Microwave-assisted synthesis and anticonvulsant activity of 5,6-bisaryl-1,2,4-triazine-3-thiolderivatives

Article abstract of DOI:10.1007/s00044-013-0843-6

A series of 5,6-bisaryl-1,2,4-triazine-3-thiol derivatives were synthesized through microwave-promoted chemistry by condensation of the aromatic 1,2-diketones and thiosemicarbazide in a mixed green solvent. Subsequently, S-alkylation of 1,2,4-triazine-3-t

Full text of DOI:10.1007/s00044-013-0843-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:2503–2514
DOI 10.1007/s00044-013-0843-6
ORIGINAL RESEARCH
Microwave-assisted synthesis and anticonvulsant activity
of 5,6-bisaryl-1,2,4-triazine-3-thiol derivatives
•
Hamid Irannejad Nima Naderi Saeed Emami
•
•
•
Roja Qobadi Ghadikolaei Alireza Foroumadi Tina Zafari
•
•
•
Ali Mazar-Atabaki Sakineh Dadashpour
Received: 23 July 2013 / Accepted: 10 October 2013 / Published online: 23 October 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of 5,6-bisaryl-1,2,4-triazine-3-thiol
derivatives were synthesized through microwave-promoted
chemistry by condensation of the aromatic 1,2-diketones
and thiosemicarbazide in a mixed green solvent. Subse-
quently, S-alkylation of 1,2,4-triazine-3-thiols afforded S-
substituted derivatives. The anticonvulsant activity of the
synthesized compounds was evaluated in vivo by electro-
shock and pentylenetetrazole (PTZ)-induced seizures tests.
Among them, compound 4a bearing 4-pyridylmethylthio
moiety on the triazine ring showed the highest protection in
both electroshock and PTZ-induced seizures tests. Com-
pound 4a showed no sign of neurotoxicity at the dose of
100 mg/kg in both rotarod and chimney tests.
Keywords Anticonvulsant activity ꢀ
Microwave synthesis ꢀ Mixed-green solvent system ꢀ
1,2,4-Triazine, distance mapping
Introduction
Approximately 1 % of the world’s population has epilepsy,
the second most common neurologic disorder after stroke.
Although standard therapy with antiepileptic drugs (AEDs)
permits control seizures in 50 % of patients developing
partial seizures and 60–70 % of those developing gen-
eralized seizures, rest are not responsive to the conven-
tionally available AEDs. Moreover, the current drug
therapy exhibits unfavorable side effects such as ataxia,
hepatotoxicity, megaloblastic anemia, gingival hyperplasia,
and hirsutism. These facts necessitate the search for new
AEDs (Landmark, 2007; Siddiqui and Ahsan, 2010).
Several heterocycle containing compounds have been
reported to have considerable antiepileptic activities.
Among them, 1,2,4-triazines have been a promising scaf-
fold which is mainly used in the novel anticonvulsant drugs
such as lamotrigine (Fig. 1). Lamotrigine shares similar
mode of action on neuronal sodium channel as phenytoin
(Kaushik et al., 2010). 1,2,4-Triazine template has also
been associated with diverse pharmacological activities
such as antiplatelet aggregation, anti-inflammatory (Ansari
et al., 2010), potent neuroprotective (Irannejad et al.,
2010), and antifungal (Sangshetti and Shinde, 2010)
activities.
H. Irannejad (&) ꢀ S. Emami ꢀ R. Q. Ghadikolaei
Department of Medicinal Chemistry, Faculty of Pharmacy and
Pharmaceutical Sciences Research Center, Mazandaran
University of Medical Sciences, Sari, Iran
e-mail: irannejadhamid@gmail.com;
irannejad.hamid@yahoo.com
N. Naderi
Department of Pharmacology and Toxicology, School of
Pharmacy, Shahid Beheshti University of Medical Sciences,
Tehran, Iran
A. Foroumadi
Department of Medicinal Chemistry, Faculty of Pharmacy and
Drug Design and Development Research Center, Tehran
University of Medical Sciences, Tehran, Iran
T. Zafari ꢀ A. Mazar-Atabaki
The reported structure–activity relationships studies on
the 5,6-diaryl-3-amino-1,2,4-triazines revealed that the
required pharmacophoric elements are lipophilic aryl rings
attached to the 1,2,4-triazine as well as presence of
hydrogen bonding capable moieties (Mallikarjuna et al.,
Faculty of Medicine, Shahid Beheshti University of Medical
Sciences, Tehran, Iran
S. Dadashpour
Student Research Committee, Faculty of Pharmacy, Mazandaran
University of Medical Sciences, Sari, Iran
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Med Chem Res (2014) 23:2503–2514
Fig. 1 Schematic
representation of anticonvulsant
drugs phenytoin and
O
NH2
lamotrigine, and the synthesized
compounds (2a–g, 3a–c, and
4a–d)
NH
O
Cl
N
N
HN
Cl
N
NH2
Lamotrigine
Phenytoin
X
X
S
S
X
N
N
SR
Y
Z
N
N
Y
N
N
S
N
N
N
X
2a-f: X= H, Me, MeO, F, Cl, Br
2g, R=H
2h, R=Me
4a: X=Me, Y=N, Z=CH
4b: X=Me, Y=CH, Z=N
4c: X=MeO, Y=CH, Z=N
4d: X=Cl, Y= N, Z=CH
Y= -SH
3a: X= Me, Y= methylthio
3b: X= MeO, Y= methylthio
3c: X= F, Y= methylthio
3d: X=Cl, Y= methylthio
3e: X=Br, Y=methylthio
2007). These findings prompted us to the synthesis of some
novel 5,6-bisaryl-1,2,4-triazine-3-thiol derivatives 2–4 as
anticonvulsant agents (Fig. 1).
Accordingly, herein we describe microwave-assisted
green synthesis and anticonvulsant activity of 5,6-bisaryl-
1,2,4-triazine-3-thiol derivatives (Fig. 1).
Microwave-assisted organic synthesis (MAOS) has
affected synthetic chemistry mainly by enabling rapid,
reproducible, and scalable chemistry development. Micro-
wave-enhanced organic synthesis can facilitate the dis-
covery of new reactions and novel pharmaceutical lead
compounds in drug discovery projects by reducing reaction
time in optimization of reactions. In addition, it serves to
expand chemical space and diversity in compound library
design and synthesis. Synthesis of heterocycles has been
significantly affected by the use of microwave in organic
chemistry (Kappe and Dallinger, 2006); (Shipe et al.,
2005).
Materials and methods
Chemistry
Chemical reagents and starting materials were purchased
from Sigma-Aldrich or Merck. Compounds 2a–e and 3a–
d were known compounds and reported in literature (An-
1
sari et al., 2010; Irannejad et al., 2010). H NMR spectra
were recorded in CDCl3 or DMSO-d6 on a Brucker DRX-
500 MHz instrument. The chemical shifts (d) were mea-
sured relative to TMS as an internal standard. Coupling
constants (J) are expressed in Hz. Mass spectra were
obtained with a Finnigan Mat TSQ-70 or Agilent 7890A
spectrometers. TLC was conducted on silica gel F254
plates. The microwave irradiation was carried out using
MicroSYNTH Laboratory System (MILESTONE S.r.l) in
Teflon closed vessel at 300 watt power.
Conventional methods for the synthesis of 5,6-bisaryl-
1,2,4-triazine-3-thiol derivatives have been reported in the
literature by condensation of aromatic 1,2-diketones with
thiosemicarbazide in refluxing conditions in absolute eth-
anol (Bhalla et al., 1995) or in acetic acid for several hours
(Khoshneviszadeh et al., 2013). Recently, we reported
synthesis of some potent neuroprotective agents based on
the 5,6-bisaryl-1,2,4-triazine-3-thiol substructure by re-
fluxing of 1,2-diketones and thiosemicarbazide in water
and 1,4-dioxane (Irannejad et al., 2010). Heravi et al. have
reported solventless synthesis of only one derivative, 5,6-
bisphenyl-1,2,4-triazine-3-thiol under microwave irradia-
tion in a domestic microwave oven that is not applicable
for laboratory synthesis and does not have reproducible
results (Heravi et al., 2006).
General procedure for preparation of 2a–g
To a mixture of the appropriate 1,2-diketone (1 eq) and
thiosemicarbazide (1.2 eq) was added a few drops of
concentrated hydrochloric acid in the reported solvent in
Table 2 and then the mixture was irradiated under micro-
wave conditions for 10 min at 120 °C (300 W). After
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2505
completion of the reaction, the precipitated solid was fil-
tered and washed with water several times to give the
desired product.
(dd, J = 8.5 Hz, 5.5 Hz, 2H, Ar), 7.59 (dd, J = 8.5 Hz,
5.5 Hz, 2H, Ar). 13C NMR (125 MHz, CDCl3) d: 115.73
(C-3 and C-5, phenyl), 116.41 (C-3 and C-5, phenyl),
129.65 (C-1, phenyl), 130.55 (C-1, phenyl), 133.14 (C-2
and C-6, phenyl), 134.81 (C-2 and C-6, phenyl), 155.79 (C-
6, triazine), 156.39 (C-5, triazine), 163.13 (C-4, phenyl),
163.90 (C-4, phenyl), 174.41 (C-3, triazine). MS m/z (%):
301 (M?, 15), 214 (100), 173 (65), 160 (34). Anal. Calcd.
For C15H9F2N3S: C, 59.79; H, 3.01; N, 13.95. Found: C,
59.42; H, 3.30; N,13.92.
5,6-Diphenyl-1,2,4-triazine-3-thiol (2a)
M.p.: 89–91 °C, 1H NMR (500 MHz, CDCl3) d: 7.32–7.45
(m, 6H, Ar), 7.51–7.56 (m, 4H, Ar). 13C NMR (125 MHz,
CDCl3) d: 128.42 (C-2 and C-6, phenyl), 128.73 (C-2 and
C-6, phenyl), 128.82 (C-4, phenyl), 129.54 (C-4, phenyl),
129.61 (C-3 and C-5, phenyl), 130.57 (C-3 and C-5, phe-
nyl), 135.53 (C-1, phenyl), 135.76 (C-1, phenyl), 155.91
(C-6, triazine), 156.31 (C-5, triazine), 173.62 (C-3, tri-
azine). MS m/z (%): 265 (M?, 34), 179 (100), 156 (33), 143
(21). Anal. Calcd for C15H11N3S: C, 67.90; H, 4.18; N,
15.84. Found: C, 68.12; H, 4.34; N, 15.66.
5,6-Bis(4-chlorophenyl)-1,2,4-triazine-3-thiol (2e)
M.p.: 158–160 °C; 1H NMR (500 MHz, CDCl3) d: 7.36 (d,
J = 9.0 Hz, 2H, Ar), 7.38 (d, J = 8.5 Hz, 2H, Ar), 7.48 (d,
J = 9.0 Hz, 2H, Ar), 7.58 (d, J = 8.5 Hz, 2H, Ar). 13C
NMR (125 MHz, CDCl3) d: 127.73 (C-2 and C-6, phenyl),
128.44 (C-2 and C-6, phenyl), 129.25 (C-3 and C-5, phe-
nyl), 129.48 (C-3 and C-5, phenyl), 133.83 (C-1, phenyl),
134.36 (C-1, phenyl), 134.68 (C-4, phenyl), 135.63 (C-4,
phenyl), 156.27 (C-6, triazine), 157.18 (C-5, triazine),
174.63 (C-3, triazine). MS m/z (%): 333 (M?, 21), 246
(100), 223 (66), 209 (48), 150 (25), 147 (23). Anal. Calcd
for C15H9Cl2N3S: C, 53.90; H, 2.71; N, 12.57. Found: C,
53.69; H, 2.84; N, 12.76.
5,6-Di-p-tolyl-1,2,4-triazine-3-thiol (2b)
M.p.: 156–157 °C, 1H NMR (500 MHz, CDCl3) d: 2.35 (s,
3H, Ar-CH3), 2.36 (s, 3H, Ar–CH3), 7.11 (d, J = 8.0 Hz,
2H, Ar), 7.14 (d, J = 8.0 Hz, 2H, Ar), 7.40 (d, J = 8.0 Hz,
2H, Ar),7.46 (d, J = 8.0 Hz, 2H, Ar). 13C NMR
(125 MHz, CDCl3) d: 20.91 (CH3), 21.44 (CH3), 125.37
(C-2 and C-6, phenyl), 128.94 (C-2 and C-6, phenyl),
129.32 (C-3 and C-5, phenyl), 129.73 (C-3 and C-5, phe-
nyl), 134.53 (C-1, phenyl), 135.51 (C-1, phenyl), 137.63
(C-4, phenyl), 138.21 (C-4, phenyl), 155.76 (C-6, triazine),
156.39 (C-5, triazine), 174.53 (C-3, triazine). MS m/z (%):
293 (M?, 38), 207 (100), 191 (36), 170 (64), 133 (15).
Anal. Calcd for C17H15N3S: C, 69.59; H, 5.15; N, 14.32.
Found: C, 69.34; H, 5.34; N, 14.76.
5,6-Bis(4-bromophenyl)-1,2,4-triazine-3-thiol (2f)
M.p.: 147–148 °C; 1H NMR (500 MHz, CDCl3) d: 7.61 (d,
J = 9.0 Hz, 2H, Ar), 7.64 (d, J = 9.0 Hz, 2H, Ar), 7.70 (d,
J = 9.0 Hz, 2H, Ar), 7.81 (d, J = 8.8 Hz, 2H, Ar). 13C
NMR (125 MHz, CDCl3) d: 121.55 (C-4, phenyl), 123.94
(C-4, phenyl), 127.68 (C-2 and C-6, phenyl), 129.73 (C-2
and C-6, phenyl), 132.02 (C-1, phenyl), 132.15 (C-1,
phenyl), 134.81 (C-3 and C-5, phenyl), 136.62 (C-3 and
C-5, phenyl), 154.66 (C-6, triazine), 159.45 (C-5, triazine),
176.42 (C-3, triazine). MS m/z (%): 425 (10), 423 (22), 421
(M?, 14), 335 (100), 333 (57), 257 (32), 255 (19). Anal.
Calcd for C15H9Br2N3S: C, 42.58; H, 2.14; N, 9.93. Found:
C, 42.26; H, 2.37; N, 9.76.
5,6-Bis(4-methoxyphenyl)-1,2,4-triazine-3-thiol (2c)
1
M.p.: 169 °C, H NMR (500 MHz, CDCl3) d: 3.81 (s, 3H,
OCH3), 3.83(s, 3H, OCH3), 6.80 (d, J = 9.0 Hz, 2H, Ar),
6.91 (d, J = 9.0 Hz, 2H, Ar), 7.52 (d, J = 9.0 Hz, 2H, Ar),
7.59 (d, J = 9.0 Hz, 2H, Ar). 13C NMR (125 MHz,
CDCl3) d: 55.31 (CH3), 55.46 (CH3), 114.33 (C-3 and C-5,
phenyl), 114.54 (C-3 and C-5, phenyl), 127.30 (C-1, phe-
nyl), 128.45 (C-1, phenyl), 134.80 (C-2 and C-6, phenyl),
135.55 (C-2 and C-6, phenyl), 155.86 (C-6, triazine),
156.77 (C-5, triazine), 159.71 (C-4, phenyl), 161.26 (C-4,
phenyl), 174.22 (C-3, triazine). MS m/z (%): 325 (M?, 22),
238 (73), 206 (100), 145 (46), 123 (16). Anal. Calcd. For
C17H15N3O2S: C, 62.75; H, 4.65; N, 12.91. Found: C,
62.55; H, 4.29; N, 12.52.
5,6-Di(thiophen-2-yl)-1,2,4-triazine-3-thiol (2g)
M.p.: 215 °C; IR (KBr): 1289 (C=S), 1630 (C=N), 1503
(N=N). 1H NMR (500 MHz, CDCl3) d (ppm): 7.0 (t,
J = 5.0 Hz, 1H, thienyl), 7,21 (t, J = 5.0 Hz, 1H, thienyl),
7.46 (d, J = 3.5 Hz, 1H, thienyl), 7.52 (d, J = 3.5 Hz, 1H,
thienyl), 7.77 (d, J = 5.0 Hz, 1H, thienyl), 7.68 (d,
J = 5.0 Hz, 1H, thienyl), 13C NMR (125 MHz, CDCl3) d:
124.62 (C-5, thienyl), 126.90 (C-5, thienyl), 127.21 (C-3,
thienyl), 127.65 (C-3, thienyl), 127.91 (C-4, thienyl),
128.30 (C-4, thienyl), 134.45 (C-2, thienyl), 138.21 (C-2,
thienyl), 145.32 (C-6, triazine), 154.64 (C-5, triazine),
5,6-Bis(4-fluorophenyl)-1,2,4-triazine-3-thiol (2d)
M.p.: 105–106 °C, 1H NMR (500 MHz, CDCl3) d: 7.10 (t,
J = 8.5 Hz, 2H, Ar), 7.15 (t, J = 8.5 Hz, 2H, Ar), 7.54
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172.71 (C-3, triazine). MS m/z (%): 277 (M?, 12), 191
(44), 161 (64), 149 (33), 91 (15). Anal. Calcd for
C11H7N3S3: C, 47.63; H, 2.54; N, 15.15. Found: C, 47.44;
H, 2.38; N, 15.32.
5,6-Bis(4-fluorophenyl)-3-(methylthio)-1,2,4-triazine (3c)
1
M.p.: 83 °C, H NMR (500 MHz, CDCl3) d: 2.76 (s, 3H,
SCH3), 7.05 (t, J = 8.5 Hz, 2H, Ar), 7.08 (t, J = 8.5 Hz,
2H, Ar), 7.5 (dd, J = 8.5 Hz, 5 Hz, 2H, Ar), 7.56 (dd,
J = 8.5 Hz, 5 Hz, 2H, Ar). 13C NMR (125 MHz, CDCl3)
d: 14.54 (SCH3), 115.70 (C-3 and C-5, phenyl), 116.38 (C-
3 and C-5, phenyl), 129.62 (C-1, phenyl), 130.57 (C-1,
phenyl), 133.12 (C-2 and C-6, phenyl), 134.79 (C-2 and
C-6, phenyl), 155.73 (C-6, triazine), 156.32 (C-5, triazine),
163.04 (C-4, phenyl), 163.89 (C-4, phenyl), 174.23 (C-3,
triazine). MS: m/z (%): 315 (M?, 5), 214 (100). Anal.
Calcd. For C16H11F2N3S: C, 60.94; H, 3.52; N, 13.33.
Found: C, 60.72; H, 3.36; N, 13.42.
General procedure for preparation of 3a–e and 2h
To a stirring solution of compounds 2a–g (1 mmol) in dry
methanol (50 ml), methyl iodide (1.2 mmol) and triethyl-
amine (0.1 ml) were added and the mixture was stirred for
2 h at room temperature. Then, the solvent was removed
under reduced pressure. Water (20 ml) and dichlorometh-
ane (50 ml) were added to the mixture. The organic phase
was separated, dried (sodium sulfate), filtered and evapo-
rated under reduced pressure. The obtained residue was
dissolved in chloroform and then precipitated by adding
petroleum ether to give the desired product.
5,6-Bis(4-chlorophenyl)-3-(methylthio)-1,2,4-
triazine (3d)
M.p.: 137–139 °C; 1H NMR (500 MHz, CDCl3) d: 2.70 (s,
3H, SCH3), 7.34 (d, J = 9.0 Hz, 2H, Ar), 7.37 (d,
J = 8.5 Hz, 2H, Ar), 7.45 (d, J = 9.0 Hz, 2H, Ar), 7.50 (d,
J = 8.5 Hz, 2H, Ar). 13C NMR (125 MHz, CDCl3) d:
14.33 (SCH3), 127.66 (C-2 and C-6, phenyl), 128.37 (C-2
and C-6, phenyl), 129.15 (C-3 and C-5, phenyl), 129.46 (C-
3 and C-5, phenyl), 133.79 (C-1, phenyl), 134.35 (C-1,
phenyl), 134.66 (C-4, phenyl), 135.59 (C-4, phenyl),
156.17 (C-6, triazine), 157.04 (C-5, triazine), 174.43 (C-3,
triazine). MS m/z (%): 347 (M?, 17), 245 (100), 176 (45),
123 (15), 63 (42). Anal. Calcd for C16H11Cl2N3S: C, 55.09;
H, 3.18; N, 12.05. Found: C, 55.39; H, 3.24; N, 11.89.
3-(Methylthio)-5,6-di p-tolyl-1,2,4-triazine (3a)
M.p.: 149–150 °C; 1H NMR (500 MHz, CDCl3) d: 2.37 (s,
3H, Ar–CH3), 2.38 (s,3H, Ar–CH3), 2.74 (s, 3H, SCH3),
7.13 (d, J = 8.0 Hz, 2H, Ar), 7.16 (d, J = 8.0 Hz, 2H, Ar),
7.42 (d, J = 8.0 Hz, 2H, Ar),7.47 (d, J = 8.0 Hz, 2H, Ar).
13C NMR (125 MHz, CDCl3) d: 13.34 (SCH3), 20.92 (Ar–
CH3), 21.54 (Ar–CH3), 125.32 (C-2 and C-6, phenyl),
128.91 (C-2 and C-6, phenyl), 129.27 (C-3 and C-5, phe-
nyl), 129.73 (C-3 and C-5, phenyl), 134.56 (C-1, phenyl),
135.54 (C-1, phenyl), 137.66 (C-4, phenyl), 138.26 (C-4,
phenyl), 155.72 (C-6, triazine), 156.32 (C-5, triazine),
174.33 (C-3, triazine). MS m/z (%): 307 (M?, 10), 206
(100), 176 (89), 190 (5), 119 (12). Anal. Calcd. For
C18H17NS: C, 70.33; H, 5.57; N, 13.67. Found: C, 70.49;
H, 5.19; N, 13.72.
5,6-Bis(4-bromophenyl)-3-(methylthio)-1,2,4-triazine (3e)
M.p.: 122–124 °C; 1H NMR (500 MHz, CDCl3) d: 2.60 (s,
3H, SCH3), 7.59 (d, J = 9.0 Hz, 2H, Ar), 7.61 (d,
J = 9.0 Hz, 2H, Ar), 7.67 (d, J = 9.0 Hz, 2H, Ar), 7.79 (d,
J = 8.5 Hz, 2H, Ar). 13C NMR (125 MHz, CDCl3) d:
14.33 (SCH3), 121.50 (C-4, phenyl), 123.91 (C-4, phenyl),
127.67 (C-2 and C-6, phenyl), 129.62 (C-2 and C-6, phe-
nyl), 131.90 (C-3 and C-5, phenyl), 132.10 (C-3 and C-5,
phenyl), 134.75 (C-1, phenyl), 136.52 (C-1, phenyl),
154.60 (C-6, triazine), 159.40 (C-5, triazine), 176.14 (C-3,
triazine). MS m/z (%): 439 (9), 437 (25), 435 (M?, 12), 338
(49), 336 (100), 333 (52). Anal. Calcd for C16H11Br2N3S:
C, 43.96; H, 2.54; N, 9.61. Found: C, 44.16; H, 2.27; N,
9.49.
5,6-Bis(4-methoxyphenyl)-3-(methylthio)-1,2,4-
triazine (3b)
1
M.p.: 134 °C, H NMR (500 MHz, CDCl3) d: 2.75 (s, 3H,
SCH3), 3.80 (s, 3H, OCH3), 3.85(s, 3H, OCH3), 6.85 (d,
J = 9.0 Hz, 2H, Ar), 6.90 (d, J = 9.0 Hz, 2H, Ar), 7.50 (d,
J = 9.0 Hz, 2H, Ar), 7.57 (d, J = 9.0 Hz, 2H, Ar). 13C
NMR (125 MHz, CDCl3) d: 14.73 (SCH3), 55.32 (OCH3),
55.45 (OCH3), 114.35 (C-3 and C-5, phenyl), 114.56 (C-3
and C-5, phenyl), 127.28 (C-1, phenyl), 128.46 (C-1,
phenyl), 134.82 (C-2 and C-6, phenyl), 135.54 (C-2 and
C-6, phenyl), 155.80 (C-6, triazine), 156.76 (C-5, triazine),
159.70 (C-4, phenyl), 161.23 (C-4, phenyl), 174.13 (C-3,
triazine). MS: m/z (%): 339 (M?, 5), 237 (100), 222 (55),
194 (10), 152 (22), 118 (18), 54 (8). Anal. Calcd. For
C18H17N3O2S: C, 63.70; H, 5.05; N, 12.38. Found: C,
63.92; H, 5.16; N, 12.42.
3-(Methylthio)-5,6-di(thiophen-2-yl)-1,2,4-triazine (2 h):
M.p.: 135 °C; IR (KBr): 3086, 2842, 1671 (C=N), 1523
(N=N). 1H NMR (500 MHz, CDCl3) d (ppm): 2.74 (s, 3H,
SCH3), 7.03 (t, J = 5.0 Hz, 1H, Ar), 7,14 (t, J = 5.0 Hz,
1H, Ar), 7.41 (d, J = 3.5 Hz, 1H, Ar), 7.47 (d, J = 3.5 Hz,
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1H, Ar), 7.56 (d, J = 5.0 Hz, 1H, Ar), 7.60 (d, J = 5.0 Hz,
1H, Ar), 13C NMR (125 MHz, CDCl3) d: 16.35 (SCH3),
125.54 (C-5, thienyl), 126.88 (C-5, thienyl), 127.33 (C-3,
thienyl), 127.67 (C-3, thienyl), 127.82 (C-4, thienyl),
128.34 (C-4, thienyl), 133.49 (C-2, thienyl), 137.82 (C-2,
thienyl), 143.35 (C-6, triazine), 154.70 (C-5, triazine),
173.40 (C-3, triazine). MS m/z (%): 291 (M?, 23), 204
(30), 189 (100), 149 (46), 95 (15), 67 (22). Anal. Calcd for
C12H9N3S3: C, 49.46; H, 3.11; N, 14.42. Found: C, 49.74;
H, 2.98; N, 14.62.
7.15–7.23 (m, 4H, phenyl), 7.16 (d, J = 8.5 Hz, 2H, phe-
nyl), 7.27 (d, J = 8.5 Hz, 2H, phenyl), 7.66 (d,
J = 8.0 Hz, 1H, pyridine), 7.75 (d, J = 8.0 Hz, 1H, pyri-
dine), 8.31 (d, J = 7.5 Hz, 1H, pyridine), 8.34 (s, 1H,
pyridine). 13C NMR (125 MHz, DMSO-d6) d: 20.90 (CH3),
21.55 (CH3), 37.33 (CH2), 123.55 (C-5, pyridine), 125.32
(C-2 and C-6, phenyl), 128.58 (C-2 and C-6, phenyl),
129.33 (C-3 and C-5, phenyl), 129.73 (C-3 and C-5, phe-
nyl), 134.51 (C-3, pyridine), 134.76 (C-1, phenyl), 135.90
(C-1, phenyl), 136.43 (C-4, pyridine), 137.72 (C-4, phe-
nyl), 138.60 (C-4, phenyl), 148.15 (C-6, pyridine), 150.41
(C-2, pyridine), 155.72 (C-6, triazine), 156.55 (C-5, tri-
azine), 172.85 (C-3, triazine). MS m/z (%): 384 (M?, 10),
326 (44), 281 (62), 221 (35), 206 (66), 147 (100). Anal.
Calcd for C23H20N4S: C, 71.85; H, 5.24; N, 14.57. Found:
C, 72.19; H, 5.43; N, 14.39.
General procedure for preparation of compound 4a–d
To a stirred solution of compound 2 (1.7 mmol) in ethanol
(20 ml), sodium hydroxide solution (170 mg in 5 ml of
water) and appropriate chloromethylpyridinium chloride
(2 mmol) were added respectively. The resulting mixture
was stirred for 3 h at room temperature. After completion
of the reaction, it was concentrated under vacuum, neu-
tralized with HCl 1N. The resulting aqueous phase was
extracted with chloroform three times, dried over sodium
sulfate and concentrated. The product was purified by
column chromatography (silica gel, mesh 230–400) eluting
with dichloromethane–methanol (9:1).
5,6-Bis(4-methoxyphenyl)-3-((pyridin-3-ylmethyl)thio)-
1,2,4-triazine (4c)
M.p.: 265–266 °C; IR (KBr): 3181, 2835, 1673 (C=N),
1
1556 (N=N). H NMR (500 MHz, DMSO-d6) d: 3.76 (s,
3H, OCH3), 3.77 (s, 3H, OCH3), 3.78 (s, 2H, SCH2), 6.90
(d, J = 8.5 Hz, 2H, phenyl), 6.91 (d, J = 8.5 Hz, 2H,
phenyl), 7.21 (d, J = 8.5 Hz, 2H, phenyl), 7.37 (d,
J = 8.5 Hz, 2H, phenyl), 7.57 (d, J = 8.5 Hz, 1H, pyri-
dine), 7.70 (d, J = 8.0 Hz, 1H, pyridine), 8.84 (d,
J = 8.0 Hz, 1H, pyridine), 8.27 (s, 1H, pyridine). 13C
NMR (125 MHz, DMSO-d6) d: 37.26 (CH2), 55.20
(OCH3), 55.35 (OCH3), 114.34 (C-3 and C-5, phenyl),
114.65 (C-3 and C-5, phenyl), 123.32 (C-5, pyridine),
127.34 (C-1, phenyl), 128.41 (C-1, phenyl), 134.55 (C-2
and C-6, phenyl), 134.89 (C-2 and C-6, phenyl), 135.55 (C-
3, pyridine), 136.75(C-4, pyridine), 148.15 (C-6, pyridine),
150.31 (C-2, pyridine), 155.72 (C-6, triazine), 156.34 (C-5,
triazine), 159.77 (C-4, phenyl), 161.15 (C-4, phenyl),
173.45 (C-3, triazine). MS m/z (%): 416 (M?, 26), 326
(45), 281 (100), 252 (27), 206 (56), 147 (96), 118 (34).
Anal. Calcd for C23H20N4O2S: C, 66.33; H, 4.84; N, 13.45.
Found: C, 66.59; H, 4.64; N, 13.79.
3-((Pyridin-4-ylmethyl)thio)-5,6-di-p-tolyl-1,2,4-
triazine (4a)
M.p.: 180–181 °C; IR (KBr): 3195, 2898, 1668 (C=N),
1
1556 (N=N). H NMR (500 MHz, DMSO-d6) d: 2.28 (s,
3H, Ar–CH3), 2.29 (s, 3H, Ar–CH3), 3.41 (s, 2H, SCH2),
7.15 (d, J = 8.5 Hz, 2H, phenyl), 7.33 (d, J = 8.5 Hz, 2H,
phenyl), 7.48 (d, J = 8.5 Hz, 2H, phenyl), 7.52 (d,
J = 9.0 Hz, 2H, phenyl), 7.70 (d, J = 9.0 Hz, 2H, pyri-
dine), 8.1 (d, J = 8.8 Hz, 2H, pyridine). 13C NMR
(125 MHz, DMSO-d6) d: 21.30 (CH3), 21.72 (CH3), 34.83
(CH2), 122.65 (C-3 and C-5, pyridine), 125.32 (C-2 and
C-6, phenyl), 128.24 (C-2 and C-6, phenyl), 129.54 (C-3
and C-5, phenyl), 130.33 (C-3 and C-5, phenyl), 134.55 (C-
1, phenyl), 135.54 (C-1, phenyl), 138.35 (C-4, phenyl),
138.60 (C-4, phenyl), 139.06 (C-4, pyridine), 148.86 (C-2
and C-6, pyridine), 155.44 (C-6, triazine), 156.55 (C-5,
triazine), 174.50 (C-3, triazine). MS m/z (%): 384 (M?,
15), 326 (55), 281 (65), 221 (25), 206 (36), 147 (100).
Anal. Calcd for C23H20N4S: C, 71.85; H, 5.24; N, 14.57.
Found: C, 72.15; H, 5.54; N, 14.45.
5,6-Bis(4-chlorophenyl)-3-((pyridin-4-ylmethyl)thio)-
1,2,4-triazine (4d)
M.p.: 205–206 °C; IR (KBr): 3125, 2888, 1672 (C=N),
1
1552 (N=N). H NMR (500 MHz, DMSO-d6) d: 3.72 (s,
2H, SCH2), 7.29 (d, J = 8.5 Hz, 2H, phenyl), 7.38 (d,
J = 8.5 Hz, 2H, phenyl), 7.43 (d, J = 8.5 Hz, 2H, phenyl),
7.46 (d, J = 8.5 Hz, 2H, phenyl), 7.57 (d, J = 9.0 Hz, 2H,
pyridine), 8.4 (d, J = 9.0 Hz, 2H, pyridine). 13C NMR
(125 MHz, DMSO-d6) d: 35.33 (CH2), 122.70 (C-3 and
C-5, pyridine), 126.5 (C-2 and C-6, phenyl), 128.27 (C-2
and C-6, phenyl), 128.92 (C-3 and C-5, phenyl), 129.34
3-((Pyridin-3-ylmethyl)thio)-5,6-di-p-tolyl-1,2,4-
triazine (4b)
M.p.: 220–221 °C; IR (KBr): 3185, 2970, 1664 (C=N),
1
1550 (N=N). H NMR (500 MHz, DMSO-d6) d: 2.30 (s,
3H, Ar–CH3), 2.31 (s, 3H, Ar–CH3), 3.32 (s, 2H, SCH2),
123

2508
Med Chem Res (2014) 23:2503–2514
(C-3 and C-5, phenyl), 133.72 (C-1, phenyl), 134.35 (C-1,
phenyl), 134.73 (C-4, phenyl), 135.52 (C-4, phenyl),
137.34 (C-4, pyridine), 148.64 (C-2 and C-6, pyridine),
155.70 (C-6, triazine), 156.44 (C-5, triazine), 175.59 (C-3,
triazine). MS m/z (%): 425 (M?, 55), 326 (43), 281 (75),
206 (16), 147 (100), 94 (15). Anal. Calcd for
C21H14Cl2N4S: C, 59.3; H, 3.32; N, 13.17. Found: C,
59.49; H, 3.54; N, 13.39.
(model 53140, Stoelting, USA) through tail vein. A
30-gauge needle with flexible tube, allows infusion of PTZ
to unrestrained animal. Infusion was stopped either when a
tonic–clonic convulsion with loss of righting reflex was
achieved or after the animal received a maximum amount
of 1 ml PTZ solution. The dose of PTZ (mg/kg) adminis-
tered to induce tonic–clonic seizures with loss of righting
¨
reflex was considered as seizures threshold (Loscher et al.,
1991).
Pharmacology
Neurotoxicity
Male NMRI mice (Pasture Institute, Tehran, Iran) weight-
ing 18–22 g were used for pharmacological study. Animals
were allowed free access to food and water except during
the experiment and housed at controlled room temperature
with 12 h light/dark schedule (lights on at: 0700 h). All
compounds were dissolved a vehicle consists of DMSO
(10 %) in distilled water with the injection volume of
10 mL/kg body weight of mice. Control group received
DMSO (10 %) in saline. Each test compound was admin-
istered to different groups of mice at the dose of 100 mg/kg.
The antiseizure effect of test compounds was also compared
with a standard antiepileptic drug (lamotrigine 12.5 mg/kg,
i.p.), which has been shown to produce protective effect in
both Electroshock and PTZ induced seizure. Each animal
was used once throughout the experiments.
The neurotoxicity of the compounds was measured in mice
by the rotarod test as well as chimney test. In rotarod test,
the mice were trained to stay on a rotarod of diameter 3.2-
cm that rotates at 10 rpm. Trained animals were given i.p.
injection of the test compounds and 30 min later were
tested again on rotarod. 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. The effects of test
compounds, at doses corresponding to their anticonvulsant
effect from the PTZ and Electroshock-induced seizure
tests, on motor performance in mice were also quantified
with the chimney test (Naderi et al., 2011). In this test,
animals had to climb backwards up a plastic tube (25-cm
length, 3-cm inner diameter). Motor impairment was
indicated by the inability of the animals to perform the test
within 60 s. Motor impairment was defined as the number
of mice who failed to climb the tube in 60 s.
Electroshock-induced seizure test
The electroshock-induced seizure test was carried out by
the methods described before. Seizures were elicited with a
50 Hz alternating current of 40 mA intensity in mice using
a generator (Borjsanat, Tehran, Iran). The current was
applied via corneal electrodes for 0.2 s. This current
intensity could produce tonic–clonic seizures in mice.
Protection against the spread of Electroshock-induced sei-
zures was defined as the abolition of the hind leg and tonic
maximal extension component of the seizure (Woodbury
and Davenport, 1952; Woodbury and Swinyard, 1952). At
30 min after the administration of the test compounds, the
activities were evaluated in electroshock-induced seizure
test. Protection against seizures was expressed as the
number of mice that did not exhibit hind-limb extension
after applying electroshock to the animal.
Statistical analysis
Data were analyzed using Graphpad Prism 5 (Graphpad
Software Inc.). In electroshock, rotarod and chimney tests,
the results of test drugs were compared with control group
using Fisher’s exact test. In PTZ-induced seizure, one-way
ANOVA followed by Dunnett’s post-test was used. Data
were shown as mean SEM and a P value \0.05 was
considered as statistically significant.
Computational study
Chemical structures were sketched in ChemSketch (ACD
Lab 12.0, Advanced Chemistry Development, Inc.) and
imported to ACD/3D Viewer version 12.0. The pharma-
cophore mapping studies, the distance between the various
groups postulated as essential pharmacophoric elements for
anticonvulsant activity, were performed on the 3D opti-
mized structures. OSIRIS Property Explorer (OSIRIS
Property Explorer, 2013) was used for the prediction of
cLogP and solubility values reported in Table 1.
PTZ-induced seizure test
PTZ-induced seizure test was performed 30 min after
intraperitoneal (i.p.) injection (at a volume of 10 ml/kg) of
therapeutic drugs or their respective vehicle. Seizures were
induced by intravenous (i.v.) infusion of 1 % PTZ in saline
at the constant rate of 0.25 ml/min using an infusion pump
123

Med Chem Res (2014) 23:2503–2514
2509
Table 1 Synthesis of 5,6-bisaryl-1,2,4-triazine-3-thiol derivatives
Thiosemicarbazide, HCl
MW, 120°C, 10 min
Ar
Ar
N
N
SH
Ar
Ar
O
O
N
2a-g
1a-g
Compound
Ar
Formula
MW
Solubilitya
cLogPb
M.p. (°C)
2a
2b
2c
2d
2e
2f
2g
a
Phenyl
C15H11N3S
265
293
325
301
334
423
277
-4.59
-5.28
-4.63
-5.22
-6.06
-6.26
-4.43
3.54
4.17
3.33
3.66
4.77
4.94
3.78
210
182
235
210
205
162
215
p-Tolyl
C
C
C
C
C
C
17H15N3S
4-Methoxyphenyl
4-Fluorophenyl
4-Chlorophenyl
4-Bromophenyl
2-Thienyl
17H15N3O2S
15H9F2N3S
15H9Cl2N3S
15H9Br2N3S
11H7N3S3
Estimated logS value is a logarithm (base 10) of the predicted solubility in mol/liter
b
Logarithm of the partition coefficient between n-octanol and water log(Coctanol/Cwater
)
Scheme 1 Synthesis of 5,6-
bisaryl-1,2,4-triazine-3-thiol
derivatives. Reagents and
conditions: a thiosemicarbazide,
HCl, MW, 120 °C, 10 min;
b methyl iodide, triethylamine,
methanol, rt; c 4-
chloromethylpyridinium
chloride or
3-chloromethylpyridinium
chloride, NaOH, EtOH, rt
X
X
X
X
b
Ar
Ar
O
O
N
SH
a
N
N
S
N
N
N
2a-f
1a-g
3a-e
X=Me, MeO, F, Cl, Br
X=H, Me, MeO, F, Cl, Br
a,b
c
X
S
S
N
N
SR
N
N
Y
N
N
X
2g, R=H
2h, R=Me
4a, X=Me, Y= 4-pyridylmethylthio
4b, X=Me, Y= 3-pyridylmethylthio
4c, X=MeO, Y= 3-pyridylmethylthio
4d, X=Cl, Y= 4-pyridylmethylthio
Results and discussion
the Scheme 1. The 5,6-bisaryl-1,2,4-triazine-3-thiols 2a–
g were obtained by the condensation of 1,2-diarylketones
1a–g with thiosemicarbazide and catalytic amount of
hydrochloric acid under microwave irradiation at 120 °C
for 10 min in the appropriate solvent system reported in
Table 1. 5,6-Bisaryl-1,2,4-triazine-3-thiols 2a–g were S-
Chemistry
The synthetic pathways leading to the formation of 5,6-
bisaryl-1,2,4-triazine-3-thiol derivatives 2–4 are outlined in
123

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Med Chem Res (2014) 23:2503–2514
Table 2 Optimization of reaction between 1,2-diketones and thiosemicarbazide in different solvent with their isolated production yield (%)
Compound
DMF
Acetonitrile
Water
Solvent systems
Water/ethanol (2:1)
Water/ethanol (1:1)
Water/ethanol (1:2)
Abs. ethanol
2a
2b
2c
2d
2e
2f
50
56
55
40
45
47
53
33
30
20
32
nta
nt
30
15
10
25
23
12
16
92
88
91
56
15
26
40
54
85
47
83
93
25
80
62
76
54
61
60
67
75
55
77
49
62
65
81
65
2g
nt
The highest isolated production yield for each derivative is highlighted in bold
a
Not tested
methylated by using methyl iodide to give 3-methylthio-
5,6-bisaryl-1,2,4-triazine derivatives 3a–e and 2h. Com-
pounds 4a–d were obtained by reacting of 5,6-bisaryl-
1,2,4-triazine-3-thiols 2a–f with appropriate chloro-
methylpyridinium chloride in ethanol at room temperature.
Some physicochemical properties of thiol compounds
2a–g, such as predicted solubility, clogP, melting point,
and MW are given in Table 1. The formation of com-
for the preparation of all derivatives (Table 1). Afterward,
choosing the best solvent among water, absolute ethanol,
different ratio of water and ethanol (2:1, 1:1, and 1:2) was
the crucial step for the optimization of reaction. Acetoni-
trile and dimethylformamide (DMF) have also been used as
solvents. The results are summarized in Table 2.
The production yield in different ratio of water and
ethanol strongly depends on the lipophilicity (LogP) or
solubility of the starting material and final compound in the
solvent. In the cases of compounds 2a and 2c, with more
polar substituents and lower cLogP (3.33 and 3.54,
respectively) and by using water and ethanol (2:1 ratio) as
solvent, the higher yields ([90 %) were obtained. The
most nonpolar substituent, para-bromo on the phenyl ring
in compound 2f with cLogP = 4.94 gave the highest yield
in absolute ethanol (81 %). Eventually, the other com-
pounds 2b, 2d, 2e, and 2g, dependent on their lipophilicity,
performed well in either 2:1 or 1:1 ratio mixtures of water
and ethanol with 88, 83, 93, and 80 % yields, respectively.
Performing reaction under microwave irradiation in
organic solvents like acetonitrile and DMF produced low
yields (40–50 % for DMF and 20–30 % for acetonitrile) as
well as much more by-product and increased impurity of
the product checked by TLC. Our observations showed that
increasing percentage of water in the solvent system
reduces the number of by-products and performs a clean
reaction. However, hydrophobic nature of the starting
material, 1,2-diketones bearing chloro, fluoro, bromo, and
methyl at the para-position of phenyl ring, and also
2-thienyl group limits the high percentage usage of water in
the reaction solvent. Usage of water alone as solvent did
not give satisfactory yields due to insolubility problem of
1,2-diketones. Adding an environmentally friendly solvent
with high safety such as ethanol for solubilizing the starting
materials and also its high intrinsic ability to absorb
microwave irradiation (dielectric loss = 22.86) makes
ethanol a prominent solvent for MAOS (Hayes, 2002).
Therefore, the best results were obtained in a mixture of
1
pounds was confirmed by H NMR, 13C NMR and MS
1
spectral data. The H NMR spectrum of the representative
compound 2g showed six doublets for the two thienyl rings
containing six protons at d 7.03, 7.14, 7.41, 7.47, 7.56, and
7.60 ppm. The IR spectrum showed bands at 1,559 and
1,503 cm-1 for C=N and N=N bonds at triazine ring. The
spectral information of compound 3a was identical to the
previously reported data in the literature (Ansari et al.,
1
2010). The H NMR spectrum of the representative com-
pound 5,6-bis(4-methoxyphenyl)-3-((pyridin-3-ylmethyl)-
thio)-1,2,4-triazine (4c) showed two singlets at d 3.76 and
3.77 ppm for the two methoxyl groups, and a singlet at
3.78 ppm for the methylene protons. Aromatic protons
appeared as doublets at d 6.90, 6.91, 7.21, and 7.37 ppm.
The pyridyl protons of compound 4c appeared at 7.57
(doublet), 7.70 (doublet), 8.44 (doublet), and 8.47 (singlet)
ppm. Also, MS spectral data showed the correct molecular
ion peak of the product.
Microwave-assisted green synthesis of 2a–g
We explored the condensation of aromatic 1,2-diketones
with thiosemicarbazide starting with the polar 4,40-di-
methoxybenzil in a different ratio of water and ethanol
under MW irradiation for obtaining 5,6-bis(4-methoxy-
phenyl)-1,2,4-triazine-3-thiol (2c). After screening a range
of various temperatures and reaction run times, we found
that the highest yield could be accessible in 120 °C for
10 min and 300 watt power in closed vessel. The same
protocol (reaction temperature and run time) was kept fixed
123

Med Chem Res (2014) 23:2503–2514
2511
Fig. 2 Possible mechanism for the formation of the 5,6-bisaryl-1,2,4-triazine-3-thiol derivatives
Table 3 The anticonvulsant effect of the test compounds (100 mg/kg) against electroshock-induced seizures, as well as muscle coordination in
rotarod and Chimney tests
X
X
N
N
Y
N
Compound
X
Y
Electroshock induced
seizure (no. of animals
protected/no. of animals tested) of animal tested)
Rotarod (no. of animal Chimney (no. of animal
exhibiting toxicity/no.
exhibiting toxicity/no.
of animal tested)
2b
p-Tolyl
–SH
1/6
0/6
3/6
1/6
2/6
3/6
3/6
3/6
1/6
0/6
0/6
0/6
1/6
0/6
0/6
0/6
0/6
0/6
0/6
1/6
0/6
1/6
0/6
0/6
2/6
1/6
0/6
1/6
2/6
0/6
1/6
1/6
0/6
0/6
2c
4-Methoxyphenyl –SH
2e
4-Chlorophenyl
2-thienyl
–SH
2g
–SH
2h
2-thienyl
–SMe
–SMe
3a
p-Tolyl
3b
4-Methoxyphenyl –SMe
3c
4-Chlorophenyl
p-Tolyl
–SMe
4a
4-Pyridylmethylthio 6/6
3-Pyridylmethylthio 4/6
4b
p-Tolyl
4c
4-Methoxyphenyl 3-Pyridylmethylthio 2/6
4d
4-Chlorophenyl
4-Pyridylmethylthio 5/6
6/6
Lamotrigine
water and ethanol with ratio of 2:1 and 1:1 for most various
hydrophobic starting materials 1 with the exception of 4,40-
dibromobenzil.
thiosemicarbazone intermediate in the presence of acid.
This reaction involves nucleophilic attack of the hydrazine
end of thiosemicarbazide as a strong nucleophile to one of
the carbonyl groups. This open chain intermediate could be
isolated and purified as a stable product. In the second step,
the less and weak nucleophilic thioamide nitrogen atom
attacks to the protonated carbonyl group to form the tri-
azine ring. This step demands much more energy and
driving force that is provided significantly by the high
energy microwaves. Moreover, usage of a solvent that is
capable to convert microwave electromagnetic energy into
heat is favorable. The best parameter that provides an
organic chemist with the coupling efficiency of a particular
solvent is defined by dielectric loss. The more dielectric
loss of a solvent, the higher converting ability of micro-
wave energy is into heat. Therefore, using of a solvent with
low dielectric loss such as DMF and acetonitrile with 6.07
and 2.32 is not recommended for performing a high energy
Previous results have also revealed that simple alcohols
like methanol and ethanol or alkanes such as heptane and
hexane are environmentally preferable solvents, whereas
the use of dioxane, acetonitrile, and tetrahydrofuran is not
recommended from an environmental perspective. Addi-
tionally, in agreement with our finding, a case study indi-
cated that methanol–water or ethanol–water mixtures are
environmentally favorable compared to pure alcohol or
propanol–water mixtures for solvolysis of p-methoxyben-
zoyl chloride (Capello et al., 2007).
Additionally, on the basis of the previous studies on the
formation of triazine ring with thiosemicarbazide and
phenylglyoxalaldoxime (Lalezari and Golgolab, 1970), a
possible mechanism could be suggested as shown in Fig. 2.
The first step is the feasible and easy formation of
123

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Med Chem Res (2014) 23:2503–2514
Table 4 Anticonvulsant effect of test compounds (100 mg/kg)
against PTZ-induced seizures in mice
X
X
N
N
Y
N
Compound
X
Y
PTZ dose
(mg/kg)a
2b
p-Tolyl
–SH
40.4 3.8
ntb
2c
4-Methoxyphenyl –SH
2e
4-Chlorophenyl
2-thienyl
–SH
nt
2g
–SH
24.42 4.63
38.23 2.34
39.4 2.9
41.4 4.2
nt
2h
2-thienyl
–SMe
–SMe
3a
p-Tolyl
3b
4-Methoxyphenyl –SMe
3c
4-Chlorophenyl
p-Tolyl
–SMe
4a
4-Pyridylmethylthio 49.4 4.4
3-Pyridylmethylthio 47.0 4.7
4b
p-Tolyl
4c
4-Methoxyphenyl 3-Pyridylmethylthio 41.5 4.1
4d
4-Chlorophenyl
4-Pyridylmethylthio 43.0 5.3
72.9 10.6
Lamotrigine
Controlc
31.5 4.4
Fig. 3 Essential pharmacophoric elements in phenytoin and lamotri-
gine as voltage-gated sodium channel blockers. Distance ranges
between the essential structural elements R, D, and HAD in
pharmacophoric model proposed by Unverferth et al., 1998
a
PTZ dose (mg/kg) to induce clonic seizures (number of mice in
each group = 6); a significant increase in required dose of PTZ to
induce seizures shows significant protective effect of the test com-
pound. Values are shown as mean SEM
b
Not tested
c
DMSO (10 %)
demanding reaction like the second step of triazine ring
closure here. Usage of an appropriate solvent with high
dielectric loss like ethanol (dielectric loss = 22.86) defi-
nitely proceeds the reaction toward completion (Hayes,
2002).
Anticonvulsant activity
The anticonvulsant effect of the test compounds (100 mg/
kg) against electroshock-induced seizure, as well as muscle
coordination in rotarod and chimney tests were listed in
Table 3.
The results of electroshock-induced seizure test revealed
that compounds 4a and d bearing 4-pyridylmethylthio
group at the position 3 of triazine ring had better antisei-
zure activity. Meanwhile, bis(p-tolyl)- analog 4a showed
100 % protection against electroshock-induced seizure.
Notably, the most effective compound 4a showed no sign
of neurotoxicity at the dose of 100 mg/kg in both rotarod
and chimney tests. Moreover, in the rotarod test, other
compounds with the exception of 2b and 4d did not show
any sign of neurotoxicity. However, in the Chimney test
Fig. 4 Distance ranges between hydrophobic unit (R), electron-donor
atom (D), and hydrogen bond acceptor (HA) in compound 4a
some compounds exhibited maximum 33 % neurological
toxicity (for example, compound 3c). The results demon-
strated that the target compounds had no significant
neurotoxicity at the dose of 100 mg/kg.
The anticonvulsant activity of test compounds was also
investigated by evaluating the protective potential of the
compounds at the dose of 100 mg/kg against PTZ-induced
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seizure in mice. The dose of PTZ (mg/kg) administered to
induce tonic–clonic seizure with loss of righting reflex was
considered as seizure threshold and the results were pre-
sented in Table 4. The control group receiving vehicle
showed tonic–clonic seizures at the PTZ dose of
31.5 4.4 mg/kg. A significant increase in required dose
of PTZ to induce seizure shows significant protective effect
of the test compounds. Again, compound 4a having
4-pyridylmethylthio group showed the most protective
effect against PTZ. The maximum required dose of PTZ
(49.4 mg/kg) to induce seizure in mice among the com-
pounds tested confirms the most protective effect of 4a.
Also, the regio-isomer of 4a (compound 4b) containing
3-pyridylmethylthio group showed significant increase in
the required dose of PTZ to induce seizure in mice (47 mg/
kg). In the case of standard drug lamotrigine, the higher
dose of PTZ (72.9 mg/kg) was required for inducing tonic–
clonic seizures.
3-thiol derivatives under MW irradiation that could be used
in chemical library synthesis in combinatorial chemistry.
Compared to the conventional synthetic methods, this
approach has the advantage of using safe, nontoxic and
environmentally benign solvents as well as being rapid,
simple and easy purification procedure. The anticonvulsant
activity evaluation of the synthesized compounds demon-
strated that compound 4a bearing 4-pyridylmethylthio
moiety on the triazine ring showed the highest protection in
both electroshock- and PTZ-induced seizures tests. The
neurotoxicity study revealed that the target compound had
no significant neurotoxicity at the dose of 100 mg/kg.
Based on the promising pharmacological properties of
compound 4a, the 3-(pyridylmethylthio)triazine core
structure with vicinal bis-aryl could be considered as a lead
for discovery of novel antiseizure drugs.
Acknowledgments We gratefully acknowledge the financial sup-
port from the Research Council of Mazandaran University of Medical
Sciences, Sari, Iran.
Structurally, the obtained results demonstrated that pre-
sence of a lipophilic group onthe vicinal bis-phenyl ringsand
a pyridylmethyl pendent group on sulfur atom connected to
triazine core could increase anticonvulsant activity. It seems
that the methyl or chloro-substituents at the para-position of
the vicinal bis-phenyl rings which increased hydrophobic
character of the molecule results in higher potency. The
4-methoxy derivative 4c has relatively a lower hydropho-
bicity resulting in lowered anticonvulsant effect.
References
ACD/Labs version 12.0. Advanced Chemistry Development Inc.
www.acdlabs.com
Ansari N, Khodagholi F, Ramin M, Amini M, Irannejad H, Dargahi L,
Dehghani Amirabad A (2010) Inhibition of LPS-induced apop-
tosis in differentiated-PC12 cells by new triazine derivatives
through NF-kB-mediated suppression of COX-2. Neurochem Int
57:958–968
Computational study (distance mapping)
Bhalla M, Srivastava VK, Bhalla TN, Shanker K (1995) Synthesis
and pharmacological evaluation of 1,2,4-triazine and its cong-
eners. Boll Chim Farm 134:9–15
According to Unverferth et al. study (Unverferth et al.,
1998) essential pharmacophoric elements that are neces-
sary for good anticonvulsant activity through blocking of
voltage-gated sodium channels are a hydrophobic unit (R),
an electron-donor atom (D), and a hydrogen acceptor/donor
unit (HAD). These structural features have to be positioned
in an allowed specified distances to each other (Fig. 3).
Distance ranges between the essential structural features
(R, D, and HAD) were measured for the most effective
compound 4a by ACD/3D Viewer software and are shown
in Fig. 4. As shown, the measured distances are in the
allowed and specified distance ranges defined by Unverf-
erth et al. for the voltage-gated sodium channel blockers.
Due to the flexibility of the 4-pyridylmethylthio side chain,
distances between hydrophobic unit (R) and HAD and also
between electron-donor atom (D) and HAD can be variable
in a range shown in the figure.
Capello C, Fischer U, Hungerbuhler K (2007) What is a green
solvent? A comprehensive framework for the environmental
assessment of solvents. Green Chem 9:927–934
Hayes BL (2002) Microwave synthesis chemistry at the speed of life.
CEM Publishing, Matthews
Heravi MM, Nami N, Oskooie HA, Hekmatshoar R (2006) One-pot
synthesis of some nitrogen and sulfur heterocycles using
thiosemicarbazide under microwave irradiation in a solventless
system. Phosphorus, Sulfur Silicon Relat Elem 181:87–91
Irannejad H, Amini M, Khodagholi F, Ansari N, Khoramian Tusi S,
Sharifzadeh M, Shafiee A (2010) Synthesis and in vitro eval-
uation of novel 1,2,4-triazine derivatives as neuroprotective
agents. Bioorg Med Chem 18:4224–4230
Kappe CO, Dallinger D (2006) The impact of microwave synthesis on
drug discovery. Nat Rev Drug Disc 5:51–63
Kaushik D, Ahmad Khan S, Chawla G (2010) Design & synthesis of
2-(substituted aryloxy)-5-(substituted benzylidene)-3-phenyl-
2,5-dihydro-1H-[1,2,4] triazin-6-one as potential anticonvulsant
agents. Eur J Med Chem 45:3960–3969
Khoshneviszadeh M, Ghahremani MH, Foroumadi A, Miri R, Firuzi
O, Madadkar-Sobhani A, Edraki N, Parsa M, Shafiee A (2013)
Design, synthesis and biological evaluation of novel anti-
cytokine 1,2,4-triazine derivatives. Bioorg Med Chem. doi:/10.
1016/j.bmc.2013.08.009
Conclusion
Lalezari I, Golgolab H (1970) A one-step synthesis of the as-triazine
ring system. J Het Chem 7:689–691
In conclusion, we have described a rapid, simple and green
procedure for the preparation of 5,6-bisaryl-1,2,4-triazine-
Landmark CJ (2007) Targets for antiepileptic drugs in the synapse.
Med Sci Monit 13:RA1–RA7
123

2514
Med Chem Res (2014) 23:2503–2514
¨
¨
Loscher W, Honack D, Fassbender CP, Nolting B (1991) The role of
technical, biological and pharmacological factors in the labora-
tory evaluation of anticonvulsant drugs. III. Pentylenetetrazole
seizure models. Epilepsy Res 8:171–189
Siddiqui N, Ahsan W (2010) Triazole incorporated thiazoles as a new
class of anticonvulsants: design, synthesis and in vivo screening.
Eur J Med Chem 45:1536–1543
Unverferth K, Engel J, Hofgen N, Rostock A, Gunther R, Lankau HJ,
Menzer M, Rolfs A, Liebscher J, Muller B, Hofmann H (1998)
Synthesis, anticonvulsant activity, and structure–activity rela-
tionships of sodium channel blocking 3-aminopyrroles. J Med
Chem 41:63–73
Woodbury LA, Davenport VD (1952) Design and use of a new
electroshock seizure apparatus, and analysis of factors altering
seizure threshold and pattern. Arch Int Pharmacodyn Ther
92:97–107
Woodbury LA, Swinyard CA (1952) Stimulus parameters for
electroshock seizures in rats. Am J Physiol 170:661–667
Mallikarjuna BP, Suresh Kumar GV, Sastry BS, Manohara KP (2007)
Synthesis and anticonvulsant activity of some potent 5,6-bis aryl
1,2,4-triazines. J Zhejiang Univ Sci B 8:526–532
Naderi N, Ahmad-Molaei L, Aziz Ahari F, Motamedi F (2011)
Modulation of anticonvulsant effects of cannabinoid compounds
by GABA-A receptor agonist in acute pentylenetetrazole model
of seizure in rat. Neurochem Res 36:1520–1525
OSIRIS Property Explorer (2013) www.organic-chemistry.org
Sangshetti JN, Shinde DB (2010) One pot synthesis and SAR of some
novel 3-substituted 5,6-diphenyl-1,2,4-triazines as antifungal
agents. Bioorg Med Chem Lett 20:742–745
Shipe WD, Wolkenberg SE, Lindsley CW (2005) Accelerating lead
development by microwave-enhanced medicinal chemistry.
Drug Disc Today 2:155–161
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