Design, synthesis, and biological evaluation of hydrazone incorporated 1,2,4-triazines as anticonvulsant agents

Article abstract of DOI:10.1002/ardp.201400045

New hydrazone incorporated triazines were designed and synthesized using an appropriate synthetic route with regard to essential pharmacophores, and evaluated for their anticonvulsant activity through maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole-induced seizure (scPTZ) screenings. Among the tested compounds, 4-[{2-(5-(3-chlorobenzyl)-3-phenyl-1,2,4-triazine-6-yl)hydrazono} methyl]-N,N-dimethylaniline 6k (MES ED₅₀ 54.31, scPTZ ED₅₀ 92.01) and 4-[{2-(5-(4-chlorobenzyl)-3-phenyl-1,2,4-triazine-6-yl)hydrazono}methyl]-N,N-dimethylaniline 6r (MES ED₅₀ 46.05, scPTZ ED₅₀ 83.90) emerged as the most active anticonvulsant agents having GABAergic effects. Compounds 6k and 6r also showed lesser CNS depressant effect than the standard drug carbamazepine. To obtain further insights into the binding interactions of these molecules, molecular docking studies were carried out.

Full text of DOI:10.1002/ardp.201400045

958
Arch. Pharm. Chem. Life Sci. 2014, 347, 958–968
Full Paper
Design, Synthesis, and Biological Evaluation of Hydrazone
Incorporated 1,2,4-Triazines as Anticonvulsant Agentsy
Mohammed Amir1, Israr Ali1, Mohd. Zaheen Hassan1, and Naveen Mulakayala2
1
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Hamdard University, New Delhi, India
Institute of Life Science, University of Hyderabad Campus, Hyderabad, India
2
New hydrazone incorporated triazines were designed and synthesized using an appropriate synthetic
route with regard to essential pharmacophores, and evaluated for their anticonvulsant activity through
maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole-induced seizure (scPTZ)
screenings. Among the tested compounds, 4-[{2-(5-(3-chlorobenzyl)-3-phenyl-1,2,4-triazine-6-yl)hydra-
zono}methyl]-N,N-dimethylaniline 6k (MES ED50 54.31, scPTZ ED50 92.01) and 4-[{2-(5-(4-chlorobenzyl)-3-
phenyl-1,2,4-triazine-6-yl)hydrazono}methyl]-N,N-dimethylaniline 6r (MES ED50 46.05, scPTZ ED50 83.90)
emerged as the most active anticonvulsant agents having GABAergic effects. Compounds 6k and 6r also
showed lesser CNS depressant effect than the standard drug carbamazepine. To obtain further insights
into the binding interactions of these molecules, molecular docking studies were carried out.
Keywords: Anticonvulsant / CNS depression / GABA / Hydrazone / Triazine
Received: April 22, 2014; Revised: August 8, 2014; Accepted: August 15, 2014
DOI 10.1002/ardp.201400045
Introduction
Therefore, the development of novel AEDs acting through
diverse mechanisms with lower toxicity requires substantial
investigations [6].
Epilepsy is a serious neurological condition characterized by
periodic and unpredictable episodes of seizures affecting
almost 2% of world population with around 2.4 million new
cases adding up each year as suggested by World Health
Organization (WHO) in partnership with International
Bureau for Epilepsy (IBE) and International League Against
Epilepsy (ILAE) [1–3]. The classical anti-epileptic drugs
comprise phenobarbital, prescribed since 1911; phenytoin,
marketed since 1939; carbamazepine used for epilepsy in
Europe from the mid 1960s; and valproic acid, available in
several European countries as of the late 1960s. Although
several newer drugs such as vigabatrin, pregabalin, zonisa-
mide, tiagabine, lamotrigine, and topiramate are promising
anticonvulsants and have proven to be effective in reducing
seizure, they have dose-related toxicity and idiosyncratic side
effects [4, 5]. Moreover regrettably it seems that antiepileptic
drugs (AEDs) are neither preventive nor curative and are
employed solely as a means of controlling symptoms.
Lamotrigine is a triazine derivative, which acts through the
prolongation of voltage-sensitive Naþ channel’s inactivation
and appears to be effective against all types of generalized
seizures [7]. Results of recent anticonvulsant drug develop-
ment investigations on related triazine analogues were also
encouraging [8, 9]. Moreover, in the course of investigations
for developing structurally novel anticonvulsants, a number
–
of hydrazones containing an azomethine –NHN CH– proton
–
were found to posses interesting seizure protective proper-
ties [10, 11]. As per the binding hypothesis, these compounds
were believed to interact mainly at the locations on a putative
binding site designated as hydrogen bonding domain,
hydrophobic aryl domain, and electron donor system [12].
Thus far, this model has become pivotal and inspired chemists
to utilize the essential pharmacophoric fragments in different
bioactive moieties to synthesize novel anticonvulsant com-
pounds. Therefore, as part of our research efforts devoted for
developing anticonvulsant agents [13–15], we envisaged that
Correspondence: Dr. Mohammad Amir, Faculty of Pharmacy, Depart-
ment of Pharmaceutical Chemistry, Hamdard University, New Delhi
110062, India.
E-mail: mamir_s2003@yahoo.co.in
Fax: þ91 11 26059663
yPart of the work was presented at the International Conference and
Exhibition on Neurology & Therapeutics held at Las Vegas, USA, May 14–
16, 2012.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 958–968
Hydrazone Incorporated 1,2,4-Triazines
959
combining triazine with hydrazone moiety would lead to the
fulfillment of aforesaid pharmacophoric requirements and
will result in the development of potential anticonvulsant
molecules.
In this paper, we report design, synthesis, and anticonvul-
sant screenings of various triazine derivatives. To get insight
into the mechanism of seizure protection, whole brain GABA
estimation was also carried out. Further, in silico molecular
docking studies were performed using Autodock 4.2 program
for the conformation of GABergic effects, and the crystal
structure of Sus scrofa g-aminobutyrate-aminotransferase
(PDB ID:1OHV) was utilized as a template to build human
homology model.
4.51 ppm and a singlet for NH proton at d 8.07–12.64 ppm.
–
–
Furthermore, the N CH or N CCH protons appeared as
–
–
3
singlet at d 8.24–8.73 and 1.98–2.43 ppm, respectively, along
with the aromatic protons at d 6.78–8.23 ppm. The 13C NMR
spectrum of a prototype compound 6k revealed two upfield
peaks at d 34.8 and 41.3 due to a CH2 and N,N-dimethyl
carbons, respectively, while three triazinyl carbons appeared
downfield at d 162.2, 155.1, and 153.6. The characteristic
–
imine carbon (N CH) appeared at d 149.4. The other aromatic
–
carbons were observed at d 124.0, 124.8, 125.2, 126.5, 127.2,
131.0, 132.2, 133.5, 134.1, 135.4, 136.4, and 137.0 confirming
the presence of 25 carbons in the compound.
Pharmacology
Results and discussion
In the preliminary anticonvulsant screening (phase1 screen-
ing), all the compounds except 6m and 6n showed some
degree of protection in both maximal electroshock seizure
(MES) as well as subcutaneous pentylenetetrazole-induced
seizure (scPTZ) screen, indicating the broad spectrum
protection of triazine analogs (Table 1). In the MES test,
compounds 6a, 6d, 6j, 6k, 6p, 6q, and 6t showed activity at
100 mg/kg after 0.5 h, whereas compounds 6a, 6d, 6j, 6p, 6q,
and 6t were active after 4.0 h at the same dose. The most
potent compounds of the series, 6k (after 4.0 h) and 6r (0.5 and
4.0 h), showed protection of hind limb tonic extension at a
dose of 30 mg/kg. Some compounds like 6f, 6g, 6h, 6i, 6l, 6o,
6s, and 6u were also active at a dose level of 300 mg/kg. In the
scPTZ screen, compounds 6k and 6r showed activity at
100 mg/kg after both 0.5 and 4.0 h. On the other hand,
compounds 6a, 6c, 6d, 6e, 6j, 6k, and 6q were active after 4.0 h
at a dose of 100 mg/kg. Hydrazones of 4-N,N-dimethylamino-
benzaldehyde, compounds 6k (3-Cl) and 6r (4-Cl) were found
to be most active anticonvulsants showing protection in both
MES and scPTZ tests at lower doses of 30 mg/kg. As observed
through structure–activity relationship, the attachment of
distal aryl rings with varied substitution in the triazine
Chemistry
The title compounds 6a–u were synthesized by the method
outlined in Scheme 1. The N-(3-hydrazinyl-3-oxo-1-substituted
phenylprop-1-en-2-yl)benzamides (2a–c) were synthesized, in
high yields, by the treatment of hippuric acid with
appropriate aldehydes in presence of pyridine and acetic
anhydride followed by refluxing with hydrazine hydrate.
Further triazinones (3a–c) were easily cyclized from 2a–c in
presence of NaOH. In next step, it was chlorinated by POCl3
and then refluxed with hydrazine hydrate to yield 5-
substituted benzyl-6-hydrazinyl-3-phenyl-1,2,3-triazines (5a–
c). Finally, 5-substituted benzyl-3-phenyl-6[2-(substituted ben-
zylidene)hydrazinyl]-1,2,4-triazines (6a–u) were synthesized
by reaction of 5a–c with substituted benzaldehydes/ketones in
ethanol having catalytic amount of GAA. The structures of the
synthesized compounds were elucidated by elemental analy-
ses and spectral data. FT-IR spectra of compounds 6a–u
–
revealed the characteristic bands for NH and C N at 3311–
–
3379 and 1539–1578 cmꢀ1, respectively. The 1H NMR spectra
of compounds 6a–u showed a singlet for CH2 proton at d 4.18–
Scheme 1. Protocols for the synthesis of the triazine derivatives.
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M. Amir et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 958–968
Table 1. Anticonvulsant and motor impairment screening of synthesized compounds (6a–u).
R1
H
N
N
N
N
N
R2
R
6a−u
MESa)
scPTZa)
Motor imp.a)
Compound
R
R1
R2
0.5 h
4.0 h
0.5 h
4.0 h
0.5 h
4.0 h
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
H
H
H
H
H
H
H
H
H
2-Cl
4-OCH3
4-N,N-Me2
4-Me
4-Cl
4-Br
H
2-Cl
4-OCH3
4-N,N-Me2
4-Me
4-Cl
4-Br
H
100
–
100
100
100
100
100
300
300
100
300
100
30
–
–
100
300
100
100
100
–
–
–
–
–
–
–
–
–
H
100
–
300
–
–
–
CH3
CH3
CH3
H
H
H
–
–
H
H
–
–
–
–
–
–
–
–
–
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
–
300
–
–
–
–
–
–
–
–
–
100
100
–
–
100
100
–
–
–
H
100
–
–
–
CH3
CH3
CH3
H
H
H
300
–
–
–
–
–
–
x
x
x
–
x
x
x
300
–
–
–
–
–
–
300
100
100
30
–
–
2-Cl
100
100
30
300
100
–
–
300
100
100
–
6q
6r
6s
6t
4-OCH3
4-N,N-Me2
4-Me
4-Cl
4-Br
–
300
100
–
–
H
–
–
CH3
CH3
CH3
–
–
–
–
100
300
30
–
–
–
300
300
100
300
6u
–
–
–
Phenytoinb)
CBZb)
30
30
–
–
100
100
–
–
–
100
100
300
a)
Doses of 30, 100, and 300 mg/kg were administered through intraperitoneal injection in mice. The figures in the table indicate the
minimum dose whereby bioactivity was demonstrated in half or more of mice. The animals were examined 0.5 and 4 h. The dash (–)
indicates an absence of activity at maximum dose administered (300 mg/kg). Cross (x) indicates compounds not tested.
Reference drugs, data for phenytoin and carbamazepine from reference [32].
b)
pharmacophore has proven to be valuable in the anticonvul-
exhibited neurological deficit at a dose of 300 mg/kg after
4.0 h.
sant activity, which was presumed to be responsible for
controlling the pharmacokinetic properties of AEDs. Howev-
er, stereo-electronic properties of the substituents at the distal
aryl ring appeared to greatly influence the anticonvulsant
properties. In general, benzaldehyde derivatives were more
potent than acetophenone derivatives. Moreover, the pres-
ence of electron releasing groups at the benzylidene moiety
and electron-withdrawing groups at the benzyl moiety lead to
more active compounds.
All 18 compounds showing good anticonvulsant activity
against MES and scPTZ screens were further evaluated for
the motor impairment effects. Among these, 15 compounds
successfully passed rotorod test without any sign of
neurological deficit, while compounds 6p, 6t, and 6u
The major therapeutic disadvantage of AEDs is their CNS
depressant effects. Therefore, some selected compounds (6a,
6d, 6k, 6q, and 6r) showing significant anticonvulsant activity
were also tested for their CNS depressant effects by Porsolts
swim pool test (Fig. 1). These compounds (6a, 6d, 6k, 6q, and
6r) showed 94.39, 120.47, 62.41, 24.83, and 57.78% increase in
immobility time with respect to control whereas the standard
drug carbamazepine showed 63.35% increase in the immo-
bility time. Thus, it was observed that 6k (62.41%), 6q (24.83%),
and 6r (57.78%) showed less CNS depressant effect than
carbamazepine (63.35%).
Two of the most potent compounds (6k and 6r) of this series
were also tested to determine the GABA levels in the whole
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Hydrazone Incorporated 1,2,4-Triazines
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Table 2. Phase II quantitative anticonvulsant evaluation in mice.
ED50
MES
PIc)
a)
b)
Compound
scPTZ
TD50
MES
scPTZ
6k
6r
54.31
46.05
9.5
8.8
272
92.01
83.90
>300
>100
149
435.32
360.43
65.5
71.6
426
8.01
7.82
6.9
8.1
1.6
4.73
4.29
<0.22
<0.22
2.9
Phenytoind)
Carbamazepined)
Sod. valproated)
a)
Dose in milligrams per kilogram body mass (n ¼ 10).
b)
Minimal toxicity was determined by rotarod test 30 min after
the test drug was administered.
c)
PI ¼ protective index (TD50/ED50). Probit analyses were done
using Statplus 2007 professional 4.0.3 program by Finney
method.
Reference drugs, data for phenytoin, carbamazepine, and
sodium valproate from reference [32].
Figure 1. CNS depressant study of the selected compounds by
Porsolt’s forced swim pool method. Each value represents the
mean ꢁ SEM of six mice compared with respect to standard drug.
ꢂp < 0.0001.
d)
mice brain in order to explore the mechanism of anticon-
vulsants activity. The tested compounds, 6k and 6r, showed
39.7 and 37.3 mg/100 mg tissue of GABA, respectively, while
standard drug clobazam and control were found to have
54.9 and 25.1 mg/100 mg of GABA, respectively (Fig. 2).
The tested compounds were found to increase the GABA
levels significantly when compared to control animals. These
results suggest that synthesized compounds exert anticon-
vulsant activity through a GABA-mediated mechanism as well.
Finally, the two most active compounds 6k and 6r were
quantitatively evaluated for their anticonvulsant activity
(ED50) and motor impairment effect (TD50) in the phase II
screening (Table 2). In the MES screen, both compounds 6k
and 6r showed protection at ED50 of 54.31 and 92.01 mg/kg,
respectively, which are higher than that of standard drug
phenytoin (ED50 9.5 mg/kg) and carbamazepine (ED50
8.8 mg/kg) and lower than sodium valproate (ED50 272 mg/kg).
While in the scPTZ screen, compounds 6k and 6r showed
protection at ED50 of 92.01 and 83.90 mg/kg, which is
considerably lower than that of standard drugs. The median
toxic doses TD50 of 435.32 and 360.43 mg/kg were also higher
than that of standard drugs. Both the compounds 6k and 6r
exhibited better profile of anticonvulsant activity with higher
protective index values (PI 18.3).
In silico studies
Molecular docking studies
Docking results obtained with AutoDock 4.2 showed that both
the compounds bound to the human GABA-AT with high
affinity and with firm binding. Docking results of 6k with
human GABA-AT showed that 6k bound to receptor in a
pocket formed of Leu147, Cys163, Gly164, Asn168, Phe217, His218
,
Gly219, Arg220, Val328, Gln329, His343, and Lys357. Among these
residues 6k had shown to form a total of six hydrogen
bonds with Asn168, Phe217, Gly219, Val328, Gln329, His343
,
and Lys357 (Fig. 3). Rest of the residues was showing
hydrophobic and aromatic interactions with 6k. Docking
energy produced by 6k was ꢀ10.26 kJ/mol. Compound 6r had
shown a different docking mode to that of 6k at binding
pocket of human 4-GABA-AT. Docking interactions of 6r with
GABA-AT had showed that it was bound to a site formed of
Glu56, Leu58, Met59, Ser149, Val150, Ala151, Gly215, Ser216
Phe217, Arg220, Gln293, Asp326, Val328, Lys357, Met358, Arg473
and Arg475 (Fig. 4). Among these Ser149, Val150, Gly215, Gln293
,
,
,
Lys357, Met358, Arg473, and Arg475 were showing hydrogen
bonding interactions with 6r whereas rest of the residues
were involved in hydrophobic and aromatic interactions.
Docking energy produced by 6r is ꢀ10.85 kJ/mol. Most of
the residues involved in the interactions with 6k and 6r
were conserved and found to be involved in the catalytic site
in the template 1OHV.
Figure 2. GABA estimation of some selected compounds in mice
whole brain. Each value represents the mean ꢁ SEM of six mice.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 958–968
Figure 3. Docked conformation of 6k with human GABA-AT.
The results of docking studies showed the involvement of
same pharmacophoric fragments of triazine derivatives (6k and
6r) in the hydrogen bonding which we assumed for the
designing of molecules according to Dimmock et al. [11] (Fig. 5).
Pharmacophore mapping
Pharmacophore modeling provides a consistent and more
precise picture of the biologically active molecules at the
receptor level thereby facilitating the designing of novel
Figure 4. Docked conformation of 6r with human GABA-AT.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 958–968
Hydrazone Incorporated 1,2,4-Triazines
963
Figure 5. Proposed binding site of hydrazones
according to Dimmock et al.
therapeutic agents. Our three point pharmacophore distance
studies showed that compounds 6k and 6r fulfilled the
essential demands of the pharmacophore compared with the
standard marketed AEDs (Table 3).
emerged as the most active anticonvulsant agents having
GABAergic effects. These compounds also satisfied all the
essential anticonvulsant pharmacophoric requirements, thus
emerging as the pilot molecules for antiepileptic drug
research. The docking study also confirmed the GABAergic
effect of these molecules; however, further studies are
required to ascertain their precise mechanism of action.
Conclusion
In summary, various triazines were synthesized having
hydrazone type pharmacophore and evaluated for their
pharmacological properties with the objective of finding better
anticonvulsant lead molecules. Among these two compounds,
4-[{2-(5-(3-chlorobenzyl)-3-phenyl-1,2,4-triazine-6-yl)hydrazono}-
methyl]-N,N-dimethylaniline and 4-[{2-(5-(4-chlorobenzyl)-3-
phenyl-1,2,4-triazine-6-yl)hydrazono}methyl]-N,N-dimethylaniline
Experimental
Chemistry
Instrumentation
The chemicals used in the synthesis were supplied by E. Merck
and S.D. Fine Chemicals. Melting points were determined by open
Table 3. Pharmacophore model of triazine derivatives.
Compound
A-HBD
A-D
D-HBD
Lamotrigine
Carbamazepine
Phenytoin
Remacemide
6k
4.26
5.10
4.98
5.08
5.92
5.92
4.02
5.30
4.68
6.72
5.13
5.13
2.48
4.44
3.40
4.34
3.91
3.91
6r
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capillary tube method and are uncorrected. Purity of the
compound was checked with thin layer chromatography using
iodine vapors as visualizing agents. IR spectra were obtained on a
5-(4-Chlorobenzyl)-3-phenyl-1,2,4-triazin-6-(1H)-one (3c)
Yield: 72%; m.p.: 181–183°C; IR (KBr) nmax cmꢀ1: 1687 (C O), 3301
–
–
(NH); 1H NMR (400MHz, CDCl3) d ppm: 10.82 (s, 1H, CONH), 8.09–
7.28 (m, 9H, ArH), 4.22 (s, 2H, CH2); MS m/z: 298 (Mþ1)þ, 300 (Mþ2)þ.
1
Perkin-Elmer 1720 FT-IR spectrometer (KBr pellets). The H NMR
spectra were measured on a Bruker Avance-400 instrument
(400 MHz) and 13C NMR spectra were measured on Bruker Avance-
400 instrument (100 MHz) with complete proton decoupling.
Chemical shifts were reported in ppm downfield from tetrame-
thylsilane (TMS) as the internal standard. Mass spectra were
obtained on Water’s Synapt LC/MS instrument using electron
impact ionization.
Synthesis of 5-substituted benzyl-6-chloro-3-phenyl-1,2,4-
triazines (4a–c)
Compounds 3a–c (0.01 mol) was dissolved in POCl3 (15 mL) and
heated up to 80°C with continuous stirring for 2–3 h. After
completion of the reaction, it was poured into ice-cold water
slowly. Brown solid thus obtained was filtered and dried. The
resulting solid was purified by column chromatography using
ethyl acetate and hexane as mobile phase (polarity: 0–10%) and
silica gel (60–120) as stationary phase.
General procedure for the synthesis of 4-substituted
benzylidene-2-phenyloxazol-5(4H)-ones (1a–c)
A mixture of hippuric acid (0.01 mol), substituted benzaldehyde
(0.01 mol), pyridine (0.01 mol) in acetic anhydride (0.03 mol) was
stirred at 80°C for 2–3 h. After completion of the reaction, it was
diluted with ethanol. The fine crystals thus obtained were filtered,
dried, and recrystallized from methanol.
5-Benzyl-6-chloro-3-phenyl-1,2,4-triazine (4a)
Yield: 50%; m.p.: 143–145°C; 1H NMR (400 MHz, CDCl3) d ppm: 8.21–
7.09 (m, 10H, ArH), 4.02 (s, 2H, CH2); MS m/z: 282 (Mþ1)þ, 284 (Mþ2)þ.
5-(3-Chlorobenzyl)-6-chloro-3-phenyl-1,2,4-triazine (4b)
Yield: 48%; m.p.: 165–167°C; 1H NMR (400MHz, CDCl3) d ppm: 8.24–
7.00 (m, 9H, ArH), 4.12 (s, 2H, CH2); MS m/z: 316 (Mþ1)þ, 318 (Mþ2)þ.
Benzylidene-2-phenyloxazol-5-(4H)-one (1a)
Yield: 63%; m.p.: 80–82°C; IR (KBr) cmꢀ1: 1705 (C O), 1543 (C N);
–
–
–
–
1
–
H NMR (DMSO-d ) d ppm: 7.19–7.59 (m, 11H, 10ArH and C CH);
–
6
MS: m/z 250 (Mþ1)þ.
5-(4-Chlorobenzyl)-6-chloro-3-phenyl-1,2,4-triazine (4c)
Yield: 52%; m.p.: 176–178°C; 1H NMR (400MHz, CDCl3) d ppm: 8.26–
7.21 (m, 9H, ArH), 4.32 (s, 2H, CH2); MS m/z: 316 (Mþ1)þ, 318 (Mþ2)þ.
3-Chlorobenzylidene-2-phenyloxazol-5(4H)-one (1b)
Yield: 60%; m.p.: 110–112°C; IR (KBr) cmꢀ1: 1709 (C O), 1548
–
–
(C N); 1H NMR (DMSO-d6) d ppm: 7.19–7.54 (m, 10H, 9ArH and
–
–
þ
þ
Synthesis of 5-substituted benzyl-6-hydrazinyl-3-phenyl-
1,2,3-triazines (5a–c)
–
C CH); MS: m/z 284 (Mþ1) , 286 (Mþ2) .
–
A mixture of compounds 4a–c (0.001 mol) and hydrazine hydrate
(0.0012 mol) dissolved in adequate amount of ethanol was
refluxed for 3–4 h. After completion of the reaction, it was
poured into ice-cold water. The solid obtained was filtered, dried,
and recrystallized with ethanol.
4-Chlorobenzylidene-2-phenyloxazol-5(4H)-one (1c)
Yield: 64%; m.p.: 121–123°C; IR (KBr) cmꢀ1: 1702 (C O), 1539
–
–
(C N); 1H NMR (DMSO-d6) d ppm: 7.11–7.51 (m, 10H, 9ArH and
–
–
þ
þ
–
C CH); MS: m/z 284 (Mþ1) , 286 (Mþ2) .
–
Synthesis of 5-substituted benzyl-3-phenyl-1,2,4-triazin-6-
(1H)-ones (3a–c)
5-Benzyl-6-hydrazinyl-3-phenyl-1,2,3-triazine (5a)
Yield: 62%; m.p.: 197–199°C; IR (KBr) nmax cmꢀ1: 3305 (NH); 1H NMR
(400MHz, CDCl3) d ppm: 8.42–7.24 (m, 10H, ArH), 5.83 (s, 2H, CH2),
5.74 (brs, 2H, NH2), 4.08 (brs, 1H, NH); MS m/z: 278 (Mþ1)þ.
A mixture of compounds 1a–c (0.01 mol) and hydrazine hydrate
(0.015 mol) in absolute ethanol (30 mL) was refluxed for 0.5 h. It
was filtered and the filtrate was cooled to room temperature and
allowed to stand at 0°C for 2 h. The white precipitate of N-(3-
hydrazinyl-3-oxo-1-arylprop-1-en-2-yl)benzamide (2a–c) was fil-
tered and washed twice with ice-cold ethanol. Thereafter,
compounds 2a–c (0.01 mol) were refluxed in sodium hydroxide
solution (1 M) for 5 min, cooled to room temperature, and
neutralized with concentrated HCl. The yellow precipitate of 3a–c
thus obtained was filtered, dried, and recrystallized from ethanol.
5-(3-Chlorobenzyl)-6-hydrazinyl-3-phenyl-1,2,3-triazine (5b)
Yield: 60%; m.p.: 203–205°C; IR (KBr) nmax cmꢀ1: 3332 (NH);
1H NMR (400 MHz, CDCl3) d ppm: 8.32–7.27 (m, 9H, ArH), 5.73 (s,
2H, CH2), 5.72 (brs, 2H, NH2), 4.10 (brs, 1H, NH); MS m/z: 312
(Mþ1)þ; 314 (Mþ2)þ.
5-(4-Chlorobenzyl)-6-hydrazinyl-3-phenyl-1,2,3-triazine (5c)
Yield: 65%; m.p.: 210–212°C; IR (KBr) nmax cmꢀ1: 3323 (NH); 1H NMR
(400 MHz, CDCl3) d ppm: 8.33–7.21 (m, 9H, ArH), 5.75 (s, 2H, CH2), 5.70
(brs, 2H, NH2), 4.14 (brs, 1H, NH); MS m/z: 312 (Mþ1)þ; 314 (Mþ2)þ.
5-Benzyl-3-phenyl-1,2,4-triazin-6-(1H)-one (3a)
Yield: 67%; m.p.: 168–170°C; IR (KBr) nmax cmꢀ1: 1689 (C O), 3305
–
–
(NH); 1H NMR (400 MHz, CDCl3) d ppm: 10.88 (s, 1H, CONH), 8.01–
7.22 (m, 10H, ArH), 4.25 (s, 2H, CH2); MS m/z: 264 (Mþ1)þ.
General procedure for the synthesis of 5-substituted
benzyl-3-phenyl-6[2-(substituted benzylidene)hydrazinyl]-
1,2,4-triazines (6a–u)
A mixture of compounds 5a–c (0.0018 mol), substituted benzal-
dehyde/acetophenone (0.0018 mol) and glacial acetic acid (1 mL)
dissolved in ethanol (20 mL) was refluxed for 1–2 h. The reaction
5-(3-Chlorobenzyl)-3-phenyl-1,2,4-triazin-6-(1H)-one (3b)
Yield: 70%; m.p.: 181–183°C; IR (KBr) nmax cmꢀ1: 1680 (C O), 3300
–
–
(NH); 1H NMR (400 MHz, CDCl3) d ppm: 10.81 (s, 1H, CONH), 8.03–
7.02 (m, 9H, ArH), 4.28 (s, 2H, CH2); MS m/z: 298 (Mþ1)þ, 300
(Mþ2)þ.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 958–968
Hydrazone Incorporated 1,2,4-Triazines
965
mixture was left overnight. Fine colored crystals thus obtained
were filtered, washed with hexane, and dried.
7.25 (m, 14H, ArH), 4.29 (s, 2H, CH2), 2.46 (s, 3H, CH3); MS m/z: 458
(Mþ1)þ, 460 (Mþ2)þ. Anal. calcd. for C24H20BrN5: C 62.89, H 4.40,
N 15.28. Found: C 62.93, H 4.45, N 15.24%.
5-Benzyl-6-[2-(benzylidene)hydrazinyl]-3-phenyl-1,2,4-
triazine (6a)
6-(2-Benzylidenehydrazinyl)-5-(3-chlorobenzyl)-3-phenyl-
Yield: 65%; m.p.: 195–197°C; IR (KBr) nmax cmꢀ1: 3311 (NH), 1543
1,2,4-triazine (6h)
1
Yield: 62%; m.p.: 193–195°C; IR (KBr) nmax cmꢀ1: 1543 (C N), 3375
–
–
(N C); H NMR (400 MHz, CDCl ) d ppm: 8.47 (s, 1H, N CH), 8.41–
–
–
–
3
–
7.26 (m, 16H, ArH and NH), 4.34 (s, 2H, CH2); 13C NMR (100 MHz,
CDCl3) d ppm: 35.5, 125.7, 127.5, 128.4, 128.9, 129.2, 130.0, 131.1,
132.5, 133.7, 135.2, 136.2, 138.5, 143.3, 149.2, 155.0, 162.0; MS m/z:
366 (Mþ1)þ. Anal. calcd. for C23H19N5: C 75.59, H 5.24, N 19.16.
Found: C 75.55, H 5.26, N 19.14%.
1
(NH); H NMR (400 MHz, CDCl3) d ppm: 11.34 (brs, 1H, NH), 8.71
–
(s, 1H, N CH), 8.04–7.12 (m, 14H, ArH), 4.29 (s, 2H, CH ); MS m/z:
–
2
400 (Mþ1)þ, 402 (Mþ2)þ. Anal. calcd. for C23H18ClN5: C 69.08,
H 4.54, N 17.51. Found: C 69.13, H 4.50, N 17.54%.
5-(3-Chlorobenzyl)-6-[2-(2-chlorobenzylidene)hydrazinyl]-
5-Benzyl-6-[2-(2-chlorobenzylidene)hydrazinyl]-3-phenyl-
3-phenyl-1,2,4-triazine (6i)
1,2,4-triazine (6b)
Yield: 65%; m.p.: 199–201°C; IR (KBr) nmax cmꢀ1: 1533 (C N), 3365
–
–
Yield: 67%; m.p.: 201–203°C; IR (KBr) nmax cmꢀ1: 3319 (NH), 1549
1
–
–
(NH); H NMR (400 MHz, CDCl ) d ppm: 8.73 (s, 1H, N CH), 8.07
1
3
–
–
(N C); H NMR (400 MHz, CDCl ) d ppm: 8.44 (s, 1H, N CH), 8.39–
–
–
3
(brs, 1H, NH), 7.33–7.13 (m, 13H, ArH), 4.26 (s, 2H, CH2); MS m/z:
434 (Mþ1)þ, 436 (Mþ2)þ. Anal. calcd. for C23H17Cl2N5: C 63.60,
H 3.95, N 16.12. Found: C 63.53, H 4.00, N 16.14%.
7.22 (m, 15H, ArH and NH), 4.36 (s, 2H, CH2); MS m/z: 400 (Mþ1)þ,
402 (Mþ2)þ. Anal. calcd. for C23H18ClN5: C 69.08, H 4.54, N 17.51.
Found: C 69.00, H 4.58, N 17.55%.
5-(3-Chlorobenzyl)-6-[2-(4-methoxybenzylidene)-
5-Benzyl-6-[2-(4-methoxybenzylidene)hydrazinyl]-3-
hydrazinyl]-3-phenyl-1,2,4-triazine (6j)
phenyl-1,2,4-triazine (6c)
Yield: 66%; m.p.: 187–189°C; IR (KBr) nmax cmꢀ1: 1543 (C N), 3374
Yield: 62%; m.p.: 192–194°C; IR (KBr) nmax cmꢀ1: 3332 (NH), 1540
–
–
(NH); 1H NMR (400 MHz, DMSO-d6) d ppm: 12.64 (brs, 1H, NH), 8.41
1
–
(N C); H NMR (400 MHz, CDCl ) d ppm: 11.90 (brs, 1H, NH), 8.30
–
3
–
(s, 1H, N CH), 8.29–6.98 (m, 13H, ArH), 4.19 (s, 2H, CH ), 3.80
–
–
2
(s, 1H, N CH), 8.10–6.88 (m, 14H, ArH), 4.23 (s, 2H, CH ), 3.78
–
2
(s, 3H, OCH3); 13C NMR (100 MHz, CDCl3) d ppm: 38.5, 55.7, 125.9,
127.0, 128.3, 128.6, 129.8, 129.9, 130.0, 130.2, 130.4, 131.0, 133.1,
134.9, 139.1, 144.0, 148.0, 155.8, 161.5, 167.9; MS m/z: 430 (Mþ1)þ,
432 (Mþ2)þ. Anal. calcd. for C24H20ClN5O: C 67.05, H 4.69, N 16.29.
Found: C 67.00, H 4.72, N 16.33%.
(s, 3H, OCH3); MS m/z: 396 (Mþ1)þ. Anal. calcd. for C24H21N5O:
C 72.89, H 5.35, N 17.71. Found: C 72.80, H 5.31, N 17.75%.
5-Benzyl-6-[2-(4-N,N-dimethylaminobenzylidene)-
hydrazinyl]-3-phenyl-1,2,4-triazine (6d)
Yield: 70%; m.p.: 210–212°C; IR (KBr) nmax cmꢀ1: 3329 (NH),
1
4-[{2-(5-(3-Chlorobenzyl)-3-phenyl-1,2,4-triazine-6-yl)-
–
–
–
1544 (N C); H NMR (400 MHz, CDCl ) d ppm: 8.39 (s, 1H, N CH),
–
3
hydrazono}methyl]-N,N-dimethyl aniline (6k)
8.10–6.70 (m, 15H, ArH and NH), 4.28 (s, 2H, CH2), 3.04 (s, 6H,
N(CH3)2); 13C NMR (100MHz, CDCl3) d ppm: 35.2, 41.5, 125.1, 127.8,
128.6, 128.2, 129.8, 131.5, 132.4, 132.9, 133.2, 135.0, 135.8, 137.5,
149.2, 153.1, 155.0, 162.0; MS m/z: 409 (Mþ1)þ. Anal. calcd. for
C25H24N6: C 73.51, H 5.92, N 20.57. Found: C 77.70, H 5.96, N 20.55%.
Yield: 66%; m.p.: 180–182°C; IR (KBr) nmax cmꢀ1: 1554 (C N), 3361
–
–
(NH); 1H NMR (400 MHz, DMSO-d6) d ppm: 12.04 (brs, 1H, NH), 8.42
–
(s, 1H, N CH), 8.23–6.88 (m, 13H, ArH), 4.29 (s, 2H, CH ), 3.25 (s,
–
2
6H, N(CH3)2); 13C NMR (100 MHz, CDCl3) d ppm: 34.8, 41.3, 124.0,
124.8, 125.2, 126.5, 127.2, 131.0, 132.2, 133.5, 134.1, 135.4, 136.4,
137.0, 149.4, 153.6, 155.1, 162.2; MS m/z: 443 (Mþ1)þ, 445 (Mþ2)þ.
Anal. calcd. for C25H23ClN6: C 67.79, H 5.23, N 18.97. Found: C
67.60, H 5.27, N 18.90%.
5-Benzyl-3-phenyl-6-[2-(1-p-tolylethylidene)hydrazinyl]-
1,2,4-triazine (6e)
Yield: 68%; m.p.: 215–217°C; IR (KBr) nmax cmꢀ1: 3340 (NH), 1541
1
–
(N C); H NMR (400 MHz, CDCl ) d ppm: 8.14–6.98 (m, 15H, ArH
–
3
and NH), 4.26 (s, 2H, CH2), 2.09 (s, 3H, CH3), 2.31 (s, 3H, CH3); MS m/z:
394 (Mþ1)þ. Anal. calcd. for C25H23N5: C 76.31, H 5.89, N 17.80.
Found: C 76.39, H 5.86, N 17.75%.
5-(3-Chlorobenzyl)-3-phenyl-6-[2-(1-p-tolylethylidene)-
hydrazinyl]-1,2,4-triazine (6l)
Yield: 65%; m.p.: 190–192°C; IR (KBr) nmax cmꢀ1: 1546 (C N), 3378
–
–
(NH); 1H NMR (400 MHz, DMSO-d6) d ppm: 12.43 (brs, 1H, NH),
8.03–7.21 (m, 13H, ArH), 4.22 (s, 2H, CH2), 2.41 (s, 3H, CH3), 2.33 (s,
5-Benzyl-6-[2-{1-(4-chlorophenyl)ethylidene}hydrazinyl]-
3H, CH3); MS m/z: 428 (Mþ1)þ 430 (Mþ2)þ. Anal. calcd. for
3-phenyl-1,2,4-triazine (6f)
,
Yield: 66%; m.p.: 220–222°C; IR (KBr) nmax cmꢀ1: 3344 (NH), 1543
C25H22ClN5: C 70.17, H 5.18, N 16.37. Found: C 70.20, H 5.21, N
16.39%.
1
–
(N C); H NMR (400 MHz, CDCl ) d ppm: 12.12 (brs, 1H, NH), 8.23–
–
3
7.01 (m, 14H, ArH), 4.23 (s, 2H, CH2), 2.25 (s, 3H, CH3); MS m/z: 414
(Mþ1)þ, 416 (Mþ2)þ. Anal. calcd. for C24H20ClN5: C 69.64, H 4.57,
N 16.92. Found: C 69.60, H 4.86, N 16.90%.
5-(3-Chlorobenzyl)-6-[2-(1-(4-chlorophenyl)ethylidene)-
hydrazinyl]-3-phenyl-1,2,4-triazine (6m)
Yield: 67%; m.p.: 207–209°C; IR (KBr) nmax cmꢀ1: 1556 (C N), 3345
–
–
(NH); 1H NMR (400 MHz, DMSO-d6) d ppm: 12.13 (brs, 1H, NH),
8.13–7.23 (m, 13H, ArH), 4.24 (s, 2H, CH2), 2.31 (s, 3H, CH3); MS m/z:
448 (Mþ1)þ, 450 (Mþ2)þ. Anal. calcd. for C24H19Cl2N5: C 64.29, H
4.27, N 15.62. Found: C 64.20, H 4.21, N 15.59%.
5-Benzyl-6-[2-{1-(4-bromophenyl)ethylidene}hydrazinyl]-
3-phenyl-1,2,4-triazine (6g)
Yield: 70%; m.p.: 217–219°C; IR (KBr) nmax cmꢀ1: 1549 (C N), 3381
–
–
(NH); 1H NMR (400 MHz, CDCl3) d ppm: 10.26 (brs, 1H, NH), 8.12–
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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966
M. Amir et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 958–968
5-(3-Chlorobenzyl)-6-[2-(1-(4-bromophenyl)ethylidene)-
7.28 (m, 13H, ArH), 4.51 (s, 2H, CH2), 2.59 (s, 3H, CH3); MS m/z: 448
(Mþ1)þ, 450 (Mþ2)þ. Anal. calcd. for C24H19Cl2N5: C 64.29, H 4.27,
N 15.62. Found: C 64.20, H 4.25, N 15.64%.
hydrazinyl]-3-phenyl-1,2,4-triazine (6n)
Yield: 64%; m.p.: 193–195°C; IR (KBr) nmax cmꢀ1: 1550 (C N), 3341
–
–
(NH); 1H NMR (400 MHz, DMSO-d6) d ppm: 12.03 (brs, 1H, NH),
8.23–7.01 (m, 13H, ArH), 4.34 (s, 2H, CH2), 2.33 (s, 3H, CH3); MS m/z:
492 (Mþ1)þ, 494 (Mþ2)þ. Anal. calcd. for C24H19BrClN5: C 58.49, H
3.89, N 14.21. Found: C 58.40, H 3.91, N 14.23%.
5-(4-Chlorobenzyl)-6-[2-{1-(4-bromophenyl)ethylidene}-
hydrazinyl]-3-phenyl-1,2,4-triazine (6u)
Yield: 68%; m.p.: 229–231°C; IR (KBr) nmax cmꢀ1: 1555 (C N), 3334
–
–
(NH); 1H NMR (400 MHz, CDCl3) d ppm: 12.04 (brs, 1H, NH), 8.00–
7.21 (m, 13H, ArH), 4.55 (s, 2H, CH2), 2.55 (s, 3H, CH3); MS m/z: 492
(Mþ1)þ, 494 (Mþ2)þ. Anal. calcd. for C24H19BrClN5: C 58.49,
H 3.89, N 14.21. Found: C 58.20, H 3.92, N 14.23%.
6-(2-Benzylidenehydrazinyl)-5-(4-chlorobenzyl)-3-phenyl-
1,2,4-triazine (6o)
Yield: 65%; m.p.: 230–232°C; IR (KBr) nmax cmꢀ1: 1554 (C N), 3339
–
–
(NH); 1H NMR (400 MHz, DMSO-d6) d ppm: 11.23 (brs, 1H, NH), 8.44
–
(s, 1H, N CH), 8.11–7.23 (m, 14H, ArH), 4.44 (s, 2H, CH ); MS m/z:
–
Pharmacology
2
400 (Mþ1)þ 402 (Mþ2)þ. Anal. calcd. for C23H18ClN5: C 69.08, H
,
All the pharmacological experiments were conducted in compli-
ance with ethical principles after Institutional Animal Ethics
Committee (IAEC) approval. The investigations were conducted
on albino mice of either sex (25–30 g). Animals were obtained
from central animal house facility, Hamdard University, New
Delhi. Registration No. of IAEC is 173/CPCSEA and date of
registration is January 28, 2000. Triazine derivatives (6a–u) were
4.54, N 17.51. Found: C 69.00, H 4.56, N 17.49%.
5-(4-Chlorobenzyl)-6-[2-(2-chlorobenzylidene)hydrazinyl]-
3-phenyl-1,2,4-triazine (6p)
Yield: 66%; m.p.: 221–223°C; IR (KBr) nmax cmꢀ1: 1549 (C N), 3342
–
–
(NH); 1H NMR (400 MHz, DMSO-d6) d ppm: 12.23 (brs, 1H, NH), 8.45
evaluated for preliminary anticonvulsant activity (phase
I
–
(s, 1H, N CH), 8.14–7.13 (m, 13H, ArH), 4.45 (s, 2H, CH ); MS m/z:
–
2
screening) at dose levels of 30, 100, and 300 mg/kg i.p. in mice
by following the established protocols of Antiepileptic Drug
Development (ADD) program by NINDS, US using two “gold
standard models” MES and scPTZ tests and motor impairment was
evaluated by rotarod test. The most potent compounds were also
434 (Mþ1)þ, 436 (Mþ2)þ. Anal. calcd. for C23H17Cl2N5: C 63.60, H
3.94, N 16.10. Found: C 63.55, H 4.00, N 16.14%.
5-(4-Chlorobenzyl)-6-[2-(4-methoxybenzylidene)-
hydrazinyl]-3-phenyl-1,2,4-triazine (6q)
evaluated at phase II quantitative screening for calculating ED50,
Yield: 62%; m.p.: 235–237°C; IR (KBr) nmax cmꢀ1: 1559 (C N), 3352
–
–
TD50, and protective index (PI). Some selected compounds were
also evaluated for their CNS depression effects by Porsolt’s swim
pool test. Levels of whole brain GABA estimations were carried out
in order to explore the mechanism of anticonvulsants activity.
(NH); 1H NMR (400 MHz, CDCl3) d ppm: 11.80 (brs, 1H, NH), 8.24 (s, 1H,
–
N CH), 8.01–6.89 (m, 13H, ArH), 4.18 (s, 2H, CH ), 2.96 (s, 3H, OCH );
–
2
3
13C NMR (100 MHz, CDCl3) d ppm: 35.6, 55.8, 114.2, 124.8, 125.7, 126.0,
126.8, 129.5, 130.6, 131.4, 133.6, 134.8, 135.8, 137.9, 149.0, 152.2, 155.0,
162.0; MS m/z: 430 (Mþ1)þ, 432 (Mþ2)þ. Anal. calcd. for C24H20ClN5O:
C 67.05, H 4.69, N 16.29. Found: C 67.35, H 4.60, N 16.24%.
Preliminary anticonvulsant screening (phase I screening)
Maximum electroshock seizure (MES) test
The MES test was carried out according to the standard protocol [16,
17]. Albino mice were stimulated through corneal electrodes to
50 mA current at a pulse of 60 Hz applied for 0.25s. Animals were
previously administered with the test drug i.p. Abolition of hind
limb tonic extension spasm was recorded as the anticonvulsant
activity. The test compounds were dissolved in polyethylene glycol
(PEG). In the preliminary screening, each compound was adminis-
tered as an i.p. injection at three dose levels (30, 100, and 300mg/kg
body mass) and the anticonvulsant activity was assessed after 0.5
and 4.0h intervals of administration.
4-[{2-(5-(4-Chlorobenzyl)-3-phenyl-1,2,4-triazine-6-yl)-
hydrazono}methyl]-N,N-dimethyl aniline (6r)
Yield: 65%; m.p.: 211–213°C; IR (KBr) nmax cmꢀ1: 1549 (C N), 3351
–
–
(NH); 1H NMR (400 MHz, CDCl3) d ppm: 11.89 (brs, 1H, NH), 8.21 (s, 1H,
–
N CH), 8.10–6.99 (m, 13H, ArH), 4.28 (s, 2H, CH ), 3.26 (s, 6H, N(CH ) );
–
2
3 2
13C NMR (100 MHz, CDCl3) d ppm: 36.2, 41.2, 111.9, 125.8, 126.7,
128.7, 129.8, 130.0, 132.0, 133.4, 134.4, 135.4, 136.6, 137.3, 149.1,
153.2, 155.3, 160.0; MS m/z: 442 (Mþ1)þ, 444 (Mþ2)þ. Anal. calcd. for
C25H23ClN6: C 67.79, H 5.23, N 18.97. Found: C 67.65, H 5.26, N 18.90%.
5-(4-Chlorobenzyl)-3-phenyl-6-[2-(1-p-tolylethylidene)-
Pentylenetetrazole-induced seizure (scPTZ) test
The subcutaneous pentylenetetrazole test was performed according
to the known protocol [18]. This method utilizes pentylenetetrazole
(75 mg/kg) that produces seizures in >95% of animals as a 0.5%
solution subcutaneously in the posterior midline. The animals
were observed for 30 min. Failure to observe even a threshold
seizure (a single episode of clonic spasms of at least 5 s duration)
was defined as protection.
hydrazinyl]-1,2,4-triazine (6s)
Yield: 60%; m.p.: 225–227°C; IR (KBr) nmax cmꢀ1: 1548 (C N), 3369
–
–
(NH); 1H NMR (400 MHz, DMSO-d6) d ppm: 12.46 (brs, 1H, NH), 8.23–
7.18 (m, 13H, ArH), 4.19 (s, 2H, CH2), 2.41 (s, 3H, CH3), 2.32 (s, 3H, CH3);
13C NMR (100 MHz, CDCl3) d ppm: 14.4, 21.4, 38.8, 126.0, 127.4, 128.6,
129.1, 129.3, 129.9, 131.7, 131.8, 134.9, 135.7, 136.0, 139.4, 142.5,
146.6, 160.6, 168.6; MS m/z: 428 (Mþ1)þ, 430 (Mþ2)þ. Anal. calcd. for
C25H22ClN5: C 70.17, H 5.18, N 16.37. Found: C 70.23, H 5.21, N 16.40%.
Neurotoxicity screening (NT)
5-(4-Chlorobenzyl)-6-[2-{1-(4-chlorophenyl)ethylidene}-
The minimal motor impairment was measured in mice by the
rotarod test. The mice were trained to stay on an accelerating
rotarod of diameter 3.2 cm that rotates at 10 rpm. Trained
animals were given i.p. injection of the test compounds 30, 100,
hydrazinyl]-3-phenyl-1,2,4-triazine (6t)
Yield: 70%; m.p.: 240–242°C; IR (KBr) nmax cmꢀ1: 1541 (C N), 3359
–
–
(NH); 1H NMR (400 MHz, CDCl3) d ppm: 11.11 (brs, 1H, NH), 7.80–
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Arch. Pharm. Chem. Life Sci. 2014, 347, 958–968
Hydrazone Incorporated 1,2,4-Triazines
967
and 300 mg/kg. Neurotoxicity was indicated by the inability of the
animal to maintain equilibrium on the rod for at least 1 min in
each of the trials [19].
the construction of 3D model. The developed model was energy
minimized using GROMACS 4.0.5. The geometry of the minimized
model was evaluated with Ramachandran plot calculations with
PROCHECK program. This result revealed 91.8% of the residues
were in the core region, 7.0% residues in the allowed regions, 1.0%
in generously allowed regions, and 0.2% residues in the disallowed
regions. Ca atoms and back bone atoms RMSD of the model and
template was 0.48 Å. Procheck and RMSD superposition of the
developed model of human GABA-AT suggested that the model is
good enough to be used for molecular docking studies.
CNS depression study
Behavioral depression was measured by evaluating the immobili-
ty time of the mice during swimming phase following the Porsolt
forced swim test [20]. Before starting test the animals were trained
for swimming (24 h before). Then the trained animals were given
an i.p. injection (100 mg/kg) of the test compounds 60 min before
the test session. The duration of passive floating without
struggling was recorded during the last 4 min of the 6 min
testing period as the immobility time.
Homology modeling and optimization
To build the human GABA-AT homology model, a BLASTp against
PDB was used to carry out the sequence homology searches.
Crystal structure of S. scrofa GABA-AT (PDB ID:1OHV) was taken
as a template to build homology model [23]. Modellar 9.11 [24]
program was employed to generate the construction of 3D
structure of human GABA-AT. The generated model was refined
by energy minimization using Gromacs 4.0.5 [25]. The energy
minimized model was subjected to validation using Ramachan-
dran plot [26] and RMSD superposition [27].
Gamma amino butyric acid estimation
The GABA estimation was performed in brain tissue extract
spectrophotometrically as described by Gokani et al. [21] after 2 h
of drug administration (100 mg/kg, i.p.). After the dosing, animals
were decapitated and brains were dropped into vials and rinsed
with ice-cold isotonic saline followed by homogenization with
0.1 mmol/L phosphate buffer (pH 7.4). The homogenate (10% w/v)
was then centrifuged at 20,000 ꢃ g for 30 min and the superna-
tant so formed was applied on silica gel 60F254 aluminum sheets
(10 cm ꢃ 10 cm) as stationary phase, using mobile phase com-
prised of water saturated phenol. TLC plates were prewashed with
methanol and activated in an oven at 50°C for 5 min prior to
chromatography. The 0.1 mL supernatant solutions were applied
on TLC plates in the form of bands. Ascending development to
80 mm was performed in a glass chamber saturated with the
mobile phase for 30 min at room temperature. The developed TLC
plates were air dried and sprayed with ninhydrin. Ten developed
spots were scraped together, mixed with 3 mL distilled water and
centrifuged. The supernatant obtained was spectrophotometri-
cally analyzed at 570 nm. The specificity of the method was
ascertained by taking standard GABA samples and confirmed by
comparing their Rf values. The calibration curve was prepared by
plotting OD versus concentration.
Molecular docking studies
Energy minimized structure of human GABA-AT was used as a
receptor for docking studies. PDB files of compounds 6k and 6r
were generated from Prodrug server [28]. AutoDock 4.2 [29] was
used for conducting docking experiments of human GABA-AT
with 6k and 6r. All docking experiments were performed with 2.5
million evaluations for 27,000 generations with population size
300 to produce 100 docked conformations [27]. After ensuring
that protein and ligands are in correct orientations for docking,
the receptor-grid files were generated using grid parameter file
program. Grid box was set to whole protein with dimensions of
80 Å ꢃ 80 Å ꢃ 80 Å with a grid center of 0.414 Å. The interactions
of the ligands with protein were visualized with AutoDock Tools
(ADT) and figures were formed using PYMOL 1.1 [30].
Pharmacophore mapping
A pharmacophore model offers a valuable tool for explanation of
plausible interactions with the putative receptors. The 3-point
pharmacophore group’s distance estimations were done on 3D
optimized structures using ACD/Chemsketch/3D viewer 2.0 version
program. Molecular model was suggested through the molecular
dynamic distance estimations of clinically available AEDs such as
lamotrigine, carbamazepine, phenytoin, and remacemide [31].
Quantitative in vivo anticonvulsant/toxicity evaluation
(phase II screening)
The most potent compound in phase I screening was evaluated
for quantification studies in phase II screening in mice and the
results of anticonvulsant activity and neurotoxicity are expressed
as median effective dose (ED50) and median toxic dose (TD50),
respectively. Different doses of test compound were administered
intraperitoneally in groups of 10 mice until three endpoints were
established in the range of 10–90% response. Finally, the probit
analysis was done by means of computer program BioStat 2009
using Finney’s method [22].
The authors are thankful to University Grants Commission (UGC) New
Delhi, India, for financial support. Authors are also thankful to
Mr. M. S. Zaman and Mr. Niyaz Ahmed, Faculty of Pharmacy,
Hamdard University for spectral analysis of compounds.
In silico studies
Homology model of g-aminobutyrate-aminotransferase
The authors have declared no conflict of interest.
(GABA-AT)
The amino acid sequence of human g-aminobutyrate-aminotrans-
ferase, GABA-AT (500 amino acids) was obtained from NCBI.
Crystal structures of GABA-AT from different species have already
been determined and available in Protein Data Bank (PDB). Among
them S. scrofa GABA-AT (PDB ID:1OHV) showed highest identity
(95%) with human GABA-AT, which was chosen as a template for
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