Design, Synthesis, and Docking Study of Pyrimidine–Triazine Hybrids for GABA Estimation in Animal Epilepsy Models

Article abstract of DOI:10.1002/ardp.201700146

A series of new pyrimidine–triazine hybrids (4a–t) was designed and synthesized, from which potent anticonvulsant agents were identified. Most of the compounds exhibited promising anticonvulsant activity against the maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) tests, along with minimal motor impairment with higher safety compared to the standard drugs, phenytoin and carbamazepine. In the series, 5-(4-(4-fluorophenyl)-6-(4-hydroxyphenyl)-2-thioxo-5,6-dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-3(6H)-one (4o) and 5-(6-(4-hydroxy-3-methoxyphenyl)-4-(4-hydroxyphenyl)-2-thioxo-5,6-dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-3(6H)-one (4s) emerged as most potent anticonvulsant agents with median doses of 22.54 and 29.40 mg/kg (MES ED₅₀), 285.02 and 293.42 mg/kg (scPTZ ED₅₀), and 389.11 and 412.16 mg/kg (TD₅₀), respectively. Docking studies were also performed for all synthesized compounds to get insight into the binding pattern toward the GABA_A receptor as a possible mechanism of their anticonvulsant action, and in silico ADME studies were carried out to predict the safety and stability of the molecules. The increased GABA level in the experimental animals in the neurochemical estimation assay confirmed their GABAergic modulating activity. The most potent compounds were also evaluated for their neurotoxic and hepatotoxic effects. Fortunately, they did not show any sign of neurotoxicity or hepatotoxicity, suggesting that they have a broad spectrum of anticonvulsant activity with a large safety margin. Together, this research suggested that 4o and 4s may serve as leads in the discovery and development of new anticonvulsant drugs.

Full text of DOI:10.1002/ardp.201700146

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Full Paper
Design, Synthesis, and Docking Study of Pyrimidine–Triazine
Hybrids for GABA Estimation in Animal Epilepsy Models
Meeta Sahu
1, Nadeem Siddiqui1, Mohd. Javed Naim1, Ozair Alam
1, Mohammad Shahar Yar1,
Vidushi Sharma2, and Sharad Wakode2
1
Department of Pharmaceutical Chemistry, School of Pharmaceutical Education and Research (Formerly
Faculty of Pharmacy), Jamia Hamdard, New Delhi, India
Department of Pharmaceutical Chemistry, Delhi Institute of Pharmaceutical Sciences and Research
(DIPSAR), New Delhi, India
2
A series of new pyrimidine–triazine hybrids (4a–t) was designed and synthesized, from which potent
anticonvulsant agents were identified. Most of the compounds exhibited promising anticonvulsant
activity against the maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) tests,
along with minimal motor impairment with higher safety compared to the standard drugs, phenytoin
and carbamazepine. In the series, 5-(4-(4-fluorophenyl)-6-(4-hydroxyphenyl)-2-thioxo-5,6-dihydropyr-
imidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-3(6H)-one (4o) and 5-(6-(4-hydroxy-3-methoxyphenyl)-4-(4-
hydroxyphenyl)-2-thioxo-5,6-dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-3(6H)-one (4s)
emerged as most potent anticonvulsant agents with median doses of 22.54 and 29.40 mg/kg (MES
ED50), 285.02 and 293.42 mg/kg (scPTZ ED50), and 389.11 and 412.16 mg/kg (TD50), respectively.
Docking studies were also performed for all synthesized compounds to get insight into the binding
pattern toward the GABAA receptor as a possible mechanism of their anticonvulsant action, and in
silico ADME studies were carried out to predict the safety and stability of the molecules. The
increased GABA level in the experimental animals in the neurochemical estimation assay confirmed
their GABAergic modulating activity. The most potent compounds were also evaluated for their
neurotoxic and hepatotoxic effects. Fortunately, they did not show any sign of neurotoxicity or
hepatotoxicity, suggesting that they have a broad spectrum of anticonvulsant activity with a large
safety margin. Together, this research suggested that 4o and 4s may serve as leads in the discovery
and development of new anticonvulsant drugs.
Keywords: Rational drug design / Structure elucidation / Synthesis
Received: May 5, 2017; Revised: June 21, 2017; Accepted: June 27, 2017
DOI 10.1002/ardp.201700146
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
infection, stroke, or head injury [1]. According to the
International League Against Epilepsy (ILAE), epilepsy is
Introduction
Epilepsy, a common neurological disorder, is characterized by
seizures because of recurrent, spontaneous events of abnor-
mal synchronism in neuronal networks which may be due to
redefined as “a disease of the brain” in contrast to its
previous categorization as a central nervous system (CNS)
disorder [2]. According to the World Health Organization,
worldwide approximately 50 million people have epilepsy,
making it one of the most common neurological diseases
globally. Anticonvulsant drugs are effective in about 70% of
the population with epilepsy and for the rest, the treatment
of epilepsy remains still inadequate [3]. In the past decade,
more than 40 antiepileptic drugs (AEDs) had been introduced.
However, till date, none of the AEDs can be considered as a
Correspondence: Nadeem Siddiqui, Department of Pharmaceu-
tical Chemistry, SPER (Formerly Faculty of Pharmacy), Jamia
Hamdard, New Delhi 110062, India.
E-mail: nadeems_03@yahoo.co.in
Fax: þ91 11 2605 9663
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“magical remedy” for the treatment of the patient suffering
from epilepsy [4]. The currently available AEDs have a variety
of side-effects with chronic toxicities like neuro- and
hepatotoxicities [5]. The mechanisms of action of available
AEDs are complex and include: (i) modification of the
excitability of nerve by blocking of voltage-activated sodium
channels; (ii) by influencing GABA receptors via direct positive
allosteric modulation (PAM) of GABAA receptor, or indirectly,
by increasing levels of GABA via inhibition of GABA
transaminase or GABA transporter [6]; (iii) blocking of
voltage-gated calcium channels [7, 8]; and (iv) activation of
peroxisome proliferator-activated receptor alpha [9]. In
relation to epilepsy, GABAergic inhibition is thought to be
the main mechanism counterbalancing glutamatergic excita-
tion and hypersynchronous neuronal discharges inhibi-
tion [10]. Furthermore, GABA acts either by activation of
ionotropic (GABAA) receptors or via metabotropic (GABAB)
receptors. GABAA receptors form a chloride-permeable ion
channel and work as fast, short-acting synaptic inhibition [11].
The GABAA receptor complex is formed by a combination of
several receptor subunits but only a small number of native
receptor subtypes are identified so far. Various classes of
chemically synthesized compounds bind to the GABAA
chloride ion channel complex such as benzodiazepines,
neurosteroids, and barbiturates [12]. GABAA receptor mod-
ulates the action of GABA on chloride ion flux and
demonstrates a broad range of pharmacological actions such
as full agonist which is responsible for sedative/hypnotic,
anxiolytic, and anticonvulsant activities; and inverse agonistic
responsible for effects such as anxiogenic, and proconvulsant
activities [13]. Phenytoin and carbamazepine are the most
commonly used standard drugs which act by inhibition of
voltage-gated sodium ion channels (VGSCs). These drugs are
also reported to enhance GABA-mediated response and
alleviate seizures [14, 15].
Pyrimidines are essential biomolecules and structural
components of various nucleotides, nucleic acids, vitamins,
pterins, and folates. Their critical participation in processes,
such as RNA and DNA synthesis, the formation of UDP-sugars
for glycosylation of proteins and lipids, and formation of CDP-
activated precursors for membrane phospholipids, makes
them attractive for study [16]. Pyrimidine derivatives are well
known for broad spectrum of interesting biological activities
such as anticancer [17, 18], anti-HIV [19], antihistaminic [20],
anti-inflammatory [21–23], antidiabetic [24, 25], antitubercu-
lar [26, 27], anticonvulsant [28], and antimicrobial [29–31].
Interestingly, 1,2,4-triazine derivatives have been reported
for their diverse biological activities such as anticancer [32–34],
antibacterial [35], antidiabetic [36, 37], antifungal [38],
antimalarial [39], neuroprotective agents [40], anticonvul-
sant [41], and antiviral [42] activities.
Results and discussion
Rationale and design
The available AEDs such as phenytoin, carbamazepine,
progabide, remacemide, ralitoline are commonly used clini-
cally to treat patients suffering from epilepsy (Fig. 1). The
structural analysis of these AEDs led to the proposal of a
general model for anticonvulsant action which suggests that a
model should contain two aromatic rings or their equivalent
in a special spatial arrangement and a distal region containing
different functional groups which form hydrogen bond-
ing [43]. Dimmock et al. [44, 45] proposed a model describing
the various pharmacophoric elements required for interaction
at the binding site which are the lipophilic aryl ring (A),
hydrogen bonding domain (HBD), electron donor moiety (D),
and distal aryl ring (C). The designed pyrimidine–triazine
hybrids 4a–t possess all these pharmacophoric elements as
described in Fig. 2.
Unverferth et al. [46] also suggested a two-dimensional
model describing pharmacophoric features essential for
anticonvulsant activity comprising at least one aryl group
acting as lipophilic domain, one or more electron donor
centers, and/or an NH group in a favorable orientation for
forming hydrogen bonding and a distal hydrophobic center.
Interestingly, some drugs such as phenobarbital, mephobar-
bital, primidone, and lamotrigine are known for their
anticonvulsant properties and contain pyrimidine and 1,2,4-
triazine, respectively, as core nucleus in them as shown in
Fig. 3. Moreover, several researchers have worked on the
anticonvulsant potential of pyrimidines [47–50] and
triazines [51–53] in subsequent years and from their studies,
it has been proved that both of these moieties contain
antiseizure properties. The distance range such as A, D, and
HBD, between the essential pharmacophoric elements of
clinically used and designed compounds 4a–t are depicted in
Table 1. Therefore, all these facts and the biological
importance of pyrimidine and 1,2,4-triazine derivatives as
anticonvulsants prompted us to design and synthesize some
hybrid structures which were believed to show promising
anticonvulsant activity.
Chemistry
The synthetic route of title compounds 4a–t is described in
Scheme 1. All the compounds were obtained in satisfactory
yield (54–79%) and their structure and purity were confirmed
by spectral and elemental analysis. The aldol condensation
products (E)-1-(substituted phenyl)-3-(4-substituted phenyl)-
prop-2-en-1-ones 1a–t were obtained by treatment of 4-
substituted acetophenone and substituted benzaldehyde in
the vicinity of ethanolic sodium hydroxide solution. The
cyclization of chalcones 1a–t with thiourea in the presence of
potassium hydroxide in ethanol resulted in the in situ
formation of 5,6-dihydropyrimidine-thiones 2a–t. The chlor-
oacetylation of this cyclized product was done in the presence
of a catalytical amount of triethylamine in dichloromethane
to get 3a–t. Further, compounds 3a–t were cyclized by
Henceforth, the aim of this research was to deliberate the
development of pyrimidine–triazine hybrids, 5-(4-(4-substi-
tuted phenyl)-6-(substituted phenyl)-2-thioxo-5,6-dihydro-
pyrimidine-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-3(6H)-ones
(4a–t), as anticonvulsant agents.
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Figure 1. Pharmacophoric features for anticonvulsant action in marketed drugs and the designed compounds 4a–t.
–
treating with semicarbazide in potassium carbonate using
N,N-dimethylformamide as solvent to obtain the target
compounds 5-(4-(4-substituted phenyl)-6-(substituted phe-
nyl)-2-thioxo-5,6-dihydropyrimidine-1(2H)-yl)-1,2-dihydro-1,
2,4-triazin-3(6H)-ones 4a–t. The recrystallization of all the
synthesized compounds was done from the appropriate
solvents. The structure elucidation of synthesized compounds
was done by FT-IR, 1H-NMR, 13C-NMR, mass spectrometry, and
elemental analysis. The physicochemical parameters of the
synthesized compounds in this series are demonstrated in
Table 2.
The FT-IR spectra of compounds 4a–t afforded C N stretching
–
(1643–1688 cmꢀ1), C S stretching of pyrimidine ring (1206–
–
–
1226 cmꢀ1), and CONH stretching of triazine (3312–
3346 cmꢀ1) bands. In the 1H NMR spectrum, CH2 protons of
pyrimidine ring resonate as a pair of doublets of doublets (dd)
at 3.42–3.52 ppm (HA), 3.50–3.61 ppm (HB). The CH proton (HX)
of pyrimidine was observed at 5.04–5.16 ppm as a pair of
doublet of doublets due to vicinal coupling with the two
magnetically non-equivalent protons of the methylene
group at position 5 of the pyrimidine ring (JAB: 16.1–17.2 Hz,
JAX: 6.3–7.6 Hz, JBX: 12.0–13.2 Hz). The CH2 protons of triazine
Figure 2. Pharmacophoric elements of Dimmock’s model and the designed compounds 4a–t.
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Figure 3. Rationale behind the designing of the new pyrimidine–triazine hybrids 4a–t.
ring appeared as a pair of doublets at 3.15–3.29 ppm (H1) and
3.79–3.91 ppm (H2). The NH proton of triazine was seen at
9.20–9.37 ppm and NH proton of CONH at 11.78–12.53 ppm as
a pair of singlets. The elemental analyses data were within
ꢁ0.4% of the theoretical values.
(3JAB ¼ 5.7) for the CH C and another doublet at 6.75–
–
–
6.78 ppm (3JAB ¼ 15.7 Hz) for the CO CH, indicating that the
–
–
ethylene moiety in the enone linkage is in the trans
conformation. All measured coupling constants for 1a–t were
between 15 Hz and 18 Hz which is typical for trans-orientated
double bond protons.
Stereochemistry of chalcones
All the chalcones 1a–t were obtained as pure E-isomers
Pharmacology
(Fig. 4). An AB two-spin system overlapping with a typical
The preclinical discovery and development of a new chemical
entity as an anticonvulsant agent is a great challenge in the
field of medicinal chemistry. The development of a new
anticonvulsant compound is completely based on the use of
trans coupling of 3JAB ¼ 12–18 Hz confirms a C C double bond
–
1
–
with protons in trans configuration [54–56]. H NMR spectra
of chalcone 1a showed one doublet at 7.58–7.61 ppm
Table 1. Distance ranges between the essential pharmacophoric elements A, D, and HBD.
Compound
A–HBDa)
A–Da)
D–HBDa)
Carbamazepine
Phenytoin
Lamotrigine
Zonisamide
Rufinamide
Dezinamide
Remacemide
Diazepam
6.517
3.042
5.807
4.058
2.407
4.481
3.211
4.793
2.465
3.931
3.868
3.301
5.651
7.474
5.909
9.811
4.827
3.792
5.554
2.497
4.598
6.729
5.209
2.948
6.635
1.49
4a–t
5.561
a) Distance calculated for 3D-optimized structures using ACD Freeware 3D Viewer 12.01 version.
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Pyrimidine–Triazine Hybrids as Anticonvulsants
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Scheme 1. Synthesis of compounds 4a–t.
Table 2. Physicochemical parameters of the synthesized pyrimidine–triazine hybrids (4a–t).
Mol.
Compound
R
R1
formulaa)
Mol. wt.
m.p. (°C)
Rfb)
%Yield
Log Pc)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
H
Cl
CH3
OH
F
H
Cl
CH3
OH
F
H
Cl
CH3
OH
F
H
Cl
CH3
OH
F
H
H
H
H
H
C19H17N5OS
C19H16ClN5OS
C20H19N5OS
C19H17N5O2S
C19H16FN5OS
C21H22N6OS
C21H21ClN6OS
C22H24N6OS
C21H22N6O2S
C21H21FN6OS
363.44
397.88
377.46
379.44
381.43
406.50
440.95
420.53
422.50
424.49
379.44
413.88
393.46
395.43
397.43
409.46
443.91
423.49
425.46
427.45
116–118
202–204
234–236
156–158
218–220
196–198
242–244
270–272
192–194
178–180
216–218
224–226
246–248
168–170
194–196
112–114
188–190
214–216
172–174
230–232
0.66
0.62
0.74
0.72
0.81
0.69
0.61
0.47
0.73
0.65
0.75
0.78
0.68
0.56
0.64
0.73
0.57
0.63
0.58
0.82
67
62
54
73
77
68
59
63
58
69
72
78
74
65
70
56
79
61
75
55
3.08
3.79
3.58
2.41
3.22
3.25
3.96
3.74
2.58
3.39
2.41
3.13
2.91
1.75
2.56
2.26
2.98
2.76
1.60
2.41
4-N(CH3)2
4-N(CH3)2
4-N(CH3)2
4-N(CH3)2
4-N(CH3)2
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH, 3-OCH3
4-OH, 3-OCH3
4-OH, 3-OCH3
4-OH, 3-OCH3
4-OH, 3-OCH3
C19H17N5O2S
C19H16ClN5O2S
C20H19N5O2S
C19H17N5O3S
C19H16FN5O2S
C20H19N5O3S
C20H18ClN5O3S
C21H21N5O3S
C20H19N5O4S
C20H18FN5O3S
4s
4t
a) Solvent of crystallization – ethanol.
b) Solvent system – toluene/ethyl acetate/formic acid (5:4:1); chloroform/methanol (9:1).
c) Log P was calculated using the software Chem Draw Ultra 8.0.
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reference standards. All the synthesized compounds were
injected intraperitoneally (i.p.) at doses of 30, 100, and
300 mg/kg body weight at 0.5 and 4.0 h. The neurologic deficit
was performed by rotarod test. The initial (phase I)
anticonvulsant evaluation showed that the title compounds
were found to have promising anticonvulsant activity and the
data are listed in Table 3.
Phase I anticonvulsant evaluation
Almost all of the synthesized compounds were found active in
MES test. Interestingly, 4e, 4g, 4k, 4o, 4p, and 4s were found
most potent against standard drugs at 0.5 h at a dose of
30 mg/kg and therefore indicative of their antiseizure
properties by preventing the seizure spread at a relatively
lower dose. In contrast, 4c, 4d, 4h, 4i, 4j, and 4n showed
protection at a moderate dose of 100 mg/kg after 0.5 h,
showing their ability to have a rapid onset and short duration
of anticonvulsant action. Thereafter, 4e, 4o, and 4s continued
to show protection even after 4.0 h of drug administration at
a dose of 30 mg/kg and from this it can be well said that these
compounds have rapid onset with long duration of action.
Compounds 4c, 4g, 4k, and 4p showed protection at 100
mg/kg after 4.0 h of drug administration in animals, which
Figure 4. The stereochemistry of chalcones 1a–t.
proposed animal models [57]. The anticonvulsant activity of
the target compounds was evaluated against two standard
animal seizure models; one is electrically induced seizure
model, that is, maximal electroshock (MES)-induced seizures,
and other is chemically induced seizure model, that is,
subcutaneous pentylenetetrazole (scPTZ)-induced seizures.
The carbamazepine (CBZ) and phenytoin (PHY) were used as
Table 3. Phase I anticonvulsant activity and minimal motor impairment of 4a–t.
Intraperitoneal injection in micea)
Minimal motor impairment
screen
MES screen
scPTZ screen
Compound
0.5 h
4.0 h
0.5 h
4.0 h
0.5 h
4.0 h
b)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
ꢀ
300
300
100
ꢀ
ꢀ
ꢀ
300
300
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
300
100
100
30
300
30
100
100
100
30
ꢀ
300
ꢀ
300
ꢀ
ꢀ
ꢀ
30
300
300
ꢀ
300
ꢀ
ꢀ
300
ꢀ
ꢀ
ꢀ
100
ꢀ
ꢀ
300
ꢀ
300
ꢀ
ꢀ
ꢀ
ꢀ
300
300
ꢀ
300
300
ꢀ
ꢀ
ꢀ
300
100
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
300
ꢀ
100
300
100
100
100
100
300
100
ꢀ
ꢀ
300
ꢀ
300
ꢀ
ꢀ
300
ꢀ
100
30
300
30
300
ꢀ
ꢀ
100
100
300
300
100
ꢀ
ꢀ
30
100
ꢀ
300
ꢀ
300
300
30
300
30
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
4s
4t
PHY
CBZ
30
ꢀ
ꢀ
300
30
100
300
100
100
ꢀ
ꢀ
ꢀ
100
300
30
100
300
a) Number of animals used ¼ 6. The data in the table indicate the minimum dose whereby bioactivity was demonstrated in half or
more of the mice.
b) The dash (ꢀ) indicates an absence of activity at the maximum dose administered (300 mg/kg).
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indicates their ability of protection at a moderate dose and for
a longer duration. Moreover, 4e, 4o, and 4s were found most
potent compounds among all at a dose of 30 mg/kg at 0.5 h
duration and continued to show protection even after 4.0 h of
drug administration at the same dose which indicated the
potential of these compounds against generalized tonic–
clonic seizure.
In the chemically induced scPTZ test, compounds 4l, 4n, 4o,
4p, 4q, and 4s showed protection at 100 mg/kg at 0.5 h of drug
administration, which indicates that these compounds have
the potency to raise seizure threshold. The compounds 4c, 4e,
4f, 4i, 4j, 4m, and 4r were found active at 300 mg/kg at 0.5 h,
exhibiting their potential of rapid onset at a higher dose. Even
after 4.0 h of test compound administration, 4o, 4p, and 4s
were found potent at a dose of 100 mg/kg. Interestingly,
compounds 4o, 4p, and 4s were most active among the series
against PTZ-induced seizures.
Most of the compounds in neurotoxicity study were devoid
of any sign of toxicity except that compounds 4c, 4g, 4l, 4n, 4t
and 4e, 4g, 4l, 4p showed neurotoxicity at the dose level of
300 mg/kg at 0.5 h and 4.0 h, respectively. All of these
compounds were found toxic at a dose of 300 mg/kg or
above but below this dose, these were found safe.
Though the compounds 4e and 4p were found active against
MES- and scPTZ-induced seizure test on another side, they were
found neurotoxic at 300 mg/kg. From all these results, it may be
concluded that 4o and 4s are lead candidates in the series.
carbamazepine (8.80 mg/kg). In the scPTZ screen, 4o and 4s
offered protection with an ED50 of 285.02 and 293.42 mg/kg,
respectively, which was comparable to phenytoin and
carbamazepine. The TD50 for 4o and 4s in rotarod test was
also higher with
a dose of 389.11 and 412.16 mg/kg,
respectively, compared to the standard drugs. The PI values
were found 17.26 and 14.01 in MES and 1.36 and 1.40 in the
scPTZ screen for 4o and 4s, respectively, showing that both of
these compounds are safer and effective anticonvulsant
agents among the series.
Serum enzyme estimation
Some anticonvulsant drugs exert serious side effects. The liver
toxicity is the major issue among all. Therefore, the most
active compounds 4o and 4s were tested for liver enzyme
estimation. The most potent compounds 4o and 4s showed
significant (p < 0.05) change in the liver enzymes level such as
serum glutamate oxaloacetate transaminase (SGOT), serum
glutamate pyruvate transaminase (SGPT), alkaline phosphates
(ALP), total albumin and total bilirubin level as compared to
toxic (PTZ) group. The change in the SGOT level in the normal
(saline) group and tested compounds was almost same. It can
be said that no considerable change in enzyme level was
observed as compared to normal saline group and the test
compounds (4o and 4s) did not show any signs of hepatic
toxicity as evidenced from the results shown in Table 5.
GABA estimation
Phase II anticonvulsant evaluation
To assess the potency of the most potent compounds (4o and
4s) on GABA receptor, neurochemical estimation for GABA
level was done on mice brain. The results showed that after
2 h of drug administration, 4o and 4s were found to
significantly enhance the concentration of GABA in the brain
On the basis of promising anticonvulsant results found in
phase I anticonvulsant evaluation, compounds 4o and 4s were
further subjected to phase II trials for quantification studies in
mice. Both of the compounds were administered i.p. at
different doses for their anticonvulsant activity and neuro-
toxicity evaluation of median effective dose (ED50), median
toxic dose (TD50), and protective index (PI).
As shown in Table 4, both of the compounds 4o and 4s
exhibited efficacy against MES screen with ED50 values of
22.54 and 29.40 mg/kg, respectively, which were higher as
compared to standard drugs, phenytoin (9.50 mg/kg) and
with
a value of 77.42 mg/100 mg and 86.17 mg/100 mg,
respectively, as compared to control (58.49 mg/100 mg). For
continuous 7-day treatment of test drugs on mice, the
concentration of GABA was increased to 78.96 mg/100 mg
and 91.47 mg/100 mg for compound 4o and 4s, respectively,
against a value of 60.55 mg/100 mg for control as presented in
Table 6 and graph presented in Fig. 5.
Table 4. Phase II quantification studies of the active compounds (4o and 4s).
ED50 (mg/kg)a)
PIc)
Compound
MES
scPTZ
TD50 (mg/kg)b)
MES
scPTZ
4o
4s
PHY
CBZ
22.54 ꢁ 1.32d)
29.40 ꢁ 0.81
9.50 ꢁ 0.77
8.80 ꢁ 1.02
285.02 ꢁ 2.42
293.42 ꢁ 2.63
>300
389.11 ꢁ 9.32
412.16 ꢁ 11.09
65.50 ꢁ 12.06
71.60 ꢁ 12.07
17.26
14.01
6.9
1.36
1.40
<0.22
<0.22
>100
8.1
a) ED50 ¼ median effective dose eliciting anticonvulsant protection in 50% of the animals.
b) TD50 ¼ median toxic dose eliciting minimal neurological toxicity in 50% of the animals.
c) PI ¼ protective index (TD50/ED50).
d) Data are in the 95% confidence limits.
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Table 5. Liver enzyme estimation of the active compounds (4o and 4s).
Compounda)
SGOT level
SGPT level
ALP
Albumin
Bilirubin
Normalb)
Toxicc)
4o
34.90 ꢁ 1.16d)
43.42 ꢁ 1.46
36.92 ꢁ 1.93
37.57 ꢁ 1.82
25.42 ꢁ 1.62
36.65 ꢁ 1.39
31.10 ꢁ 1.51
30.64 ꢁ 0.82
20.39 ꢁ 2.68
32.16 ꢁ 2.67
22.40 ꢁ 2.12
21.19 ꢁ 2.45
1.91 ꢁ 0.29
3.44 ꢁ 0.34
2.12 ꢁ 0.28
2.99 ꢁ 0.25
1.72 ꢁ 0.24
3.28 ꢁ 0.30
2.08 ꢁ 0.19
2.18 ꢁ 0.29
4s
a) Number of animals tested (n ¼ 6).
b) Normal group treated with normal saline for 2 weeks.
c) Toxic group treated with PTZ for 2 weeks.
d) p < 0.05 versus toxic (control). Data analyzed by using ANOVA followed by Dunnett’s test. The data are in mean ꢁ SEM.
Structure–activity relationships
at the fourth position of lipophilic aryl ring and 4-OH or (4-
OH,3-OCH3) at distal aryl ring. From this outcome, it can be
said that substituent at the fourth position of lipophilic aryl
ring may be responsible for hydrophobic interactions of
ligands with the target protein and the substituent at the
fourth and third position of distal aryl ring may involve in
intermolecular hydrogen bonding with the receptor sites and
hence supports the anticonvulsant action.
The synthesized pyrimidine–triazine hybrids (4a–t) were
found as a lead toward efficacious anticonvulsant drug
development. Their potential as anticonvulsant agents
enabled us to make some general conclusions about the
relation between their structure and activity. The protection
offered by these compounds was indicative of their ability as
antiseizure agents and this property is supposed to be due to
various electron donating and withdrawing groups at the
fourth position of lipophilic aryl ring and unsubstituted or
substituted distal aryl ring. The compounds 4e, 4o, and 4s
were found most active in preliminarily in vivo anticonvulsant
activity against MES test. From this, it may be observed that
the presence of electron withdrawing group (F) or electron
releasing group (OH) at the fourth position of substituted
lipophilic aryl ring and unsubstituted or substituted distal aryl
with 4-OH or (4-OH,3-OCH3) are responsible for the excellent
anticonvulsant activity. In the scPTZ model, unsubstituted or
substituted with an electron withdrawing group such as F, or
electron releasing group (OH) on the fourth position of
lipophilic aryl ring with 4-OH or (4-OH,3-OCH3) substituted
distal aryl ring in compounds 4p, 4o, and 4s favors the
anticonvulsant activity. Fortunately, compounds 4o and 4s
were found to enhance the GABA concentration in the brain.
Moreover, it may be concluded that promising anticonvulsant
effect of these compounds was supposed to be due to F or OH
Molecular docking study
Computer-aided drug design assists in designing the selective
and potent inhibitors [58]. The availability of 3D structure of
the receptor further improvises the process and techniques
like molecular docking can be used to optimize novel drug
candidates and to study the molecular interaction at protein–
ligand interface. To understand the molecular basis of
anticonvulsant activity of the newly designed series of
pyrimidine–triazine derivatives (4a–t) and to explore the
binding mechanisms of these molecules, we docked these
molecules into the active site of GABAA receptor (PDB ID-
4COF). The active site of GABAA receptor consists of Arg142,
Pro144, Leu145, Asp146, Glu147, Gln185, Ser187, Lys215,
Arg216, Asn217, Ile218, Asn439, Arg142, Glu147, Asn217,
Leu145, Lys215, Asp146, Trp443, Tyr446, and Val447 [56, 57].
Table 6. GABA level estimation of active compounds.
GABA concentration in mice whole
brain (mg/100 mg tissue)a)
2-h
7-day
post-treatment
Compound
post-treatment
Control
4o
4s
58.49 ꢁ 1.77b)
77.42 ꢁ 2.00
86.17 ꢁ 2.21
60.55 ꢁ 2.03
78.96 ꢁ 2.13
91.47 ꢁ 2.17
a) Tested at a dose of 30 mg/kg i.p.
Figure 5. Effects of compounds 4o and 4s on GABA levels in
mice brain tissues (mg/100 mg tissue). Each value represents the
mean ꢁ SEM of six mice, significantly different from the control
at ꢂꢂp < 0.01 (Student’s t-test).
b) Each value represents the mean ꢁ SEM of six mice,
significantly different from the control at p < 0.01 (Student’s
t-test).
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Similar to carbamazepine, a marketed GABAA inhibitor, 16
of our synthesized molecules also showed the conserved
H-bond interaction with Arg216 which is a gate-keeper
residue in the GABAA active site. The Glide score (in kcal/mol)
of all our pyrimidine–triazine analogs ranged within ꢀ7.4 and
ꢀ4.9. The Glide score of molecules 4s (ꢀ7.4) and 4o (ꢀ7.1)
were even better than the standard drugs (phenytoin: ꢀ6.9;
carbamazepine: ꢀ6.3) as depicted in Table 7. The docking
score for 4e was ꢀ6.9 which is similar to phenytoin but higher
than carbamazepine. The results of all molecules docked in
GABAA active sites are shown in Table 7 in terms of different
interactions (H-bonds, p–cation, p–stacking, and CH–p).
Besides H-bonding, the substituted phenyl ring in 11 of the
synthesized pyrimidine–triazine derivatives made p–cation
interaction with Lys215, another key residue in GABAA active
site. In addition, 4d and 4q showed p–p stacking interaction
Table 7. Glide scores, H-bond, p-cation, CH-p, and p-stacking interactions of compounds (4a–t) with GABAA receptor
(PDB ID: 4COF).
Type of interactions
Hydrogen bonds
Atom
p Interactions
Glide
score
(kcal/
mol)
Serial
no.
of
ligand
Amino
acids
Dist.
Amino
acids
Dist.
(A)
̊
̊
Compd.
4s
(A)
Type
Ring/group
1
ꢀ7.4
N
O
O
O
N
O
N
O
N
O
O
N
N
O
O
N
N
O
N
O
O
N
N
N
O
N
N
O
O
O
O
N
O
O
N
O
O
N
O
Arg142
Glu147
Arg216
Ile218
Arg216
Ile218
Arg216
Ile218
Arg216
Ile218
Arg216
Arg216
Arg216
Ile218
Arg216
Lys215
Lys215
Arg216
Lys215
Arg216
Lys215
Arg216
Arg216
Arg216
Ile 218
Asn217
Arg216
Asn439
Asn439
Asn439
Arg216
Arg216
Arg216
Ile218
2.92
2.03
1.74
2.80
1.77
2.11
2.07
2.06
1.83
2.07
2.16
2.74
1.83
2.12
1.60
2.13
2.00
1.85
2.16
2.08
2.07
1.67
2.25
1.88
2.11
3.01
2.03
1.76
1.75
1.77
1.84
1.75
1.89
3.07
2.08
2.24
2.04
1.96
2.89
p–cation
p–cation
4-OH-Phenyl
4-OH,3-OCH3Phenyl
Lys215
Arg142
4.46
4.70
2
4o
4e
4g
4k
4p
4r
ꢀ7.1
ꢀ6.9
ꢀ6.5
ꢀ6.5
ꢀ6.5
ꢀ6.4
ꢀ6.4
ꢀ6.3
ꢀ6.2
p–cation
CH–p
4-OH-Phenyl
Lys215
Val447
Lys215
Lys215
Lys215
Lys215
Lys215
4.72
3.73
4.67
4.99
4.38
5.03
4.78
3
4-F-Phenyl
4
p–cation
p–cation
p–cation
p–cation
p–cation
4-N(CH3)2Phenyl
4-OH-Phenyl
5
6
4-OH,3-OCH3Phenyl
4-OH,3-OCH3Phenyl
4-OH,3-OCH3Phenyl
7
8
4t
9
4q
4n
p–cation
p–stacking
p–cation
4-OH, 3-OCH3Phenyl
4-Cl-Phenyl
4-OH-Phenyl
Lys215
Trp443
Lys215
4.86
3.93
4.81
10
11
12
4f
4a
ꢀ5.9
ꢀ5.7
p–cation
4-N(CH3)2Phenyl
Lys215
ꢀ
5.15
ꢀ
ꢀ
ꢀ
13
4b
ꢀ5.7
p–cation
Phenyl
Lys215
4.73
14
15
16
17
18
19
4c
4m
4l
4d
4j
ꢀ5.5
ꢀ5.5
ꢀ5.4
ꢀ5.4
ꢀ5.4
ꢀ5.2
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
p–stacking
Phenyl
Trp443
5.18
ꢀ
–
ꢀ
ꢀ
4i
ꢀ
ꢀ
ꢀ
ꢀ
20
21
4h
PHY
ꢀ4.9
ꢀ6.9
Asp146
Lys215
Ile218
Arg216
Ile218
ꢀ
ꢀ
ꢀ
ꢀ
p–cation
Phenyl
Lys215
4.73
22
CBZ
ꢀ6.3
p–cation
Fused Phenyl
Lys215
4.43
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Figure 6. Predicted binding pose of compound a) 4s and b) 4o with GABAA receptor (PDB ID: 4COF). Compounds represented as tube,
H-bonding as blue line, p-cation interactions as green dashed line, and hydrophobic interactions as violet dashed lines.
with Trp443 through phenyl (4d) or chlorophenyl (4q)
substitution. Compound 4e exhibited CH–p interaction with
Val447.
Molecules 4a, 4g, and 4i also formed H-bond with Ile218 which
was also exhibited by standard drugs such as phenytoin and
carbamazepine (Fig. 7). In addition to p–cation interaction,
Lys215 also formed H-bond with 4n, 4q, 4r, 4t, and phenytoin.
Molecules 4c, 4l, and 4m showed H-bond with Asn439. In
addition, residues Asn217 (4k, 4b) and Asp146 (4h) were also
involved in H-bond interactions. In this docking study (Table 7),
we observed that 4-hydroxy-3-methoxyphenyl-substituted
distal aryl ring molecules (4p–t) showed most favorable
interactions (H-bond, p–cation, and p–p) with GABAA. In this
group, three molecules (4q, 4r, and 4t) showed H-bond with
Among all, the most effective molecule, 4s, showed
hydrogen bonds with Arg216, Ile218, Arg142, and Glu147.
In addition, para-hydroxy phenyl ring of 4s showed two
p–cation interactions with Lys215 and Arg142 (Fig. 6a).
Compound 4o (docking score: ꢀ7.1) made crucial H-bonds
with Arg216 and Ile218 of GABAA active site and a p–cation
interaction with Lys215 through its para-hydroxy phenyl ring
(Fig. 6b).
Figure 7. Predicted binding interactions of standard drugs a) phenytoin and b) carbamazepine with the GABAA receptor (PDB ID:
4COF). Compounds represented as tube, H-bonding as blue line, p-cation interactions as green dashed line, and hydrophobic
interactions as violet dashed lines.
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Lys215. In compound 4r, methyl-substituted lipophilic aryl ring
developedH-bondwithArg216,whileothermoleculeswiththis
substitution (4c, 4h, and 4m) failed to form this H-bond.
Among molecules containing unsubstituted distal aryl ring
(4a–e), an electronegative substitution at lipophilic aryl ring
(4b, 4d, and4e)ledtop–cation,p–p, andCH–pinteractionwith
GABAA receptor. Among these, molecule 4e having most
electronegative substitution (fluoro) was found most promis-
ing (Glide score: ꢀ6.9) within this series. In group containing
4-(dimethylamino)phenyl-substituted distal aryl ring (4f–j), 4f
(unsubstituted lipophilic aryl ring) and 4g (weak electronega-
tive group-substituted lipophilic aryl ring) interacted through
H-bond and p–cation interactions. In group containing 4-
hydroxyphenyl-substituteddistalarylringderivatives(4k–o),4l
(electron-donating substituents) and 4m (weak electronega-
tive substituent) on lipophilic aryl ring failed to show p–cation
and strong H-bonds, while unsubstituted lipophilic aryl ring
(4k) and strong electronegative groups derivatives (4n and 4o)
possessed p–cation and strong H-bonds. These docking results
were in concordance with the observed animal studies.
screening of compounds for activity on HERG channels during
preclinical safety studies reduces the risk of failure of
compounds because of QT prolongation [66]. The desirable
ADME properties of these compounds provide confidence in
their safety, stability promising pharmacokinetic behavior,
and their druggable candidature (Supporting Information
Table S1).
Conclusion
A
series of 25 5-(4-(4-substituted phenyl)-6-(substituted
phenyl)-2-thioxo-5,6-dihydropyrimidine-1(2H)-yl)-1,2-dihydro-
1,2,4-triazin-3(6H)-ones (4a–t) was synthesized and envisaged
as potential anticonvulsants in this unprecedented study. The
initial anticonvulsant screening was performed in mice using
the “classical” MES and scPTZ tests, and acute neurological
toxicity was determined using rotarod test. Compounds 4o and
4s showed the broad spectra of activity across all the preclinical
seizure models. Additionally, quantification study results
showed that 4o and 4s were found to have median doses of
22.54 and 29.40 mg/kg (MES ED50), 285.02 and 293.42 mg/kg
(scPTZ ED50), and 389.11 and 412.16 mg/kg (TD50), respectively.
To derive a mechanistic approach toward their mode of action,
the study on GABA channel was carried out based on their
structural resemblance to the reference AEDs. The molecular
docking study and GABA estimation in rat whole brain also
supported the potential of most active compounds as
anticonvulsant agents. In summary, it is justified that 4o and
4s emerged as the most promising molecules with favorable
protective indexes compared with standard AEDs and can play
an essential role in the search for new anticonvulsants.
Absorption, distribution, metabolism, and excretion
(ADME) studies
ADME descriptors analysis provides significant insight into the
properties that are pharmaceutically relevant for organic
molecules. Therefore, this analysis helps to exclude weak or
toxic molecule at initial stage of drug discovery and
development process and thus prevent the cost and man-
power efforts associated with the late stage failures in the
drug discovery process [59–63].
The synthesized small molecules were analyzed for ADME
parameters by QikProp 4.0. The Qikprop calculates the vital
properties of a molecules which provide insight into its stability
(number of reactive functional groups, number of hydrogen
bond donor and acceptors, number of metabolic reactions),
druggable behavior, and its pharmacokinetics (molecular
weight, CNS activity, violation of Lipinski’s rule of five,
octanol/water coefficient, brain/blood partition coefficient,
aqueous solubility, skin permeability, binding to human serum
albumin, human oral absorption, IC50 value for HERG Kþ
channel, Caco-2 cell permeability, MDCK cell permeability) [64].
The human colon adenocarcinoma (Caco-2) and Madin–
Darby canine kidney (MDCK) epithelial cell lines are the in
vitro probes to evaluate a drug’s permeability and transporter
interactions during drug discovery and development process.
This study has critical significance in predicting absorption,
permeability mechanism, effect of formulation on perme-
ability, and probability for transport-mediated drug–drug
interactions [65]. Human ether-a-go-go-related gene (HERG)
is the name of a human gene. The protein encoded by this
gene is the HERG channel which is voltage-gated potassium
channel. These channels are actively involved in cardiac action
potential repolarization. The reduced activity of HERG leads
to lengthening of ventricular action potentials and extension
of the QT interval in an electrocardiogram which in turn
increases the risk for fatal ventricular arrhythmias. Therefore,
Experimental
Chemistry
All the chemicals used were procured from commercial sources
and used in their original form. The progress of reactions was
monitored by TLC on silica gel plates. The melting points of
synthesized compounds were determined in open capillary
tubes in a Hicon melting point apparatus (Hicon, India) and are
uncorrected. All the Fourier transform infrared (FT-IR) spectra
were recorded on a Shimadzu FTIR spectrometer in KBr pellets.
1H NMR spectra were recorded on Bruker model DRX 400 NMR
spectrometer in CDCl3/DMSO-d6 using tetramethylsilane (TMS)
as an internal standard with the resonant frequency of
400MHz. 13C NMR spectra were also measured on Bruker
model DRX 400 NMR at 100MHz with complete proton
decoupling. Splitting patterns are nominated as follows: s,
singlet; brs, broad singlet; d, doublet; dd, double doublet; m,
multiplet; and J, coupling constant. The NH protons were
D2Oexchangedfortheirspectralcharacterization. Mass spectra
were recorded on Waters Synapt Mass spectrometer. The
elemental analyses (C, H, N, S) of all compounds were
performed on the CHNS Elementar (Analysensysteme, GmbH,
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Germany) Vario EL III and data were within ꢁ0.4% of the
theoretical values.
The InChI codes of the investigated compounds are
provided as Supporting Information.
400 MHz) d ppm: 3.21 (d, J ¼ 16.1 Hz, 1H, H1-triazine), 3.42 (dd,
JAB ¼ 16.4 Hz, JAX ¼ 6.8 Hz, 1H, HA), 3.51 (dd, JBA ¼ 16.4 Hz,
JBX ¼ 12.4 Hz, 1H, HB), 3.87 (d, J ¼ 16.1 Hz, 1H, H2-triazine), 5.04
(dd, JXA ¼ 6.8 Hz, JXB ¼ 12.4 Hz, 1H, HX), 7.04–8.08 (m, 10H, Ar-
H), 9.25 (s, 1H, NH, D2O exchangeable), 12.23 (s, 1H, CONH,
D2O exchangeable); 13C NMR (CDCl3, 100 MHz) d ppm: 42.3,
49.2, 59.8, 126.5, 128.2, 128.6, 128.7, 128.8, 128.9, 136.4, 140.3,
153.4, 153.5, 167.3, 182.6; MS m/z: 364 (Mþþ1); Anal. calcd. for
Synthesis of 1-(4-substituted phenyl)-3-(substituted
phenyl)prop-2-en-1-ones 1a–t
The 4-substituted acetophenone (0.01 mol) and appropriate
aromatic aldehyde (0.01 mol) were stirred in ethanol (10–
20 mL) at 0–5°C for 2 h, followed by addition of 10 mL of 40%
sodium hydroxide solution in the same vessel. This mixture
was kept overnight at room temperature for completion of
the reaction. The contents were then poured onto crushed ice
and acidified with concentrated hydrochloric acid. The
precipitated crude product (chalcone) was filtered off, dried,
and recrystallized from ethanol to obtain 1a–t.
C
19H17N5OS: C, 62.79; H, 4.71; N, 19.27; S, 8.82%. Found: C,
62.82; H, 4.74; N, 19.30; S, 8.80%.
5-(4-(4-Chlorophenyl)-6-phenyl-2-thioxo-5,6-
dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-
3(6H)-one (4b)
IR (KBr) nmax cmꢀ1: 3311 (cyclic CONH, NHstr.), 3278 (NHstr.),
3030 (C–H Arstr.), 1656 (C N ), 1220 (C Sstr.); 1H NMR (CDCl3,
–
–
–
–
str.
400 MHz) d ppm: 3.22 (d, J ¼ 16.2 Hz, 1H, H1-triazine), 3.43 (dd,
JAB ¼ 16.8 Hz, JAX ¼ 6.8 Hz, 1H, HA), 3.52 (dd, JBA ¼ 16.8 Hz,
JBX ¼ 12.8 Hz, 1H, HB), 3.89 (d, J ¼ 16.2 Hz, 1H, H2-triazine), 5.06
(dd, JXA ¼ 6.8 Hz, JXB ¼ 12.8 Hz, 1H, HX), 7.10–8.09 (m, 9H,
Ar-H), 9.20 (s, 1H, NH, D2O exchangeable), 12.16 (s, 1H, CONH,
D2O exchangeable); 13C NMR (CDCl3, 100 MHz) d ppm: 42.4,
49.3, 59.9, 126.4, 128.5, 128.6, 128.7, 128.8, 135.6, 136.5, 140.4,
153.5, 153.7, 167.1, 182.7; MS m/z: 398 (Mþþ1), 399 (Mþþ2);
Anal. calcd. for C19H16ClN5OS: C, 57.35; H, 4.05; N, 17.60; S,
8.06%. Found: C, 57.38; H, 4.09; N, 17.63; S, 8.09%.
Synthesis of 4-(4-substituted phenyl)-6-(substituted
phenyl)-5,6-dihydropyrimidine-2(1H)-thiones 2a–t
An equimolar quantity of synthesized chalcone (1a–t) and
thiourea was dissolved in ethanol. Then, 0.017 mol of
potassium hydroxide was added and the contents were
refluxed for 18–20 h. After completion of the reaction, the
reaction mixture was poured on ice and the precipitated solid
was collected and recrystallized from appropriate solvent to
afford the 4-(4-substituted phenyl)-6-(substituted phenyl)-
5,6-dihydropyrimidine-2(1H)-thiones 2a–t.
5-(6-Phenyl-2-thioxo-4-p-tolyl-5,6-dihydropyrimidin-
1(2H)-yl)-1,2-dihydro-1,2,4-triazin-3(6H)-one (4c)
IR (KBr) nmax cmꢀ1: 3344 (cyclic CONH, NHstr.), 3289 (NHstr.),
Synthesis of 2-chloro-1-(4-(4-substituted phenyl)-6-
(substituted
phenyl)-2-thioxo-5,6-dihydropyrimidine-
1(2H)-yl)ethanones 3a–t
3033 (C–H Arstr.), 1688 (C N ), 1211 (C Sstr.); 1H NMR (CDCl3,
–
str.
–
–
–
The appropriate pyrimidinethione (2a–t) (0.01 mol) was mixed
with 20 mL dichloromethane (DCM) and two to three drops of
triethylamine. This mixture was stirred at 0–5°C in ice for half
an hour while adding 0.14 mol of chloroacetylchloride drop
by drop. The reaction mixture was stirred overnight. Excess of
DCM was evaporated and the leftover residue was dried and
recrystallized from ethanol to get the 2-chloro-1-(4-(4-
substituted phenyl)-6-(substituted phenyl)-2-thioxo-5,6-dihy-
dropyrimidine-1(2H)-yl)ethanones 3a–t.
400 MHz) d ppm: 2.36 (s, 3H, CH3), 3.25 (d, J ¼ 16.4 Hz, 1H, H1-
triazine), 3.46 (dd, JAB ¼ 16.4 Hz, JAX ¼ 6.4 Hz, 1H, HA), 3.53
(dd, JBA ¼ 16.4 Hz, JBX ¼ 12.0 Hz, 1H, HB), 3.88 (d, J ¼ 16.4 Hz,
1H, H2-triazine), 5.10 (dd, JXA ¼ 6.4 Hz, JXB ¼ 12.0 Hz, 1H, HX),
6.80–8.25 (m, 9H, Ar-H), 9.23 (s, 1H, NH, D2O exchangeable),
12.19 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3,
100 MHz) d ppm: 21.2, 42.5, 49.3, 59.7, 126.6, 128.2, 128.4,
128.8, 129.3, 136.4, 139.7, 140.5, 153.5, 153.6, 167.2, 182.5;
MS m/z: 378 (Mþþ1); Anal. calcd. for C20H19N5OS: C, 63.64; H,
5.07; N, 18.55; S, 8.49%. Found: C, 63.66; H, 5.09; N, 18.59; S,
8.52%.
Synthesis of 5-(4-(4-substituted phenyl)-6-(substituted
phenyl)-2-thioxo-5,6-dihydropyrimidine-1(2H)-yl)-1,2-
dihydro-1,2,4-triazin-3(6H)-ones 4a–t
The synthesized product (3a–t) (0.01 mol) and potassium
carbonate (0.01 mol) were mixed with N,N-dimethylforma-
mide (20 mL). An equimolar quantity of semicarbazide was
added to the reaction mixture and refluxed for 12–16 h. The
resultant mixture was left to cool, poured onto ice-cold water,
filtered, and recrystallized from appropriate solvent to obtain
targeted compounds 4a–t.
5-(4-(4-Hydroxyphenyl)-6-phenyl-2-thioxo-5,6-
dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-
3(6H)-one (4d)
IR (KBr) nmax cmꢀ1: 3326 (cyclic CONH, NHstr.), 3190 (NHstr.),
3026 (C–H Arstr.), 1682 (C N ), 1222 (C Sstr.); 1H NMR (CDCl3,
–
–
–
–
str.
400 MHz) d ppm: 3.26 (d, J ¼ 16.1 Hz, 1H, H1-triazine), 3.44 (dd,
JAB ¼ 17.2 Hz, JAX ¼ 7.2 Hz, 1H, HA), 3.52 (dd, JBA ¼ 17.2 Hz,
JBX ¼ 12.8 Hz, 1H, HB), 3.88 (d, J ¼ 16.1 Hz, 1H, H2-triazine), 5.05
(dd, JXA ¼ 7.2 Hz, JXB ¼ 12.8 Hz, 1H, HX), 6.82–8.24 (m, 9H, Ar-
H), 9.24 (s, 1H, NH, D2O exchangeable), 10.03 (s, 1H, OH), 12.22
(s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3, 100 MHz) d
ppm: 42.5, 49.4, 59.9, 116.9, 126.5, 128.4, 128.6, 128.9, 136.6,
140.4, 153.7, 153.8, 157.8, 167.3, 182.4; MS m/z: 380 (Mþþ1);
5-(4,6-Diphenyl-2-thioxo-5,6-dihydropyrimidin-1(2H)-yl)-
1,2-dihydro-1,2,4-triazin-3(6H)-one (4a)
IR (KBr) nmax cmꢀ1: 3328 (cyclic CONH, NHstr.), 3291 (NHstr.),
3028 (C–H Arstr.), 1643 (C N ), 1224 (C Sstr.); 1H NMR (CDCl3,
–
–
–
–
str.
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Arch. Pharm. Chem. Life Sci. 2017, 350, e1700146
Pyrimidine–Triazine Hybrids as Anticonvulsants
Archiv der Pharmazie
Anal. calcd. for C19H17N5O2S: C, 60.14; H, 4.52; N, 18.46; S,
8.45%. Found: C, 60.17; H, 4.56; N, 18.42; S, 8.47%.
400 MHz) d ppm: 2.34 (s, 3H, CH3), 2.94 (s, 6H, N(CH3)2), 3.18 (d,
J ¼ 16.4 Hz, 1H, H1-triazine), 3.42 (dd, JAB ¼ 16.4 Hz, JAX ¼ 6.8
Hz, 1H, HA), 3.50 (dd, JBA ¼ 16.4 Hz, JBX ¼ 12.0 Hz, 1H, HB), 3.82
(d, J ¼ 16.4 Hz, 1H, H2-triazine), 5.10 (dd, JXA ¼ 6.8 Hz, JXB
¼ 12.0 Hz, 1H, HX), 7.11–8.31 (m, 8H, Ar-H), 9.37 (s, 1H, NH,
D2O exchangeable), 11.91 (s, 1H, CONH, D2O exchangeable);
13C NMR (CDCl3, 100 MHz) d ppm: 21.3, 40.7, 42.9, 49.1, 59.6,
112.9, 127.3, 128.2, 129.3, 136.7, 139.7, 140.5, 151.5, 153.7,
153.9, 167.5, 181.6; MS m/z: 421 (Mþþ1); Anal. calcd. for
5-(4-(4-Fluorophenyl)-6-phenyl-2-thioxo-5,6-
dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-
3(6H)-one (4e)
IR (KBr) nmax cmꢀ1: 3330 (cyclic CONH, NHstr.), 3288 (NHstr.),
3036 (C–H Arstr.), 1644 (C N ), 1208 (C Sstr.); 1H NMR (CDCl3,
–
–
–
–
str.
400 MHz) d ppm: 3.21 (d, J ¼ 16.2 Hz, 1H, H1-triazine), 3.49 (dd,
JAB ¼ 17.2 Hz, JAX ¼ 7.6 Hz, 1H, HA), 3.58 (dd, JBA ¼ 17.2 Hz,
JBX ¼ 13.2 Hz, 1H, HB), 3.89 (d, J ¼ 16.2 Hz, 1H, H2-triazine), 5.08
(dd, JXA ¼ 7.6 Hz, JXB ¼ 13.2 Hz, 1H, HX), 7.08–8.14 (m, 9H,
Ar-H), 9.25 (s, 1H, NH, D2O exchangeable), 12.22 (s, 1H, CONH,
D2O exchangeable); 13C NMR (CDCl3, 100 MHz) d ppm: 42.6,
49.2, 59.6, 115.9, 126.5, 127.7, 128.6, 128.9, 136.7, 140.6, 153.7,
153.8, 163.3, 167.6, 182.8; MS m/z: 382 (Mþþ1); Anal. calcd.
for C19H16FN5OS: C, 59.83; H, 4.23; N, 18.36; S, 8.41%. Found:
C, 59.86; H, 4.26; N, 18.39; S, 8.44%.
C
22H24N6OS: C, 62.83; H, 5.75; N, 19.98; S, 7.62%. Found: C,
62.85; H, 5.74; N, 19.81; S, 7.66%.
5-(6-(4-(Dimethylamino)phenyl)-4-(4-hydroxyphenyl)-2-
thioxo-5,6-dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-
triazin-3(6H)-one (4i)
IR (KBr) nmax cmꢀ1: 3346 (cyclic CONH, NHstr.), 3274 (NHstr.),
3034 (C–H Arstr.), 1682 (C N ), 1210 (C Sstr.); 1H NMR (CDCl3,
–
–
–
–
str.
400 MHz) d ppm: 2.92 (s, 6H, N(CH3)2), 3.20 (d, J ¼ 16.1 Hz, 1H,
H1-triazine), 3.42 (dd, JAB ¼ 16.4 Hz, JAX ¼ 6.4 Hz, 1H, HA), 3.51
(dd, JBA ¼ 16.4 Hz, JBX ¼ 12.8 Hz, 1H, HB), 3.91 (d, J ¼ 16.1 Hz,
1H, H2-triazine), 5.11 (dd, JXA ¼ 6.4 Hz, JXB ¼ 12.8 Hz, 1H, HX),
6.92–8.29 (m, 8H, Ar-H), 9.22 (s, 1H, NH, D2O exchangeable),
10.12 (brs, 1H, OH), 11.78 (s, 1H, CONH, D2O exchangeable);
13C NMR (CDCl3, 100 MHz) d ppm: 40.5, 42.7, 49.3, 59.9,
112.8, 116.9, 127.7, 128.7, 136.7, 140.6, 151.6, 153.4, 153.8,
157.8, 167.6, 182.5; MS m/z: 423 (Mþþ1); Anal. calcd. for
5-(6-(4-(Dimethylamino)phenyl)-4-phenyl-2-thioxo-5,6-
dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-
3(6H)-one (4f)
IR (KBr) nmax cmꢀ1: 3324 (cyclic CONH, NHstr.), 3256 (NHstr.), 3027
(C–H Arstr.), 1646 (C N ), 1206 (C Sstr.); 1H NMR (DMSO-d6,
–
–
–
–
str.
400MHz) d ppm: 2.84 (s, 6H, N(CH3)2), 3.24 (d, J ¼ 16.2 Hz, 1H,
H1-triazine), 3.46 (dd, JAB ¼ 16.8 Hz, JAX ¼ 6.4 Hz, 1H, HA), 3.55
(dd, JBA ¼ 16.8 Hz, JBX ¼ 12.8 Hz, 1H, HB), 3.91 (d, J ¼ 16.2 Hz, 1H,
H2-triazine), 5.09 (dd, JXA ¼ 6.4 Hz, JXB ¼ 12.8 Hz, 1H, HX), 7.16–
8.29 (m, 9H, Ar-H), 9.35 (s, 1H, NH, D2O exchangeable), 12.46 (s,
1H, CONH, D2O exchangeable); 13C NMR (DMSO-d6, 100MHz) d
ppm: 40.3, 42.5, 49.3, 59.9, 112.9, 127.4, 128.2, 128.8, 128.9,
136.6, 140.5, 151.4, 153.7, 153.8, 167.3, 182.7; MS m/z: 407
(Mþþ1); Anal. calcd. for C21H22N6OS: C, 62.05; H, 5.45; N, 20.67;
S, 7.89%. Found: C, 62.09; H, 5.49; N, 20.70; S, 7.91%.
C
21H22N6O2S: C, 59.70; H, 5.25; N, 19.89; S, 7.59%. Found:
C, 59.72; H, 5.29; N, 19.91; S, 7.62%.
5-(6-(4-(Dimethylamino)phenyl)-4-(4-fluorophenyl)-2-
thioxo-5,6-dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-
triazin-3(6H)-one (4j)
IR (KBr) nmax cmꢀ1: 3327 (cyclic CONH, NHstr.), 3293 (NHstr.),
3038 (C–H Arstr.), 1645 (C N ), 1226 (C Sstr.); 1H NMR (CDCl3,
–
–
–
–
str.
400 MHz) d ppm: 2.88 (s, 6H, N(CH3)2), 3.15 (d, J ¼ 16.3 Hz, 1H,
H1-triazine), 3.48 (dd, JAB ¼ 16.8 Hz, JAX ¼ 6.8 Hz, 1H, HA), 3.56
(dd, JBA ¼ 16.8 Hz, JBX ¼ 13.2 Hz, 1H, HB), 3.87 (d, J ¼ 16.3 Hz,
1H, H2-triazine), 5.06 (dd, JXA ¼ 6.8 Hz, JXB ¼ 13.2 Hz, 1H, HX),
6.80–8.23 (m, 8H, Ar-H), 9.32 (s, 1H, NH, D2O exchangeable),
12.14 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3,
100 MHz) d ppm: 40.6, 42.8, 49.5, 59.3, 112.9, 115.6, 127.6,
127.8, 136.6, 140.8, 151.4, 153.9, 167.4, 182.2; MS m/z: 425
(Mþþ1); Anal. calcd. for C21H21FN6OS: C, 59.42; H, 4.99; N,
19.80; S, 7.55%. Found: C, 59.46; H, 5.02; N, 19.83; S, 7.58%.
5-(4-(4-Chlorophenyl)-6-(4-(dimethylamino)phenyl)-2-
thioxo-5,6-dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-
triazin-3(6H)-one (4g)
IR (KBr) nmax cmꢀ1: 3312 (cyclic CONH, NHstr.), 3258 (NHstr.), 3032
(C–H Arstr.), 1658 (C N ), 1225 (C Sstr.); 1H NMR (CDCl3,
–
–
–
–
str.
400 MHz) d ppm: 2.87 (s, 6H, N(CH3)2), 3.29 (d, J ¼ 16.3 Hz, 1H,
H1-triazine), 3.45 (dd, JAB ¼ 17.2 Hz, JAX ¼ 7.2Hz, 1H, HA), 3.52
(dd, JBA ¼ 17.2 Hz, JBX ¼ 12.8Hz, 1H, HB), 3.88 (d, J ¼ 16.3 Hz, 1H,
H2-triazine), 5.07 (dd, JXA ¼ 12.8 Hz, JXB ¼ 7.2 Hz, 1H, HX), 6.92–
8.20 (m, 8H, Ar-H), 9.41 (s, 1H, NH, D2O exchangeable), 12.53 (s,
1H,CONH,D2Oexchangeable);13CNMR(CDCl3,100 MHz)dppm:
40.4, 42.4, 49.6, 59.8, 112.8, 127.6, 128.6, 128.7, 135.68, 136.6,
140.4, 151.5, 153.6, 153.8, 167.4, 181.9; MS m/z: 441 (Mþþ1), 442
(Mþþ2);Anal.calcd.for C21H21ClN6OS:C, 57.20;H, 4.80;N, 19.06;
S, 7.27%. Found: C, 57.23; H, 4.82; N, 19.08; S, 7.29%.
5-(6-(4-Hydroxyphenyl)-4-phenyl-2-thioxo-5,6-
dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-
3(6H)-one (4k)
IR (KBr) nmax cmꢀ1: 3348 (cyclic CONH, NHstr.), 3266 (NHstr.),
3033 (C–H Arstr.), 1686 (C N ), 1212 (C Sstr.); 1H NMR (CDCl3,
–
–
–
–
str.
400 MHz) d ppm: 3.24 (d, J ¼ 16.2 Hz, 1H, H1-triazine), 3.47
(dd, JAB ¼ 16.9 Hz, JAX ¼ 7.1 Hz, 1H, HA), 3.55 (dd, JBA ¼ 12.7
Hz, JBX ¼ 16.9 Hz, 1H, HB), 3.87 (d, J ¼ 16.2 Hz, 1H, H2-
triazine), 5.04 (dd, JXA ¼ 7.1 Hz, JXB ¼ 12.7 Hz, 1H, HX), 6.79–
8.24 (m, 9H, Ar-H), 9.28 (s, 1H, NH, D2O exchangeable), 10.20
(brs, 1H, OH), 12.44 (s, 1H, CONH, D2O exchangeable);
5-(6-(4-(Dimethylamino)phenyl)-2-thioxo-4-p-tolyl-5,6-
dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-
3(6H)-one (4h)
IR (KBr) nmax cmꢀ1: 3314 (cyclic CONH, NHstr.), 3244 (NHstr.),
3029 (C–H Arstr.), 1648 (C N ), 1217 (C Sstr.); 1H NMR (CDCl3,
–
–
–
–
str.
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Arch. Pharm. Chem. Life Sci. 2017, 350, e1700146
M. Sahu et al.
Archiv der Pharmazie
13C NMR (CDCl3, 100 MHz) d ppm: 42.7, 49.3, 59.6, 115.3,
125.1, 128.3, 128.6, 128.8, 136.7, 153.5, 153.8, 157.8, 167.4,
182.7; MS m/z: 380 (Mþþ1); Anal. calcd. for C19H17N5O2S:
C, 60.14; H, 4.52; N, 18.46; S, 8.45%. Found: C, 60.17; H, 4.55;
N, 18.50; S, 8.47%.
5-(4-(4-Fluorophenyl)-6-(4-hydroxyphenyl)-2-thioxo-5,6-
dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-
3(6H)-one (4o)
IR (KBr) nmax cmꢀ1: 3318 (cyclic CONH, NHstr.), 3245 (NHstr.),
3035 (C–H Arstr.), 1687 (C N ), 1225 (C Sstr.); 1H NMR (CDCl3,
–
–
–
–
str.
400 MHz) d ppm: 3.16 (d, J ¼ 16.1 Hz, 1H, H1-triazine), 3.50 (dd,
JAB ¼ 16.5 Hz, JAX ¼ 6.8 Hz, 1H, HA), 3.58 (dd, JBA ¼ 16.5 Hz,
JBX ¼ 12.8 Hz, 1H, HB), 3.84 (d, J ¼ 16.1 Hz, 1H, H2-triazine), 5.04
(dd, JXA ¼ 6.8 Hz, JXB ¼ 12.8 Hz, 1H, HX), 6.84–8.22 (m, 8H, Ar-
H), 9.27 (s, 1H, NH, D2O exchangeable), 10.01 (brs, 1H, OH),
12.18 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3,
100 MHz) d ppm: 42.5, 49.7, 59.8, 115.7, 115.9, 125.4, 127.8,
136.8, 140.8, 153.4, 153.9, 157.9, 163.1, 167.8, 182.6; MS m/z:
398 (Mþþ1); Anal. calcd. for C19H16FN5O2S: C, 57.42; H, 4.06;
N, 17.62; S, 8.07%. Found: C, 57.40; H, 4.02; N, 17.65; S, 8.09%.
5-(4-(4-Chlorophenyl)-6-(4-hydroxyphenyl)-2-thioxo-5,6-
dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-
3(6H)-one (4l)
IR (KBr) nmax cmꢀ1: 3342 (cyclic CONH, NHstr.), 3257 (NHstr.),
3027 (C–H Arstr.), 1649 (C N ), 1208 (C Sstr.); 1H NMR (CDCl3,
–
–
–
–
str.
400 MHz) d ppm: 3.17 (d, J ¼ 16.2 Hz, 1H, H1-triazine), 3.47 (dd,
JAB ¼ 16.9 Hz, JAX ¼ 6.3 Hz, 1H, HA), 3.56 (dd, JBA ¼ 16.9 Hz,
JBX ¼ 12.3 Hz, 1H, HB), 3.86 (d, J ¼ 16.2 Hz, 1H, H2-triazine), 5.10
(dd, JXA ¼ 6.3 Hz, JXB ¼ 12.3 Hz, 1H, HX), 6.75–8.29 (m, 8H, Ar-
H), 9.33 (s, 1H, NH, D2O exchangeable), 10.23 (brs, 1H, OH),
12.46 (s, 1H, CONH, D2O exchangeable); 13C NMR (CDCl3,
100 MHz) d ppm: 42.9, 49.6, 59.9, 115.1, 125.2, 128.2, 128.7,
135.6, 136.6, 140.2, 153.4, 153.8, 157.8, 167.1, 182.9; MS m/z:
414 (Mþþ1), 415 (Mþþ2); Anal. calcd. for C19H16ClN5O2S:
C, 55.14; H, 3.90; N, 16.92 S, 7.75%. Found: C, 55.18; H, 3.94;
N, 16.90; S, 7.79%.
5-(6-(4-Hydroxy-3-methoxyphenyl)-4-phenyl-2-thioxo-5,6-
dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-
3(6H)-one (4p)
IR (KBr) nmax cmꢀ1: 3329 (cyclic CONH, NHstr.), 3256 (NHstr.),
3030 (C–H Arstr.), 1658 (C N ), 1220 (C Sstr.); 1H NMR (CDCl3,
–
–
–
–
str.
400 MHz) d ppm: 3.23 (d, J ¼ 16.2 Hz, 1H, H1-triazine), 3.45 (dd,
JAB ¼ 16.1 Hz, JAX ¼ 6.4 Hz, 1H, HA), 3.53 (dd, JBA ¼ 16.1 Hz,
JBX ¼ 12.4 Hz, 1H, HB), 3.89 (d, J ¼ 16.2 Hz, 1H, H2-triazine), 4.02
(s, 3H, OCH3), 5.12 (dd, JXA ¼ 6.4 Hz, JXB ¼ 12.4 Hz, 1H, HX),
7.15–8.23 (m, 8H, Ar-H), 9.33 (s, 1H, NH, D2O exchangeable),
10.08 (brs, 1H, OH), 11.86 (s, 1H, CONH, D2O exchangeable);
13C NMR (CDCl3, 100 MHz) d ppm: 42.8, 49.7, 56.5, 59.8, 108.6,
116.5, 125.4, 128.1, 128.4, 128.9, 136.1, 136.5, 147.8, 149.4,
153.8, 153.9, 167.1, 181.7; MS m/z: 410 (Mþþ1); Anal. calcd.
for C20H19N5O3S: C, 57.67; H, 4.68; N, 17.10; S, 7.83%. Found:
C, 57.70; H, 4.64; N, 17.11; S, 7.86%.
5-(6-(4-Hydroxyphenyl)-2-thioxo-4-p-tolyl-5,6-
dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-
3(6H)-one (4m)
IR (KBr) nmax cmꢀ1: 3329 (cyclic CONH, NHstr.), 3248 (NHstr.),
3034 (C–H Arstr.), 1655 (C N ), 1216 (C Sstr.); 1H NMR (CDCl3,
–
–
–
–
str.
400 MHz) d ppm: 2.37 (s, 3H, CH3), 3.27 (d, J ¼ 16.3 Hz, 1H, H1-
triazine), 3.49 (dd, JAB ¼ 16.1 Hz, JAX ¼ 6.8 Hz, 1H, HA), 3.57 (dd,
JBA ¼ 16.1 Hz, JBX ¼ 12.3 Hz, 1H, HB), 3.88 (d, J ¼ 16.3 Hz, 1H,
H2-triazine), 5.05 (dd, JXA ¼ 6.8 Hz, JXB ¼ 12.3 Hz, 1H, HX), 7.03–
8.19 (m, 8H, Ar-H), 9.37 (s, 1H, NH, D2O exchangeable), 10.44
(brs, 1H, OH), 12.42 (s, 1H, CONH, D2O exchangeable);
13C NMR (CDCl3, 100 MHz) d ppm: 21.4, 42.5, 49.7, 59.7,
115.2, 125.4, 128.2, 129.3, 136.7, 139.7, 140.5, 153.2, 153.6,
157.7, 167.4, 182.1; MS m/z: 394 (Mþþ1); Anal. calcd. for
5-(4-(4-Chlorophenyl)-6-(4-hydroxy-3-methoxyphenyl)-2-
thioxo-5,6-dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-
triazin-3(6H)-one (4q)
IR (KBr) nmax cmꢀ1: 3340 (cyclic CONH, NHstr.), 3277 (NHstr.),
3032 (C–H Arstr.), 1672 (C N ), 1223 (C Sstr.); 1H NMR (CDCl3,
–
str.
–
–
C
20H19N5O2S: C, 61.05; H, 4.87; N, 17.80; S, 8.15%. Found:
–
C, 61.08; H, 4.91; N, 17.83; S, 8.19%.
400 MHz) d ppm: 3.25 (d, J ¼ 16.4 Hz, 1H, H1-triazine), 3.51 (dd,
¼ 16.7 Hz, JAX ¼ 7.2 Hz, 1H, HA), 3.59 (dd, JBA ¼ 16.7 Hz,
JAB
5-(4,6-Bis(4-hydroxyphenyl)-2-thioxo-5,6-
dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-
3(6H)-one (4n)
JBX ¼ 12.4 Hz, 1H, HB), 3.87 (d, J ¼ 16.4 Hz, 1H, H2-triazine), 4.04
(s, 3H, OCH3), 5.06 (dd, JXA ¼ 7.2 Hz, JXB ¼ 12.4 Hz, 1H, HX),
6.90–8.04 (m, 7H, Ar-H), 9.28 (s, 1H, NH, D2O exchangeable),
10.21 (brs, 1H, OH), 11.89 (s, 1H, CONH, D2O exchangeable);
13C NMR (CDCl3, 100 MHz) d ppm: 42.9, 49.7, 56.1, 60.1, 108.6,
116.5, 125.2, 128.1, 128.7, 135.6, 136.4, 136.7, 147.9, 149.5,
153. 8, 154.1, 167.4, 183.2; MS m/z: 444 (Mþþ1), 445 (Mþþ2);
Anal. calcd. for C20H18ClN5O3S: C, 54.11; H, 4.09; N, 15.78;
S, 7.22%. Found: C, 54.12; H, 4.12; N, 15.80; S, 7.24%.
IR (KBr) nmax cmꢀ1: 3316 (cyclic CONH, NHstr.), 3276 (NHstr.),
3028 (C–H Arstr.), 1647 (C N ), 1220 (C Sstr.); 1H NMR (CDCl3,
–
–
–
–
str.
400 MHz) d ppm: 3.21 (d, J ¼ 16.2 Hz, 1H, H1-triazine), 3.46
(dd, JAB ¼ 16.7 Hz, JAX ¼ 7.2 Hz, 1H, HA), 3.54 (dd, JBA ¼ 16.7
Hz, JBX ¼ 12.1 Hz, 1H, HB), 3.85 (d, J ¼ 16.2 Hz, 1H,
H2-triazine), 5.11 (dd, JXA ¼ 7.2 Hz, JXB ¼ 12.1 Hz, 1H, HX),
6.77–8.13 (m, 8H, Ar-H), 9.36 (s, 1H, NH, D2O exchangeable),
10.12 (brs, 2H, OH), 12.44 (s, 1H, CONH, D2O exchangeable);
13C NMR (CDCl3, 100 MHz) d ppm: 42.8, 49.9, 59.7, 115.5,
116.8, 125.8, 128.9, 136.7, 140.3, 153.7, 153.9, 157.2, 157.8,
167.5, 182.2; MS m/z: 396 (Mþþ1); Anal. calcd. for
5-(6-(4-Hydroxy-3-methoxyphenyl)-2-thioxo-4-p-tolyl-5,6-
dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-
3(6H)-one (4r)
IR (KBr) nmax cmꢀ1: 3310 (cyclic CONH, NHstr.), 3269 (NHstr.),
3031 (C–H Arstr.), 1666 (C N ), 1206 (C Sstr.); 1H NMR (CDCl3,
–
str.
–
–
–
C
19H17N5O3S: C, 57.71; H, 4.33; N, 17.71; S, 8.11%. Found:
C, 57.74; H, 4.32; N, 17.75; S, 8.15%.
400 MHz) d ppm: 2.38 (s, 3H, CH3), 3.20 (d, J ¼ 16.1 Hz, 1H,
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H1-triazine), 3.48 (dd, JAB ¼ 16.4 Hz, JAX ¼ 6.8 Hz, 1H, HA), 3.56
(dd, JBA ¼ 16.4 Hz, JBX ¼ 6.8 Hz, 1H, HB), 3.79 (d, J ¼ 16.19 Hz,
1H, H2-triazine), 4.03 (s, 3H, OCH3), 5.16 (dd, JXA ¼ 16.4 Hz,
JXB ¼ 6.8 Hz, 1H, HX), 6.98–8.12 (m, 7H, Ar-H), 9.26 (s, 1H, NH,
D2O exchangeable), 10.09 (brs, 1H, OH), 12.12 (s, 1H, CONH,
D2O exchangeable); 13C NMR (CDCl3, 100 MHz) d ppm: 21.6,
42.5, 49.7, 56.1, 60.1, 108.8, 116.5, 125.6, 128.2, 129.3, 136.2,
136.4, 139.7, 147.8, 149.5, 153.5, 153.8, 167.9, 181.8; MS m/z:
424 (Mþþ1); Anal. calcd. for C21H21N5O3S: C, 59.56; H, 5.00; N,
16.54; S, 7.57%. Found: C, 59.57; H, 5.02; N, 16.55; S, 7.60%.
food and water. Each test and control group consisted of at
least four animals. The test compounds were suspended in
30% v/v PEG-200 and were administered i.p. at doses of 30,
100, and 300 mg/kg body weight. The standard drugs used
were phenytoin and carbamazepine. The bioactivity was
observed as protection in half or more of the animals. The
compounds found potent in phase I were further screened for
phase II quantitative evaluation followed by serum enzyme
estimation in liver and GABA level estimation in the whole
brain of mice.
5-(6-(4-Hydroxy-3-methoxyphenyl)-4-(4-hydroxyphenyl)-
2-thioxo-5,6-dihydropyrimidin-1(2H)-yl)-1,2-dihydro-
1,2,4-triazin-3(6H)-one (4s)
Phase I anticonvulsant screening
Maximal electroshock-induced seizure (MES) test: The MES
screen is generally done for generalized tonic–clonic seizures
and identifies compounds which prevent seizure spread [68].
An electrical stimulus of 0.2 s in duration (50 mA, 60 Hz) was
delivered to prescreened animals via ear clip electrodes
primed with an electrolyte solution (0.9% NaCl). The test
compounds were administered i.p. in each animal at 30, 100
and 300 mg/kg. After 0.5 h and 4.0 h of drug administration,
the electroshock was given and the observations were made.
The end point observed was abolition of hind limb tonic
extensor (HLTE) in at least half of the animals and was defined
as protection.
IR (KBr) nmax cmꢀ1: 3338 (cyclic CONH, NHstr.), 3192 (NHstr.),
1
–
–
3037 (C–H Arstr.), 1680 (C N ), 1214 ( Sstr.); H NMR (CDCl3,
–
–
str.
400 MHz) d ppm: 3.18 (d, J ¼ 16.1 Hz, 1H, H1-triazine), 3.52 (dd,
JAB ¼ 16.9 Hz, JAX ¼ 6.8 Hz, 1H, HA), 3.61 (dd, JBA ¼ 16.9 Hz,
JBX ¼ 12.4 Hz, 1H, HB), 3.84 (d, J ¼ 16.19 Hz, 1H, H2-triazine),
4.05 (s, 3H, OCH3), 5.08 (dd, JXA ¼ 6.8 Hz, JXB ¼ 12.4 Hz, 1H, HX),
7.11–8.09 (m, 7H, Ar-H), 9.33 (s, 1H, NH, D2O exchangeable),
10.05 (brs, 2H, OH), 12.30 (s, 1H, CONH, D2O exchangeable);
13C NMR (CDCl3, 100 MHz) d ppm: 41.9, 49.1, 56.5, 60.3, 108.7,
116.5, 116.8, 125.2, 128.7, 135.9, 136.2, 147.3, 149.4, 153.7,
154.5, 157.6, 167.8, 183.2; MS m/z: 426 (Mþþ1); Anal. calcd.
for C20H19N5O4S: C, 56.46; H, 4.50; N, 16.46; S, 7.54%. Found:
C, 56.47; H, 4.52; N, 16.48; S, 7.53%.
Subcutaneous pentylenetetrazole-induced seizure (scPTZ)
test: The pentylenetetrazole-induced seizures identify com-
pounds that raise seizure threshold [69]. Seizures were
induced in the animals by pentylenetetrazole (85 mg/kg;
i.p.) that produces clonic seizures in animals lasting for at least
5 s in more than 95% of animals tested. The test compounds
were injected i.p. at the dose levels of 30, 100, and 300 mg/kg.
The animals were examined for a period of 0.5 h and 4 h. The
absence of clonic spasms in the observed time period for at
least 5-s duration was defined as protection and observed as
end-point.
5-(4-(4-Fluorophenyl)-6-(4-hydroxy-3-methoxyphenyl)-2-
thioxo-5,6-dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-
triazin-3(6H)-one (4t)
IR (KBr) nmax cmꢀ1: 3320 (cyclic CONH, NHstr.), 3256 (NHstr.),
3031 (C–H Arstr.), 1646 (CNstr.), 1218 (CSstr.); 1H NMR (CDCl3,
400 MHz) d ppm: 3.19 (d, J ¼ 16.2 Hz, 1H, H1-triazine), 3.51 (dd,
JAB ¼ 16.2 Hz, JAX ¼ 6.9 Hz, 1H, HA), 3.60 (dd, JBA ¼ 16.8 Hz,
JBX ¼ 12.8 Hz, 1H, HB), 3.81 (d, J ¼ 16.2 Hz, 1H, H2-triazine), 4.01
(s, 3H, OCH3), 5.07 (dd, JXA ¼ 6.9 Hz, JXB ¼ 12.8 Hz, 1H, HX),
6.89–8.19 (m, 7H, Ar-H), 9.32 (s, 1H, NH, D2O exchangeable),
10.10 (brs, 1H, OH), 11.98 (s, 1H, CONH, D2O exchangeable);
13C NMR (CDCl3, 100 MHz) d ppm: 42.5, 49.2, 56.1, 59.9, 108.6,
115.9, 116.5, 125.6, 127.7, 136.1, 136.5, 147.8, 149.5, 153.6,
153.9, 163.3, 167.2, 182.7; MS m/z: 428 (Mþþ1); Anal. calcd. for
Minimal motor impairment test (rotarod test)
The neurological toxicity (NT) was detected in mice using
standardized rotarod test [70]. The acute motor impairment
was defined as the inability of the animals to maintain
equilibrium for 1 min in each of three successive trials. The
mice were pre-trained to maintain equilibrium on an
accelerating rotarod (diameter 3.2 cm) that rotated at a
speed of 6 rpm. Trained animals were given i.p. injection of
the test compounds at three graded doses of 30, 100, and
300 mg/kg.
C
20H18FN5O3S: C, 56.20; H, 4.24; N, 16.38; S, 7.50%. Found:
C, 57.24; H, 4.22; N, 16.35; S, 7.52%.
Pharmacology
The anticonvulsant evaluation was performed as per the
Antiepileptic Drug Development (ADD) Program in Epilepsy
Branch, National Institutes of Health, National Institute of
Neurological Disorders and Stroke (NIH/NINDS), Rockville,
MD, USA [67]. The Swiss albino mice (28–30 g) of either sex
were used as experimental animals. The animals were
obtained from the Central Animal House Facility, Jamia
Hamdard, New Delhi-62, India after approval from the
Institutional Animal Ethics Committee (1264/CPCSEA, May 2,
2016). All the animals were housed in cages with free access to
Phase II anticonvulsant screening
The compounds which showed promising anticonvulsant
activity in phase I were tested further for phase II screening
according to the protocol described elsewhere [71]. In this
phase, the anticonvulsant activity was quantified in terms of
median effective dose (ED50) and neurotoxicity as the median
toxic dose (TD50). The protective index (PI) was calculated by
PI ¼ TD50/ED50. Groups of at least eight mice were used for a
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range (10–90%) of test compound until at least three points
were established for seizure protection or minimal neurotox-
icity. All observations were made from 0.5–4.0-h time period.
structure using Glide 6.3 XP docking [81]. The 3D complex
structures of all hits were analyzed for Glide score and
H-bonding interactions.
Biochemical estimation (serum enzyme assay)
One of the authors, Meeta Sahu, is thankful to Department of
Science and Technology (DST), Ministry of Science and
Technology, New Delhi, India for funding this work under
Women Scientist-A Scheme (Grant No. SR/WOS-A/CS-1076/
2014). Vidushi Sharma is also indebted to DST for providing
financial assistance for computational facility (Grant No. SB/
FT/CS-013/2012). Gift sample of PTZ from Shrenik Pharma Pvt.
Ltd., Mumbai is gratefully acknowledged.
Many anticonvulsant drugs have shown considerable side
effect such as hepatotoxicity. Therefore, to find out the toxic
effects of the most potent compounds, if any, the liver
enzyme biochemical estimation was performed on mice. The
animals were divided into groups of six and were adminis-
tered with the test drugs in a dose of 30 mg/kg/day i.p. for
2 weeks. The normal group was treated with saline only; the
toxic group was treated with PTZ; and test groups with
respective test drugs. After the stipulated period, each
animal was anesthetized and blood was collected to assess
serum glutamate oxaloacetate transaminase, serum gluta-
mate pyruvate transaminase, alkaline phosphates, total
protein, and total albumin according to the methods
reported [72, 73]. The data are shown as mean ꢁ SEM
(units/dL).
The authors have declared no conflicts of interest.
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