Phthalazine-1,4-dione derivatives as non-competitive AMPA receptor antagonists: design, synthesis, anticonvulsant evaluation, ADMET profile and molecular docking

Article abstract of DOI:10.1007/s11030-018-9871-y

In view of the anticonvulsant activity reported for phthalazine derivatives as non-competitive AMPA receptor antagonists, a new series of phthalazine-1,4-diones (2–12) were designed and synthesized. The neurotoxicity was assessed using rotarod test. The molecular docking was performed for the synthesized compounds to assess their binding affinities toward AMPA receptor as non-competitive antagonists. The molecular modeling data were strongly interrelated to biological screening data. Compounds 8, 7_b, 7_a, 10 and 3_a exhibited the highest binding affinities as non-competitive AMPA receptor antagonists and also showed the highest relative potencies of 1.78, 1.66, 1.60, 1.59 and 1.29, respectively, as anticonvulsants in comparison with diazepam. The most active compounds 8, 7_b, 7_a, 10 and 3_a were further tested against maximal electroshock seizure (MES). Compounds 8 and 7_b and 3_a showed 100% protection at a dose level of 125?μgm/kg, while compounds 7_a and 10 exhibited 83.33% protection at the same dose level. These agents exerted low neurotoxicity and high safety margin in comparison with valproate as a reference drug. Most of our designed compounds exhibited good ADMET profile.

Full text of DOI:10.1007/s11030-018-9871-y

Molecular Diversity
https://doi.org/10.1007/s11030-018-9871-y
ORIGINAL ARTICLE
Phthalazine-1,4-dione derivatives as non-competitive AMPA receptor
antagonists: design, synthesis, anticonvulsant evaluation, ADMET
profile and molecular docking
Abdel-Ghany A. El-Helby1 · Rezk R. A. Ayyad1 · Khaled El-Adl1,2 · Hazem Elkady1
Received: 14 October 2017 / Accepted: 25 August 2018
© Springer Nature Switzerland AG 2018
Abstract
In view of the anticonvulsant activity reported for phthalazine derivatives as non-competitive AMPA receptor antagonists, a
new series of phthalazine-1,4-diones (2–12) were designed and synthesized. The neurotoxicity was assessed using rotarod
test. The molecular docking was performed for the synthesized compounds to assess their binding affinities toward AMPA
receptor as non-competitive antagonists. The molecular modeling data were strongly interrelated to biological screening data.
Compounds 8, 7b, 7a, 10 and 3a exhibited the highest binding affinities as non-competitive AMPA receptor antagonists and
also showed the highest relative potencies of 1.78, 1.66, 1.60, 1.59 and 1.29, respectively, as anticonvulsants in comparison
with diazepam. The most active compounds 8, 7b, 7a, 10 and 3a were further tested against maximal electroshock seizure
(MES). Compounds 8 and 7b and 3a showed 100% protection at a dose level of 125 μgm/kg, while compounds 7a and
10 exhibited 83.33% protection at the same dose level. These agents exerted low neurotoxicity and high safety margin in
comparison with valproate as a reference drug. Most of our designed compounds exhibited good ADMET profile.
Keywords Phthalazine · Molecular docking · Non-competitive AMPA antagonists · Anticonvulsant agents
Introduction
2,3-benzodiazepines are thought to bind to the transducer
domains and inhibit channel gating [5, 13], thus having broad
spectrum anticonvulsant activity. Talampanel (III, GYKI
53773) the analog of GYKI 52466 established efficacy in
a clinical trial for refractory partial seizure treatment. The
antagonists inhibit the ability of the agonist to open the chan-
nel by disrupting conformational changes which may occur
in the ligand-binding domains [5]. The ion channel is con-
nected to the ligand-binding domains through linkers. The
allosteric binding site was found at the interface between the
ion channel and these linkers. Thus, inhibitors act as wedges
between the transmembrane segments to prevent ion channel
opening (Fig. 1) [14]. The non-competitive AMPA receptor
antagonists remain effective independently of the level of
glutamate or the synaptic membrane polarization state dur-
ing neurological diseases. Moreover, they do not influence
the normal glutamatergic activity also after prolonged use
[3].
Approximately 30% of all epilepsies have a drug-resistant
course and require new treatment options [1]. One new direc-
tion in antiepileptic drug development is aimed at inhibiting
excitatory neurotransmission, which plays a key role in
epileptogenesis and seizure spread [2]. Consequently, AMPA
receptors, which mediate the majority of excitatory neuro-
transmission, have emerged as a promising new target for
epilepsy therapy [3–8]. The most potent and well-tolerated
inhibitors of AMPA receptors, those with fewer side effects,
act via a non-competitive (negative allosteric) mechanism.
The originally discovered non-competitive AMPA receptor
antagonist GYKI 52466 (I) became a prototype for the devel-
opment of more potent and selective 2,3-benzodiazepines
[9–11], such as GYKI 53655 (II, GYKI) [12]. Several
Khaled El-Adl
eladlkhaled74@yahoo.com
B
It was reported that clinical development of competi-
tive AMPA receptor antagonists as anticonvulsant agents
was hampered due to severe side effects, lack of sol-
ubility and poor biodistribution profiles [15]. Therefore,
non-competitive AMPA receptor antagonists were pre-
1
Pharmaceutical Chemistry Department, Faculty of Pharmacy,
Al-Azhar University, Cairo, Egypt
2
Pharmaceutical Organic Chemistry Department, Faculty of
Pharmacy, Nahda University, Benisuef, Egypt
123

Molecular Diversity
aqueous solubility and lipophilicity in order to investigate
their anticonvulsant activity.
Rationale and structure-based design
Our target compounds were designed as hybrids to main-
tain the basic features of the lead compounds 2,3-
benzodiazepines (I–IV) and in the same time the basic
features of the reported phthalazines SYM 2189 (V), VI
and VII with anticonvulsant activity. Figure 3 represents the
structure of highly active final compounds fulfilling all the
pharmacophoric structural requirements taking compounds
SYM 2189 (V), VI and VII as lead compounds. These
requirements include: the presence of phthalazine moiety as
hydrophobic domain, the phenyl ring at position-4 replaced
by carbonyl group hoping to obtain higher binding affinity
with AMPA receptor and also more water-soluble deriva-
tives.
The large space in the receptor active site motives us to
use long linker containing hydrogen bond acceptor (HBA)
and/or hydrogen bond donor (HBD) to occupy the hydrogen
bond area (high electron density) (Fig. 4) and also to increase
water solubility which is the main problem hampered AMPA
receptor antagonists. On the other hand, different hydropho-
bic distal groups, attached to the N-2 through the long linker,
allow new hydrophobic interactions. These distal moieties
have different incorporated substitutions with different elec-
tronic environments in order to study and compare the effect
of each substituent in the SAR. Finally, these modifica-
tions were carried out to obtain more hydrogen bonding and
hydrophobic interaction sites to increase the binding affinity
toward the AMPA receptor. Figure 4 shows the electronic
environment of AMPA receptor around the title compounds.
Fig. 1 Model of AMPA receptor non-competitive binding antagonists
ferred. The phthalazine bioisoster lead compounds, 2,3-
benzodiazepines, GYKI52466(I), GYKI53655(II), GYKI
53773 (III) and CFM-2 (IV) (Fig. 2), inhibit AMPA receptor
and have anticonvulsant activity [16]. Moreover, Talampanel
(III) aroused great interest as an anticonvulsant agent and
whose phase II/III clinical trials are underway [17].
Taking the 2,3-benzodiazepine derivatives as a mold, dif-
ferent arylphthalazines have been developed as selective
non-competitive negative AMPA-R modulators [14]. Many
phthalazine derivatives such as SYM 2189 (V), VI and VII
(Fig. 3) were reported to possess inhibitory properties at the
AMPA receptor with anticonvulsant activity [3, 15, 18–21].
On the other hand, sedative–hypnotic (neurotoxicity) prop-
erties of phthalazines are well documented [22, 23].
Materials and methods
The potent and selective activity of the phthalazines cou-
pled with their relative ease of synthesis and improved
solubility properties [20] prompted us to synthesize struc-
tural analogs of phthalazine-1,4-diones as selective non-
competitive AMPA receptor antagonists with controlled
Chemistry
2,3-Dihydrophthalazine-1,4-dione (1) was obtained accord-
ing to the reported procedures [22].
Fig. 2 Reported 2,3-benzodiazepines as non-competitive AMPA receptor antagonists
123

Molecular Diversity
Fig. 3 Structural similarities and pharmacophoric features of reported potent AMPA antagonists and our designed compounds specially 8, 10, 7a–b
and 3a as anticonvulsants
,
Ethyl 4-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-
(3H, t, CH2CH3), 2.08 (2H, m, CH2CH2CH2), 2.52 (2H, t,
yl)butanoate (2)
CH2CꢀO), 4.05 (2H, q, CH2CH3), 4.26 (2H, t, N-CH2),
7.86–7.93 (2H, m, H-6 and H-7 phthalazine), 7.95–7.96 (1H,
d, J=7.2 Hz, H-8 phthalazine), 8.20–8.22 (1H, d, J=8 Hz,
H-5 phthalazine), 11.88 (1H, s, NH) (D2O exchangeable);
13CNMR (DMSO, 400 MHz): δ ꢀ173.02 (C, COO), 159.15
(C, C-4), 150.07 (C, C-1), 133.88 (C, C-6), 132.63 (CH,
C-7), 129.13 (CH, C-4a), 126.57 (CH, C-8a), 124.89 (CH,
C-5),123.80 (CH, C-8), 66.02 (CH2, CH2CH3), 60.30 (CH2,
NCH2), 30.84 (CH2, CH2CO), 24.27 (CH2, CH2CH2CH2),
14.51 (CH3, CH2CH3); EIMS m/z 276 [M+] (1.52), 114
(45.53), 104 (100); Anal. Calcd. for C14H16N2O4 (276): C,
60.86; H, 5.84; N, 10.14. Found: C, 61.13; H, 5.91; N, 10.37.
White crystals (C2H5OH) (This compound was prepared
by heating a mixture of 2,3-dihydrophthalazine-1,4-dione
(1) (4.86 g, 0.03 mol) and ethyl 4-bromobutanoate (5.85 g,
0.03 mol) in dry acetone (50 ml) in the presence of anhy-
drous K2CO3 (4.14 g, 0.03 mol) under reflux for 13 h
while stirring. The reaction mixture was filtered, the sol-
vent was evaporated, and the resulting product was collected
by filtration and recrystallized from ethanol to give the tar-
get compound (2). It was obtained as white solid); Yield,
75%; mp 170–172 °C; IR (KBr) νmax 3167, 3014, 2907,
1739, 1658 cm−1; HNMR (DMSO, 400 MHz): δ ꢀ1.15
1
123

Molecular Diversity
N-Benzyl-4-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-
yl)butanamide (3b)
Light yellow powder (C2H5OH) (This compound was pre-
pared by heating a mixture of the ethyl ester (2) (0.28 g,
0.001 mol) and benzyl amine (0.22 g, 0.002 mol) in ethanol
(30 ml) under reflux in ethanol (25 ml) for 5 h. The reaction
mixture was cooled and poured onto ice water (200 ml) and
the formed solid was washed with dil. HCl and water, fil-
tered and crystallized from ethanol. It was obtained as light
yellow solid); Yield, 81%; mp 287–289 °C; IR (KBr) νmax
3239, 3078, 2952, 1632 cm ;
−1 13CNMR (DMSO, 400 MHz):
δ ꢀ174.28 (C, CONH), 168.51 (C, C-4), 159.16 (C, C-
1), 141.71 (C, C-1ꢁ), 133.88 (C, C-6), 132.57 (CH, C-7),
131.79 (CH, C-3ꢁ), 131.51 (CH, C-5ꢁ), 129.13 (CH, C-4a),
126.57 (CH, C-8a), 125.82 (CH, C-2ꢁ), 125.48 (CH, C-6ꢁ),
124.98 (CH, C-4ꢁ), 124.02 (CH, C-5), 122.71 (CH, C-8),
56.47 (CH2, NCH2), 57.07 (CH2, NHCH2), 29.20 (CH2,
CH2CO), 24.23 (CH2, NCH2CH2); EIMS m/z 337 [M+]
(2.5), 171 (45.5), 91 (100); Anal. Calcd. for C19H19N3O3
(337): C, 67.64; H, 5.68; N, 12.46. Found: C, 67.90; H, 5.77;
N, 12.72.
Fig. 4 Electronic environment of 1FTL around the title compounds. The
red color indicates hydrophobic area (low electron density), and the blue
color indicates the hydrogen bond area (high electron density)
4-(1,4-Dioxo-3,4-dihydrophthalazin-2(1H)-
yl)butanehydrazide (4)
N-Butyl-4-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-
yl)butanamide (3a)
White powder (C2H5OH) (This compound was prepared
by heating under reflux a mixture of the ester (2) (2.76 g,
0.01 mol) and hydrazine hydrate (10 ml, 80%) in ethanol
(50ml)whilestirringfor8h. Thereactionmixturewascooled
and the solid produced was collected by filtration, dried and
recrystallized from ethanol. It was obtained as white solid);
Yield, 80%; mp 220–221 °C; IR (KBr) νmax 3286, 319,
302, 289, 1647 cm−1; 1HNMR (DMSO, 400 MHz): δ ꢀ2.02
(m, 2H, CH2CH2CH2), 2.24 (t, 2H, CH2C=O), 4.16 (s, 2H,
NH2, D2O exchangeable), 4.22 (t, 2H, N-CH2), 7.87–7.94
(m, 2H, H-6 and H-7 phthalazine), 7.97–7.98 (d, 1H, H-8
phthalazine, J ꢀ7.6 Hz), 8.20–8.22 (d, 1H, H-5 phthalazine,
J ꢀ7.6 Hz), 9.01 (s, 1H, NHNH2, D2O exchangeable),
11.87 (s, 1H, NH phthalazine, D2O exchangeable). 13CNMR
(DMSO, 400 MHz): δ ꢀ171.53 (C, NH2NHCO), 159.16
(C, C-4), 150.16 (C, C-1), 133.88 (CH, C-6), 132.62 (CH,
C-7), 129.13 (C, C-4a), 126.56 (C, C-8a), 124.95 (CH, C-
5), 123.89 (CH, C-8), 66.30 (CH2, NCH2), 30.52 (CH2,
COCH2), 24.96 (CH2, NCH2CH2); EIMS m/z 264 [M++2]
(4.11), 262 [M+] (37.17), 104 (45.35), 146 (100); Anal.
Calcd. for C12H14N4O3 (262): C, 54.96; H, 5.38; N, 21.36.
Found: C, 55.17; H, 5.42; N, 21.64.
Yellowish white powder (C2H5OH) (This compound was
prepared by heating a mixture of the ethyl ester (2) (0.28 g,
0.001 mol) and butyl amine (0.15 g, 0.002 mol) in ethanol
(30 ml) under reflux in ethanol (25 ml) for 5 h. The
reaction mixture was cooled and poured onto ice water
(200 ml) and the formed solid was washed with dil. HCl
and water, filtered and crystallized from ethanol. It was
obtained as light yellow solid); Yield, 85%; mp 262–264 °C;
IR (KBr) νmax3301, 3175, 3018, 2910, 1656 cm−1; 1HNMR
(DMSO, 400 MHz): δ ꢀ0.82 (3H, t, CH2CH3), 1.22 (2H,
m, CH2CH2CH3), 1.33 (2H, m, CH2CH2CH3), 2.04 (2H,
m, CH2CH2CH2), 2.28 (2H, t, CH2C=O), 3.04 (2H, s,
NHCH2), 4.23 (2H, t, N–CH2), 7.80 (1H, s, NH, D2O
exchangeable), 7.87–7.95 (2H, m, H-6 and H-7 phthalazine),
7.96–7.99 (1H, d, J=8.8 Hz, H-8 phthalazine), 8.20–8.23
(1H, d, J=8.4 Hz, H-5 phthalazine), 11.88 (1H, s, NH, D2O
exchangeable); 13CNMR (DMSO, 400 MHz): δ ꢀ171.72
(C, CONH), 159.15 (C, C-4), 150.18 (C, C-1), 133.83 (C,
C-6), 132.60 (CH, C-7), 129.14 (CH, C-4a), 126.55 (CH,
C-8a), 124.97 (CH, C-5), 123.91 (CH, C-8), 66.41(CH2,
NCH2), 38.56 (CH2, NHCH2), 32.45 (CH2, CH2CO), 31.70
(CH2, CH2CH2CH3), 24.99 (CH2, NCH2CH2), 19.99 (CH2,
CH2CH3), 14.08 (CH3, CH2CH3); EIMS m/z 303 [(M+]
(3.4), 86 (60.63), 142 (100); Anal. Calcd. for C16H21N3O3
(303): C, 63.35; H, 6.98; N, 13.85. Found: C, 63.62; H, 7.09;
N, 14.12.
123

Molecular Diversity
2-(3-(5-Mercapto-1,3,4-oxadiazol-2-yl)propyl)-2,3-
dihydrophthalazine-1,4-dione (5)
2-(3-(5-(Propylthio)-1,3,4-oxadiazol-2-yl)propyl)-2,3-
dihydrophthalazine-1,4-dione (6b)
Dark yellow powder (C2H5OH) (This compound was pre-
pared by dissolving the acid hydrazide (4) (2.62 g, 0.01 mol)
in a solution of potassium hydroxide (0.56 g, 0.01 mol) in
ethanol (50 ml) and water (4 ml). Carbon disulfide (1.52 g,
0.02 mol) was then added while stirring, and the reaction
mixture was refluxed for 20 h. The reaction mixture was
concentrated, cooled to room temperature and acidified with
diluted hydrochloric acid. The obtained solid was filtered,
washed with water and recrystallized from ethanol. It was
obtained as dark yellow solid); Yield, 90%; mp 246–248 °C;
IR (KBr) νmax3160, 3015, 2904, 2610, 1619 cm−1; 1HNMR
(DMSO, 400 MHz): δ ꢀ2.20 (2H, m, CH2CH2CH2), 2.97
(2H, t, CH2CH2CH2), 4.32 (2H, t, N-CH2), 7.85–7.90 (2H,
m, H-6 and H-7 phthalazine), 7.91 (1H, d, J ꢀ7.6, H-8
phthalazine), 8.19 (1H, d, J ꢀ8.9, H-5 phthalazine), 11.89
(1H, s, NH phthalazine) (D2O exchangeable), 14.29 (1H,
s, SH) (D2O exchangeable); 13CNMR (DMSO, 400 MHz):
δ ꢀ178.21 (C, C-4), 164.38 (C, C-2ꢁ), 159.16 (C, C-5ꢁ),
149.99 (C, C-1), 133.88 (CH, C-6), 132.65 (CH, C-7), 129.11
(C, C-4a), 126.58 (C, C-8a), 124.79 (CH, C-5), 123.80
(CH, C-8), 65.76 (CH2, NCH2), 24.87 (CH2, CH2CꢀN),
24.68 (CH2, NCH2CH2); EIMS m/z 304 [M+] (31.33), 163
(86.36), 143 (100).; Anal. Calcd. for C13H12N4O3S (304):
C, 51.31; H, 3.97; N, 18.41. Found: C, 51.53; H, 3.93; N,
18.67.
Yellowish white powder (C2H5OH) (This compound was
prepared by heating a mixture of 5 (0.61 g, 0.002 mol) and
propyl bromide (0.25 g, 0.002 mol) under reflux in dry ace-
tone (50 ml) in the presence of anhydrous K2CO3 (0.83 g,
0.006mol)for6hwhilestirring. Thereactionmixturewasfil-
tered, the solvent was evaporated, and the resulting product
was collected by filtration and recrystallized from ethanol.
It was obtained as yellowish white solid); Yield, 80%; mp
167–169 °C; IR (KBr) νmax 3170, 3019, 2920, 1660 cm−1
;
1HNMR(DMSO, 400MHz):δ ꢀ0.94(t, 3H, CH2CH3), 1.69
(m, 2H, CH2CH3), 2.24 (m, 2H, CH2CH2CH2), 3.08 (t, 2H,
CH2CH2CH2), 3.13 (t, 2H, S-CH2), 4.34 (t, 2H, N-CH2),
7.85–7.90 (m, 2H, H-6 and H-7 phthalazine), 7.92 (d, 1H,
H-8 phthalazine, J ꢀ7.9 Hz), 8.19 (d, 1H,H-5 phthalazine, J
ꢀ6.6 Hz) and 11.89 (s, 1H, NH phthalazine, D2O exchange-
able); Anal. Calcd. for C16H18N4O3S (346): C, 55.48; H,
5.24; N, 16.17. Found: C, 55.74; H, 5.31; N, 16.34.
2-(3-(5-(Butylthio)-1,3,4-oxadiazol-2-yl)propyl)-2,3-
dihydrophthalazine-1,4-dione (6c)
Light yellow powder (C2H5OH) (This compound was pre-
pared by heating a mixture of 5 (0.61 g, 0.002 mol) and
butyl bromide (0.27 g, 0.002 mol) under reflux in dry ace-
tone (50 ml) in the presence of anhydrous K2CO3 (0.83 g,
0.006mol)for6hwhilestirring. Thereactionmixturewasfil-
tered, the solvent was evaporated, and the resulting product
was collected by filtration and recrystallized from ethanol.
It was obtained as light yellow solid); Yield, 85%; mp
2-(3-(5-(Ethylthio)-1,3,4-oxadiazol-2-yl)propyl)-2,3-
dihydrophthalazine-1,4-dione (6a)
White powder (C2H5OH) (This compound was prepared by
heating a mixture of 5 (0.61 g, 0.002 mol) and ethyl bro-
mide (0.22 g, 0.002 mol) under reflux in dry acetone (50 ml)
in the presence of anhydrous K2CO3 (0.83 g, 0.006 mol)
for 6 h while stirring. The reaction mixture was filtered,
the solvent was evaporated, and the resulting product was
collected by filtration and recrystallized from ethanol. It
was obtained as white solid); Yield, 83%; mp 153–155 °C;
175–177 °C; IR (KBr) νmax 3168, 3018, 2927, 1661 cm−1
;
1HNMR (DMSO, 400 MHz): δ ꢀ0.89 (t, 3H, CH2CH3),
1.35 (m, 2H, CH2CH3), 1.64 (t, 2H, CH2CH2CH3), 2.24
(m, 2H, CH2CH2CH2), 3.08 (t, 2H, CH2CH2CH2), 3.15 (t,
2H, S-CH2) 4.34 (t, 2H, N-CH2), 7.85–7.90 (m, 2H, H-6
and H-7 phthalazine), 7.92–7.94 (d, 1H, H-8 phthalazine, J
ꢀ8.8 Hz), 8.20–8.22 (d, 1H,H-5 phthalazine, J ꢀ8.4 Hz),
11.90 (s, 1H, NH phthalazine, D2O exchangeable); Anal.
Calcd. for C17H20N4O3S (360): C, 56.65; H, 5.59; N, 15.54.
Found: C, 56.81; H, 5.67; N, 15.82.
IR (KBr) νmax 3175, 3022, 2922, 1660 cm−1 13CNMR
;
(DMSO, 400 MHz): δ ꢀ167.93 (C, C-4), 163.56 (C, C-2ꢁ),
159.15 (C, C-5ꢁ), 149.98 (C, C-1), 133.85 (CH, C-6), 132.66
(CH, C-7), 129.11 (C, C-4a), 126.57 (C, C-8a), 124.79 (CH,
C-5), 123.77 (CH, C-8), 65.81 (CH2, NCH2), 26.87 (CH2,
CH2C=N), 25.51 (CH2, SCH2), 22.33 (CH2, NCH2CH2),
15.23 (CH3, CH2CH3); EIMS m/z 332 [M+] (1.3), 298
(25.2), 171 (100); Anal. Calcd. for C15H16N4O3S (332):
C, 54.20; H, 4.85; N, 16.86. Found: C, 54.37; H, 4.88; N,
17.09.
Methyl 2-((5-(3-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-
yl)propyl)-1,3,4-oxadiazol-2-yl)sulfanyl)acetate (7a)
White powder (C2H5OH) (This compound was prepared by
heating under reflux a mixture of 5 (0.61 g, 0.002 mol)
and methyl chloroacetate (0.22 g, 0.002 mol) in dry ace-
tone (50 ml) in the presence of anhydrous K2CO3 (0.83 g,
0.006 mol) for 8 h while stirring. The reaction mixture was
filtered, the solvent was evaporated, and the resulting product
was collected by filtration and recrystallized from ethanol. It
123

Molecular Diversity
was obtained as white solid); Yield, 70%; mp 140–142 °C; IR
(KBr) νmax 3166, 3010, 2896, 1727, 1654 cm−1 13CNMR
(418): C, 54.53; H, 5.30; N, 13.39. Found: C, 54.70; H, 5.39;
N, 13.58.
;
(DMSO, 400 MHz): δ ꢀ168.17 (C, C-4), 162.79 (C, C-2ꢁ),
159.16 (C, C-5ꢁ), 149.99 (C, C-1), 133.88 (CH, C-6), 132.66
(CH, C-7), 129.13 (C, C-4a), 126.59 (C, C-8a), 124.81 (CH,
C-5), 123.79 (CH, C-8), 65.70 (CH2, NCH2), 61.97 (CH3,
SCH3), 34.15 (CH2, SCH2), 25.49 (CH2, CH2CꢀN), 22.24
(CH2, NCH2CH2); EIMS m/z 376 [M+] (2.5), 215 (54.4),
162 (100); Anal. Calcd. for C16H16N4O5S (376): C, 51.06;
H, 4.28; N, 14.89. Found: C, 51.32; H, 4.34; N, 15.06.
2-(4-(3,5-Dimethyl-1H-pyrazol-1-yl)-4-oxobutyl)-2,3-
dihydrophthalazine-1,4-dione (9)
White powder (C2H5OH) (This compound was prepared by
the addition of acetylacetone (0.40 g, 0.004 mol) to a solution
of the acid hydrazide (4) (1.05 g, 0.004 mol) in dioxan (20 ml)
and few drops of TEA. The reaction mixture was refluxed for
6 h, concentrated and cooled to room temperature, and the
formed precipitate was filtered and crystallized from ethanol.
It was obtained as white solid); Yield, 65%; mp 182–184 °C;
Ethyl 2-((5-(3-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-
yl)propyl)-1,3,4-oxadiazol-2-yl)sulfanyl)acetate (7b)
White powder (C2H5OH) (This compound was prepared by
heating under reflux a mixture of 5 (0.61 g, 0.002 mol) and
ethyl chloroacetate (0.25 g, 0.002 mol) in dry acetone (50 ml)
in the presence of anhydrous K2CO3 (0.83 g, 0.006 mol)
for 8 h while stirring. The reaction mixture was filtered, the
solvent was evaporated, and the resulting product was col-
lected by filtration and recrystallized from ethanol. It was
obtained as white solid); Yield, 65%; mp 154–156 °C; IR
(KBr) νmax 3165, 3006, 2909, 1718, 1671 cm−1; 1HNMR
(DMSO, 400 MHz): δ ꢀ1.18 (t, 3H, CH2CH3), 2.25 (m,
2H, CH2CH2CH2), 3.09 (t, 2H, CH2CH2CH2), 4.12 (t, 2H,
N-CH2), 4.15 (q, 2H, O-CH2CH3), 4.33 (t, 2H, S-CH2),
7.86–7.90 (m, 2H, H-6 and H-7 phthalazine), 7.91–7.93 (d,
1H, H-8 phthalazine, J ꢀ7.9 Hz), 8.20–8.22 (d, 1H, H-
5 phthalazine, J ꢀ7.8 Hz), 11.89 (s, 1H, NH phthalazine,
D2O exchangeable); Anal. Calcd. for C17H18N4O5S (390):
C, 52.30; H, 4.65; N, 14.35. Found: C, 52.48; H, 4.72; N,
14.54.
IR (KBr) νmax 3169, 3012, 2917, 1653 cm−1 1HNMR
;
(DMSO, 400 MHz): δ ꢀ2.11 (s, 3H, CH3-3 pyrazole), 2.19
(m, 2H, CH2CH2CH2), 2.43(s, 3H, CH3-5pyrazole), 3.26(t,
2H, CH2C=O), 4.34 (t, 2H, N-CH2), 6.12 (s, 1H, CH pyra-
zole), 7.86–7.90 (m, 3H, H-6, H-7 and H-8 phthalazine),
8.19–8.21 (d, 1H, H-5 phthalazine, J ꢀ8.4 Hz), 11.88 (s,
1H, NH phthalazine, D2O exchangeable); Anal. Calcd. for
C17H18N4O3 (326): C, 62.57; H, 5.56; N, 17.17. Found: C,
62.67; H, 5.61; N, 17.42.
Ethyl 3-(2-(4-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-
yl)butanoyl)hydrazono)butanoate (10)
Yellowish white powder (C2H5OH) (This compound was
prepared by addition of ethyl acetoacetate (0.26 g, 0.002 mol)
to a solution of the acid hydrazide (4) (0.52 g, 0.002 mol)
in ethanol (20 ml). The reaction mixture was refluxed
for 6 h, concentrated and cooled to room temperature
and the solid product was filtered and recrystallized from
ethanol. It was obtained as yellowish white solid); Yield,
68%; mp 167–169 °C; IR (KBr) νmax 3187, 3029, 2923,
Ethyl 4-((5-(3-(1,4-dioxo-3,4-dihydrophthalazin-2(1H)-
yl)propyl)-1,3,4-oxadiazol-2-yl)thio)butanoate (8)
1
White powder (C2H5OH) (This compound was prepared by
heating under reflux a mixture of 5 (0.61 g, 0.002 mol)
and ethyl 4-bromobutanoate (0.39 g, 0.002 mol) in dry ace-
tone (50 ml) in the presence of anhydrous K2CO3 (0.83 g,
0.006 mol) for 10 h while stirring. The reaction mixture was
filtered, the solvent was evaporated, and the resulting product
was collected by filtration and recrystallized from ethanol. It
was obtained as white solid); Yield, 78%; mp 185–187 °C; IR
(KBr) νmax 3168, 2989, 2907, 1732, 1671 cm−1; 1HNMR
(DMSO, 400 MHz): δ ꢀ1.17 (t, 3H, CH2CH3), 1.96 (m,
2H, S-CH2CH2CH2), 2.26 (m, 2H, N–CH2CH2CH2), 2.41
(t, 2H, CH2CH2CH2C=O), 3.08 (t, 2H, N–CH2CH2CH2),
3.20 (t, 2H, S-CH2), 4.04 (q, 2H, O-CH2CH3), 4.34 (t,
2H, N-CH2), 7.85–7.88 (m, 2H, H-6 and H-7 phthalazine),
7.90–7.92 (d, 1H, H-8 phthalazine, J ꢀ8 Hz), 8.20–8.22 (d,
1H, H-5 phthalazine, J ꢀ8 Hz) and 11.89 (s, 1H, NH phtha-
lazine, D2O exchangeable); Anal. Calcd. for C19H22N4O5S
1730, 1660 cm−1; HNMR (DMSO, 400 MHz): δ ꢀ1.18
(t, 3H, CH3CH2), 1.87 (s, 3H, CH3C=N), 2.07 (m, 2H,
CH2CH2CH2), 2.47 (t, 2H, CH2CONH), 3.29 (s, 2H,
CH2COO), 4.06 (q, 2H, CH2CH3), 4.27 (t, 2H, N-CH2),
7.86–7.93 (m, 2H, H-6 and H-7 phthalazine), 7.96–7.98
(d, 1H, H-8 phthalazine, J ꢀ7.3 Hz), 8.20–8.22 (d, 1H,
H-5 phthalazine, J ꢀ7.3 Hz), 10.23 (s, 1H, CONHN,
D2O exchangeable), 11.87 (s, 1H, NH phthalazine, D2O
exchangeable); 13CNMR (DMSO, 400 MHz): δ ꢀ174.25
(C, COO), 170.10 (C, C-4), 168.50 (C, CONH), 159.16 (C,
C-1), 150.17 (C, CH3C=N), 133.89 (CH, C-6), 132.59 (CH,
C-7), 129.14 (C, C-4a), 126.56 (C, C-8a), 124.95 (CH, C-
5), 123.83 (CH, C-8), 66.40 (CH2, CH2CH3), 60.76 (CH2,
NCH2), 44.38 (CH2, CH2COO), 31.09 (CH2, CH2CONH),
29.43 (CH2, NCH2CH2), 16.61 (CH3, CH2CH3), 14.44
(CH3, CH3); Anal. Calcd. for C18H22N4O5 (374): C, 57.75;
H, 5.92; N, 14.96. Found: C, 58.02; H, 5.99; N, 15.08.
123

Molecular Diversity
Nꢀ-(2-Chloroacetyl)-4-(1,4-dioxo-3,4-dihydrophthalazin-
2(1H)-yl)butanehydrazide (11)
Anticonvulsant screening
Thebiologicalscreeningforourdesignedcompoundsasanti-
convulsant agents was obtained according to the reported
procedures [23, 24].
White powder (C2H5OH) (This compound was prepared
by drop-wise addition of chloroacetyl chloride (0.23 g,
0.002 mol) to the stirred solution of the acid hydrazide (4)
(0.52 g, 0.002 mol) in dry DMF (20 ml) in the presence of
potassium carbonate (0.55 g, 0.004 mol) using ice bath. After
addition, the solution was stirred at room temperature for 1 h.
The contents of the reaction flask were then poured slowly
into crushed ice with continuous stirring and the resulting
precipitate was filtered, washed with water, dried and crys-
tallized from ethanol. It was obtained as white solid); Yield,
72%; mp 236–238 °C; IR (KBr) νmax 3172, 3020, 2915,
Results and discussion
Chemistry
The synthetic strategy for preparation of the target com-
pounds (2–12) is depicted in Schemes 1 and 2. Follow-
ing the reported procedure [22], 2,3-dihydrophthalazine-
1,4-dione 1 was synthesized by reaction of hydrazine
hydrate with phthalic anhydride. Ethyl 4-(1,4-dioxo-3,4-
dihydrophthalazin-2(1H)-yl)butanoate 2 was then synthe-
sized by heating 1 with ethyl 4-bromobutanoate. Heating
the ethyl ester 2 under reflux with the appropriate amine
furnished the corresponding acid amide 3a–b. On the other
hand, the corresponding acid hydrazide 4 was obtained by
refluxing the ethyl ester 2 with hydrazine hydrate. The acid
hydrazide 4 underwent cyclization with carbon disulfide
giving the corresponding 5-sulfanyloxadiazole 5 deriva-
tive. Treatment of 5-sulfanyloxadiazole derivative 5 with
the appropriate alkyl bromide, alkyl 2-chloroacetate and/or
ethyl 4-chlorobutanoate yielded the corresponding alkyl
derivatives 6a–c, ester derivatives 7a–b and/or 8, respectively
(Scheme 1). Condensation of acid hydrazide 4 with acetyl
acetone and/or ethyl acetoacetate afforded the corresponding
pyrazole derivative 9 and/or ethyl hydrazonobutanoate 10.
Stirring of the acid hydrazide 4 with chloroacetyl chloride
yielded the corresponding 2-chloroacetohydrazide deriva-
tive 11, while when using heat afforded the corresponding
3,6-dioxohexahydropyridazine derivative 12, respectively
(Scheme 2).
1
1727, 1657 cm−1; HNMR (DMSO, 400 MHz): δ ꢀ2.06
(m, 2H, CH2CH2CH2), 2.52 (t, 2H, CH2CꢀO), 4.04 (t, 2H,
N-CH2), 4.26 (s, 2H, CH2-Cl), 7.88–7.90 (m, 2H, H-6 and
H-7 phthalazine), 7.95 (d, 1H, H-8 phthalazine, J ꢀ8.2 Hz),
8.19–8.21 (d, 1H, H-5 phthalazine, J=8.2 Hz), 10.15 (s, 1H,
NH, D2O exchangeable), 10.41 (s, 1H, NH, D2O exchange-
able), 11.88 (s, 1H, NH phthalazine, D2O exchangeable);
EIMS m/z 340 [M++1] (3.59), 339 [M+] (11.72), 86 (100);
Anal. Calcd. for C14H15ClN4O4 (338.5): C, 49.64; H, 4.46;
N, 16.54. Found: C, 49.81; H, 4.48; N, 16.78.
2-(2-(3,6-Dioxohexahydropyridazin-4-yl)ethyl)-2,3-
dihydrophthalazine-1,4-dione (12)
Yellowish white powder (C2H5OH) (This compound was
prepared by drop-wise addition of chloroacetyl chloride
(0.23 g, 0.002 mol) to the stirred solution of the acid
hydrazide (4) (0.52 g, 0.002 mol) in dry DMF (20 ml) in
the presence of potassium carbonate (0.55 g, 0.004 mol)
using ice bath. After addition, the solution was stirred at
room temperature for 1 h and then refluxed for 4 h. The
contents of the reaction flask were then poured slowly into
crushed ice with continuous stirring, and the resulting precip-
itate was filtered, washed with water, dried and crystallized
fromethanol. Itwasobtainedasyellowishwhitesolid);Yield,
65%; mp 157–159 °C; IR (KBr) νmax 3185, 3055, 2925,
Docking studies
In the present work, all modeling experiments were per-
formed using Molsoft software. Each experiment used
AMPA [25], downloaded from the Brookhaven Protein Data-
bank(http://www.rcsb.org/pdb/explore/explore.do?structure
Id=1FTL).
All studied ligands have similar position and orientation
inside the supposed AMPA receptor binding site. The active
site showed a large space which serves as entry channel
for the docked compounds (Fig. 5). There is a relationship
between the affinity of organic molecules and the free energy
of binding (ꢀG) [26–28]. This relationship enables us to
predict and interpret the activity of the organic compounds
toward the specific target protein. The most of the designed
compounds exhibited good binding affinity toward AMPA
1670, 1620 cm−1
;
13CNMR (DMSO, 400 MHz): δ ꢀ173.03
(C, C-3ꢁ, C-6ꢁ), 159.15 (C, C-4), 150.07 (C, C-1), 133.89
(CH, C-6), 132.64 (CH, C-7), 129.14 (C, C-4a), 126.58 (C,
C-8a), 124.90 (CH, C-5), 123.82 (CH, C-8), 60.30 (CH2,
NCH2), 30.84 (CH2, C-5ꢁ), 24.27 (CH, C-4ꢁ), 14.53 (CH2,
NCH2CH2); EIMS m/z 302 [M+] (1.7), 104 (64), 162 (100);
Anal. Calcd. for C14H14N4O4 (302): C, 55.63; H, 4.67; N,
18.53. Found: C, 55.72; H, 4.81; N, 18.53.
Docking studies
In the present work, all modeling experiments were carried
out according to the reported procedure [6].
123

Molecular Diversity
Scheme 1 Synthetic route for preparation of the target compounds 2–8
receptor and the computed values reflected the overall trend
(Table 1).
bic pocket formed by Leucine138, Glycine141, Serine142,
Threonine143, Tyrosine190, Leucine191, Lucine192, Gluta-
mate193, Threonine173 and Threonine174. The oxadiazole
moiety occupied the hydrophobic pocket formed by Tyro-
sine61, Leucine90, Threonine91 and Tyrosine220. The distal
propyl linker occupied the hydrophobic cleft formed by Tyro-
sine220, Glutamate193, Proline89 and Glutamate13. The
ethyl group of the distal ester moiety occupied the hydropho-
bic pocket formed by Thereonine195, Methionine196 and
Tyrosine16 (Fig. 6). These interactions of compound 8 may
explain the highest anticonvulsant activity.
The proposed binding mode of compound 7b revealed
affinity value of − 101.68 kcal/mol and 9 H-bonds. The
carbonyl group at position-1 was stabilized by formation
of 2 H-bonds with Serine142 (2.78 Å) and Threonine143
(1.84 Å). The other carbonyl group at position-4 formed 1
The proposed binding mode of compound 8 revealed
affinity value of − 112.27 kcal/mol and 9 H-bonds. The car-
bonyl group at position-1 was stabilized by formation of 2
H-bonds with Threonine143 (2.50 Å and 2.68 Å). The other
carbonyl group at position-4 formed 1 H-bond with Thre-
onine174 (2.37 Å). The nitrogen atom at position-3 of the
oxadiazole linker moiety formed one H-bond with Thre-
onine91 (1.91 Å), while the other nitrogen at position-4
formed3H-bondswithThreonine91(2.44Å)andArginine96
(2.09 Å and 2.58 Å). The carbonyl group of the ester moiety
formed 1 H-bond with Tyrosine16 (–OH group) with a dis-
tance of 1.48 Å, and the oxygen atom also formed another
H-bond with Tyrosine16 (–OH group) with a distance of
2.43 Å. The phthalazine nucleus occupied the hydropho-
123

Molecular Diversity
Scheme 2 Synthetic route for preparation of the target compounds 9–12
Table 1 Calculated ꢀG (free energy of binding) and binding affinities
for the ligands (ꢀG in Kcal/mol)
Compound
ꢀG
Compound
ꢀG
[kcal mol−1
]
[kcal mol−1
]
2
−75.67
−100.12
−97.92
−67.12
−76.18
−83.83
−88.52
−91.27
−101.12
−101.68
8
−112.27
−76.43
−100.77
−75.55
−75.86
−73.49
−73.99
−58.04
−73.41
−62.59
3a
3b
4
9
10
11
5
12
6a
6b
6c
7a
7b
Comp. (V)
Comp. VI
Comp. VII
GYKI 53655
Diazepam
Fig. 5 Superimposition of some docked compounds inside the binding
pocket of 1FTL
Glutamate193, Threonine173 and Threonine174. The oxa-
diazole moiety occupied the hydrophobic pocket formed
by Tyrosine61, Leucine90 and Threonine91. The ethyl
group of the distal ester moiety occupied the hydrophobic
pocket formed by Glutamate13, Tyrosine16, Proline89, Tyro-
sine220, Thereonine195 and Methionine196 (Fig. 7). These
interactions of compound 7b may explain the high anticon-
vulsant activity.
The proposed binding mode of compound 7a revealed
affinity value of -101.12 kcal/mol and 9 H-bonds. The
carbonyl group at position-1 was stabilized by formation
of 2 H-bonds with Serine142 (2.53 Å) and Threonine143
H-bond with Threonine174 (2.00 Å). The two nitrogen atoms
of the oxadiazole linker moiety formed 4 H-bonds with Argi-
nine96 (1.31 Å, 1.87 Å, 2.23 Å and 2.39 Å). The carbonyl
group of the ester moiety formed 1 H-bond with Tyrosine61
(–OH group) with a distance of 2.73 Å, and the oxygen atom
formed another H-bond with Tyrosine220 (–OH group) with
a distance of 1.84 Å. The phthalazine nucleus occupied the
hydrophobicpocketformedbyLeucine138, Glycine141, Ser-
ine142, Threonine143, Tyrosine190, Leucine191, Lucine192,
123

Molecular Diversity
Fig. 8 Predicted binding mode for 7a with 1FTL
Tyrosine61, Leucine90 and Threonine91. The methyl group
of the distal ester moiety occupied the hydrophobic pocket
formed by Glutamate13, Proline89, Tyrosine220, Thereo-
nine195 and Methionine196 (Fig. 8). These interactions of
compound 7a may explain the high anticonvulsant activity.
The proposed binding mode of compound 10 revealed
affinity value of −100.77 kcal/mol and 7 H-bonds. The car-
bonyl group at position-1 was stabilized by formation of 2 H-
bonds with Serine142 (2.50 Å) and Threonine143 (1.61 Å).
The other carbonyl group at position-4 formed 1 H-bond
with Glutamate193 (2.77 Å). The carbonyl group of the acid
hydrazonelinkerformed2H-bondswithArginine96 (2.73Å)
and Threonine91 (1.88 Å), while NH formed another H-bond
with Threonine91 (2.25 Å). The oxygen atom of the distal
ester formed H-bond with Tyrosine220 (–OH group) with
a distance of 2.78 Å. The phthalazine nucleus occupied the
hydrophobicpocketformedbyLeucine138, Glycine141, Ser-
ine142, Threonine143, Tyrosine190, Leucine191, Lucine192,
Glutamate193, Threonine173 and Threonine174. The distal
2-propyl linker occupied the hydrophobic cleft formed by
Tyrosine61, Proline89, Leucine90, Threonine91 and Tyro-
sine220. The ethyl group of the distal ester moiety occupied
the hydrophobic pocket formed by Glutamate13, Tyrosine16,
Tyrosine61, Thereonine195 and Methionine196 (Fig. 9).
These interactions of compound 10 may explain the high
anticonvulsant activity.
Fig. 6 Predicted binding mode for 8 with 1FTL
Fig. 7 Predicted binding mode for 7b with 1FTL
(1.91 Å). The other carbonyl group at position-4 formed 1
H-bond with Threonine174 (2.31 Å). The two nitrogen atoms
of the oxadiazole linker moiety formed 4 H-bonds with Argi-
nine96 (1.92 Å, 1.97 Å, 2.40 Å and 2.49 Å). The carbonyl
group of the ester moiety formed 1 H-bond with Tyrosine61
(–OH group) with a distance of 2.80 Å, and the oxygen atom
formed another H-bond with Tyrosine220 (–OH group) with
a distance of 2.02 Å. The phthalazine nucleus occupied the
hydrophobicpocketformedbyLeucine138, Glycine141, Ser-
ine142, Threonine143, Tyrosine190, Leucine191, Lucine192,
Glutamate193, Threonine173 and Threonine174. The oxadi-
azole moiety occupied the hydrophobic pocket formed by
Anticonvulsant screening
The anticonvulsant activity of the target phthalazine deriva-
tives was evaluated against pentylenetetrazole-induced con-
vulsions in mice following the reported procedure [29].
The most active compounds were evaluated against maxi-
mal electroshock (MES) seizure tests by the use of standard
techniques [30], while the acute neurotoxicity was done
123

Molecular Diversity
the distal ester (COO) as hydrogen bond acceptor (HBA)
to reach the other hydrogen bond area (high electron den-
sity) and easily form hydrogen bonding interactions with
the amino acid residue Tyrosine16. These long chain link-
ers impart a good distance for the distal ethyl ester moiety to
occupy a new hydrophobic cleft formed by Thereonine195,
Methionine196 and Tyrosine16 in the active site. As planed
these modifications may explain the highest binding affinities
and the highest anticonvulsant activities. The fixed oxadi-
azole HBA as in compounds 8 and 7a–b exhibited higher
activities than the free rotated C=O as HBA and NH as HBD
as in compounds 10 and 3a. In addition, the carbonyl group at
position-4 increased water solubility and the affinity toward
the AMPA receptor by formation of H-bond commonly with
the amino acid residue Threonine174. The ester derivatives
showed higher activities than the amide ones. From the
structure of the substituted compounds at N-2 and the data
shown in Table 2, we can divide these tested compounds
into four groups. The first group is ester group derivatives
which include 7a, 7b, 8 and 10. In this group, the presence of
oxadiazole moiety as in 7a, 7b and 8 showed more activities
than the acid hydrazone derivative 10. Additionally, the pres-
ence of long chain linker propylene as in 8 exhibited higher
activities than the methylene one as in 7a–b. Furthermore, the
more electron-releasing ethyl ester group 7b showed higher
activity than the methyl one 7a. The second group is the
amide derivatives 3a and 3b. The electron-releasing butyl
moiety at 3a highly increased the activity than the elec-
tron withdrawing benzyl moiety at 3b in spite of nearly the
same lipophilicity. The third group is the unsubstituted oxa-
diazole 5 and S-alkyl 6a–c group. The S-Alkyl derivatives
6a-c exhibited higher activities than the unsubstituted one as
in compound 5. Among these compounds, the presence of
highly lipophilic electron-releasing butyl group at 6c highly
increasedthe activitythanthe propyl groupat 6b and theethyl
one at 6a. The fourth group is the unsubstituted heterocyclic
group as in compounds 9 and 12. Among these compounds,
the presence of three carbon linker was preferred than the two
carbon one. Moreover, the more lipophilic heterocycle 3,5-
dimethylpyrazole at 9 highly increased the activity than the
less lipophilic heterocycle 3,6-dioxohexahydropyridazine at
12. These findings enable us to conclude that the presence
of lipophilic electron donating groups highly increased the
activity than the less lipophilic and/or the electron withdraw-
ing one. The long chain linker is preferred than the short one.
The substituted heterocyclic ring with another long chain
linker displayed higher potency than the short one and/or the
unsubstituted ones.
Fig. 9 Predicted binding mode for 10 with 1FTL
according to the method described by Dunham and Miya
[31]. The scPTZ and MES screens were the good predictors
for anticonvulsants against absence and generalized tonic—
clonic seizures, respectively [32].
In the present study, twelve phthalazine derivatives were
screened in vivo for their anticonvulsant activity against
pentylenetetrazole-induced convulsions in mice [29]. The
selected drugs were given i.p. with a range of doses to groups
of mice until four points were established in the range of
10–90%seizureprotectionatleast. ED50 wascalculatedfrom
the obtained data. Diazepam was used as a standard anticon-
vulsant drug. Compounds 8, 7b, 7a, 10 and 3a showed the
highest anticonvulsant activities in experimental mice with
relative potencies of 1.78, 1.66, 1.60, 1.59 and 1.29, respec-
tively. Compounds 3b and 6c exhibited relative potencies
of 0.72 and 0.77 of that of diazepam, respectively. Other
compounds exhibited relative potencies less than 50% of
diazepam, where compounds 5, 12 and 9 exhibited nearly
the same lowest relative potency of 0.32, 0.32 and 0.35,
respectively. Compounds 6a and 6b exhibited nearly the same
relative potency of 0.47 and 0.49, respectively.
Structure activity relationship (SAR) studies showed that
substitution on the phthalazine ring and the electronic nature
and lipophilicity of these substituents led to a consider-
able variation in the anticonvulsant activity. The presence
of oxadiazole ring spacer attached to long chain linker
(CH2CH2CH2) enables the two nitrogen atoms, as hydro-
gen bond acceptor (HBA), to reach the hydrogen bond area
(high electron density) and easily form hydrogen bonding
interactions with the essential amino acid residue Argi-
nine96. Moreover, the presence of another long chain linker
(SCH2CH2CH2) attached to the oxadiazole moiety enables
Compounds 8, 7b, 7a, 10 and 3a were subjected to max-
imal electroshock seizure (MES) test. Results of the MES
model revealed that compounds 8, 7b and 3a showed 100%
protection at a dose level of 125 μgm/kg, while compounds
123

Molecular Diversity
Table 2 Anticonvulsant activity of the selected compounds
Test
comp.
Dose
(mcg/kg)
No. of
protected
animal
PTZ protection
(%)
M. wt
ED50 (mcg/kg)
125
ED50
(μmol/kg)
Relative
potency
3a
250
500
5
6
6
3
5
6
1
3
6
2
4
5
2
4
6
3
4
6
5
6
6
5
6
6
5
6
6
1
3
5
5
6
6
1
3
5
1
3
6
83.33
100.00
100.00
50.00
83.33
100.00
16.67
50.00
100.00
33.33
66.67
83.33
33.33
66.67
100.00
50.00
66.67
100.00
83.33
100.00
100.00
83.33
100.00
100.00
83.33
100.00
100.00
16.67
50.00
83.33
83.33
100.00
100.00
16.67
50.00
83.33
16.67
50
303.36
337.38
304.33
332.38
346.41
360.44
376.39
390.42
418.48
326.36
374.4
0.41
1.29
1000
250
3b
250
0.74
1.64
1.13
1.08
0.69
0.33
0.32
0.30
1.53
0.33
1.65
0.53
0.72
0.32
0.47
0.49
0.77
1.60
1.66
1.78
0.35
1.59
0.32
1.00
500
1000
250
5
500
500
1000
250
6a
375
500
1000
250
6b
375
500
1000
250
6c
250
500
1000
250
7a
125
500
1000
250
7b
125
500
1000
250
8
125
500
1000
250
9
500
500
1000
250
10
125
500
1000
250
12
302.29
284.74
500
500
1000
75
Diazepam
150
150
300
100
7a and 10 exhibited 83.33% protection at the same dose level
(Table 3).
Neurotoxicity, the median toxic dose producing minimal
neurological toxicity in 50% of mice (TD50), protective index
(PI)ꢀ(TD50/ED50), the margin of safety and tolerability
between anticonvulsant doses and doses of anticonvulsant
drugs exerting acute adverse effects like sedation, motor
coordination impairment, ataxia, or any other neurotoxic
manifestations [33] are calculated and listed in Table 3.
Sodium valproate with ED50 value of 200 mg/kg was used
as a reference drug. The (ED50) values of the selected
compounds were found to be smaller than the reference anti-
123

Molecular Diversity
Table 3 MES (protection %), ED50, TD50, LD50, therapeutic index (TI) and protective index (PI) for the most active compounds 5a, 9b and 9h
compared to the reference drug Valproate
Compound
MES
protection %
ED50
(mcg/kg)
TD50
(mcg/kg)
LD50
(mcg/kg)
Therapeutic
index (TI)
Protective
index (PI)
8
100
125
125
125
125
125
200
550
500
450
450
500
450
950
850
850
750
700
500
7.60
6.80
6.80
6.00
5.60
2.50
4.40
4.00
3.60
3.60
4.00
2.25
7b
7a
10
3a
100
83.33
83.33
100
Valproate
(mg/kg)
100
ED50 ꢀmedian effective dose providing anticonvulsant protection in 50% of mice against pentylenetetrazole (PTZ)-induced seizures
TD50 ꢀmedian toxic dose producing minimal neurological toxicity in 50% of mice
LD50 ꢀmedian lethal dose that causes 50% mortality in mice
Therapeutic indexꢀLD50/ED50
Protective indexꢀTD50/ED50
convulsant drugs at molar doses. The most potent compounds
8, 7b, 7a, 10 and 3a had (TD50) values of 550, 500, 450,
450 and 500 mcg/kg, respectively. These values revealed that
compounds 8, 7b and 3a agents exerted lower neurological
disorders, while 7a and 10 exerted low neurological disor-
ders as that of valproate. From the results of ED50 and TD50,
compounds 8, 7b, 7a, 10 and 3a had higher (PI) values of
4.40, 4.00, 3.60, 3.6 and 4.00, respectively. This means our
synthesized compounds have higher safety margin than the
reference drug. The therapeutic index (TI)ꢀ(LD50/ED50)
[34] the comparison of the amount of a therapeutic agent
that causes the therapeutic effect to the amount that causes
death in mice was calculated. Compounds 8, 7b, 7a, 10 and
3a had (LD50) values of 950, 850, 850, 750 and 700mcg/kg
with (TI) values of 7.60, 6.80, 6.80, 6.00 and 5.60, respec-
tively. It is worthwhile to note that (TI) values of the most
potent compounds were found to be higher than those of the
reference anticonvulsant drug as shown in Table 3.
Table 4 C log P, total molecular polar surface area and molecular vol-
ume of the synthesized compounds and reference ligands
Comp. no.
C log Pa
TPSAb
M.V.c
3a
1.78
1.74
0.79
1.37
1.88
2.43
0.86
1.24
1.47
1.63
0.83
0.34
3.89
2.95
5.66
3.02
2.74
2.80
83.97
83.97
93.79
93.79
93.79
93.79
120.10
120.10
120.10
89.76
122.63
113.06
79.96
108.49
63.99
89.19
32.67
37.30
285.11
306.36
249.09
282.92
299.72
316.52
310.88
327.69
361.29
391.49
336.10
255.52
332.43
333.21
359.84
314.01
246.54
156.79
3b
5
6a
6b
6c
7a
7b
8
9
10
12
Comp. V
Comp. VI
Comp. VII
GYKI 53655
Diazepam
Valproate
C log P, total molecular polar surface area
and molecular volume correlation
aOctanol–water partition coefficient C log P
bTotal molecular polar surface area
cMolecular volume
Most AMPA receptor antagonists showed limited water
solubility which restricted efforts to formulate acceptable
parenteral solutions [35].
C log P values were calculated as a measurement for the
lipophilicity factor which could be attributed in their anti-
convulsant activity. Lipophilic substances are able to cross
blood brain barrier (BBB) in a relatively easy way [36, 37].
The C log P values played an important role in the activities
among this derivative series. The C log P data for all selected
anticonvulsant compounds are explained in Table 4 ranging
from 0.34 to 2.43. C log P for SYM 2189 (V), VI, VII and
GYKI 53655 were calculated and were found to be 3.89,
2.95, 5.66 and 3.02, respectively. It is worthwhile to note
that the C log P values for our designed compounds were
less than those for the reference ligands SYM 2189 (V), VI,
VII and GYKI 53655 which means that the water solubility
of our designed compounds was higher than that of the refer-
ence ligands. The improved aqueous solubility may explain
the higher affinity and selectivity toward AMPA receptor.
Although compounds 3a and 3b have nearly the same C log
P values (1.78 and 1.74, respectively), it showed high anti-
convulsant potency difference (1.29 and 0.72, respectively),
123

Molecular Diversity
Table 5 Predicted ADMET for the designed compounds and reference drugs
Comp. no.
BBBa
HIA
(%)b
PPB
Hepatotoxicity
probabilityd
Hepatotoxicity
level
CYP2D6e
Solubility
(mg/L)f
levelc
3a
0.066
0.035
0.088
0.023
0.018
0.016
0.051
0.055
0.062
0.028
0.056
0.045
0.135
0.029
0.558
0.024
2.582
0.912
92.08
93.65
86.33
94.17
94.94
95.60
86.77
88.35
90.99
96.33
86.77
80.07
94.57
95.82
97.37
94.90
99.49
93.72
1
2
2
2
2
2
0
0
0
1
1
2
2
2
2
0
2
0
0.470
0.430
0.741
0.536
0.569
0.576
0.675
0.470
0.331
0.264
0.198
0.735
0.536
0.682
0.947
0.721
0.708
0.006
0
0
1
1
1
1
1
0
0
0
0
1
1
1
1
1
1
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
682.42
179.80
109.71
112.61
33.68
14.36
123.45
76.48
89.28
938.62
2104.07
5340.14
1.52
3b
5
6a
6b
6c
7a
7b
8
9
10
12
Comp. V
Comp. VI
Comp. VII
GYKI 53655
Diazepam
Valproate
0.14
0.003
3.31
6.18
810.56
aBBB, in vivo blood brain barrier penetration (C. brain/C. blood)
bHuman intestinal absorption (%)
cPBB, plasma protein binding, 0 means less than 90%, 1 means more than 90%, 2 means more than 95%
dHepatotoxicity probability, value>0.5 means toxic, value<0.5 means non-toxic
eCYP2D6, cytochrome P2D6, 0ꢀnon-inhibitor, 1ꢀinhibitor
fCalculated water solubility in pure water by SK atomic types (mg/L)
with slight effect due to the lipophilicity factor, while com-
pounds 6c had higher C log P values than 6b and 6a (2.43,
1.88 and 1.77, respectively) and had good correlation with
the lipophilicity factor. Interestingly, compounds 8, 7b, 7a
and 10 which exhibited higher potency values of 1.78, 1.66,
1.60 and 1.59, respectively, had C log P values of 1.47, 1.24,
0.86 and 0.83, respectively, with good correlation with the
lipophilicity factor. On the other hand, although the values of
C log P for compounds 5, 9 and 12 were 0.79, 1.63 and 0.34,
respectively, they exhibited nearly the same relative potency
values of 0.32, 0.35 and 0.32, respectively, with no significant
effect on biological activity.
The molecules with total polar surface area (TPSA) more
than 140 have low oral bioavailability [38]. Our synthesized
compounds showed acceptable values of TPSA. Moreover,
molecular volume (M.V) descriptor determines transport
characteristics of molecules, such as intestinal absorption
[39]. It was observed that the synthesized compounds exhib-
ited good molecular volume values when compared with
reference ligands. The C log P, TPSA and M.V values are
calculated at (http://www.molinspiration.com) and summa-
rized in Table 4.
In silico ADMET analysis
ADMET (absorption, distribution, metabolism, excretion
and toxicity) prediction was calculated using discovery stu-
dio 2.5 and at (https://preadmet.bmdrc.kr/adme/) for both
the designed compounds and the reference drugs. Predicted
ADMET for all the tested compounds is listed in Table 5.
It was found that BBB penetration of the designed com-
pounds is expected to be high when compared to compounds
VI and GYKI 53655 except compounds 6b and 6c which
showed lower BBB penetration penetrations. Compounds
6a and 9 exhibited nearly the same BBB penetrations of
VI and GYKI 53655, respectively. Additionally, most of
the designed compounds were predicted to have very good
human intestinal absorption percentage which may due to
hydrophobic character of the designed compounds (C log
P). The plasma protein-binding model predicts the bind-
ing ability of a compound to plasma proteins. Most of the
synthesized compounds have very good PPB levels. Hence,
there is high probability that these compounds can reach
the desired targets. Moreover, the closer the hepatotoxicity
scores to one, the more probability to be toxic, while the
closer the hepatotoxicity scores to zero, the more probabil-
ity to be non-toxic. The most active compounds 8, 7b, 10
123

Molecular Diversity
and 3a were found to have hepatotoxicity probability val-
ues ranging from 0.198 to 0.470. Also, compounds 3b and
9 displayed hepatotoxicity probability values of 0.430 and
0.264, respectively. Hence, these derivatives were likely to
be non-hepatotoxic, while the other compounds showed hep-
atotoxicity probability values ranging from 0.536 to 0.741.
This indicates that these derivatives are likely to possess hep-
atotoxicity level one. These findings enable us to conclude
that some of the designed compounds are predicted to be
safer than reference ligands, and the others are predicted to
be nearly with the same toxicity. The CYP2D6 score predicts
the inhibitory and non-inhibitory behavior of particular com-
poundonCytochromeP4502D6enzyme. Allthesynthesized
members are predicted as non-inhibitors of CYP2D6 except
6a and 6b. Hence, the side effects (i.e., liver dysfunction) are
not expected upon administration of these compounds. It is
well known that numerous drug candidates have failed during
clinical tests because of problems related to their absorption
properties [40]. Up on computational investigation of the
synthesized compound, it was found that ADME aqueous
solubility of most compounds exhibited very good solubility
in pure water. Therefore, the main problem restricted efforts
to formulate acceptable parenteral solutions of AMPA recep-
tor antagonists was predicted to be overcome.
antagonists with good physicochemical properties and higher
anticonvulsant analogs.
Acknowledgements The authors extend their appreciation and thank-
ing to Prof. Dr. Ahmed M. Mansour, Pharmacology and Toxicology
Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt
for helping in the pharmacological screening.
Compliance with ethical standards
Conflict of interest There is no conflict of interest to declare.
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