.43-14.60 ppm (upward in DEPT) corresponding to CH3 group and at δ 60.37-61.96 ppm
(downward in DEPT) corresponding to CH2. Compound (3e); the methyl ester carbon appeared at

δ 52.64 ppm (upward in DEPT) and the carbon of CH2 between N and the phenyl moiety appeared
at δ 42.10 ppm (downward in DEPT). Spiro carbon in all compounds appeared at δ 77.56 ppm and
absent in DEPT.
IR spectra for compounds 4a-e showed significant stretching broad peaks at 3476-2563 cm-1 due
to hydroxyl group of carboxylic acid and three sharp intense stretching bands at 1775-1620 cm-1
1
due the three carbonyl groups; two imidic and one carboxylic carbonyl. H-NMR spectra
confirmed the absence of the ester protons in addition to the appearance of broad singlet peak at δ
12.36-11.94 ppm due to the carboxylic proton and a singlet peak at δ 8.84-8.82 ppm due to amidic
NH proton. The aromatic and aliphatic moieties exhibited their proton signals at the expected δ
values. 13C-NMR and DEPT spectra of compounds 4a-e confirmed the absence of ester carbons.
The imidazoles (9a-e), (10a-e) and (11a-e), [ɛ-(2-(substituted phenyl derivatives)-4,5-diphenyl-
1H-imidazol-1-yl)alkanoic acids], were obtained in a high yield by reaction of benzil, aromatic
aldehyde, amino acids (with a various spacer between the amino and carboxylic groups) and
ammonium acetate by adding the catalytic efficiency of the ionic liquid, diethyl ammonium
hydrogen sulfate (DEAHS) (Scheme 2). The reaction was found to be more facile under solvent-
free conditions with higher yield and relatively in a very short time (35minutes) [23-26].
IR spectra of compounds 9a–e, 10a–e & 11a-e showed significant stretching broad bands due to
carboxylic hydroxyl groups at 3400-2766 cm-1, one sharp intense stretching band at 1607-1601
cm-1 due to (C=N) group and one carbonyl of the carboxylic group at 1732-1675 cm-1. In 1H-NMR;
the aromatic and the aliphatic protons moieties exhibited their proton signals at the expected δ
values. In the case of (9a–e), 1H-NMR spectra of compounds 9a–e showed singlet signal at 3.93-
3.74 ppm due to methoxy group. 13C-NMR and DEPT spectra of all compounds showed a signal
at δ 169.42-178.55 ppm (absence in DEPT) indicates carbon of carboxylic carbonyl group.
13
Compounds 9b–e, 10b–e and 11b-e C-NMR and DEPT spectra confirmed the presence of an
aliphatic side chain attached to N of imidazole by significant signals at δ 44.33-44.55 ppm
13
(downward in DEPT). In the case of (9a–e), C-NMR spectra of compounds 9a-e showed the
appearance of a signal at 55.65-55.74 ppm (upward in DEPT) corresponding to the carbon of
methoxy group. The structure and purity of target compounds were also confirmed by elemental
analyses.

2.2. Anticonvulsant and neurotoxicity screening
The anticonvulsant activity and neurotoxicity of the target imidazolidinedione derivatives (3a-e)
and (4a-e)] and imidazole derivatives (9a-e), (10a-e) and (11a-e)] are given in Tables 1-4. Two
primary anticonvulsant screening models; MES and s.c. PTZ were performed in mice at a dose of
100 mg/kg. Diphenylhydantoin sodium and valproate sodium were used as reference standards for
MES and PTZ, respectively. The neurotoxicity screening (rotarod test) was performed in mice at
a dose of 100 mg/kg. Furthermore, the most active compounds were evaluated at different doses
for determining the ED50 of their anticonvulsant and neurotoxic activities [27].
2.2.1. Maximal electroshock seizure (MES) test.
The target compounds showed lesser protection against seizures than that of reference standard
phenytoin. Compounds (3a, 3b, 3d, 4c, and 4e) were found to be effective with protection
percentage (16.66, 33.33, 33.33, 16.66, 33.33%) respectively, on the other hand, imidazole series
(9a-e), (10a-e) and (11a-e) were found inactive in this test, however, it was observed that most of
the imidazole compounds showed variable protection in mice from mortality against the effect of
MES induced seizures (Table 1).
Table 1: The anticonvulsant activity and percentage mortality prevention of the target compounds
against MES induced seizures in mice in comparison with phenytoin.
Compound
Dose
Electric shock Mortality
protection
(%)
prevention
(%)
mg/kg mmol/kg
2
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
9a
9b
9c
9d
9e
10a
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
0.400
0.295
0.285
0.274
0.255
0.251
0.324
0.310
0.297
0.274
0.260
0.224
0.250
0.242
0.234
0.227
0.222
50
100
16.66
33.33
0.00
33.33
0.00
0.00
0.00
16.66
0.00
33.33
0.00
66.66
33.33
100
100
100
100
33.33
33.33
100
66.66
100
50
50
66.66
66.66
100
0.00
0.00
0.00
0.00
0.00

10b
10c
10d
10e
11a
11b
11c
11d
11e
100
100
100
100
100
100
100
100
100
0
0.248
0.240
0.232
0.225
0.240
0.271
0.261
0.252
0.243
0
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0
100
100
100
100
100
33.33
33.33
33.33
66.66
0
Control
Phenytoin
100
0.396
100
100
2.2.2 Subcutaneous pentylenetetrazole (s.c. PTZ) test.
The initial anticonvulsant evaluation indicated that all compounds were effective in s.c. PTZ
screening. In the imidazolidinedione series I (3a-e), compound 3d showed complete protection
(100%) against s.c. PTZ induced seizures illustrating higher potency than valproate sodium
reference drug, which only achieved 83.33% protection at the same dosage level. Meanwhile,
compound 3b exhibited maximum protection of 83.33% as valproate sodium reference drug.
Compound 3d showed ED50 of 55 mg/ kg (Table 3). The results of all compounds of series I (3a-
e) showed the anticonvulsant potential in the following decreasing order 3d > 3b=2 > 3e > 3c >
3a.
The results of series I (3a-e) against s.c. PTZ induced seizures showed that aliphatic substitution
on N1 with short-chain (3a) decreases the activity. But substitution with long aliphatic chain
increased the anticonvulsant activity (maybe due to the increase in lipophilicity) and exceeded the
anticonvulsant activity of unsubstituted parent compound (2) with exception to compound (3c).
Also, when the aliphatic chain decreases with phenyl moiety (3e) (restricted free rotation), no
change in the activity was found.
In the Imidazolidinediones series II (4a-e), compound 4e showed complete protection (100%)
against s.c. PTZ induced seizures compared to the reference drug valproate sodium, which
achieved 83.33% protection at the same dose level. Meanwhile, compound 4c exhibited maximum
protection of 83.33% as valproate sodium. Also, the potency of compound 4e was assured by ED50
of 60 mg/kg, (Table 3). The members of series 4a-e showed the anticonvulsant activity in the
following decreasing order: 4e > 4c=2 >4b = 4d > 4a.
The results from series II (4a-e) data also showed that by increasing the aliphatic side chain length
(lipophilicity and partition coefficient) by the substitution on N1, the anticonvulsant activity

increased against s.c. PTZ induced seizures till a certain length after which increase the length
decreases the activity (parabolic relationship). Also, when part of the aliphatic chain was replaced
with phenyl moiety (4e), which restricts free rotation, the activity increased.
By comparing the anticonvulsant activity of (3a-e) to (4a-e), it was found that the ester compounds
were more active than that of carboxylic compounds which could be due to decreased lipophilicity
affecting the crossing of the blood-brain barrier.
In the imidazoles (9a-e), (10a-e), and (11a-e) , only compound 9d emerged as the most potent
member in the series (9a-e) as it displayed 83% protection against s.c. PTZ induced seizures as
valproate sodium reference drug, and compounds 9a-e showed the anticonvulsant potential in the
following decreasing order: 9d > 9a > 4b = 4c = 9e. While compound 10e emerged as the most
potent member in the series (10a-e), it displayed 83% protection against s.c. PTZ induced seizures
compared to the reference drug valproate sodium. Series 10a-e showed the anticonvulsant potential
in the following decreasing order: 10e > 10a = 10b > 10c > 10d. Compounds 11b and 11e
displayed complete protection (100%) against s.c. PTZ induced seizures compared to valproate
sodium, the reference drug, which achieved 83.33% protection at the same dose level. Meanwhile,
compound 11a exhibited maximum protection of 83.33% as valproate sodium reference drug.
Compound 11b showed ED50 of 50 mg/kg. Series 10a-e showed the anticonvulsant potential in
the following decreasing order: 11b > 11e > 11a > 11c > 11d. Furthermore, the resulted data from
tetra substituted imidazole derivatives, series IV (10a-e) and series V (11a-e), showed that the
longer the length of the aliphatic chain (N1 substituted imidazole), the lower the anticonvulsant
activity with the exception of 10e and 11e.
The carboxylic group on the side chain represents the HBD required for anticonvulsant activity.
Thus, the length of this chain has two effects on anticonvulsant activity: an effect on the distance
between the HBD and the structure of the whole molecule and an effect on lipophilicity (log p).
When the aliphatic chain was replaced with phenyl moiety, an improvement was noticed in the
anticonvulsant activity (10a) and (11a).
In conclusion, the three series [(9a-e), (10a-e) and (11a-e)] (with the exception of d members),
which were substituted with an electron-withdrawing group (Cl), are more active than those
substituted with an electron-donating group (OCH3). On the other hand, the unsubstituted ones
were more active than both the electron-withdrawing group (Cl) and the electron-donating group
(OCH3) (Table 2).

Table 2: Anticonvulsant activity of the target compounds against s.c. PTZ-induced seizures in
comparison with valproate sodium.
Compound
Dose
PTZ- Protection
(%)
Relative
potency to
standard
100
mg/kg
mmol/kg
2
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
0
0.400
0.295
0.285
0.274
0.255
0.251
0.324
0.310
0.297
0.274
0.260
0.224
0.250
0.242
0.234
0.227
0.222
0.248
0.240
0.232
0.225
0.240
0.271
0.261
0.252
0.243
0
83.33
33.33
83.33
50.00
100.00
66.66
16.67
50.00
83.33
50.00
100.00
66.66
50
3a
40.00
100
3b
3c
60.00
120.00
80.00
20.00
60.00
100
3d
3e
4a
4b
4c
4d
60.00
120.00
80.00
60.00
60.00
100
4e
9a
9b
9c
50
9d
83.33
50
9e
60.00
80.00
80.00
60.00
40.00
100
10a
66.66
66.66
50
10b
10c
10d
33.33
83.33
83.33
100
10e
11a
100
11b
120.00
80.00
20.00
120.00
0
11c
11d
66.66
16.66
100
11e
Control
0
Valproate sodium
100
0.602
83.33
100
The selected highest potent compounds (3d, 4e and 11b) from all series were subjected to the
quantitative estimation (median effective dose, ED50) through using different doses in s.c. PTZ
induced seizures screening (Table 3).

Table 3: The median effective dose (ED50) for compounds 3d, 4e and 11b against s.c. PTZ induced
seizures.
Compound
3d
Dose
PTZ-
PTZ-
Mortality
Onset of convulsion per
unprotected animal
ED50
(mg/kg) Protection Protection prevention
(%)
100
(%)
100
100
80
(6/6)
(5/6)
(3/6)
(2/6)
(6/6)
(5/6)
(4/6)
(1/6)
(6/6)
(4/6)
(4/6)
(3/6)
(2/6)
55
83.33
50
83.33
83.33
66.66
100
after 6 min.
60
after 4, 8 & 23 min.
after 6, 10, 15 & 25 min.
40
33.33
100
4e
100
80
60
50
83.33
66.66
16.67
100
100
after 10 min.
after 6 & 8 min.
60
83.33
83.33
100
40
after 8, 10, 12, 18 & 2 min.
11b
100
80
66.66
66.66
50
100
after 10 &14 min.
after 10 & 23 min.
60
83.33
50
40
after 5, 11 & 15 min.
after 8, 12, 15 & 15 min.
20
33.33
50
2.2.3. Neurotoxicity screening (rotarod test):
The neurotoxicity screening (rotarod test) was performed in mice at a dose of 100 mg/kg. The
results indicated that the ester compounds, as in series I(3a-e), increased the neurotoxicity. While
carboxylic acid derivatives, as in series II (4a-e), decreased or prevented neurotoxicity. These
findings motivated us to prepare carboxylic acid derivatives instead of ester derivatives as in series
III (9a-e), series IV (10a-e), and series V (11a-e), whose neurotoxicity screening was dramatically
abolished (Table 4) [28].
Table 4: Neurotoxicity (Rotarod test) screening data of (3a-e), (4a-e), (9a-e), (10a-e) and (11a-e)
in mice with comparison to that of phenytoin.
Cmpd Neurotoxicity Neurotoxicity%
Cmpd
Neurotoxicity Neurotoxicity%
2
(4/6)
(6/6)
(6/6)
(6/6)
(5/6)
66.66
100
9d
9e
(3/6)
(6/6)
(0/6)
(0/6)
(0/6)
50
100
0
3a
3b
3c
3d
100
10a
10b
10c
100
0
83.33
0
3e
4a
(5/6)
(0/6)
83.33
0
10d
10e
(0/6)
(0/6)
0
0
4b
4c
4d
(0/6)
(0/6)
(0/6)
0
0
0
11a
11b
11c
(0/6)
(0/6)
(0/6)
0
0
0

4e
9a
9b
9c
(0/6)
(0/6)
(0/6)
(0/6)
0
0
0
0
11d
11e
(0/6)
(0/6)
(0/6)
(3/6)
0
0
Control
Phenytoin
0
50
2.2.4. Cytotoxicity studies
The selected highest potent compounds (3d, 4e and 11b) from all the series were subjected to the
quantitative estimation (median toxic dose eliciting minimal neurological toxicity in 50% of
animals and the dose calculated in mg/kg, TD50) through different doses (Table 5).
Table 5: Cytotoxicity studies for compounds (3d, 4e and 11b).
Compound ED50(S-PTZ, mg/kg) LD50 (mg/kg)
PI
3d
4e
11b
55
60
50
630
622
610
11.45
10.3
12.2
ED50= median effective dose in 50% of animals
LD50= median toxic dose eliciting minimal neurological toxicity in 50% of animals
PI= protective index (TD50/ED50)
2.3. Molecular modeling and Insilco study
2.3.1. ROCS application
ROCS is a virtual screening application used to perceive the similarity between chemical
compounds based on their three-dimensional shape (3D)[29-31]. The 3D chemical shape
represented good neighboring behavior, in which high similarity in shape reflects the high
similarity in biology where the high similarity in biology is not reflected in the similarity in
structure. ROCS technique has different applications including lead-hopping, molecular
alignment, pose generation, and structural predictions[29].
The approach of this implemented work was to explain the activity of the compounds based on
their 3D structures similarity to reported drug candidate (the query) and subsequently increase the
chance of “scaffold hopping” or “lead hopping”.
ROCS alignment requires a) query molecules and here are phenytoin and valproic acid standard
drugs, and b) the database molecules that our final compounds. The quality of alignment between
query and the database was estimated using Tanimoto Combo (TC) scores [32]. Applying ROCS
shape and color model for quires (phenytoin and valproic acid) was depicted in figures 2A and 2B,
respectively. In the case of phenytoin, its shapes showed volume with 2 acceptors, 2 donors, and
3 rings, as in Figure 2A. Similarly, in the case of valproic acid, it adopted the shape with 2
acceptors, 1 donor, and 3 hydrophobic species, as depicted in Figure 2B. The structures of valproic

acid and phenytoin, as presented in figure 2C, overlaid and perceived TC scores of 0.7, as shown
in Table 6.
2.3.2. ROCS study using valproic acid as a query:
Both hydantoin derivatives (series a) and imidazole derivatives (series b) aligned and overlaid
with the valproic acid query shape with lower TC scores in comparison to phenytoin, as in the
query Table 5. Compound 3d, with TS 0.51, as an example of hydantoin series, overlaid with
valproic acid and its butyl side chain which occupies the hydrophobic color of valproic acid as
shown in Figure 4A. The compound 4e, with TS 0.44, adopted different poses with valproic acid,
but the benzyl side chain located outside the volume, as illustrated in Figure 4B. In the case of the
imidazole series, it adopted dissimilarity with valproic acid shape. As shown in Figure 4C,
compound 11e exhibited dissimilarity with valproic acid with Tanimoto score 0.52, as in Table 6.
Table 6: Tanimoto Combo scores for all compounds in comparison to phenytion and valproic acid.
Tanimoto Combo Scores,
Tanimoto Combo Scores,
Phenytoin as query
Valproic acid as query
Phenytoin
Valproic
3a
2
0.73
1.2
0.71
1.14
1.2
1.16
0.717
0.713
1.2
0.70
1.12
1.07
0.69
1.20
0.72
0.69
0.68
0.69
0.76
0.72
1.01
0.75
1.08
0.704
2
1
2
3
4
5
6
7
8
0.53
0.53
0.52
0.52
0.54
0.52
0.52
0.51
0.51
0.51
0.50
0.50
0.50
0.50
0.49
0.49
0.48
0.47
0.45
0.44
0.44
0.42
0.37
0.36
0.35
10c
4c
4a
4b
10e
11b
3b
10b
3d
4d
11c
3c
11e
9e
9c
9b
10d
9d
4e
11d
3e
11a
10a
9a
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
0.716
0.70
0.68

A
B
C
Figure 2: vROCS representation shape and color atoms of: A) Phenytoin B) Valporic acid by
vROCS application; C) Overlay and alignment valproic acid on phenytoin.
A
B
C
Figure 3: A) Representation shape and color of 3a using phenytoin as query by vROCS
application; B) Representation shape and color of 3b using phenytoin as query by vROCS
application; C) Representation shape and color of 3d using phenytoin as query by vROCS
application.
A
B
C
Figure 4: A) Representation shape and color of 3d using valproic acid as query by vROCS
application; B) Representation shape and color of 4e using valproic acid as query by vROCS
application; C) Representation shape and color of 11b using valproic acid as query by vROCS
application.
Based on ROCS visualization and TC scores, these compounds have high similarity to hydantoin.
In compounds with the general formula of series a, the 9H-fluorene moiety illustrated color and
shape similarity for both hydantoin and valporic acid. These results will guide us to move forward
in the development of new anticonvulsant candidate that have general formula from series a.

II.3.3. In silico prediction of pharmacokinetics and pharmacodynamics parameters and
drug-likeness data for the active compounds:
Over the past few decades, scientists found that some bioactive molecules cannot be prioritized as
a good drug candidate due to limited bioavailability[7]. About 30% of oral drug subjected to
stopping their process in drug development and manufacturing due to poor pharmacokinetics.
Thus, an in-silico model for predicting oral bioavailability is very important to picking the most
promising compounds for further optimization and clinical development[8].
In the present study, the final compounds were subjected to molecular properties prediction and
drug-likeness by Molinspiration & Mol-Soft, and Preadmet software [33-35].
Table 6 contains the calculated topological molecular polar surface area (TPSA) for the
investigated compounds. All compounds have TPSA in the region of 37.3-86.7. It is known that
for molecules to penetrate the blood–brain barrier (BB), a PSA should be less than 90 angstroms
squared and TPSA values, for most known drugs, have to be below 140-150 Å2 [9, 10].
Compounds 11b, 11c, 10b, 9b, and 9c have BBB values of 5.23-1.13, which are higher than both
standard drugs, phenytoin and valproic acid (BBB = 0.9). Compounds 9d and 11d have BBB
values 0.65 and 0.53 respectively. All other compounds have lower BBB in comparison to standard
drugs used here.
In general, the compounds in the present study possess a high number of rotatable bonds (NRT),
2-9, except compound 9e, which has 10 NRT as shown in Table 6. Therefore, these compounds
exhibit large conformational flexibility. In addition, all compounds obey Lipinski’s rule of five
except compounds 9d, 9e, 10b, 10c, 10e, and 11e have higher log p (5.6-6.9).
The drug-likeness model score is defined as a combined effect of physicochemical properties,
pharmacodynamics and pharmacokinetics of a compound and it is represented by a numerical
value[11]. Compounds with zero or negative value could not be considered as drug-like. In this
study, 12 compounds acquired drug-likeness scores from 0.14-0.79, while other compounds
showed negative values in comparison to phenytoin and valproic acids (12-20) respectively), table
6.
Plasma protein binding (PPB) and percentages of human intestinal absorption (HIA) were
calculated as examples of pharmacokinetic parameters. All compounds exhibited high PPB values
(more than 86) except compounds 4c, and 3c, which had low values (54, 63 respectively).
Regarding HIA, all compounds showed high values.

Table 7: Predicted of pharmacokinetic and pharmacodynamic parameters compounds and
standard drugs
Compound
Lipniski rule of five
TPSA
No.
Drug- BBB PPB
HIA
violations likeness
model
score
Mwt NRB HBA HBD LogP
Phenytoin 252.27
Valproic 144.21
2
5
4
7
4
2
3
9
6
5
6
8
6
7
6
9
2
2
2
1
1
1
2
2
2
1
1
1
1
1
2
1
1
1
1
1
1
1
1
2
1
1
1
1
1
2.18 58.20
2.80 37.30
2.15 75.71
5.72 55.12
2.15 86.71
0.69 86.71
1.88 86.71
6.93 55.12
4.97 55.12
2.56 75.71
5.65 55.12
3.84 75.71
3.16 86.71
5.24 55.12
2.83 75.71
6.25 55.12
6.31 64.36
5.30 64.36
5.03 64.36
6.43 55.12
5.80 64.36
3.67 86.71
5.75 55.12
6.70 64.36
3.93 75.71
7.32 55.12
6.64 55.12
0
0
0
1
0
0
0
1
1
1
1
0
0
1
0
1
1
1
1
1
1
0
1
1
0
1
1
0.12
0.20
-1.43
0.79
-0.83
-1.02
-0.95
0.83
-0.12
-1.30
0.66
-1.09
-0.79
0.10
-1.13
0.14
0.39
0.32
0.18
0.83
0.39
-0.06
0.14
-0.97
0.53
-0.58
-0.94
0.92 96.58 92.53
0.91 100.00 93.71
0.05 86.30 96.44
0.44 100.00 97.62
0.26 54.62 96.27
0.36 90.58 95.32
0.36 92.58 95.85
0.21 100.00 97.73
5.25 92.94 97.39
0.16 94.23 96.52
2.10 100.00 97.57
0.04 87.27 96.41
0.026 86.19 96.86
3.12 98.06 97.41
0.13 63.38 96.50
0.13 100.00 97.49
0.19 97.86 97.46
1.13 94.88 97.46
2.82 91.48 97.48
0.36 100.00 97.68
0.65 96.20 97.45
0.36 97.73 97.08
0.53 99.52 97.45
0.23 100.00 97.64
0.10 96.32 96.31
0.40 100.00 97.97
0.43 100.00 97.73
1
2
3
4
5
6
7
8
3a
10c
4c
4a
4b
10e
11b
3b
10b
3d
4d
11c
3c
336.35
416.91
336.35
308.29
322.32
444.96
368.44
350.37
402.88
392.45
364.40
382.46
364.40
410.52
5
3
4
4
4
3
3
4
3
4
4
3
4
3
4
4
4
3
4
4
3
4
3
3
3
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
11e
9e
440.54 10
9c
9b
10d
9d
4e
11d
9a
3e
10a
11a
412.49
398.46
430.94
426.52
384.39
396.49
446.51
398.42
450.93
416.48
8
7
8
9
3
8
6
4
5
5
LogP: logarithm of compound partition coefficient
between n-octanol and water.
M.Wt: molecular weight.
HBA: number of hydrogen bond acceptors.
HBD: number of hydrogen bond donors.
HBA: number of hydrogen bond acceptors.
TPSA: polar surface area.
BBB: Blood Brain Barrier.
PPB: plasma protein binding
NRB: number of rotation bonds.
HIA: Percentages of human intestinal absorption

To conclude, results showed that the most active compounds exhibited reasonable physicochemical
properties with good predicted drug-likeness values, which might elevate them to be drug candidates.
2.4. Conclusion:
A group of new imidazolidinedione and tetra-substituted imidazole derivatives were synthesized and
characterized by their spectral data. They were compromised in two series: imidazolidinedione (3a-e),
(4a-e), and imidazole derivatives (9a-e), (10a-e) and (11a-e). These compounds showed from promising
to moderate anticonvulsant activities. The obtained results revealed that compound (3d), which has a
longer aliphatic side chain (n=5) linked to the imidazolidinedione nucleolus, showed complete protection
(100%) against s.c. PTZ induced seizures compared to valproate sodium reference drug, which achieved
83.33% protection at the same dose level. Also, compound (4e), which has methyl benzoic acid moiety
linked to the imidazolidinedione, exhibited complete protection (100%) against s.c. PTZ induced seizures.
On the other hand, imidazole compounds as 3-(2,4,5-triphenyl-1H-imidazol-1-yl)propanoic acid (11b)
and 5-(2,4,5-triphenyl-1H-imidazol-1-yl)pentanoic acid (11e) exhibited complete protection (100%)
against s.c. PTZ induced seizures. The neurotoxicity screening showed the absence of toxic effects at the
tested dose level for all series, which has a free carboxylic group. Moreover, the neurotoxicity increased
after replacing the carboxylic group by ester moiety as in series II that may be due to the increasing in the
hydrophilic properties of the compounds. Finally, it was found that these compounds could decrease the
threshold of seizures rather than the prevention of their spread. This was concluded from their
anticonvulsant activity against s.c. PTZ induced seizures and inactivity of these compounds against the
MES test. Furthermore, these active compounds may be valuable in the treatment of petit mal seizures
more than grand mal seizures as (4e), (3d), (11b) and (11e) revealed promising anticonvulsant agents.
Shape similarity study and physicochemical calculations advocates future work with potent and safe
anticonvulsant drugs.
3. Experimental
Stuart electro-thermal melting point equipment was used for melting points determination and was
uncorrected. Nicolet iS5 FT-IR spectrometer was used for recording IR spectra while a Bruker Advance
400 MHz NMR spectrometer was used for recording NMR spectra, using TMS as an internal reference,
at Sohag University, Egypt. Chemical shifts (δ) values were given in parts per million (ppm) relative to
CDCl3 (7.29 for proton and 76.9 for carbon) or DMSO-d6 (2.50 for proton and 39.50 for carbon) and

coupling constants (J) in Hertz. Vario El Elementar CHN Elemental analyzer in the Regional Center for
Mycology and Biotechnology, Naser City, Cairo, Egypt was used for elemental analysis and the results
were within ± 0.4% of the theoretical values. Thin-layer chromatography (TLC) using Merck 9385 pre-
coated aluminum plate silica gel (Kieselgel 60) 5 x 20 cm plates with a layer thickness of 0.2 mm were
used for monitoring the progress of reactions and the purity of the prepared compounds. All solvents were
dried by standard methods. 9-fluorenone 1 and methyl 4-bromomethyl benzoate were purchased from
Sigma Aldrich, other materials and solvent used in the preparation of intermediate and target compounds
were purchased from El-Nasr pharmaceutical chemicals company, Egypt.
3.1. Synthesis of Spiro[fluorene-9,4ʹ-imidazolidine]-2ʹ,5ʹ-dione (2)
The Spiro[fluorene-9,4ʹ-imidazolidine]-2ʹ,5ʹ-dione (2) was synthesized as reported[22] giving dark yellow
to brown crystals (92% yield) mp.353 °C, reported more than 300 °C.
3.1.1. General procedure for synthesis of ethyl ɛ-(2ʹ,5ʹ-dioxospiro[fluorene-9,4ʹ-imidazolidine]-1ʹ-
yl)alkanoate
(3a-d)
and
methyl
4-((2ʹ,5ʹ-dioxospiro[fluorene-9,4ʹ-imidazolidine]-1ʹ-
yl)methyl)benzoate (3e)
A mixture of the imidazolidinedione 2 (1.251 g, 5 mmol), (5.5 mmol) of an appropriate ester [methyl 4-
bromomethyl benzoate (1.259 g), ethyl bromoacetate (0.918 g), ethyl 3-bromopropanoate (0.995 g), ethyl
4-bromobutyrate (1.073 g), ethyl 6-bromohexanoate (1.228 g) and anhydrous potassium carbonate (1.382
g, 10 mmol) in DMF (20 mL) was allowed to stir at room temperature for 12 hrs. The reaction mixture
was diluted with ice water and the resulting precipitate was filtered, washed with water and dried. The
crude product was purified by crystallization from aqueous ethanol to form the corresponding compound
(3a-e).
Methyl 4-((2ʹ, 5ʹ-dioxospiro[fluorene-9,4ʹ-imidazolidine]-1ʹ-yl)methyl)benzoate (3e):
Reaction of 2 with methyl 4-bromomethylbenzoate, white powder; Yield (98%); mp. 248-250 °C; IR (cm-
1
1): 3307 (NH), 1772 (C=O), 1710 (C=O), 1611 (C=O), 1104 (C-O); H-NMR (400 MHz, DMSO-d6) δ
(ppm): 4.79 (s, 2H, N-CH2), 3.87 (s, 3H, O-CH3), 7.38 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.47 (t, 2H, J = 8.00
Hz, 2 Ar-H), 7.50 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.51 (d, 2H, J = 8.00 Hz, 2 Ar-H), 8.01 (d, 2H, J = 8.00
13
Hz, 2 Ar-H), 8.03 (d, 2H, J = 8.00 Hz, 2 Ar-H), 9.11 (s, 1H, NH); C-NMR (100 MHz, DMSO-d6) δ
(ppm): 42.10 (CH2), 52.64 (OCH3), 71.56 (C(spiro)), 121.24, 121.34, 124.10, 124.76, 127.84, 127.99,
128.59, 128.94, 129.28, 130.13, 130.50, 130.80, 140.59, 141.17, 141.37, 142.50, 142.88, 157.02, 162.78,
166.43, 172.85; Elemental analysis for C24H18N2O4 (Calcd./Found); C, 72.35/72.57; H, 4.55/4.58; N,
7.03/7.11.

Ethyl 2-(2ʹ,5ʹ-dioxospiro[fluorene-9,4ʹ-imidazolidine]-1ʹ-yl)acetate 3a:
Reaction of 2 with ethyl bromoacetate; white powder (yield 90%); mp. (196) °C : IR (cm-1): 3399
1
(NH), 1770 (C=O), 1734 (C=O), 1627 (C=O), 1145 (C-O); H-NMR (400 MHz, DMSO-d6) δ
(ppm): 1.26 (t, 3H, J = 7.20 Hz, CH2-CH3), 4.35 (s, 2H, N-CH2), 4.22 (q, 2H, J = 7.20 Hz, O-CH2-
CH3), 7.39 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.42 (t, 2H, J = 8.00 Hz, 2 Ar-H), 7.51 (d, 2H, J = 8.00
Hz, 2 Ar-H), 7.93 (d, 2H, J = 8.00 Hz, 2 Ar-H), 9.03 (s, 1H, NH); 13C-NMR (100 MHz, DMSO-
d6) δ (ppm): 14.47 (CH3), 40.30 (CH2), 61.96 (CH2), 71.68 (C(spiro)), 121.26, 124.25, 125.31,
128.83, 130.50, 131.13, 139.39, 141.21, 141.88, 142.89, 155.52, 156.52 ,162.76, 167.19, 172.61;
Elemental analysis for C19H16N2O4 (Calcd./Found); C, 67.85/68.02; H, 4.79/4.86; N, 8.33/8.51.
Ethyl 3-(2ʹ,5ʹ-dioxospiro[fluorene-9,4ʹ-imidazolidine]-1ʹ-yl)propanoate (3b): reaction of 2
with ethyl 3-bromopropionate; white powder (yield: 90%); mp.178-180 °C; IR (cm-1): 3174 (NH),
1
1774 (C=O), 1713 (C=O), 1618 (C=O), 1181 (C-O); H NMR (400 MHz, DMSO-d6) δ (ppm);
1.19 (t, 3H, J = 7.20 Hz, CH2-CH3), 2.68 (t, 2H, J = 6.80 Hz, CH2-CO), 3.78 (t, 2H, J = 6.80 Hz,
N-CH2), 4.09 (q, 2H, J = 7.20 Hz, O-CH2-CH3), 7.37 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.48 (t, 2H, J
= 8.00 Hz, 2 Ar-H), 7.51 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.93 (d, 2H, J = 8.00 Hz, 2 Ar-H), 8.9 (s,
13
1H, NH); C-NMR (100 MHz, DMSO-d6) δ (ppm): 14.43 (CH3), 32.71 (CH2), 40.30 (NCH2),
60.72 (OCH2), 71.32 (C(spiro)), 121.18, 121.58, 124.19, 124.56, 128.74, 128.74, 130.37, 130.90,
140.54, 141.20, 141.67, 143.10, 156.43, 171.06, 172.68.; Elemental analysis for C20H18N2O4
(Calcd./Found); C, 68.56/68.79; H, 5.18/5.23; N, 8.00/8.23.
Ethyl 4-(2ʹ,5ʹ-dioxospiro[fluorene-9,4ʹ-imidazolidine]-1ʹ-yl)butanoate (3c): Reaction of 2 with
ethyl 4-bromobutyrate; white powder (yield 97%); mp.145-150 °C: IR (cm-1): 3330 (NH), 1779
(C=O), 1707 (C=O), 1680 (C=O), 1095 (C-O); 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 1.19 (t,
3H, J = 7.20 Hz, CH2-CH3), 1.88-1.91 (m, 2H, CH2-CH2-CO), 2.40 (t, 2H, J = 6.80 Hz, CH2-CO),
3.58 (t, 2H, J = 6.80 Hz, N-CH2), 4.09 (q, 2H, J = 7.20 Hz, O-CH2-CH3), 7.37 (d, 2H, J = 8.00
Hz, 2 Ar-H), 7.48 (t, 2H, J = 8.00 Hz, 2 Ar-H), 7.52 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.94 (d, 2H, J
13
= 8.00 Hz, 2 Ar-H), 8.95 (s, 1H, NH); C-NMR (100 MHz, DMSO-d6) δ (ppm): 14.43 (CH3),
23.52 (CH2), 30.90 (CH2), 40.30 (NCH2), 60.37 (OCH2), 71.40 (C(spiro)), 121.26, 121.56, 124.08,
124.48, 128.84, 130.40, 130.82, 140.86, 141.19, 141.65, 143.12, 157.37, 172.45, 172.71, 173.07.;
Elemental analysis for C21H20N2O4 (Calcd./Found); C, 69.22/69.47; H, 5.53/5.59; N, 7.69/7.88.
Ethyl 6-(2ʹ,5ʹ-dioxospiro[fluorene-9,4ʹ-imidazolidine]-1ʹ-yl)hexanoate (3d): reaction of 2 with
ethyl 6-bromohexanoate; white powder (yield 86.7%); mp.104-106 °C; IR (cm-1): 3175 (NH),
19

1
1770 (C=O), 1703 (C=O), 1680 (C=O), 1098 (C-O); H-NMR (400 MHz, DMSO-d6) δ (ppm):
1.19 (t, 3H, J = 7.20 Hz, CH2-CH3), 1.34-1.35 (m, 2H, CH2-CH2-CH2-CO), 1.58-1.66 (m, 4H,
CH2-CH2-CH2-CH2-CO), 2.32 (t, 2H, J = 7.20 Hz, CH2-CO), 3.53 (t, 2H, J = 7.20 Hz, N-CH2),
4.07 (q, 2H, J = 7.20 Hz, O-CH2-CH3), 7.37 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.45 (t, 2H, J = 8.00
Hz, 2 Ar-H), 7.52 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.94 (d, 2H, J = 8.00 Hz, 2 Ar-H), 8.93 (s, 1H,
13
NH); C-NMR (100 MHz, DMSO-d6) δ (ppm): 14.60 (CH3), 24.50 (CH2), 26.01 (CH2), 27.71
(CH2), 33.79 (CH2), 38.76 (NCH2), 60.37 (OCH2), 71.38 (C(spiro)), 121.28, 121.59, 123.98,
124.36, 128.79, 128.84, 130.38, 130.81, 140.80, 141.20, 141.80, 143.15, 157.42 (NCO), 172.96
(NCO), 173.28.; EI-MS (70 eV) m/z (%): 392 (28) (M+), 347 (11), 305 (17), 291 (1), 264 (2), 206
(17), 192 (27), 179 (100), 140 (60); Elemental analysis for C23H24N2O4 (Calcd./Found); C,
70.39/70.51; H, 6.16/6.25; N, 7.14/7.32.
3.1.2. General procedure for the synthesis of ɛ-(2ʹ,5ʹ-dioxospiro[fluorene-9,4ʹ-imidazolidine]-
1ʹ-yl)alkanoic acids (4a-d) and 4-((2ʹ,5ʹ-dioxospiro[fluorene-9,4ʹ-imidazolidine]-1ʹ-
yl)methyl)benzoic acid (4e).
To (1 mmol) of an appropriate ester (3a-e) add 20 mL of 1N NaOH then stir at room temperature
for 16 hrs. The reaction mixture was filtered off and the filtrate was cooled to 0 °C. Then the
reaction mixture was acidified with hydrochloric acid. The obtained solid was separated, washed
with water, dried and crystallized from ethanol.
4-((2ʹ,5ʹ-Dioxospiro[fluorene-9,4ʹ-imidazolidine]-1ʹ-yl)methyl) benzoic acid (4e): white
powder (88%); mp. 220-222 °C; IR (cm-1): 3476-2641 (OH), 3290.83 (NH), 1775 (C=O), 1707
(C=O), 1612 (C=O), 1106 (C-O); 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 4.80 (s, 2H, N-CH2),
7.36 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.42 (t, 2H, J = 8.00 Hz, 2 Ar-H), 7.49 (d, 2H, J = 8.00 Hz, 2
Ar-H), 7.53 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.91 (d, 2H, J = 8.00 Hz, 2 Ar-H), 8.01 (d, 2H, J = 8.00
Hz, 2 Ar-H), 8.99 (s, 1H, NH), 12.59 (br s, 1H, OH); 13C-NMR (100 MHz, DMSO-d6) δ (ppm):
42.24 (CH2), 71.67 (C(spiro)), 121.25, 121.34, 123.98, 124.66, 127.88, 127.99, 128.47, 128.87,
129.35, 130.21, 130.26, 130.43, 140.59, 141.22, 141.37, 142.50, 142.88, 143.02, 157.02, 166.43,
172.85; Elemental analysis for C23H16N2O4 (Calcd./Found); C, 71.87/72.05; H, 4.20/4.24; N,
7.29/7.38.
2-(2ʹ,5ʹ-Dioxospiro[fluorene-9,4ʹ-imidazolidine]-1ʹ-yl)acetic acid (4a): white powder (yield
86%); mp. 212-215 °C; IR (cm-1): 3070-2563 (OH), 3358 (NH), 1775 (C=O), 1705.71 (C=O),
1622 (C=O); 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 4.24 (s, 2H, N-CH2), 7.39 (d, 2H, J = 8.00
20

Hz, 2 Ar-H), 7.50 (t, 2H, J = 8.00 Hz, 2 Ar-H), 7.53 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.91 (d, 2H, J
= 8.00 Hz, 2 Ar-H), 8.92 (s, 1H, NH), 13.19 (br s, 1H, OH); 13C-NMR (100 MHz, DMSO-d6) δ
(ppm): 40.30 (CH2), 71.68 (C(spiro)), 121.17, 124.26, 125.31, 128.77, 130.42, 131.13, 139.39,
141.23, 141.88, 143.05, 155.52, 156.52, 162.76, 167.19, 172.61; Elemental analysis for
C17H12N2O4 (Calcd./Found); C, 70.39/70.60; H, 6.16/6.24; N, 7.14/7.19.
3-(2ʹ,5ʹ-Dioxospiro[fluorene-9,4ʹ-imidazolidine]-1ʹ-yl)propanoic acid (4b): white powder
(yield 80%); mp.185 °C; IR (cm-1): 3200-2670 (OH), 3345 (NH), 1772 (C=O), 1700 (C=O), 1683
(C=O); 1H-NMR (400 MHz, DMSO-d6) δ (ppm); 2.67 (t, 2H, J = 6.80 Hz, CH2-CO), 3.79 (t, 2H,
J = 6.80 Hz, N-CH2), 7.37 (d, 2H, J = 8.00, 2 Ar-H), 7.49 (t, 2H, J = 8.00 Hz, 2 Ar-H), 7.52 (d,
2H, J = 8.00 Hz, 2 Ar-H), 7.90 (d, 2H, J = 8.00 Hz, 2 Ar-H), 8.8 (s, 1H, NH) , 11.89 (br s, 1H,
OH); 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 32.57 (CH2), 40.30 (NCH2), 71.38 (C(spiro)),
121.10 121.58, 124.24, 125.56, 127.97, 128.66, 128.73, 130.30, 140.54, 141.23, 141.67, 143.22,
157.06, 172.53, 172.71.; Elemental analysis for C18H14N2O4 (Calcd./Found); C, 67.07/67.34; H,
4.38/4.36; N, 8.69/8.78.
4-(2ʹ,5ʹ-Dioxospiro[fluorene-9,4ʹ-imidazolidine]-1ʹ-yl)butanoic acid (4c): white powder (yield
83%); mp.183 °C; IR ν (cm-1): 3153-2629 (OH), 3340 (NH), 1766 (C=O), 1696 (C=O), 1620
(C=O); 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 1.87-1.93 (m, 2H, CH2-CH2-CO), 2.32 (t, 2H, J
= 6.80 Hz, CH2-CO), 3.58 (t, 2H, J = 6.80 Hz, N-CH2), 7.36 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.49 (t,
2H, J = 8.00 Hz, 2 Ar-H), 7.53 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.93 (d, 2H, J = 8.00 Hz, 2 Ar-H),
8.80 (s, 1H, NH), 12.11 (br s, 1H, OH); 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 23.64 (CH2),
31.43 (CH2), 38.60 (NCH2), 71.49 (C(spiro)), 121.26, 121.56, 124.08, 124.48, 128.77, 128.87,
130.40, 130.82, 141.22, 141.65, 143.24, 157.38, 172.45, 173.01, 174.08.; Elemental analysis for
C19H16N2O4 (Calcd./Found): C, 67.85/68.09; H, 4.79/4.88; N, 8.33/8.51.
6-(2ʹ,5ʹ-Dioxospiro[fluorene-9,4ʹ-imidazolidine]-1ʹ-yl)hexanoic acid (4d): yellowish white
powder (yield 93%); mp.145-147 °C; IR (cm-1): 3058-2864 (OH), 3393 (NH), 1775 (C=O), 1707
(C=O), 1689 (C=O); 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 1.36-1.38 (m, 2H, CH2-CH2-CH2-
CO), 1.58-1.68 (m, 4H, CH2-CH2-CH2-CH2-CO), 2.25 (t, 2H, J = 7.20 Hz, CH2-CO), 3.54 (t, 2H,
J = 7.20 Hz, N-CH2), 7.37 (d, 2H, J = 8.00 Hz, 2 Ar-H), 7.49 (t, 2H, J = 8.00 Hz, 2 Ar-H), 7.53
(d, 2H, J = 8.00 Hz, 2 Ar-H), 7.91 (d, 2H, J = 8.00 Hz, 2 Ar-H), 8.80 (s, 1H, NH), 11.94 (br s, 1H,
13
OH); C-NMR (100 MHz, DMSO-d6) δ (ppm): 24.55 (CH2), 26.16 (CH2), 27.72 (CH2), 34.00
(CH2), 38.85 (NCH2), 71.48 (C(spiro)), 121.28, 121.59, 123.98, 124.36, 128.79, 128.84, 130.38,
21

130.81, 140.80, 141.23, 141.80, 143.25, 157.44, 172.94, 174.69.;EI-MS (70 eV) m/z (%): 364 (1)
(M+), 321 (3), 319 (1), 308 (3), 293 (4), 267 (3), 221 (2), 207 (10), 206 (15), 178(30), 165 (80). 89
(100).; Elemental analysis for C21H20N2O4 (Calcd./Found): C, 69.22/69.40; H, 5.53/5.56; N,
7.69/7.84.
3.2.1 General procedure for synthesis of 1,2,4,5-tetrasubstituted imidazoles. [Ɛ-(2-
(substituted phenyl)-4,5-diphenyl-1H-imidazol-1-yl)alkanoic acid] and [4-(2-(substituted
phenyl)-4,5-diphenyl-1H-imidazol-1-yl)benzoic acid][25, 26].
In a rounded flask 50 mL add benzil (1.05 g, 5.0 mmol), an equivalent amount of the aldehyde (5
mmol), ammonium acetate (0.385g, 5 mmol), amino acid (5 mmol) and diethyl ammonium
hydrogen sulfate catalyst (0.510 g, 3 mmol). The mixture was refluxed in an oil bath with stirring
at 110 °C for about 35 min with following up the reaction with TLC using chloroform: ethanol
(9:1) till reaction completes. The reaction mixture was poured on an iced water and the resulting
precipitate was filtered off, washed with water and dried. The crude product was crystallized from
ethanol.
3.2.2. Synthesis of diethyl ammonium hydrogen sulfate (DEAHS) [23].
Diethyl amine (22.2 g, 0.3 mol) was added into a 250 mL three-necked flask with a magnetic
stirrer. Concentrated sulfuric acid (98%), 29.4 g (0.3 mol) was added dropwise to the flask at room
temperature then heated to 80 °C for 12 h. The product was washed with diethyl ether three times
to remove the traces of non-ionic material. The residue was dried under vacuum using a rotary
evaporator to obtain the clear viscous product, diethyl ammonium hydrogen sulfate. Yield 98 %,
pH 1.6.
4-(2-(4-Methoxyphenyl)-4,5-diphenyl-1H-imidazol-1-yl)benzoic acid (9a): white crystals;
1
mp.328-330 °C (yield 92%) IR (cm-1): 3214 (OH), 1687 (C=O), 1605 (C=N), 1068 (C-O); H-
NMR (400 MHz, DMSO-d6) δ (ppm): 3.74 (s, 3H,O-CH3), 6.86 (d, 2H, J = 8.80 Hz, 2 Ar-H),
6.88-7.50 (m, 10H, Ar-H), 7.51 (d, 2H, J = 8.80 Hz, 2 Ar-H), 7.82 (d, 2H, J = 8.80 Hz, 2 Ar-H),
7.84 (d, 2H, J = 8.80 Hz, 2 Ar-H); 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 55.65 (O-CH3),
114.56, 124.17, 126.40, 126.57, 128.39, 129.22, 129.54, 130.58, 131.34, 131.76, 135.37, 136.58,
147.17, 160.122, 174.69 (OCO).; Elemental analysis for C29H22N2O3 (Calcd./Found): C,
78.01/78.27; H, 4.97/4.98; N, 6.27/6.42.
3-(2-(4-Methoxyphenyl)-4,5-diphenyl-1H-imidazol-1-yl)propanoic acid (9b): white crystals
1
mp.200-202 °C (yield 87%); IR (cm-1): 3398 (OH), 1717 (C=O), 1605 (C=N), 1040 (C-O); H-
22

NMR (400 MHz, DMSO-d6) δ (ppm); 2.87 (t, 2H, J = 6.80 Hz, CH2-CO), 4.10 (t, 2H, J = 6.80
Hz, N-CH2), 3.85 (s, 3H,O-CH3), 7.08 (d, 2H, J = 8.80, 2 Ar-H), 7.68-7.10 (m, 10H, Ar-H), 8.00
(d, 2H, J = 8.80 Hz, 2 Ar-H); 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 32.51 (CH2), 41.69
(NCH2), 55.72 (O-CH3), 114.54, 123.92, 126.52, 127.21, 128.44, 128.80, 129.34, 129.60, 129.74,
130.64, 131.35, 131.52, 135.24, 136.87, 147.09, 160.13, 171.95.; Elemental analysis for
C25H22N2O3 (Calcd./Found): C, 75.36/75.64; H, 5.57/5.65; N, 7.03/7.19.
4-(2-(4-Methoxyphenyl)-4,5-diphenyl-1H-imidazol-1-yl)butanoic acid (9c): White crystals;
1
mp.188-190 °C (yield 84%); IR (cm-1): 3371 (OH), 1705 (C=O), 1607 (C=N), 1068 (C-O); H-
NMR (400 MHz, DMSO-d6) δ (ppm): 1.53-1.54 (m, 2H, CH2-CH2-CO), 2.04 (t, 2H, J = 6.80 Hz,
CH2-CO), 3.85(t, 2H, J = 6.80 Hz, N-CH2), 3.93 (s, 3H,O-CH3), 7.09 (d, 2H, J = 8.80, 2 Ar-H),
7.18-7.53(m, 10H, Ar-H), 7.68 (d, 2H, J = 8.80 Hz, 2 Ar-H); 13C-NMR (100 MHz, DMSO-d6) δ
(ppm): 25.87 (CH2), 31.40 (CH2), 44.33 (NCH2), 55.72 (O-CH3), 114.56, 123.40, 126.43, 126.59,
128.39, 129.22, 129.54, 130.58, 131.34, 131.40, 134.62, 136.01, 145.09, 158.32, 174.08.;
Elemental analysis for C26H24N2O3 (Calcd./Found): C, 75.71/75.94; H, 5.86/5.92; N, 6.79/6.84.
5-(2-(4-Methoxyphenyl)-4,5-diphenyl-1H-imidazol-1-yl)pentanoic acid (9d): White crystals;
1
mp.156-158 °C (yield 71%); IR (cm-1): 3373 (OH), 1703 (C=O), 1604 (C=N), 1076 (C-O); H-
NMR (400 MHz, DMSO-d6) δ (ppm): 1.16-1.31 (m, 2H, CH2-CH2-CH2-CO), 1.84 (m, 2H, CH2-
CH2-CH2-CO), 2.62 (t, 2H, J = 6.80 Hz, CH2-CO), 3.85 (t, 2H, J = 6.80 Hz, N-CH2), 3.85 (s,
3H,O-CH3), 7.10 (d, 2H, J = 8.80, 2 Ar-H), 7.53-7.18 (m, 10H, Ar-H), 7.65 (d, 2H, J = 8.80 Hz, 2
13
Ar-H); C-NMR (100 MHz, DMSO-d6) δ (ppm): 22.06 (CH2), 29.81(CH2), 33.97 (CH2), 44.55
(NCH2), 55.74(O-CH3), 114.57, 123.40, 126.42, 126.59, 128.39, 129.22, 129.54, 130.58, 131.34,
131.40, 134.62, 136.01, 145.09, 158.32, 174.08.; Elemental analysis for C27H26N2O3
(Calcd./Found): C, 76.03/76.26; H, 6.14/6.20; N, 6.57/6.71.
6-(2-(4-Methoxyphenyl)-4,5-diphenyl-1H-imidazol-1-yl)hexanoic acid (9e): white crystals;
1
mp.160-162 °C (yield 93%); IR (cm-1): 3392 (OH), 1708 (C=O), 1606 (C=N), 1070 (C-O); H-
NMR (400 MHz, DMSO-d6) δ (ppm): 1.17-1.30 (m, 2H, CH2-CH2-CH2-CO), 1.30-1.52 (m, 4H,
CH2-CH2-CH2-CH2-CO), 1.94 (t, 2H, J = 7.20 Hz, CH2-CO), 3.84 (t, 2H, J = 7.20 Hz, N-CH2),
3.84 (s, 3H,O-CH3), 7.09 (d, 2H, J = 8.80, 2 Ar-H), 7.10-7.52 (m, 10H, Ar-H), 7.65 (d, 2H, J =
8.80 Hz, 2 Ar-H); 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 24.16 (CH2), 25.71 (CH2), 29.80
(CH2), 34.03 (CH2), 44.58 (NCH2), 55.72 (O-CH3), 114.56, 124.17, 126.40, 126.57, 128.39,
129.22, 129.54, 130.58, 131.34, 131.76, 135.37, 136.58, 147.17, 160.12, 174.69; EI-MS (70 eV)
23

m/z (%): 440 (70) (M+), 395 (1), 381 (3), 354 (9), 339 (14), 325 (29), 311 (12), 119 (66), 104
(100); Elemental analysis for C28H28N2O3 (Calcd./Found): C, 76.34/76.59; H, 6.41/6.48; N,
6.36/6.49.
4-(2-(4-Chlorophenyl)-4,5-diphenyl-1H-imidazol-1-yl)benzoic acid (10a): Yellowish white
crystals; mp. 233-235 °C (yield 87%); IR ν (cm-1): 3348 (OH), 1675 (C=O), 1625 (C=N), 1076
(C-O); 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 6.58 (d, 2H, J = 8.80, 2 Ar-H), 7.59-7.31(m, 10H,
Ar-H), 7.61 (d, 2H, J = 8.80 Hz, 2 Ar-H), 7.68 (d, 2H, J = 8.80 Hz, 2 Ar-H), 7.98 (d, 2H, J = 8.80
Hz, 2 Ar-H); 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 113.13, 118.56, 121.24, 129.46, 129.80,
130.94, 131.59, 134.00, 134.76, 135.37, 139.58, 151.17, 154.19, 161.17, 169.42; Elemental
analysis for C28H19ClN2O2 (Calcd./Found): C, 74.58/74.80; H, 4.25/4.23; N, 6.21/6.34.
3-(2-(4-Chlorophenyl)-4,5-diphenyl-1H-imidazol-1-yl)propanoic acid (10b): Brownish white
crystals mp.226-228 °C (yield 89%); IR ν (cm-1): 3375-2766 (OH), 1709 (C=O), 1601 (C=N),
1081 (C-O); 1H-NMR (400 MHz, DMSO-d6) δ (ppm); 2.29 (t, 2H, J = 6.80 Hz, CH2-CO), 4.15 (t,
2H, J = 6.80 Hz, N-CH2), 7.13 (d, 2H, J = 8.80, 2 Ar-H), 7.15-7.63 (m, 10H, Ar-H), 7.80 (d, 2H,
13
J = 8.80 Hz, 2 Ar-H); C-NMR (100 MHz, DMSO-d6) δ (ppm): 32.57 (CH2), 41.75 (NCH2),
126.60, 126.66, 127.7, 127.85, 128.46, 129.14, 129.48, 129.63, 130.43, 130.99, 131.33, 132.74,
135.00, 137.29, 148.42, 174.08 (OCO).; Elemental analysis for C24H19ClN2O2 (Calcd./Found): C,
71.55/71.82; H, 4.75/4.79; N, 6.95/7.13.
4-(2-(4-Chlorophenyl)-4,5-diphenyl-1H-imidazol-1-yl)butanoic acid (10c): Brown crystals;
mp.193-195 °C (yield 85%); IR ν (cm-1): 3371 (OH), 1698 (C=O), 1604 (C=N), 1082 (C-O); 1H-
NMR (400 MHz, DMSO-d6) δ (ppm): 1.74-1.83 (m, 2H, CH2-CH2-CO), 2.84 (t, 2H, J = 6.80 Hz,
CH2-CO), 3.94 (t, 2H, J = 6.80 Hz, N-CH2), 7.13 (d, 2H, J = 8.80, 2 Ar-H), 7.14-7.59 (m, 10H,
Ar-H), 7.81 (d, 2H, J = 8.80 Hz, 2 Ar-H); 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 25.96 (CH2),
31.51 (CH2), 44.51 (NCH2), 126.63, 126.66, 127.70, 127.85, 128.44, 129.14, 129.37, 129.58,
130.43, 130.88, 131.32, 132.74, 135.00, 137.29, 148.42, 174.08 (OCO). EI-MS (70 eV) m/z
(%):418 (39) (M+2), 416 (100) (M+), 371 (1), 357 (3), 343 (7), 330 (36), 178 (19). Elemental
analysis for C25H21ClN2O2 (Calcd./Found): C, 72.02/72.23; H, 5.08/5.17; N, 6.72/6.89.
5-(2-(4-Chlorophenyl)-4,5-diphenyl-1H-imidazol-1-yl)pentanoic acid (10d): White crystals;
mp.158-160 °C; (yield 70%); IR (cm-1): 3374 (OH), 1700 (C=O), 1600 (C=N), 1072 (C-O); 1H-
NMR (400 MHz, DMSO-d6) δ (ppm): 1.32-1.33 (m, 2H, CH2-CH2-CH2-CO), 1.87-1.89 (m, 2H,
CH2-CH2-CH2-CO), 2.83 (t, 2H, J = 6.80 Hz, CH2-CO), 3.91 (t, 2H, J = 6.80 Hz, N-CH2), 7.12
24

(d, 2H, J = 8.80 Hz, 2 Ar-H), 6.14-7.60 (m, 10H, Ar-H), 7.76-7.78 (d, 2H, J = 8.80 Hz, 2 Ar-H);
13C-NMR (100 MHz, DMSO-d6) δ (ppm): 21.93 (CH2), 29.75(CH2), 41.79 (CH2), 44.67 (NCH2),
126.61, 126.66, 127.7, 127.85, 128.44, 129.17, 129.38, 129.57, 130.65, 130.84, 131.34, 132.74,
135.00, 137.96, 148.42, 178.55.; Elemental analysis for C26H23ClN2O2 (Calcd./Found): C,
72.47/72.65; H, 5.38/5.44; N, 6.50/6.67.
6-(2-(4-Chlorophenyl)-4,5-diphenyl-1H-imidazol-1-yl)hexanoic acid(10e): White crystals;
mp.166-168 °C: (yield 93%); IR (cm-1): 3400 (OH), 1703 (C=O), 1600 (C=N), 1087 (C-O); 1H-
NMR (400 MHz, DMSO-d6) δ (ppm): 1.19-1.32 (m, 4H, CH2-CH2-CH2-CO), 1.52-1.60 (m, 2H,
CH2-CH2-CH2-CH2-CO), 1.94 (t, 2H, J = 7.20 Hz, CH2-CO), 3.88 (t, 2H, J = 7.20 Hz, N-CH2),
7.12 (d, 2H, J = 8.80, 2 Ar-H), 7.17-7.60 (m, 10H, Ar-H), 7.75-7.77 (d, 2H, J = 8.80 Hz, 2 Ar-H);
13C-NMR (100 MHz, DMSO-d6) δ (ppm): 24.66 (CH2), 26.55 (CH2), 29.79 (CH2), 34.67 (CH2),
44.71 (NCH2), 126.57, 127.7, 128.47, 128.85, 129.19, 129.40, 129.59, 130.58, 130.87, 131.32,
134.00, 135.04, 137.06, 137.24, 145.96, 174.82.; Elemental analysis for C27H25ClN2O2
(Calcd./Found): C, 72.88/73.21; H, 5.66/5.73; N, 6.30/6.46.
4-(2,4,5-Triphenyl-1H-imidazol-1-yl)benzoic acid (11a): White crystals; mp.286-290 °C; (yield
91%); IR (cm-1): 3357 (OH), 1690 (C=O), 1601 (C=N), 1066 (C-O); 1H-NMR (400 MHz, DMSO-
d6) δ (ppm): 7.26 (d, 2H, J = 8.80, 2 Ar-H), 7.31-7.41 (m, 13H, Ar-H), 7.54 (d, 2H, J = 8.80 Hz,
2 Ar-H), 7.84 (d, 2H, J = 8.80 Hz, 2 Ar-H); 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 113.13,
126.90, 126.96, 128.57, 128.65, 128.82, 128.86, 129.06, 130.35, 131.60, 132.12, 134.37, 136.58,
139.12, 147.17, 168.69; EI-MS (70 eV) m/z (%): 416 (5) (M+), 347 (4), 339 (8), 325 (18), 311 (7)
281 (5), 264 (4), 222 (8), 140 (19), 89 (53), 69 (100).; Elemental analysis for C28H20N2O2
(Calcd./Found): C, 80.75/81.02; H, 4.84/4.87; N, 6.73/6.95.
3-(2,4,5-Triphenyl-1H-imidazol-1-yl)propanoic acid (11b): White crystals; mp.149-152°C
(yield 84%); IR ν (cm-1): 3380 (OH), 1711 (C=O), 1601(C=N), 1071 (C-O); 1H-NMR (400 MHz,
DMSO-d6) δ (ppm); 2.22 (t, 2H, J = 6.80 Hz, CH2-CO), 4.12 (t, 2H, J = 6.80 Hz, N-CH2), 7.13-
7.54 (m, 13H, Ar-H), 7.74 (d, J = 7.8 Hz, 2H, 2 Ar-H),; 13C-NMR (100 MHz, DMSO-d6) δ (ppm):
35.78 (CH2), 41.67 (NCH2), 125.73, 126.54, 126.58, 128.43, 128.63, 128.80, 129.04, 129.23,
129.38, 129.59, 130.43, 131.36, 135.40, 137.29, 148.42, 173.55 (OCO). EI-MS (70 eV) m/z (%):
368 (66) (M+), 323 (9), 309 (3), 296 (100), 165 (68); Elemental analysis for C24H20N2O2
(Calcd./Found): C, 78.24/78.41; H, 5.47/5.54; N, 7.60/7.76.
25

4-(2,4,5-Triphenyl-1H-imidazol-1-yl)butanoic acid (11c): White crystals; mp.179-181°C;
(yield 87%); IR (cm-1): 3370 (OH), 1709 (C=O), 1604 (C=N), 1069 (C-O); 1H-NMR (400 MHz,
DMSO-d6) δ (ppm): 1.82-1.89 (m, 2H, CH2-CH2-CO), 2.85 (t, 2H, J = 6.80 Hz, CH2-CO), 3.96 (t,
2H, J = 6.80 Hz, N-CH2), 7.13 (d, J = 7.8 Hz, 2H, 2 Ar-H), 7.19-7.75 (m, 13H, Ar-H); 13C-NMR
(100 MHz, DMSO-d6) δ (ppm): 26.07 (CH2), 31.73 (CH2), 44.47 (NCH2), 125.73, 126.53, 126.62,
128.43, 128.63, 128.42, 129.05, 129.16, 129.30, 129.56, 130.43, 131.34, 135.40, 137.29, 148.42,
173.60; Elemental analysis for C25H22N2O2 (Calcd./Found): C, 78.51/78.67; H, 5.80/5.86; N,
7.32/7.49.
5-(2,4,5-Triphenyl-1H-imidazol-1-yl)pentanoic acid (11d): White crystals; mp.130-132°C
(yield 78%); IR (cm-1): 3380 (OH), 1732 (C=O), 1601 (C=N), 1072 (C-O); 1H-NMR (400 MHz,
DMSO-d6) δ (ppm): 1.32-1.36 (m, 2H, CH2-CH2-CH2-CO), 1.86 (m, 2H, CH2-CH2-CH2-CO),
2.83 (t, 2H, J = 6.80 Hz, CH2-CO), 3.91 (t, 2H, J = 6.80 Hz, N-CH2), 7.13 (d, J = 7.8 Hz, 2H, 2
Ar-H), 7.19-7.73 (m, 13H, Ar-H); 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 21.88 (CH2), 29.75
(CH2), 33.57 (CH2), 44.57 (NCH2), 126.52, 126.59, 128.42, 128.63, 128.80, 129.06, 129.16,
129.19, 129.30, 129.55, 131.35, 131.55, 135.40, 137.29, 148.42, 173.55; Elemental analysis for
C26H24N2O2 (Calcd./Found): C, 78.76/78.98; H, 6.10/6.19; N, 7.07/7.30.
6-(2,4,5-Triphenyl-1H-imidazol-1-yl)hexanoic acid (11e): White crystals; mp.146-148°C (yield
94%); IR (cm-1): 3393 (OH), 1698 (C=O), 1601 (C=N), 1069 (C-O); 1H-NMR (400 MHz, DMSO-
d6) δ (ppm): 1.19-1.29 (m, 2H, CH2-CH2-CH2-CO), 1.52-1.78 (m, 4H, CH2-CH2-CH2-CH2-CO),
1.94 (t, 2H, J = 7.20 Hz, CH2-CO), 3.88 (t, 2H, J = 7.20 Hz, N-CH2), 7.12 (d, J = 7.8 Hz, 2H, 2
Ar-H), 7.19-7.72 (m, 13H, Ar-H); 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 24.14 (CH2), 25.70
(CH2), 29.80 (CH2), 34.05 (CH2), 44.64 (NCH2), 126.50, 126.58, 128.41, 128.63, 128.80, 129.05,
129.19, 129.29, 129.55, 131.34, 131.55, 135.40, 137.29, 148.42, 173.55; Elemental analysis for
C27H26N2O2 (Calcd./Found): C, 79.00/79.23; H, 6.38/6.46; N, 6.82/6.98.
4. Biological evaluation
4.1. Anticonvulsant activity screening
Evaluating the preliminary anticonvulsant activities and neurotoxicity of all newly synthesized
compounds with respect to that of valproate sodium and diphenylhydantoin sodium as reference
drugs. The biological evaluation was done according to the protocol given by the epilepsy section
of National Institute of Neurological Disorders and Stroke (NINDS) using the standard protocol
adopted by the Antiepileptic Drug Development (ADD) program [27]which includes the maximal
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electroshock seizure (MES) screen, the subcutaneous pentylenetetrazole (s.c.PTZ) screen, and the
rotarod (neurotoxicity) test. In addition, the selected highest potent compounds were subjected to
the quantitative estimation (median effective dose, ED50).
4.1.1. Materials and methods
In this study, adult male Swiss albino mice, weighing 19-25g, were used. Animals were purchased
from Animal House Colony of the National Research Centre, Cairo, Egypt. The mice were housed
under standardized conditions (room temperature 23 ± 2°C; relative humidity 55 ± 5%; 12 h light
/ dark cycle) and allowed free access to tap water and standard mice chow throughout the whole
experimental period. All animal procedures were performed in conformity with the
recommendations for the proper care and use of laboratory animals following the regulations of
the ethical committee of the National Research Centre, Cairo, Egypt. The animals were randomly
assigned to test, reference and control experimental groups consisting of 6-8 mice each after 7 days
of their adaptation to laboratory conditions.
Tween-80 (7%) was used as a vehicle in which all the test compounds were suspended. The two
primary anticonvulsant screens MES and s.c. PTZ were performed in mice at the dose 100 mg/kg.
Further investigations were done on the most active compounds in mice i.p. at different doses for
the quantification of their anticonvulsant activity and neurotoxicity.
4.1.2. Chemicals and drugs
Tween-80, pentylenetetrazol and valproate sodium were purchased from Sigma (Sigma, St. Lewis,
USA), diphenylhydantoin sodium obtained from (DPHS, Nasr Co., Egypt).
4.2. Methods
4.2.1. Maximal electroshock seizure (MES) test
The maximal electroshock seizure (MES) screen is indicative of the ability of the test compounds
to prevent seizure attack and their ability to protect against generalized tonic-clonic seizure [27].
In this test, after half an hour of intraperitoneal injection of the tested compounds by a current
(fixed current intensity of 25 mA, 0.2s stimulus duration) delivered via ear clip electrodes by a
Roden Shocker Generator (constant-current stimulator Type 221, Hugo Sachs Elektronik,
Freiburg, Germany) [19, 36], the electro convulsions were produced.
The principle for the occurrence of seizure activity was the tonic hind limb extension (i.e. the hind
limbs of animals outstretched 180° to the plane of the body axis). The anticonvulsant potential of
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the compounds under investigation was compared with that of diphenylhydantoin sodium as a
reference drug.
4.2.2. Subcutaneous pentylenetetrazol (s.c. PTZ) test
PTZ- induced clonic seizures are widely used as a model that predicts drugs effectiveness in
decreasing the seizure threshold and active against generalized seizures of the petit mal (absence)
type [37, 38].
Aqueous solution of PTZ at a dose level of 85 mg/kg [39] was injected subcutaneously under a
loose fold of skin on the back of the mouse’s neck. Tonic-extensor convulsions in at least 97% of
controls were induced by this dose. The test compounds were injected intraperitoneally to the mice
30 minutes before PTZ administration. In single cages, the mice were placed and observed for 30
minutes for the loss of righting reflexes lasting for at least 3 second. After PTZ treatment [40, 41],
as the absence of loss of righting reflexes was chosen as an index for protective effect. The dose
of compounds and reference standard that induced maximum percentage protection was evaluated.
The studied biological activity of the tested compounds was compared with the reference standard,
valproate sodium. The ED50 of the most potent compounds were calculated according to the
method of Litchfield and Wilcoxon [20, 42].
4.3. Rotarod (neurotoxicity) test
Neurological toxicity (NT) in mice was determined by rotarod test mentioned in the method
reported by Dunham and Miya. In brief, a group of animals (groups of 6 mice) were trained to
balance on a rotating rod (3 cm diameter and 6 rpm speed) and they were allowed three attempts
to remain on the rotating rod for 1 minute. The trained animals were treated with the tested
compounds at dose level by intraperitoneal injection.
The test compounds were neurotoxic at a particular dose level if the trained animal showed lack
of Rolling Roller Performance [i.e the animals showing neurological deficits (ataxia and sedation),
as indicated by the inability to keep equilibrium on the rod for at least 1 minute in each of three
trials]. The trained animals were tested, in this manner, after 30 minute of the drug administration,
and the neurotoxic effect was recorded and compared with those animals given phenytoin [43].
5. Virtual screening using shape similarity by ROCS
The principal of representing shape and color features in ROCS is using ROCS application
OpenEye scientific software [29]. Phenytoin, and valporic acid were chosen as query molecules.
A library of final compounds was adopted as the database file. Omega application in OpenEye
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scientific software was used for energy minimizing of both query and database files. vROCS was
employed to run and analyze/visualize the results. Compounds conformers were scored based upon
the Gaussian overlap to the query and the best scoring parameters is Tanimoto Combo scores
(shape + color) the highest score is the best matched with query compound. The quality of
alignment between database and query was calculated using Tanimoto Combo. Tanimoto Combo
is the summation of Shape Tanimoto and Color Tanimoto. Shape Tanimoto represents the shared
volume and mismatch volume and has scale from 0 to 1. Color Tanimoto (scale from 0 to 1) is
reflective of the degree of matching or mismatching of light chemical features in 3 dimensions.
Using fragment disconnected non-chemically meaning pieces of a molecule, both query and
database molecules are combined into a single species.
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