Design, synthesis, molecular modeling and biological evaluation of novel 2,3-dihydrophthalazine-1,4-dione derivatives as potential anticonvulsant agents

Article abstract of DOI:10.1016/j.molstruc.2016.10.052

In view of their expected anticonvulsant activity, some novel derivatives of 2,3-dihydrophthalazine-1,4-dione 4–22 were designed, synthesized and evaluated using pentylenetetrazole (PTZ) and picrotoxin as convulsion-inducing models. Moreover, the most active compounds were tested against electrical induced convulsion using maximal electroshock (MES) models of seizures. Most of the tested compounds showed considerable anticonvulsant activity in at least one of the anticonvulsant tests. Compounds 13 and 14g were proved to be the most potent compounds of this series with relatively low toxicity in the median lethal dose test when compared with the reference drug. Molecular modeling studies were done to verify the biological activity. The obtained results showed that the most potent compounds could be useful as a template for future design, optimization, and investigation to produce more active analogues.

Full text of DOI:10.1016/j.molstruc.2016.10.052

Accepted Manuscript
Design, synthesis, molecular modeling and biological evaluation of novel 2,3-
dihydrophthalazine-1,4-dione derivatives as potential anticonvulsant agents
Abdel Ghany A. El-Helby, Rezk R. Ayyad, Helmy M. Sakr, Adel S. Abdelrahim, K. El-
Adl, Farag S. Sherbiny, Ibrahim H. Eissa, Mohamed M. Khalifa
PII:
S0022-2860(16)31093-6
10.1016/j.molstruc.2016.10.052
MOLSTR 23053
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 25 August 2016
Revised Date: 13 October 2016
Accepted Date: 15 October 2016
Please cite this article as: A.G.A. El-Helby, R.R. Ayyad, H.M. Sakr, A.S. Abdelrahim, K. El-Adl, F.S.
Sherbiny, I.H. Eissa, M.M. Khalifa, Design, synthesis, molecular modeling and biological evaluation
of novel 2,3-dihydrophthalazine-1,4-dione derivatives as potential anticonvulsant agents, Journal of
Molecular Structure (2016), doi: 10.1016/j.molstruc.2016.10.052.
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ACCEPTED MANUSCRIPT
Design, synthesis, molecular modeling and biological evaluation of novel
2,3-dihydrophthalazine-1,4-dione derivatives as potential anticonvulsant agents
Abdel Ghany A. El-Helbya, Rezk R. Ayyada,b, Helmy M. Sakra, Adel S. Abdelrahima, K. El-Adla, Farag S.
a
Sherbinyc,d, Ibrahim H. Eissa*a and Mohamed M. Khalifa
*
a
Pharmaceutical Chemistry Department, Faculty of Pharmacy (Boys), Al-Azhar University, Cairo,
11884, Egypt
b
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Delta University for Science and
Technology, Gamasa, Dakahlia, Egypt
c Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar University (Boys), Cairo, 11884, Egypt
d Organic Chemistry Department, Faculty of Pharmacy, October University for Modern Science and Arts
(MSA), 11787, 6th October City, Egypt
*
Corresponding author; Pharmaceutical Chemistry Department, Faculty of Pharmacy (Boys), Al-Azhar
University, Cairo, 11884, Egypt.
E-mail address; ibrahimeissa@azhar.edu.eg
khalifa_mohamed22@yahoo.com
1

ACCEPTED MANUSCRIPT
Abstract
In view of their expected anticonvulsant activity, some novel derivatives of 2,3-
dihydrophthalazine-1,4-dione 4-22 were designed, synthesized and evaluated using
pentylenetetrazole (PTZ) and picrotoxin as convulsion-inducing models. Moreover, the most
active compounds were tested against electrical induced convulsion using maximal electroshock
(MES) models of seizures. Most of the tested compounds showed considerable anticonvulsant
activity in at least one of the anticonvulsant tests. Compounds 13 and 14g were proved to be the
most potent compounds of this series with relatively low toxicity in the median lethal dose test
when compared with the reference drug. Molecular modeling studies were done to verify the
biological activity. The obtained results showed that the most potent compounds could be useful
as a template for future design, optimization, and investigation to produce more active
analogues.
Key words: 2,3-Dihydrophthalazine-1,4-dione, Molecular modeling, Anticonvulsant, GABA-A
1. Introduction
About 1% of the total world’s population (~50 million people) is affected by epilepsy, a serious
neurological disorder that typically manifests as spontaneous convulsions and/or a loss of
consciousness [1]. Epilepsy results in 42000 deaths in the US annually. Epidemiological studies
suggest that between 70- 80% of people developing epilepsy will go into remission, while the
rest of patients will continue to have seizures and are refractory to treatment with the currently
available therapies [2]. Despite the development of several new anticonvulsants, the treatment of
epilepsy remains still inadequate and the patients experience many side effects like
neurotoxicity, depression, and different CNS disorders. Therefore, it is necessary to search for
more secure and more effective anticonvulsants [3].
Gamma-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter of many
CNS pathways regulating various neurological functions including convulsions, anxiety and
sleep activity as well as memory and learning processes. Molecular biology studies have
illustrated that several receptor subunits combine to form GABA-A receptor complex with a
number of native receptor subtypes recognized up to this point. Numerous structural classes of
chemical compounds bind to the GABA-A chloride ion channel complex including
benzodiazepines, neurosteroids and barbiturates. Ligands acting at the benzodiazepine binding
site of the GABA-A receptor allosterically modulate the action of GABA on neuronal chloride
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ion flux. They show a wide variety of pharmacological actions ranging in a continuum from full
agonists (sedative/hypnotic, anxiolytic and anticonvulsant activities) to inverse agonists [4].
Cook and co-workers [5-7] identified the essential structural features which could be
responsible for an interaction with the active site of GABA-A receptors. These features consist
of a hydrophobic unit (L1, L3), a hydrophobic aliphatic unit (L2), a hydrogen bond acceptor unit
(H1) and a hydrogen bond donor or acceptor unit (H2/A3) (Fig. 1).
Fig. 1. pharmacophore model of the benzodiazepine binding site on GABA-A
receptor. White sticks represent diazepam, while green sticks represent the non-
benzodiazepine, CGG-9896. H1 and H2/A3 are, respectively, hydrogen bond
donating and accepting sites in the receptors, while L1, L2 and L3 denote lipophilic
binding sites [7]
Based on the literature review, the sedative-hypnotic (neurotoxicity) properties of
phthalazines are well documented [8,9]. An important fact was analyzed before chemical
synthesis of our target compounds; the phthalazine nucleus was reported to elicit a
pharmacological response by interacting with the benzodiazepine-binding site of GABA-A
receptors. They display a continuous range of effects, from being positive allosteric
modulators that enhance the GABAergic inhibition exhibiting anxiolytic, anticonvulsant,
sedative-hypnotic and myorelaxant activities [10].
On the other hand, it has been reported that a lot of compounds containing amine,
amide, hydrazone, oxadiazole, pyrazole, semicarbazide, thiosemicarbazide, phthalimide,
and/or Schiff base moieties possessed good anticonvulsant activity [11]. Based on that, it was
decided to synthesize the titled compounds as hybrid molecules formed of phthalazine
nucleus joined with some of the previously mentioned moieties in hope of developing potent
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and safe new effective anticonvulsant agents with low dose-related toxicity and without
idiosyncratic side effects.
2. Experimental
2.1 Chemistry
2.1.1 General
Melting points were determined with a Gallen kamp melting point apparatus and were
uncorrected. IR spectra were recorded on pye Unicam SP 1000 IR spectrophotometer and
1
expressed in wave number (cm-1). The H NMR spectra were recorded on a jeol 300 MHZ-
NMR spectrometer or a Bruker spectrophotometer at 400 MHz in deuterated
dimethylsulphoxide (DMSO-d6). Chemical shifts (dH) were reported relative to TMS as
internal standard. All coupling constant (J) values were given in hertz. The abbreviations
used are as the following: s, singlet; d, doublet; m, multiplet. Mass spectra were measured on
a GCMS-QP1000 EX spectrometer at 70 eV. Analytical thin layer chromatography (TLC) on
silica gel plates containing UV indicator was routinely used to follow the course of reactions
and to check the purity of products. All reagents and solvents were purified and dried by
standard techniques. Compounds 2 and 3 are previously reported.
2.1.2. 2,3-Dihydrophthalazine-1,4-dione (2)
Compound 2 was prepared according to the reported procedure [12]. Yield 95%; mp
>300 °C as reported.
2.1.3. Potassium salt of 2,3-dihydrophthalazine-1,4-dione (3)
Compound 3 was prepared according to the reported procedure [12]. Yield 92%; mp
>350 °C as reported.
2.1.4 Methyl 2-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-yl)propanoate (4)
Equimolar quantities of the potassium salt 3 (2.00 g, 0.01 mol) and methyl 2-
chloropropionate (1.22 g, 0.01 mol) in DMF (20 ml) was heated on water bath for 5 h. The
reaction mixture was poured onto ice-water (200 ml) and stirred for 30 min. The obtained
solid was filtered and crystallized from ethanol to give compound 4.
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Yield 75%; mp 180-182 °C; IR (KBr) ν cm-1: 3171 (NH), 1757 (C=O), 1656 (C=O); 1H
NMR (DMSO-d6) δ ppm: 1.60 (d, 3H, J = 7.2 Hz, -CHCH3), 3.68 (s, 3H, -OCH3), 5.20 (q,
1H, J = 7.2 Hz, -CHCH3), 7.81 (m, 2H, Ar-H, H-6 and H-7 of phthalazine), 7.90 (d, 1H, J =
8.1 Hz, Ar-H, H-5 of phthalazine), 8.12 (d, 1H, J = 7.8 Hz, Ar-H, H-8 of phthalazine), 11.85
(s, 1H, exchangeable with D2O, -NH phthalazine); Mass m/z [%]: 248 [M+ , 100]; Anal.
calcd. for C12H12N2O4 (248.24): C, 58.06; H, 4.87; N, 11.29. Found: C, 58.32; H, 4.94; N,
11.42.
2.1.5 General procedure for preparation of target compounds (5a-d)
Compound 4 (2.48 g, 0.01 mol) was dissolved in ethanol then an excess of the
appropriate alkyl amine, namely methyl, ethyl, cyclopentyl or benzyl amine, was added. The
mixture was then refluxed for 3 h. The reaction mixture was cooled, poured onto ice-water
(200 ml) and the formed precipitate was washed with water, filtered and recrystallized from
ethanol to afford the corresponding target compounds 5a-d respectively.
2.1.5.1 2-(1,4-Dioxo-3,4-dihydrophthalazin-2(1H)-yl)-N-methylpropanamide (5a)
Yield 83%; mp 273-275 °C; IR (KBr) ν cm-1: 3293, 3177 (2NH), 1664 (C=O); 1H NMR
(DMSO-d6) δ ppm: 1.56 (d, 3H, J = 6.9 Hz, -CHCH3), 2.61 (s, 3H, -NHCH3), 5.23 (q, 1H, J
= 6.9 Hz, -CHCH3), 7.88 (s, 1H, exchangeable with D2O, -NHCH3), 7.90 (m, 2H, Ar-H, H-6
and H-7 of phthalazine), 8.10 (d, 1H, J = 7.5 Hz, Ar-H, H-5 of phthalazine), 8.20 (d, 1H, J =
7.8 Hz, Ar-H, H-8 of phthalazine), 11.89 (s, 1H, exchangeable with D2O, -NH phthalazine);
Mass m/z [%]: 247 [M+ , 32.79], 189 [100]; Anal. calcd. for C12H13N3O3 (247.25): C, 58.29;
H, 5.30; N, 17.00. Found: C, 58.46; H, 5.39; N, 17.21.
2.1.5.2 N-Butyl-2-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-yl)propanamide (5b)
Yield 86%; mp 262-264 °C; IR (KBr) ν cm-1: 3283, 3170 (2NH), 1670 (C=O); 1H NMR
(DMSO-d6) δ ppm: 0.81 (t, 3H, J = 7.2 Hz, -CH2CH3), 1.20 (m, 2H, -CH2CH2CH3), 1.35 (m,
2H, -CH2CH2CH2-), 1.52 (d, 3H, J = 6.6 Hz, -CHCH3), 3.04 (t, 2H, J = 6.3 Hz, -NHCH2CH2-
), 5.11 (q, 1H, J = 6.6 Hz, -CHCH3), 7.86 (s, 1H, exchangeable with D2O, -NHCH2), 7.95 (m,
2H, Ar-H, H-6 and H-7 of phthalazine), 8.09 (d, 1H, J = 8.4 Hz, Ar-H, H-5 of phthalazine),
8.22 (d, 1H, J = 7.8 Hz, Ar-H, H-8 of phthalazine), 11.82 (s, 1H, exchangeable with D2O, -
NH phthalazine); Mass m/z [%]: 289 [M+ , 26], 189 [100]; Anal. calcd. for C15H19N3O3
(289.34): C, 62.27; H, 6.62; N, 14.52. Found: C, 62.43; H, 6.71; N, 14.90.
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2.1.5.3 N-Cyclopentyl-2-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-yl)propanamide (5c)
Yield 82%; mp 270-272 °C; IR (KBr) ν cm-1: 3284, 3172 (2NH), 1657 (C=O); 1H NMR
(DMSO-d6) δ ppm: 1.48 (d, 3H, J = 6.9 Hz, -CHCH3), 1.50 (m, 2CH2 of cyclopentyl group),
1.61 (m, 2CH2 of cyclopentyl group), 4.00 (m, 1H, CH of cyclopentyl group), 5.07 (q, 1H, J
= 6.9 Hz, -CHCH3), 7.43 (s, 1H, NH-cyclopentyl), 7.88 (m, 2H, Ar-H, H-6 and H-7 of
phthalazine), 8.02 (d, 1H, J = 8.4 Hz, Ar-H, H-5 of phthalazine), 8.21 (d, 1H, J = 7.5 Hz, Ar-
H, H-8 of phthalazine), 11.89 (s, 1H, exchangeable with D2O, -NH phthalazine); Mass m/z
[%]: 301 [M+ , 3.8], 161 [100]; Anal. calcd. for C16H19N3O3 (301.35): C, 63.77; H, 6.36; N,
13.94. Found: C, 63.94; H, 6.42; N, 14.07.
2.1.5.4 N-Benzyl-2-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-yl)propanamide (5d)
Yield 87%; mp 287-289 °C; IR (KBr) ν cm-1: 3290, 3175 (2NH), 1672 (C=O); 1H NMR
(DMSO-d6) δ ppm: 1.56 (d, 3H, J = 6.6 Hz, -CHCH3), 4.29 (s, 2H, -NHCH2-), 5.22 (q, 1H, J
= 6.6 Hz, -CHCH3), 7.20-7.31 (m, 5H, Ar-H, C6H5), 7.91 (m, 2H, Ar-H, H-6 and H-7 of
phthalazine), 8.08 (d, 1H, J = 7.8 Hz, Ar-H, H-5 of phthalazine), 8.23 (d, 1H, J = 8.1 Hz, Ar-
H, H-8 of phthalazine), 8.52 (s, 1H, exchangeable with D2O, NH-benzyl), 11.89 (s, 1H,
exchangeable with D2O, NH phthalazine); Anal. calcd. for C18H17N3O3 (323.35): C, 66.86; H,
5.30; N, 13.00. Found: C, 67.02; H, 5.38; N, 13.24.
2.1.6 2-(1,4-Dioxo-3,4-dihydrophthalazin-2(1H)-yl)propanehydrazide (6)
A solution of compound 4 (2.48 g, 0.01 mol) in absolute ethanol (50 ml) was treated
with hydrazine hydrate (95%, 20 mL). The reaction mixture was stirred well and heated to
50°C for 2 h, then cooled. The crude product was collected by filtration, washed with water
and then crystallized from ethanol to afford compound 6.
Yield 80%; mp 220-222 °C; IR (KBr) ν cm-1: 3315 (NH2), 3168 (NH), 1669 (C=O); 1H
NMR (DMSO-d6) δ ppm: 1.51 (d, 3H, J = 6.6 Hz, -CHCH3), 4.24 (s, 2H, exchangeable with
D2O, -NH2), 5.21 (q, 1H, J = 6.6 Hz, -CHCH3), 7.86 (m, 2H, Ar-H, H-6 and H-7 of
phthalazine), 8.07 (d, J = 7.5, 1H, Ar-H, H-5 of phthalazine), 8.22 (d, 1H, J = 7.8 Hz, Ar-H,
H-8 of phthalazine), 9.29 (s, 1H, exchangeable with D2O, -NHNH2), 11.85 (s, 1H,
exchangeable with D2O, NH phthalazine); Mass m/z [%]: 248 [M+, 100], 175 [99.9]; Anal.
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calcd. for C11H12N4O3 (248.24): C, 53.22; H, 4.87; N, 22.57. Found: C, 53.41; H, 4.92; N,
22.83.
2.1.7 2-[1-(5-Sulfanyl-1,3,4-oxadiazol-2-yl)ethyl]-2,3-dihydrophthalazine-1,4-dione (7)
Compound 6 (2.48 g, 0.01 mol) was dissolved in a solution of potassium hydroxide
(0.56 g, 0.01 mol) in ethanol (40 ml) and water (4 ml). Carbon disulphide (3 ml) was then
added dropwise while stirring and the reaction mixture was refluxed for 24 h. The reaction
mixture was concentrated, cooled to room temperature and acidified with diluted
hydrochloric acid. The obtained precipitate was filtered, washed with water and recrystallized
from ethanol to give compound 7.
1
Yield 80%; mp 246-248 °C; IR (KBr) ν cm-1: 3170 (NH), 2724 (SH), 1669 (C=O); H
NMR (DMSO-d6) δ ppm: 1.73 (d, 3H, J = 6.6 Hz, -CHCH3), 6.05 (q, 1H, J = 6.6 Hz, -
CHCH3), 7.90 (m, 2H, Ar-H, H-6 and H-7 of phthalazine), 7.98 (d, 1H, J = 7.5 Hz, Ar-H, H-
5 of phthalazine), 8.22 (d, J = 8.1 Hz, 1H, Ar-H, H-8 of phthalazine), 11.99 (s, 1H,
exchangeable with D2O, NH phthalazine), 14.22 (s, 1H, exchangeable with D2O, SH); Mass
m/z [%]: 290 [M+, 10.78], 162 [100]; Anal. calcd. for C12H10N4O3S (290.30): C, 49.65; H,
3.47; N, 19.30. Found: C, 49.71; H, 3.52; N, 19.47.
2.1.8 General procedure for preparation of target compounds (8a-d)
A mixture of compound 6 (1.48 g, 0.006 mol) and appropriate isothiocyanate namely,
ethyl, propyl, cyclohexyl and phenyl isothiocyanate (0.006 mol) in 20 ml absolute ethanol
was heated under reflux for 3 h. After cooling, the precipitate was collected and crystallized
from ethanol to afford compounds 8a-d respectively.
2.1.8.1
2-[2-(1,4-Dioxo-3,4-dihydrophthalazin-2(1H)-yl)propanoyl]-N-ethylhydrazine-1-
carbothioamide (8a).
Yield 69%; mp 197-199 °C; IR (KBr) ν cm-1: 3290, 3170 (4NH- overlapped), 1674
(C=O); 1H NMR (DMSO-d6) δ ppm: 1.00 (t, 3H, J = 6.9 Hz, -CH2CH3), 1.56 (d, 3H, J = 6.6
Hz, -CHCH3), 3.46 (q, 2H, J = 6.9 Hz, -CH2CH3), 5.25 (q, 1H, J = 6.6 Hz, -CHCH3), 7.31 (s,
1H, exchangeable with D2O, -NHCH2-), 7.90 (m, 2H, Ar-H, H-6 and H-7 of phthalazine),
8.10 (d, 1H, J = 7.8 Hz, Ar-H, H-5 of phthalazine), 8.23 (d, 1H, J = 8.1 Hz, Ar-H, H-8 of
phthalazine), 9.21 (s, 1H, exchangeable with D2O, -CONHNH-), 10.07 (s, 1H, exchangeable
with D2O, -NHNHCS), 11.92 (s, 1H, exchangeable with D2O, NH phthalazine); Mass m/z
7

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[%]: 335 [M+, 1.28], 217 [98]; Anal. calcd. for C14H17N5O3S (335.38): C, 50.14; H, 5.11; N,
20.88. Found: C, 50.23; H, 5.18; N, 21.17.
2.1.8.2
2-[2-(1,4-Dioxo-3,4-dihydrophthalazin-2(1H)-yl)propanoyl]-N-propylhydrazine-1-
carbothioamide (8b)
Yield 74%; mp 212-214 °C; IR (KBr) ν cm-1: 3249, 3177 (4NH- overlapped), 1674
1
(C=O); H NMR (DMSO-d6) δ ppm: 0.75 (t, 3H, J = 7.5 Hz, -CH2CH3), 1.40 (m, 2H, -
CH2CH2CH3), 1.56 (d, 3H, J = 6.9 Hz, -CHCH3), 3.43 (t, 2H, -NHCH2CH2-), 5.24 (q, 1H, J
= 6.9 Hz, -CHCH3), 7.29 (s, 1H, exchangeable with D2O, -NHCH2-), 7.90 (m, 2H, Ar-H, H-6
and H-7 of phthalazine), 8.10 (d, 1H, J = 7.8, Ar-H, H-5 of phthalazine), 8.22 (d, 1H, J = 7.5,
Ar-H, H-8 of phthalazine), 9.23 (s, 1H, exchangeable with D2O, -CONHNH-), 10.11 (s, 1H,
exchangeable with D2O, -NHNHCS), 11.93 (s, 1H, exchangeable with D2O, NH
phthalazine); Mass m/z [%]: 349 [M+, 5.47], 217 [99]; Anal. calcd. for C15H19N5O3S
(349.41): C, 51.56; H, 5.48; N, 20.04. Found: C, 51.73; H, 5.56; N, 20.19.
2.1.8.3 N-Cyclohexyl-2-[2-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-yl)propanoyl]hydrazine-
1-carbothioamide (8c)
Yield 72%; mp 227-229 °C; IR (KBr) ν cm-1: 3243, 3144 (4NH- overlapped), 2930,
1
2858 (C-H aliphatic), 1656 (C=O); H NMR (DMSO-d6) δ ppm: 1.10-1.20 (m, 4H, 2CH2 of
cyclohexyl group), 1.56 (d, 3H, J = 6.6 Hz, -CHCH3), 1.62 (m, 2H, CH2 of cyclohexyl
group), 1.79 (m, 4H, 2CH2 of cyclohexyl group), 4.01 (m, 1H, of cyclohexyl group), 5.26 (q,
1H, J = 6.6 Hz, -CHCH3), 7.29 (s, 1H, exchangeable with D2O, NH-cyclohexyl), 7.90 (m,
2H, Ar-H, H-6 and H-7 of phthalazine), 8.12 (d, 1H, J = 7.8 Hz, Ar-H, H-5 of phthalazine),
8.23 (d, 1H, J = 8.1 Hz, Ar-H, H-8 of phthalazine), 9.13 (s, 1H, exchangeable with D2O, -
CONHNH-), 10.08 (s, 1H, exchangeable with D2O, -CONHNH), 11.91 (s, 1H, exchangeable
with D2O, NH phthalazine); Mass m/z [%]: 389.4 [M+, 3.15], 161 [99]; Anal. calcd. for
C18H23N5O3S (389.47): C, 55.51; H, 5.95; N, 17.98. Found: C, 55.84; H, 5.97; N, 18.14.
2.1.8.4
2-[2-(1,4-Dioxo-3,4-dihydrophthalazin-2(1H)-yl)propanoyl]-N-phenylhydrazine-1-
carbothioamide (8d)
Yield 69%; mp 222-224 °C; IR (KBr) ν cm-1: 3247, 3181 (4NH- overlapped), 3018,
1
3059 (CH aromatic), 1665 (C=O); H NMR (DMSO-d6) δ ppm: 1.59 (d, 3H, J = 6 Hz, -
CHCH3), 5.39 (q, 1H, J = 6 Hz, -CHCH3), 7.11 (dd, 1H, J = 6.9 Hz, Ar-H, H-4 of phenyl),
7.31 (t, 2H, J = 6.9 Hz, Ar-H, H-3 and H-5 of phenyl), 7.41 (d, 2H, J = 6.9 Hz, Ar-H, H-2
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and H-6 of phenyl), 7.87 (m, 2H, Ar-H, H-6 and H-7 of phthalazine), 8.14 (d, 1H, J = 7.2 Hz,
Ar-H, H-5 of phthalazine), 8.22 (d, 1H, J = 7.8 Hz, Ar-H, H-8 of phthalazine), 9.41 (s, 1H,
exchangeable with D2O, -CONHNH-), 9.64 (s, 1H, exchangeable with D2O, -CONHNH),
10.26 (s, 1H, exchangeable with D2O, NH-phenyl), 11.89 (s, 1H, exchangeable with D2O,
NH phthalazine); Mass m/z [%]: 383 [M+, 7.02], 217 [99]; Anal. calcd. for C18H17N5O3S
(383.43): C, 56.39; H, 4.47; N, 18.27. Found: C, 56.64; H, 4.49; N, 18.58.
2.1.9 General procedure for preparation of target compounds (9a-c)
A mixture of acid hydrazide 6 (0.5 g, 0.002 mol) and the appropriate isocyanate namely,
butyl, cyclohexyl and phenyl isocyanate (0.002 mol) was refluxed in absolute ethanol (25 ml)
for 12 h. the solution was cooled and the solid was filtered and recrystallized from ethanol to
produce compounds 9a-c respectively.
2.1.9.1
N-Butyl-2-[2-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-yl)propanoyl]hydrazine-1-
carboxamide (9a)
Yield 61%; mp 240-242 °C; IR (KBr) ν cm-1: 3250, 3172 (4NH- overlapped), 2962 (C-
H aliphatic), 1677 (C=O); 1H NMR (DMSO-d6) δ ppm: 0.90 (t, 3H, J = 8 Hz, -CH2CH3), 1.35
(m, 2H, -CH2CH3), 1.38 (m, 2H, -CH2CH2CH2-), 1.47 (d, 3H, J = 6.6 Hz, -CHCH3), 3.10 (t,
2H, J = 7.1 Hz, -NHCH2-), 5.21 (q, 1H, J = 6 Hz, -CHCH3), 6.06 (s, 1H, exchangeable with
D2O, NH-butyl), 7.95 (m, 2H, Ar-H, H-6 and H-7 of phthalazine), 8.22 (d, 1H, J = 7.2 Hz,
Ar-H, H-5 of phthalazine), 8.22 (d, 1H, J = 7.8 Hz, Ar-H, H-8 of phthalazine), 10.09 (s, 1H,
exchangeable with D2O, -CONHNH-), 10.56 (s, 1H, exchangeable with D2O, -CONHNH),
11.89 (s, 1H, exchangeable with D2O, NH phthalazine); Mass m/z [%]: 348 [M+, 1.31], 349
[M+1, 0.74] 104 [100]; Anal. calcd. for C16H21N5O4 (347.38): C, 55.32; H, 6.09; N, 20.16.
Found: C, 55.47; H, 6.18; N, 20.54.
2.1.9.2 N-Cyclohexyl-2-[2-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-yl)propanoyl]hydrazine-
1-carboxamide (9b)
Yield 68%; mp 265-267 °C; IR (KBr) ν cm-1: 3242, 3141 (4NH- overlapped), 2931 (C-
1
H aliphatic), 1655 (C=O); H NMR (DMSO-d6) δ ppm: 1.11-1.20 (m, 4H, 2CH2 of
cyclohexyl group), 1.59 (d, 3H, J = 6.6 Hz, -CHCH3), 1.67 (m, 2H, CH2 of cyclohexyl
group), 1.76 (m, 4H, 2CH2 of cyclohexyl group), 4.12 (m, 1H, of cyclohexyl group), 5.20 (q,
1H, J = 6.6 Hz, -CHCH3), 7.29 (s, 1H, exchangeable with D2O, NH-cyclohexyl), 7.90 (m,
2H, Ar-H, H-6 and H-7 of phthalazine), 8.12 (d, 1H, J = 7.8 Hz, Ar-H, H-5 of phthalazine),
9

ACCEPTED MANUSCRIPT
8.23 (d, 1H, J = 8.1 Hz, Ar-H, H-8 of phthalazine), 9.13 (s, 1H, exchangeable with D2O, -
CONHNH-), 10.08 (s, 1H, exchangeable with D2O, -CONHNH), 11.91 (s, 1H, exchangeable
with D2O, NH phthalazine); Mass m/z [%]: 373 [M+, 4.85], 104 [100]; Anal. calcd. for
C18H23N5O4 (373.41): C, 57.90; H, 6.21; N, 18.76. Found: C, 58.13; H, 6.28; N, 19.02.
2.1.9.3
2-[2-(1,4-Dioxo-3,4-dihydrophthalazin-2(1H)-yl)propanoyl]-N-phenylhydrazine-1-
carboxamide (9c)
Yield 78%; mp 258-260 °C; IR (KBr) ν cm-1: 3435, 3333, 3210 (4NH- overlapped),
1
3055, 3076 (C-H aromatic), 1668 (C=O); H NMR (DMSO-d6) δ ppm: 1.61 (d, 3H, J = 6.9
Hz, -CHCH3), 5.8 (q, 1H, J = 6.9 Hz, -CHCH3), 6.97 (t, 1H, Ar-H, H-4 of phenyl), 7.24 (t,
2H, Ar-H, H-3 and H-5 of phenyl), 7.39 (d, 2H, Ar-H, H-2 and H-6 of phenyl), 7.90 (m, 2H,
Ar-H, H-6 and H-7 of phthalazine), 8.14 (d, 1H, J = 8.4 Hz, Ar-H, H-5 of phthalazine), 8.23
(d, 1H, J = 7.5, Ar-H, H-8 of phthalazine), 8.60 (s, 1H, exchangeable with D2O, -CONHNH-
), 9.60 (s, 1H, exchangeable with D2O, NH-phenyl), 9.98 (s, 1H, exchangeable with D2O, -
CONHNH), 11.91 (s, 1H, exchangeable with D2O, NH phthalazine); Mass m/z [%]: 367 [M+,
3.44], 104 [100]; Anal. calcd. for C18H17N5O4 (367.37): C, 58.85; H, 4.66; N, 19.06. Found:
C, 59.04; H, 4.69; N, 19.31.
2.1.10
2-(1,4-Dioxo-3,4-dihydrophthalazin-2(1H)-yl)-N-(1,3-dioxoisoindolin-2-yl)propan-
amide (10)
A mixture of the acid hydrazide 6 (2.48 g, 0.01 mol) and phthalic anhydride (2.22 g,
0.015 mol) was refluxed in glacial acetic acid (25 ml) for 18 h. The solution was cooled and
then poured onto crushed ice. The solid product was filtered and crystallized from ethanol to
yield compound 10. Yield 81%; mp 237-239 °C; IR (KBr) ν cm-1: 3217 (NH), 3061 and 3033
1
(CH- aromatic), 1710, 1658 (C=O); H NMR (DMSO-d6) δ ppm: 1.67 (d, 3H, J = 6.8 Hz, -
CHCH3), 5.45 (q, 1H, J = 6.8 Hz, -CHCH3), 7.89-7.99 (m, 6H, Ar-H, 4H of isoindoline, H-6
and H-7 of phthalazine), 8.11 (d, 1H, J = 8.4 Hz, Ar-H, H-5 of phthalazine), 8.23 (d, 1H, J =
7.6 Hz, Ar-H, H-8 of phthalazine), 10.94 (s, 1H, exchangeable with D2O, -CONH-) and 12
(s, 1H, exchangeable with D2O, NH phthalazine); Anal. calcd. for C19H14N4O5 (378.34): C,
60.32; H, 3.73; N, 14.81. Found: C, 60.49; H, 3.71; N, 14.97.
2.1.11
2-(1,4-Dioxo-3,4-dihydrophthalazin-2(1H)-yl)-N-(2,5-dioxopyrrolidin-1-yl)propan-
amide (11)
10

ACCEPTED MANUSCRIPT
Compound 11 was obtained by refluxing acid hydrazide 6 (2.48 g, 0.01 mol) with
succinic anhydride (1.5 g, 0.015 mol) in glacial acetic acid (25 ml) for 15 h. The solution was
cooled and then poured onto crushed ice. The solid product was filtered and crystallized from
ethanol. Yield 77%; mp 213-215 °C; IR (KBr) ν cm-1: 3223 (2NH), 2960 (CH- aliphatic),
1
1722, 1660 (C=O); H NMR (DMSO-d6) δ ppm: 1.67 (d, 3H, J = 6.8 Hz, -CHCH3), 2.64 (t,
4H, J = 6.4 Hz, 2CH2 of pyrrolidin), 5.45 (q, 1H, J = 6.8 Hz, -CHCH3), 7.91 (m, 2H, Ar-H,
H-6 and H-7 of phthalazine), 8.11 (d, 1H, J = 8.4 Hz, Ar-H, H-5 of phthalazine), 8.22 (d, 1H,
J = 7.6 Hz, Ar-H, H-8 of phthalazine), 10.8 ( s, 1H, exchangeable with D2O, -CONH), 12 (s,
1H, exchangeable with D2O, NH phthalazine); Mass m/z [%]: 330 [M+, 1.97], 163 [100];
Anal. calcd. for C15H14N4O5 (330.30): C, 54.55; H, 4.27; N, 16.96. Found: C, 54.81; H, 4.32;
N, 16.83.
2.1.12
2-(1,4-Dioxo-3,4-dihydrophthalazin-2(1H)-yl)-N-(4,5,6,7-tetrabromo-1,3-dioxo-
isoindolin-2-yl)propanamide (12)
A mixture of the acid hydrazide 6 (2.48 g, 0.01 mol) and tetrabromophthalic anhydride
(6.94 g, 0.015 mol) was refluxed in glacial acetic acid (25 ml) for 20 h. The solution was
cooled and then poured onto crushed ice. The solid product was filtered and crystallized from
ethanol to yield compound 12. Yield 62%; mp 254-256 °C; IR (KBr) ν cm-1: 3231, 3189
1
(2NH), 1722, 1660 (C=O); H NMR (DMSO-d6) δ ppm: 1.51 (d, 3H, J = 6.6 Hz, -CHCH3),
5.21 (q, 1H, J = 6.6 Hz, -CHCH3), 7.86 (m, 2H, Ar-H, H-6 and H-7 of phthalazine), 8.07 (d,
1H, J = 7.5 Hz, Ar-H, H-5 of phthalazine), 8.22 (d, 1H, J = 7.8 Hz, Ar-H, H-8 of
phthalazine), 9.69 (s, 1H, exchangeable with D2O, -CONH), 11.86 (s, 1H, exchangeable with
D2O, NH phthalazine); Anal. calcd. for C19H10Br4N4O5 (693.93): C, 32.89; H, 1.45; N, 8.07.
Found: C, 32.97; H, 1.46; N, 8.16.
2.1.13 Ethyl 3-{2-[2-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-yl)propanoyl]hydrazono}butan-
oate (13)
Ethyl acetoacetate (1.3 g, 0.01 mol) was added to a solution of compound 6 (2.48 g,
0.01 mol) in ethanol (20 ml). The reaction mixture was refluxed for 4 h, concentrated, cooled
to room temperature and the solid product obtained was filtered and crystallized from ethanol
to afford compound 13. Yield 75%; mp 167-169 °C; IR (KBr) ν cm-1: 3196 (2NH), 1730,
1
1660 (C=O), 1597 (C=N); H NMR (DMSO-d6) δ ppm: 1.20 (t, 3H, J = 7.2 Hz, -CH2CH3),
1.56 (d, 3H, J = 6.8 Hz, -CHCH3), 1.91 (s, 3H, -N=C-CH3), 3.32 (s, 2H, -N=C-CH2-), 4.10
(q, 2H, J = 7.2 Hz, -CH2CH3), 5.3 (q , 1H , J = 6.8 Hz, -CHCH3), 7.88 (m, 2H, Ar-H, H-6
11

ACCEPTED MANUSCRIPT
and H-7 of phthalazine), 8.03 (d, 1H, J = 8.00 Hz, Ar-H, H-5 of phthalazine), 8.22 (d, 1H, J =
7.2 Hz, Ar-H, H-8 of phthalazine), 10.44 (s, 1H, exchangeable with D2O, CONH-), 11.82 (s ,
1H , exchangeable with D2O, NH phthalazine); Anal. calcd. for C17H20N4O5 (360.37): C,
56.66; H, 5.59; N, 15.55. Found: C, 56.87; H, 5.64; N, 15.83.
2.1.14 General procedure for preparation of target compounds (14a-g)
Equimolar amounts of 6 (0.5 g, 0.002 mol) and the appropriate aldehyde namely, 2-
chloro, 4-chloro, 2,4-dichloro, 2,6-dichloro, 4-hydroxy, 4-methoxy and 3-nitro bezaldehyde
(0.002 mol) were refluxed in absolute ethanol (25 ml) with catalytic amount of glacial acetic
acid for 6 h and the reaction was followed up by TLC, the mixture was cooled, filtered and
recrystallized from ethanol to afford compounds 14a-g respectively.
2.1.14.1
N'-(2-Chlorobenzylidene)-2-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-yl)propane
hydrazide (14a)
Yield 83%; mp 250-252 °C; IR (KBr) ν cm-1: 3192 (NH), 3058 (C-H aromatic), 1674
(C=O), 1593 (C=N); 1H NMR (DMSO-d6) δ ppm: 1.62 (d, 3H, J = 7.5 Hz, -CHCH3), 5.19 (q,
1H, J = 7.5 Hz, -CHCH3), 7.41-7.51 (m, 4H, Ar-H, H-3, 4, 5 and 6 of phenyl), 7.90 (m, 2H,
Ar-H, H-6 and H-7 of phthalazine), 8.07 (d, 1H, J = 8.1 Hz, Ar-H, H-5 of phthalazine), 8.20
(d, 1H, J = 7.5 Hz, Ar-H, H-8 of phthalazine), 8.66 (s, 1H, -N=CH), 11.7 (s, 1H,
exchangeable with D2O, NHN=CH), 11.8 (s, 1H, exchangeable with D2O, NH phthalazine);
Mass m/z [%]: 370 [M+, 1.97], 372 [M+2, 0.66], 217 [100]; Anal. calcd. for C18H15ClN4O3
(370.79): C, 58.31; H, 4.08; N, 15.11. Found: C, 58.53; H, 4.17; N, 15.23.
2.1.14.2
N'-(4-Chlorobenzylidene)-2-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-yl)propane
hydrazide (14b)
Yield 84%; mp 247-249 °C; IR (KBr) ν cm-1: 3188 (NH), 3028 (C-H aromatic), 1658
(C=O), 1591 (C=N); 1H NMR (DMSO-d6) δ ppm: 1.53 (d, 3H, J = 7.2 Hz, -CHCH3), 5.20 (q,
1H, J = 7.2 Hz, -CHCH3), 6.79 (2d, 2H, J = 8.7 Hz, H-2 and H-6 of phenyl), 7.47 (2d, 2H, J
= 8.7 Hz, H-3 and H-5 of phenyl), 7.87 (m, 2H, Ar-H, H-6 and H-7 of phthalazine), 8.10 (d,
1H, J = 8.1 Hz, Ar-H, H-5 of phthalazine), 8.20 (d, 1H, J = 7.5 Hz, Ar-H, H-8 of
phthalazine), 8.41 (s, 1H, -N=CH), 11.79 (s, 1H, exchangeable with D2O, NHN=CH), 11.8
(s, 1H, exchangeable with D2O, NH phthalazine); Mass m/z [%]: 370 [M+, 0.47], 372 [M+2,
0.15], 217 [100]; Anal. calcd. for C18H15ClN4O3 (370.79): C, 58.31; H, 4.08; N, 15.11.
Found: C, 58.49; H, 4.05; N, 15.34.
12

ACCEPTED MANUSCRIPT
2.1.14.3 N'-(2,4-Dichlorobenzylidene)-2-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-yl)propane
hydrazide (14c)
Yield 82%; mp 258-259 °C; IR (KBr) ν cm-1: 3173 (NH), 3035 (C-H aromatic), 1658
(C=O), 1598 (C=N); 1H NMR (DMSO-d6) δ ppm: 1.63 (d, 3H, J = 7.6 Hz, -CHCH3), 5.20 (q,
1H, J = 7.6 Hz, -CHCH3), 7.48 (d, 1H, J = 6.4 Hz, H-5 of phenyl), 7.70 (s, 1H, H-3 of
phenyl), 7.85 (d, 1H, J = 6.4 Hz, H-6 of phenyl), 7.97 (m, 2H, Ar-H, H-6 and H-7 of
phthalazine), 8.04 (d, 1H, J = 8.1 Hz, Ar-H, H-5 of phthalazine), 8.22 (d, 1H, J = 7.5 Hz, Ar-
H, H-8 of phthalazine), 8.60 (s, 1H, -N=CH), 11.74 (s, 1H, exchangeable with D2O,
NHN=C), 11.81 (s, 1H, exchangeable with D2O, NH phthalazine); Mass m/z [%]: 405 [M+,
4.53], 407 [M+2, 2.97], 407 [M+4, 0.48], 217 [100]; Anal. calcd. for C18H14Cl2N4O3 (405.24):
C, 53.35; H, 3.48; N, 13.83. Found: C, 53.57; H, 3.53; N, 14.02.
2.1.14.4 N'-(2,6-Dichlorobenzylidene)-2-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-yl)propane
hydrazide (14d)
Yield 87%; mp 260-262 °C; IR (KBr) ν cm-1: 3148 (NH), 3092 (C-H aromatic), 1672
(C=O), 1591 (C=N); 1H NMR (DMSO-d6) δ ppm: 1.61 (d, 3H, J = 6.8 Hz, -CHCH3), 5.18 (q,
1H, J = 6.8 Hz, -CHCH3), 7.50 (dd, 1H, J = 8.4 Hz, H-4 of phenyl), 7.71 (d, 2H, J = 9.2 Hz,
H-3 and H-5 of phenyl), 7.91 (m, 2H, Ar-H, H-6 and H-7 of phthalazine), 8.06 (d, 1H, J = 8.1
Hz, Ar-H, H-5 of phthalazine), 8.20 (d, 1H, J = 7.5 Hz, Ar-H, H-8 of phthalazine), 8.60 (s,
1H, -CH=N-), 11.78 (s, 1H, exchangeable with D2O, NHN=CH), 11.92 (s, 1H, exchangeable
with D2O, NH); Anal. calcd. for C18H14Cl2N4O3 (405.24): C, 53.35; H, 3.48; N, 13.83.
Found: C, 53.48; H, 3.56; N, 13.98.
2.1.14.52-(1,4-Dioxo-3,4-dihydrophthalazin-2(1H)-yl)-N'-(4-hydroxybenzylidene)propane-
hydrazide (14e)
Yield 81%; mp 272-274 °C; IR (KBr) ν cm-1: 3337 broad peak (OH), 3206 (NH), 3029
(C-H aromatic), 1645 (C=O), 1590 (C=N); 1H NMR (DMSO-d6) δ ppm: 1.59 (d, 3H, J = 7.2
Hz, -CHCH3), 5.18 (q, 1H, J = 7.2 Hz, -CHCH3), 6.79 (2d, 2H, J = 8.7 Hz, H-2 and H-6 of
phenyl), 7.85 (2d, 2H, J = 8.7 Hz, H-3 and H-5 of phenyl), 7.90 (m, 2H, Ar-H, H-6 and H-7
of phthalazine), 8.06 (d, 1H, J = 7.8 Hz, Ar-H, H-5 of phthalazine), 8.15 (s, 1H, - N=CH-),
8.22 (d, 1H, J = 7.8 Hz, Ar-H, H-8 of phthalazine), 9.8 (s, 1H, exchangeable with D2O, OH)
11.2 (s, 1H, exchangeable with D2O, NHN=CH), 11.8 (s, 1H, exchangeable with D2O, NH
13

ACCEPTED MANUSCRIPT
phthalazine); Mass m/z [%]: 352 [M+, 1.78], 217 [100]; Anal. calcd. for C18H16N4O4 (352.35):
C, 61.36; H, 4.58; N, 15.90. Found: C, 61.58; H, 4.63; N, 16.07.
2.1.14.62-(1,4-Dioxo-3,4-dihydrophthalazin-2(1H)-yl)-N'-(4-methoxybenzylidene)propane-
hydrazide (14f)
Yield 84%; mp 260-262 °C; IR (KBr) ν cm-1: 3180 (NH), 3002 (C-H aromatic), 1657
(C=O), 1574 (C=N); 1H NMR (DMSO-d6) δ ppm: 1.63 (d, 3H, J = 6.8 Hz, -CHCH3), 3.79 (s,
3H, -OCH3), 5.18 (q, 1H, J = 6.8 Hz, -CHCH3), 6.96 (2d, 2H, J = 6.8 Hz, H-2 and H-6 of
phenyl), 7.62 (2d, 2H, J = 6.8 Hz, H-3 and H-5 of phenyl), 7.92 (m, 2H, Ar-H, H-6 and H-7
of phthalazine), 7.97 (s, 1H, -N=CH-), 8.04 (d, 1H, J = 7.8 Hz, Ar-H, H-5 of phthalazine),
8.21 (d, 1H, J = 7.8 Hz, Ar-H, H-8 of phthalazine), 11.41 (s, 1H, exchangeable with D2O,
NHN=CH), 11.90 (s, 1H, exchangeable with D2O, NH phthalazine); Anal. calcd. for
C19H18N4O4 (366.38): C, 62.29; H, 4.95; N, 15.25. Found: C, 62.43; H, 4.98; N, 15.53.
2.1.14.7 2-(1,4-Dioxo-3,4-dihydrophthalazin-2(1H)-yl)-N'-(3-nitrobenzylidene)propanehydr-
azide (14g)
Yield 81%; mp 262-264 °C; IR (KBr) ν cm-1: 3189 (NH), 3067 (C-H aromatic), 1660
(C=O), 1592 (C=N); 1H NMR (DMSO-d6) δ ppm: 1.58 (d, 3H, J = 6.6 Hz, -CHCH3), 5.22 (q,
1H, J = 6.6 Hz, -CHCH3), 7.62 (t, 1H, J = 7.8 Hz, H-5 of phenyl), 7.71 (d, 1H, J = 7.8 Hz, H-
6 of phenyl), 7.74 (d, 1H, J = 7.8 Hz, H-4 of phenyl), 7.87 (m, 2H, Ar-H, H-6 and H-7 of
phthalazine), 7.96 (s, 1H, - N=CH-), 8.03 (d, 1H, J = 7.8 Hz, Ar-H, H-5 of phthalazine), 8.21
(d, 1H, J = 7.8 Hz, Ar-H, H-8 of phthalazine), 8.36 (s, 1H, H-2 of phenyl), 11.41 (s, 1H,
exchangeable with D2O, NHN=C), 11.90 (s, 1H, exchangeable with D2O, NH phthalazine);
Mass m/z [%]: 381 [M+, 6.22], 217 [100]; Anal. calcd. for C18H15N5O5 (381.35): C, 56.69; H,
3.96; N, 18.37. Found: C, 56.93; H, 3.98; N, 18.51.
2.1.15 2-[1-(3,5-Dimethyl-1H-pyrazol-1-yl)-1-oxopropan-2-yl]-2,3-dihydrophthalazine-1,4-
dione (15)
Acetylacetone (1.00 g, 1.03 ml, 0.01 mol) was added to a solution of compound 6 (2.48
g, 0.01 mol) in dioxan (20 ml) and few drops of TEA. The reaction mixture was refluxed for
6 h, concentrated, cooled to room temperature and the precipitate was filtered and crystallized
from ethanol to give compound 15. Yield 77%; mp 182-184 °C; IR (KBr) ν cm-1: 3167 (NH),
1
1669 (C=O), 1590 (C=N); H NMR (DMSO-d6) δ ppm: 1.53 (d, 3H, J = 6.6 Hz, -CHCH3),
1.70 (s, 3H, N=C-CH3), 2.10 (s, 3H, C=C-CH3), 5.72 (q, 1H, J = 6.6 Hz, -CHCH3), 6.20 (s,
14

ACCEPTED MANUSCRIPT
1H, -CH of pyrazole), 7.88 (m, 2H, Ar-H, H-6 and H-7 of phthalazine), 8.03 (d, 1H, J = 8 Hz,
Ar-H, H-5 of phthalazine), 8.22 (d, 1H, J = 7.2 Hz, Ar-H, H-8 of phthalazine), 11.7 (s, 1H,
exchangeable with D2O, NH phthalazine); Mass m/z [%]: 312 [M+, 14.09], 162 [100]; Anal.
calcd. for C16H16N4O3 (312.33): C, 61.53; H, 5.16; N, 17.94. Found: C, 61.75; H, 5.19; N,
18.13.
2.1.16
2-(1,4-Dioxo-3,4-dihydrophthalazin-2(1H)-yl)-N'-(4-oxopentan-2-ylidene)propane-
hydrazide (16)
To a solution of compound 6 (2.48 g, 0.01 mol) in ethanol (20 ml), acetylacetone (1.00
g, 1.03 ml, 0.01 mol) was added. The reaction mixture was refluxed for 4 h, concentrated,
cooled to room temperature and the precipitate was filtered and crystallized from ethanol to
give compound 16. Yield 75%; mp 170-172 °C; IR (KBr) ν cm-1: 3170 (NH), 1645 (C=O),
1
1609 (C=N); H NMR (DMSO-d6) δ ppm: 1.54 (d, 3H, J = 4.8 Hz, -CHCH3), 1.72 (s, 3H, -
C=NCH3), 2.00 (s, 3H, -C=OCH3), 2.78 (s, 2H, CH2C=O), 5.71 (q, 1H, J = 4.8 Hz, -CHCH3),
6.37 (s, 1H, exchangeable with D2O, -NHN=C), 7.89 (m, 2H, Ar-H, H-6 and H-7 of
phthalazine), 8.02 (d, 1H, J = 8 Hz, Ar-H, H-5 of phthalazine), 8.20 (d, 1H, J = 7.5 Hz, Ar-H,
H-8 of phthalazine), 11.7 (s, 1H, exchangeable with D2O, NH phthalazine); Anal. calcd. for
C16H18N4O4 (330.34): C, 58.17; H, 5.49; N, 16.96. Found: C, 58.43; H, 5.57; N, 17.28.
2.1.17 N'-(2-Chloroacetyl)-2-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-yl)propanehydrazide
(17)
Chloroacetyl chloride (1 g, 0.01mol) was added dropwise to a solution of compound 6
(2.48 g, 0.01 mol) in DMF (20 mL) and stirred at room temperature for 1 h, then poured onto
ice-water. The solid product obtained was filtered, dried, and crystallized from ethanol to
give compound 17. Yield 87%; mp 238-240 °C; IR (KBr) ν cm-1: 3244, 3195 (NH), 2059
(CH aliphatic), 1665 (C=O); 1H NMR (DMSO-d6) δ ppm: 1.57 (d, 3H, J = 6.4 Hz, -CHCH3),
4.10 (s, 2H, -CH2Cl), 5.23 (q, 1H, J = 6.4 Hz, -CHCH3), 7.88 (m, 2H, Ar-H, H-6 and H-7 of
phthalazine), 8.07 (d, 1H, J = 7.6 Hz, Ar-H, H-5 of phthalazine), 8.20 (d, 1H, J = 7.6 Hz, Ar-
H, H-8 of phthalazine), 10.31 (s, 1H, -C=ONHNH), 10.37 (s, 1H, exchangeable with D2O, -
C=ONHNH), 11.91 (s, 1H, exchangeable with D2O, NH phthalazine); Mass m/z [%]: 324
[M+, 3.83], 326 [M+2, 1.64], 217 [100]; Anal. calcd. for C13H13ClN4O4 (324.72): C, 48.09; H,
4.04; N, 17.25. Found: C, 48.28; H, 4.11; N, 17.43.
15

ACCEPTED MANUSCRIPT
2.1.18
2-(4-Methyl-3,6-dioxohexahydropyridazin-4-yl)-2,3-dihydrophthalazine-1,4-dione
(18)
Chloroacetyl chloride (1 g, 0.01mol) was added dropwise to a solution of compound 6
(2.48 g, 0.01 mol) in DMF (20 mL). The reaction mixture was heated under reflux for 4 h.
The solid obtained upon cooling was filtered, dried, and crystallized from ethanol. Yield
82%; mp 158-160 °C; IR (KBr) ν cm-1: 3164, 3195 (NH), 2059 (CH aliphatic), 1669 (C=O);
1H NMR (DMSO-d6) δ ppm: 1.87 (s, 3H, -CH3), 2.61 (d, 2H, -CH2CO), 7.90 (m, 2H, Ar-H,
H-6 and H-7 of phthalazine), 8.09 (d, 1H, J = 7.6 Hz, Ar-H, H-5 of phthalazine), 8.22 (d, 1H,
J = 7.6 Hz, Ar-H, H-8 of phthalazine), 11.27 (s, 1H, exchangeable with D2O, -NH-2 of
pyridazine), 11.29 (s, 1H, exchangeable with D2O, NH-1 of pyridazine), 11.91 (s, 1H,
exchangeable with D2O, NH phthalazine); Anal. calcd. for C13H12N4O4 (288.26): C, 54.17; H,
4.20; N, 19.44. Found: C, 54.29; H, 4.35; N, 19.52.
2.1.19 Potassium 5-[1-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-yl)ethyl]-1,3,4-oxadiazole-2-
thiolate (19)
Compound 7 was added portion wise to a solution of potassium hydroxide (0.56 g, 0.01
mol) in absolute ethanol (20 ml) then the reaction mixture was refluxed for 0.5 h. Upon
cooling to the room temperature, a yellow solid product was obtained, collected and washed
with diethyl ether. Yield: 90%; mp> 300 °C.
2.1.20 General procedure for preparation of target compounds (20a,b)
A mixture of the potassium 5-[1-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-yl)ethyl]-
1,3,4-oxadiazole-2-thiolate 19 (0.65 g, 0.002 mol) and appropriate alkyl halid namely, methyl
iodide and ethyl bromide (0.002 mol) in DMF (20ml) was heated on a water bath for 2 h.
After cooling to the room temperature, the reaction mixture was cooled while stirring with ice
cold water (150 ml). The white solid product was filtered, washed with water and crystallized
from ethanol to afford compounds 20a,b.
2.1.20.1
2-{1-[5-(Methylsulfanyl)-1,3,4-oxadiazol-2-yl]ethyl}-2,3-dihydrophthalazine-1,4-
dione (20a). Yield 83%; mp 153-155 °C; IR (KBr) ν cm-1: 3179 (NH), 2911 (C-H aliphatic),
1
1654 (C=O), 1599 (C=N); H NMR (DMSO-d6) δ ppm: 1.59 (d, 3H, J = 6.8 Hz, -CHCH3),
3.67 (s, 3H, -SCH3), 5.20 (q, 1H, J = 6.8 Hz, -CHCH3), 7.89 (m, 2H, Ar-H, H-6 and H-7 of
phthalazine), 8.00 (d, 1H, J = 7.6 Hz, Ar-H, H-5 of phthalazine), 8.23 (d, 1H, J = 7.6 Hz, Ar-
H, H-8 of phthalazine), 11.91 (s, 1H, exchangeable with D2O, NH phthalazine); Mass m/z
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[%]: 304 [M+, 5.08], 201 [98]; Anal. calcd. for C13H12N4O3S (304.32): C, 51.31; H, 3.97; N,
18.41. Found: C, 51.38; H, 3.99; N, 18.72.
2.1.20.2
2-{1-[5-(Ethylsulfanyl)-1,3,4-oxadiazol-2-yl]ethyl}-2,3-dihydrophthalazine-1,4-
dione (20b). Yield 83%; mp 153-155 °C; IR (KBr) ν cm-1: 3427 (NH), 2967 (C-H aliphatic),
1
1647 (C=O), 1597 (C=N); H NMR (DMSO-d6) δ ppm: 1.31 (t, 3H, J = 7.2 Hz, -CH2CH3),
1.80 (d, 3H, J = 6.8 Hz, -CHCH3), 3.20 (t, 2H, J = 7.2 Hz, -CH2CH3), 6.17 (q, 1H, J = 6.8
Hz, -CHCH3), 7.89 (m, 2H, Ar-H, H-6 and H-7 of phthalazine), 7.99 (d, 1H, J = 7.6 Hz, Ar-
H, H-5 of phthalazine), 8.25 (d, 1H, J = 6.8 Hz, Ar-H, H-8 of phthalazine), 11.91 (s, 1H,
exchangeable with D2O, NH phthalazine); Anal. calcd. for C14H14N4O3S (318.35): C, 52.82;
H, 4.43; N, 17.60. Found: C, 52.97; H, 4.49; N, 17.83.
2.1.21 Methyl 2-{[5-(1-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-yl)ethyl)-1,3,4-oxadiazol-2-
yl]sulfanyl}acetate (21)
Equimoler quantity of the potassium salt 19 (3.28 g, 0.01 mol) and methyl
chloroacetate (1.08 g, 0.01 mol) in DMF (20 ml) was heated on water bath for 3 h. The
reaction mixture was poured onto ice water (200 ml) and stirred for 30 minutes. The obtained
solid was filtered and crystallized from absolute ethanol to afford compound 21. Yield 76%;
mp 140-142 °C; IR (KBr) ν cm-1: 3169 (NH), 2903 (C-H aliphatic), 1749, 1660 (C=O), 1594
(C=N); 1H NMR (DMSO-d6) δ ppm: 1.50 (d, 3H, J = 6.6 Hz, -CHCH3), 3.66 (s, 3H, -OCH3),
4.32 (q, 1H, -SCH2), 5.43 (q, 1H, J = 6.6 Hz, -CHCH3), 7.86 (m, 2H, Ar-H, H-6 and H-7 of
phthalazine), 8.01 (d, 1H, J = 7.6 Hz, Ar-H, H-5 of phthalazine), 8.23 (d, 1H, J = 7.8 Hz, Ar-
H, H-8 of phthalazine), 12.00 (s, 1H, exchangable with D2O, NH phthalazine); Mass m/z [%]:
362 [M+, 3.22], 201 [100]; Anal. calcd. for C15H14N4O5S (362.36): C, 49.72; H, 3.89; N,
15.46. Found: C, 49.95; H, 3.94; N, 15.63.
2.1.22 Methyl 2-{[5-(1-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-yl)ethyl)-1,3,4-oxadiazol-2-
yl]sulfanyl}propanoate (22)
Methyl 2-chloropropanoate (1.22 g, 0.01 mol) was heated with compound 19 (3.28 g,
0.01 mol) in DMF (20ml) on a water bath for 3 h. The reaction mixture was poured onto ice
water (200 ml) and stirred for 30 minutes. The obtained solid was filtered and crystallized
from absolute ethanol to give compound 22. Yield 74%; mp 154-156 °C; IR (KBr) ν cm-1:
1
3172 (NH), 2897 (C-H aliphatic), 1757, 1655 (C=O), 1599 (C=N); H NMR (DMSO-d6) δ
ppm: 1.03 (d, 3H, J = 6.8 Hz, -NCHCH3), 1.67 (d, 3H, J = 6.8 Hz, -SCHCH3), 3.42 (s, 3H, -
17

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OCH3), 4.34 (q, 1H, -SCHCH3), 5.4 (q, 1H, J = 6.8 Hz, -NCHCH3), 7.89 (m, 2H, Ar-H, H-6
and H-7 of phthalazine), 8.11 (d, 1H, J = 8.4 Hz, Ar-H, H-5 of phthalazine), 8.22 (d, 1H, J =
7.6 Hz, Ar-H, H-8 of phthalazine), 12 (s, 1H, exchangable with D2O, NH phthalazine); Anal.
calcd. for C16H16N4O5S (376.39): C, 51.06; H, 4.28; N, 14.89. Found: C, 51.32; H, 4.34; N,
15.15.
2.2 Molecular modeling
2.2.1 Docking
Molecular docking studies were done to provide insights of molecular binding modes of the
tested molecules inside the pocket of GABA-A receptor suggested by the homology
hypothesis. The compounds were docked against GABA-A receptor using Discovery Studio
2.5 to evaluate the free energies and mode of binding of the proposed molecules with the
active site of GABA-A. The choice of the most promising molecules depends on the correct
binding mode and the binding free energy (ꢀG).
The homology crystal structure of GABA-A receptor was downloaded from the Nature
Chemical Biology (www.nature.com/ Protein data bank files/ nchembio.917) using Discovery
Studio 2.5 software. Water molecules were removed from downloaded protein.
Crystallographic disorders and unfilled valence atoms were corrected using alternate
conformations and valence monitor options. Protein was subjected to energy minimization by
applying CHARMm force fields for charge, and MMFF94 force field for partial charge.
Inflexibility of structure is obtained by creating fixed atom constraint. The binding site of the
protein was defined and prepared for docking.
Ligand (Diazepam) and the designed compounds 2D structures were sketched using
ChemBioDraw Ultra 14.0 and saved in MDL-SDfile format. SDfile was opened, 3D
structures were protonated and energy was minimized by applying CHARMm force fields for
charge, and MMFF94 force field for partial charge and then prepared for docking by
optimization of the parameters. Docking was accomplished using CDOCKER-CHARMm-
based technique in the interface of Accelry’s discovery studio 2.5. After that the docking
scores (-CDOCKER interaction energy) of the best-fitted conformation of each of the docked
molecules with the amino acids at the GABA-A binding pocket were recorded, Table 1.
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2.2.2 Generation of GABA-A modulators hypothesis
The training set was selected as described above [10]. The generation of the pharmacophore
model for GABA-A modulators was accomplished using Discovery Studio 2.5 software.
Molecules were built and conformational models for each compound were generated
automatically using the prepare ligand protocol. The prepared training set molecules were
used for generation 3D pharmacophore using common features hypothesis generation
protocol (Hip-Hop module). In this study, hydrogen bonding acceptor, hydrogen bonding
donor, hydrophobic, hydrophobic aliphatic and ring aromatic features, were used as the
chemical features, which were reported to be crucial for the GABA-A modulating activity.
By this process we specified the crucial features required for binding with the GABA-A
receptor.
The structures of the titled compounds were built using the Discovery studio 2.5
software, and their conformational models were generated in the energy range of 20 kcal/mol
above the estimated global energy minima. The fitting of each tested compound was
performed using best fit during the compare/fit process.
2.3 Pharmacological tests
All the trials were carried out according to the respective internationally guidelines. Swiss
albino adult male mice, weighing 20–25 g, were utilized as experimental animals. They were
obtained from an animal facility (Animal house, Department of Pharmacology and
Toxicology, Faculty of Pharmacy, Al-Azhar University). Mice were housed in stainless steel
wire-floored cages without any stressful stimuli. Animals were kept under well-ventilated
conditions at room temperature (25–30 °C). They were fed on an adequate standard
laboratory chow (El-Nasr Co., Abou-Zabal, Egypt) and allowed to acclimatize with free
access to food and water for 24 h period before testing except during the short time they were
removed from the cages for testing. Albino mice were randomly arranged in groups, each of
six animals. Sodium valproate (Sigma-Aldrich Chemical Co, Milwaukee, WI, USA) was
used as a reference drug for comparison.
2.3.1 Pentylenetetrazole (PTZ) and Picrotoxin-induced convulsion tests
PTZ and picrotoxin (Sigma-Aldrich Chemical Co, Milwau-kee, WI, USA) were used to
induce convulsions in the experimental animals. The test compounds were solubilized in
DMSO and were administered IP. PTZ and picrotoxin were dissolved in normal saline in 2 %
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concentration and were given IP in a dose of 100 mg/kg and 10 mg/kg body weight (dose that
could induce convulsions in at least 80 % of the animals without death during the following
24 h), respectively. Sodium valproate (Sigma, USA) was dissolved in normal saline in 2 %
concentration and it was IP given in doses of 200 mg/kg using the same dosing volume. The
time needed for PTZ or picrotoxin to induce convulsions or cause death was calculated. The
active anticonvulsant compound was able to prevent such episodes or delayed this time
pattern and such compound will be considered to possess anticonvulsant activity.
2.3.2 Maximal electroshock seizure (MES)
The test compounds and sodium valporate were injected IP at a dose of 100 mg/kg before
electrical induction of convulsions with maximal electroshocks (MES). The electrical
stimulus produced from electroconvulsiometer was 50 mA, 60 Hz. Current was applied for
0.2 s via auricular electrodes. Protection against the spread of MES-induced seizures was
identified by the abolition of the hind leg and tonic maximal extension component of the
seizure [13].
2.3.3 Bicuculline (Bic)-induced convulsion test
1.5 mmol of each compound were injected i.p. in 10 mice. 30 min later, animals were
injected s.c. with Bic (2.7 mg/kg) and observed for 30 min and the percentage protection
against convulsive seizures for each compound was calculated [14].
3. Results and discussion
3.1 Rationale drug design
The design and synthesis of the titled compounds (Fig. 2) were carried out with an objective;
that was the molecular hybridization of 2,3-dihydrophthalazine-1,4-dione moiety with
effective anticonvulsant moieties such as hydrazones, semicarbazones, thiosemicarbazones
and oxadiazole, followed by the chemical modification and substitutions on functional group
of the synthesized compounds by two pathways, the first pathway was by replacement of
hydrazones, semicarbazones and thiosemicarbazones groups of synthesized compounds by
phthalimide, succinamide, pyrazole or pyridazine groups, while the second pathway was by
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the insertion of different alkyl and alkyl ester moieties to the sulfanyl group of the oxadiazole
ring. The choice of substituents was based on their relatively high lipophilicity to pass the
blood–brain barrier aiming to have strong anticonvulsant activity. Moreover all the
synthesized compounds comprised of the essential pharmacophoric elements that are
necessary for good binding with GABA-A receptors as suggested by Cook. This study was
carried out to obtain new compounds with different chemical isosteres in order to study the
SAR of these compounds and the effect of each substituent on their anticonvulsant activity.
In addition, we hope to get new, potent, safe and effective anticonvulsant agents with lower
side effects.
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* HP = Hydrophobic
*HB = Hydrogen bonding site
*HA = Hydrophobic aliphatic
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3.2 Chemistry
The intermediates 2 and 3 that were used to introduce methyl 2-(1,4-dioxo-3,4-
dihydrophthalazin-2(1H)-yl)propanoate 4, were synthesized according to the reported
strategies [12]. For synthesis of the target compounds 4-22, the following straightforward
pathways were pursued; the starting material phthalic anhydride 1 reacted with hydrazine
hydrate in ethanol to yield 2,3-dihydrophthalazine-1,4-dione 2 which reacted further with
alcoholic potassium hydroxide to afford the corresponding potassium salt 3. Upon reacting
compound 3 with methyl 2-chloropropanoate in heated DMF with catalytic amount of
potassium iodide, compound 4 was afforded. The IR spectrum of the latter product exhibited
1
a characteristic band at 1757 cm-1 corresponding to the carbonyl of the ester group. The H
NMR spectrum showed a singlet at δ 3.68 assignable to the OCH3 protons. Its mass spectrum
revealed a peak at m/z 248 corresponding to its molecular ion. The amide derivatives 5a-d
were prepared in good yields by reacting compound 4 with the appropriate amines in ethanol.
The reactivity of methyl 2-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-yl)propanoate 4 towards
some nitrogen nucleophiles was investigated. Thus, the treatment of compound 4 with
hydrazine hydrate, in refluxing ethanol, afforded white crystals of 2-(1,4-dioxo-3,4-
dihydrophthalazin-2(1H)-yl)propanehydrazide 6 followed the reported procedure [15]. The
1
spectral data of the product confirmed its structure, as its H NMR spectrum displayed two
broad singlets (D2O-exchangeable) at δ 4.24 and at δ 9.29 indicating the protons of NH2 and
NH respectively. Mass spectrum of compound 6 showed prominent molecular ion peak at m/z
248 as the base peak. Cyclization of compound 6 using carbon disulfide yielded the
1
oxadiazole derivative 7 in a good yield. The H NMR of the later compound showed
appearance of a singlet at δ 14.22 characteristic to the SH proton of oxadiazole moiety.
Compound 7 showed thiol–thione tautomerism. In solution, there is an evident thiol singlet
1
signal at δ 14.22 ppm in H NMR spectra while in the solid state, this compound presents in
thione (–C=S) form, as indicated in their IR spectrum by the absence of absorption band at
2500 cm−1 (–SH stretching) and the presence of absorption bands at 1163 cm−1, characteristic
of the –C=S group (Scheme 1).
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The reaction of the acid hydrazide 6 with appropriate isothiocyanates or isocyanates
afforded the corresponding thiosemicarbazide 8a-d and semicarbazide 9a-c derivatives,
respectively. The spectral and microanalytical data for compounds 8a-d and 9a-c were
1
consistent with their chemical structures. For example, their H NMR spectra were
characterized by disappearance of the the NH2 signal and appearance of two new highly
deshielded NH protons. The condensation of 6 with phthalic anhydride, tetrabromophthalic
24

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anhydride, succinic anhydride, and ethyl acetoacetate, afforded the corresponding
isoindolines 10 and 12, pyrrolidine 11 and ester 13 respectively (Scheme 2).
The reactivity of acid hydrazide 6 towards some aromatic aldehydes was also
investigated. Thus, the treatment of compound 6 with different benzaldehyde derivatives in
the presence of a catalytic amount of glacial acetic acid afforded the corresponding Schiff’s
1
bases 14a-g. The H NMR spectrum of compound 14a, taken as a typical example of the
prepared series, revealed a sharp singlet at δ 8.66 corresponds to the azomethine (N=CH)
proton. The previously prepared acid hydrazide 6 reacted with acetylacetone in boiling
1
dioxan to give the pyrazole derivative 15. The H NMR spectrum showed two singlets at
δ1.70 and δ 2.10 corresponding to pyrazole-attached methyl groups. Other important signal
appeared at δ 6.20 due to the pyrazole proton that confirmed the cyclic structure. On the other
hand when the reaction was carried out in boiling ethanol, the acetone derivative 16 was
afforded. Reaction of acid hydrazide 6 with chloroacetyl chloride at room temperature
produced the chloroacetyl derivative 17. Mass spectrum of compound 17 showed prominent
molecular ion peak at m/z 324. Repetition of this reaction under refluxing conditions gave the
cyclized product 18 (Scheme 3).
The treatment of 5-sulfanyloxadiazole derivative 7 with alcoholic potassium hydroxide
resulted in the corresponding S-potassium salt 19 which was then reacted with different alkyl
bromides, methyl chloroacetate and methyl 2-chloropropanoate to give the corresponding S-
alkyl 20a,b, sulfanyl acetate 21 and sulfanyl propanoate 22 derivatives respectively (Scheme
4).
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O
CH3
S
H
N
R
R N C S
N
N
H
N
H
NH
O
Ethanol/reflux/3h
8a, R= C2H5
8a-d
O
O
8b, R= nꢁC3H7
8c, R= Cyclo C6H11
8d, R= C6H5
CH3
O
H
N
R1
N
R1 N C S
N
N
H
H
Ethanol/reflux/12h
NH
O
9a, R1= nꢁC4H9
9b, R1= Cyclo C6H11
9c, R1= C6H5
O
O
CH3
9a-c
H
O
O
N
N
NH2
O
O
CH3
O
NH
O
H
N
N
N
O
NH
O
gl. acetic acid/
reflux/18h
6
O
O
10
O
O
O
CH3
O
H
N
N
N
O
gl. acetic acid/
reflux/15h
NH
O
O
11
O
O
Br
O
Br
CH3
O
O
H
N
Br
Br
O
N
N
Br
NH
O
gl. acetic acid/
reflux/20h
Br
O
O
O
Br
12
Br
CH3 OC2H5
O
CH3
H
N
O
O
N
N
H3C
OC2H5
NH
O
Ethanol/reflux/4h
13
O
Scheme 2: Synthesis of the target compounds 8-13
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3.3 Molecular modeling
Considering ligand–receptor interaction, one usually takes into account two points: strength
of the interaction (what is known as ‘affinity’) and kind of an effect at the biochemical,
electrophysiological and behavioral level triggered by the ligand (what we refer to as
‘intrinsic activity’). To get an insight into the type of chemical forces involved in the
interaction, one may build models of a specific pharmacophore or construct models of a
receptor protein and examine the binding forces. Interaction of a ligand with a receptor may
result in receptor activation (agonists) [16].
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The most popular approach to predict the correct binding pose and binding affinity
(BA) is protein-based modeling (docking) in which physicochemical interactions between a
ligand and receptor are deduced from the 3D structures of both molecules. This in silico
method is opposed to the alternative approach, ligand-based (pharmacophore), in which only
ligands that are biochemically similar to the ones known to bind to the target are screened
[17]. In this work, both docking and pharmacophore methods were performed to predict the
anticonvulsant activity of the synthesized compounds.
3.3.1 Docking study
Molecular docking studies of the titled compounds were performed in order to rationalize the
obtained biological results as well as to help us to understand the various interactions
between the ligands and the active site of GABA-A homology model in details.
The GABA-A receptor complex has distinct, high-affinity binding sites which are able
to modulate their channel function, such as benzodiazepine binding site, BzR. The
benzodiazepines are of prime importance since these ligands have been employed widely as
anxiolytic and anticonvulsant agents [18].
A series of conformational searches followed by energy minimization for the titled
compounds allowed the determination of the most biologically active conformers.
Docking study was conducted to evaluate the orientation of the titled compounds inside
the BzR-binding site using the GABA-A homology model demonstrated by Richter et al [19].
Diazepam was also docked for validation purposes. Fig. 3 displays the important amino acids
interacting with diazepam (Thr142, Thr206, His101, Phe77, Tyr159 and Ser204); one
nitrogen atom formed a hydrogen bond with the acidic part of Ser204 at 2.67 Å, the carbonyl
group near the nitrogen atom formed two hydrogen bond interactions with Thr206 and
Thr142 at 2.08 and 2.31 Å respectively.
29