Synthesis of 6-(3-substituted-4H-1,2,4-triazol-4-yl)-2- phenylbenzo[d]oxazoles as potential anticonvulsant agents

Article abstract of DOI:10.1007/s00044-009-9239-z

A series of 6-(3-substituted-4H-1,2,4-triazol-4-yl)-2-phenylbenzo[d] oxazoles was synthesized. The anticonvulsant effect and neurotoxicity of the compounds were calculated with maximal electroshock (MES) test, subcutaneous pentylenetetrazole (Sc-PTZ), and

Full text of DOI:10.1007/s00044-009-9239-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:925–935
DOI 10.1007/s00044-009-9239-z
ORIGINAL RESEARCH
Synthesis of 6-(3-substituted-4H-1,2,4-triazol-4-yl)-2-
phenylbenzo[d]oxazoles as potential anticonvulsant
agents
•
Cheng-Xi Wei Di Wu Zhi-Gang Sun
•
•
•
Kyu-Yun Chai Zhe-Shan Quan
Received: 8 January 2009 / Accepted: 25 August 2009 / Published online: 14 October 2009
¨
Ó Birkhauser Boston 2009
Abstract A series of 6-(3-substituted-4H-1,2,4-triazol-4-yl)-2-phenylbenzo[d]
oxazoles was synthesized. The anticonvulsant effect and neurotoxicity of the
compounds were calculated with maximal electroshock (MES) test, subcutaneous
pentylenetetrazole (Sc-PTZ), and rotarod tests in intraperitoneally injected mice.
Among the synthesized compounds, compound 2-phenyl-6-(4H-1,2,4-triazol-4-
yl)benzo[d]oxazole (3a) could be considered the potentially most useful and safe
therapeutic compound, with ED50 = 29.6 mg/kg, TD50 = 285 mg/kg, and PI =
9.7. Its neurotoxicity was the lowest of all the synthesized compounds and was also
markedly lower than that of the reference drug carbamazepine with PI value of 6.4.
Keywords Synthesis ꢀ Benzo[d]oxazole ꢀ Triazole ꢀ Anticonvulsant activity
Introduction
Epilepsy is a syndrome of different cerebral disorders of the central nervous system
(CNS) and is characterized by paroxysmal, excessive, and hypersynchronous
discharges of large numbers of neurons (Gasior et al., 1997). Despite the optimal
use of available antiepileptic drugs (AEDs), many patients with epilepsy fail to
C.-X. Wei ꢀ Z.-G. Sun (&)
Institute of Neurosurgery, Inner Mongolia University for Nationalities, No. 1742, Holin River Street,
Tongliao, Inner Mongolia Autonomous Region 028007, People’s Republic of China
e-mail: sunzhigang101@yahoo.cn
C.-X. Wei ꢀ D. Wu ꢀ Z.-S. Quan (&)
College of Pharmacy, Yanbian University, No. 121, JuZi Street, Yanji, Jilin 133000,
People’s Republic of China
e-mail: zsquan@ybu.edu.cn
K.-Y. Chai
Department of Chemistry, Wonkwang University, Iksan 570-749, Korea

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Med Chem Res (2010) 19:925–935
N
R
R
Ph
N
O
O
N
R
N
N
N
R'
N
N
Fig. 1 Structure of compounds I, II, and 3a–q
experience seizure control, and others experience seizure control only at the expense
of significant toxic effects (Leppik, 1994; Al-Soud et al., 2003). The limitations of
conventional AEDs highlight the need to develop newer agents for epilepsy.
In recent years, the field of antiepileptic drug development has been quite
dynamic, affording many promising research opportunities. As shown in Fig. 1,
derivatives of benzo[d]oxazole I have been shown to possess a variety of biological
activities such as antitumor (Shi et al., 1996; Reynolds et al., 1999), antibiotic
(Temiz et al., 1998), antimicrobial (Yildiz et al., 2004), anti-inflammatory (Unlu¨
´
et al., 2003), and anticonvulsant (Dıaz-Molina et al., 2005). Also, the derivatives of
triazole II have been known to possess a variety of biological activities such as
antibacterial properties (Pandeya et al., 1999; Gu¨lerman et al., 2001), HIV-RT
inhibitory (Silva et al., 2009), and anti-inflammatory (Abdel-Megeed et al., 2009).
Moreover, the triazole nucleus is present in many compounds that have anticon-
vulsant activity (Ku¨c¸u¨kgu¨zel et al., 2004; Dawood et al., 2006; Chen et al., 2007).
In the study, the triazole moiety was connected to the 2-substituted benzo[d]oxa-
zole moiety at its 6-position (3a–q in Fig. 1), as the combination of different
pharmacophores in one frame may lead to compound with interesting pharmaco-
logical profiles. In view of the pharmacological profiles of the two chemical
moieties, as described above, we considered it interesting to further explore the
biological properties of compounds 3a–q. In the present work, a series of 6-(4H-
1,2,4-triazol-4-yl)benzo[d]oxazoles were synthesized and tested for their anticon-
vulsant activity and neurotoxicity with maximal electroshock (MES) test, subcu-
taneous pentylenetetrazole (Sc-PTZ), and rotarod tests in intraperitoneally injected
mice.
Experimental
Melting points were determined in open capillary tubes and are reported
1
uncorrected. Infrared (IR) spectra were recorded (in KBr) on a FT-IR1730. H
nuclear magnetic resonance (NMR) spectra were measured on a AV-300 (Bruker,
Switzerland), and all chemical shifts are given in ppm relative to tetramethylsilane.
Mass spectra were measured on an HP1100LC (Agilent Technologies, USA).
Elemental analyses were performed on a 204Q CHN (Perkin Elmer, USA).
Microanalyses of C, N, and H were performed using a Heraeus CHN rapid analyzer.
The major chemicals were purchased from Aldrich Chemical Corporation. All other
chemicals were of analytical grade.

Med Chem Res (2010) 19:925–935
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Synthesis of compounds (1)
Starting material of organic acid (20 mmol) and 2-amino-5-nitrophenol (20 mmol)
was heated in 48 g polyphosphoric acid with stirring for 8 h. At the end of the
reaction period, the residue was poured into ice–water mixture and counteracted
with excess 10% NaOH solution. The crude product was collected through filtration
and dried in vacuo.
6-Nitro-2-phenylbenzo[d]oxazole (1)
Yield: 85%; m.p. = 138–142°C; 1H-NMR (d ppm, CDCl3): 8.52 (1H, d,
J7,5 = 2.1 Hz, 7-H), 8.31-8.37 (3H, m, Ar–H), 7.87 (1H, d, J4,5 = 8.8 Hz, 4-H),
7.57-7.62 (3H, m, Ar–H); IR (KBr) cm-1: 2235, 1558; MS m/z 241 (M ? 1).
General procedures for the synthesis the compounds (2)
Compound 1 (15 mmol) and iron powder (45 mmol) were placed into a three-
necked round-bottomed flask containing 150 ml ethanol (95%). The mixture was
stirred and heated with a blade stirrer and oil bath until the temperature attained 80–
85°C. Then 4 ml hydrochloric acid (1.2 M) was added and the mixture was stirred
at 80–85°C for 3–4 h. The mixture was filtered immediately with a hot filter under
reduced pressure. Adjusting the filtrate pH value to 7–8 using saturated NaOH
resulted in precipitation of solid. The crude product was collected through filtration
and dried in vacuo.
2-Phenylbenzo[d]oxazol-6-amine (2)
1
Yield: 77%; m.p. = 172–174°C; H-NMR (d ppm, CDCl3): 8.20 (2H, s, Ar–H),
7.39-7.52 (4H, m, Ar–H), 6.90 (H, s, 7-H), 6.73 (1H, d, J4, 5 = 8.6 Hz, 4-H), 3.86
(2H, s, –NH2); IR (KBr) cm-1: 3333, 1499; MS m/z 211 (M ? 1).
General procedures for the synthesis the compounds (3a–q)
Dimethoxy-N,N-dimethylmethanaminel (11 mmol) was added to a solution of
corresponding hydrazide (11 mmol) in acetonitrile (10 ml) in a 100-ml round-
bottomed flask equipped with a reflux condenser. The reaction mixture was warmed
to 50°C for 30 min, and then solid compound 2a (10 mmol) in acetonitrile (10 ml)
was added as well as acetic acid (1 ml) as a catalyst at the same time. The reaction
temperature was raised to 120°C for 3 h. After being cooled and concentrated, the
product was recrystallized in ice–water. The precipitate was collected through
filtration and dried in vacuo.
2-Phenyl-6-(4H-1,2,4-triazol-4-yl)benzo[d]oxazole (3a)
1
Yield: 66%; m.p. = 203°C dec; H-NMR (d ppm, CDCl3): 8.56 (2H, s, (–CH–)2),
8.27 (1H, s, Ar–H), 7.99 (1H, d, J4,5 = 8.46 Hz), 7.71 (1H, s, 7-H), 7.29–7.62 (4H,

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Med Chem Res (2010) 19:925–935
m, Ar–H); IR (KBr) cm-1: 3111, 1518, 1076; MS m/z 263 (M ? 1), Anal. Calcd. for
C15H10N4O: C, 68.69; H, 3.84; N, 21.36; Found: C, 68.59; H, 3.95; N, 21.44.
6-(3-Methyl-4H-1,2,4-triazol-4-yl)-2-phenylbenzo[d]oxazole (3b)
Yield: 65%; m.p. = 199–202°C dec; 1H-NMR (d ppm, CDCl3): 8.56 (1H, s, –CH–),
8.29 (2H, d, J = 6.3 Hz, Ar–H), 7.92 (1H, d, J = 8.4 Hz, 4-H), 7.71 (1H, s, 7-H),
7.28–7.68 (4H, m, Ar–H), 2.75 (3H, s, –CH3); IR (KBr) cm-1: 3099, 1517, 1066;
MS m/z 277 (M ? 1), Anal. Calcd. for C16H12N4O: C, 69.55; H, 4.38; N, 20.28;
Found: C, 69.33; H, 4.45; N, 20.08.
6-(3-Ethyl-4H-1,2,4-triazol-4-yl)-2-phenylbenzo[d]oxazole (3c)
1
Yield: 61%; m.p. = 188–190°C dec; H-NMR (d ppm, CDCl3): 8.27–8.54 (3H, m,
–CH– and Ar–H), 7.90 (1H, d, J = 8.4 Hz, 4-H), 7.28–7.59 (5H, m, Ar–H), 2.78
(2H, q, J = 7.5 Hz, –CH2),1.32 (3H, t, J = 7.5 Hz, –CH3); IR (KBr) cm-1: 3121,
1518, 1106; MS m/z 291 (M ? 1), Anal. Calcd. for C17H14N4O: C, 70.33; H, 4.86;
N, 19.30; Found: C, 70.08; H, 4.68; N, 19.11.
2-Phenyl-6-(3-propyl-4H-1,2,4-triazol-4-yl)benzo[d]oxazole (3d)
1
Yield: 54%; mp. = 127–129°C dec; H-NMR (d ppm, CDCl3): 8.27–8.30 (3H, m,
–CH– and Ar–H), 7.91 (1H, d, J = 8.2 Hz, 4-H), 7.28–7.60 (5H, m, Ar–H), 2.75
(2H, t, J = 7.2 Hz, –CH2CH2CH3), 1.74 (2H, m, –CH2CH2CH3), 0.95 (3H, t,
J = 7.5 Hz, –CH3); IR (KBr) cm-1: 3099, 1512, 1088; MS m/z 305 (M ? 1), Anal.
Calcd. for C18H16N4O: C, 71.04; H, 5.30; N, 18.41; Found: C, 70.94; H, 5.12; N,
18.28.
6-(3-Butyl-4H-1,2,4-triazol-4-yl)-2-phenylbenzo[d]oxazole (3e)
1
Yield: 44%; m.p. = 108–111°C dec; H-NMR (d ppm, CDCl3): 8.27–8.30 (3H, m,
–CH– and Ar–H), 7.90 (1H, d, J = 8.2 Hz, 4-H), 7.26–7.58 (5H, m, Ar–H), 2.87
(2H, t, J = 7.2 Hz, –CH2(CH2)2CH3), 1.30–1.74 (4H, m, –CH2(CH2)2CH3), 0.85
(3H, t, J = 7.5 Hz, –CH3); IR (KBr) cm-1: 3079, 1498, 1166; MS m/z 319
(M ? 1), Anal. Calcd. for C19H18N4O: C, 71.68; H, 5.70; N, 17.60; Found: C,
71.44; H, 5.57; N, 17.46.
6-(3-Pentyl-4H-1,2,4-triazol-4-yl)-2-phenylbenzo[d]oxazole (3f)
1
Yield: 45%; m.p. = 152–155°C dec; H-NMR (d ppm, CDCl3): 8.27–8.31 (3H, m,
–CH– and Ar–H), 7.91 (1H, d, J = 8.1 Hz, 4-H), 7.28–7.62 (5H, m, Ar–H), 2.76
(2H, t, J = 7.4 Hz, –CH2(CH2)3CH3), 1.28–1.75 (6H, m, –CH2(CH2)3CH3), 0.84
(3H, t, J = 7.4 Hz, –CH3); IR (KBr) cm-1: 3121, 1500, 1066; MS m/z 333
(M ? 1), Anal. Calcd. for C20H20N4O: C, 72.27; H, 6.06; N, 16.86; Found: C,
72.01; H, 5.99; N, 16.66.

Med Chem Res (2010) 19:925–935
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6-(3-Nonyl-4H-1,2,4-triazol-4-yl)-2-phenylbenzo[d]oxazole (3g)
1
Yield: 45%; m.p. = 70–71°C dec; H-NMR (d ppm, CDCl3): 8.27–8.31 (3H, m,
–CH– and Ar–H), 7.90 (1H, d, J = 8.0 Hz, 4-H), 7.28–7.62 (5H, m, Ar–H), 2.76
(2H, t, J = 7.4 Hz, –CH2(CH2)7CH3), 1.20–1.74 (14H, m, –CH2(CH2)7CH3), 0.84
(3H, t, J = 7.2 Hz, –CH3); IR (KBr) cm-1: 3096, 1501, 1055; MS m/z 389
(M ? 1), Anal. Calcd. for C24H28N4O: C, 74.20; H, 7.26; N, 14.42; Found: C,
74.01; H, 7.34; N, 14.18.
2-Phenyl-6-(3-phenyl-4H-1,2,4-triazol-4-yl)benzo[d]oxazole (3h)
Yield: 45%; m.p. = 159–164°C dec; 1H-NMR (d ppm, CDCl3): 8.54 (2H, s, Ar–H),
8.28 (2H, d, J = 8.5 Hz, Ar–H), 7.92 (1H, d, J = 7.2 Hz, Ar–H), 7.41–7.67 (9H, m,
Ar–H); IR (KBr) cm-1: 3002, 1503, 1056; MS m/z 339 (M ? 1), Anal. Calcd. for
C21H14N4O: C, 74.54; H, 4.17; N, 16.56; Found: C, 74.34; H, 4.31; N, 16.75.
6-(3-(3-Fluorophenyl)-4H-1,2,4-triazol-4-yl)-2-phenylbenzo[d]oxazole (3i)
Yield: 46%; m.p. = 159–164°C dec; 1H-NMR (d ppm, CDCl3): 8.42 (1H, s, –CH),
8.27 (2H, d, J = 7.6 Hz, Ar–H), 7.88 (1H, d, J = 8.4 Hz, Ar–H), 7.12–7.90 (9H, m,
Ar–H); IR (KBr) cm-1: 3089, 1499, 1107; MS m/z 357 (M ? 1), Anal. Calcd. for
C21H13FN4O: C, 70.78; H, 3.68; N, 15.72; Found: C, 70.52; H, 3.49; N, 15.49.
6-(3-(4-Fluorophenyl)-4H-1,2,4-triazol-4-yl)-2-phenylbenzo[d]oxazole (3j)
1
Yield: 57%; mp. = 90–93°C dec; H-NMR (d ppm, CDCl3): 8.42 (1H, s, –CH),
8.27 (2H, d, J = 7.6 Hz, Ar–H), 7.88 (1H, d, J = 8.4 Hz, Ar–H), 7.12–7.90 (9H, m,
Ar–H); IR (KBr) cm-1: 3089, 1498, 1100; MS m/z 357 (M ? 1), Anal. Calcd. for
C21H13FN4O: C, 70.78; H, 3.68; N, 15.72; Found: C, 70.57; H, 3.76; N, 15.53.
6-(3-(2-Chlorophenyl)-4H-1,2,4-triazol-4-yl)-2-phenylbenzo[d]oxazole (3k)
Yield: 57%; m.p. = 152–155°C; 1H-NMR (d ppm, CDCl3): 8.44 (1H, s, –CH), 8.27
(2H, d, J = 7.8 Hz, Ar–H), 7.75 (1H, d, J = 7.8 Hz, Ar–H), 7.27–7.66 (9H, m, Ar–
H); IR (KBr) cm-1: 3111, 1488, 1176; MS m/z 373 (M ? 1), Anal. Calcd. for
C21H13ClN4O: C, 67.66; H, 3.51; N, 15.03; Found: C, 67.44; H, 3.29; N, 1533.
6-(3-(3-Chlorophenyl)-4H-1,2,4-triazol-4-yl)-2-phenylbenzo[d]oxazole (3l)
Yield: 56%; m.p. = 182–185°C; 1H-NMR (d ppm, CDCl3): 8.42 (1H, s, –CH), 8.27
(2H, d, J = 7.5 Hz, Ar–H), 7.88 (1H, d, J = 8.4 Hz, Ar–H), 7.25–7.63 (9H, m, Ar–
H); IR (KBr) cm-1: 3121, 1518, 1076; MS m/z 373 (M ? 1), Anal. Calcd. for
C21H13ClN4O: C, 67.66; H, 3.51; N, 15.03; Found: C, 67.35; H, 3.35; N, 1521.

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Med Chem Res (2010) 19:925–935
6-(3-(4-Chlorophenyl)-4H-1,2,4-triazol-4-yl)-2-phenylbenzo[d]oxazole (3m)
Yield: 64%; m.p. = 213–216°C; 1H-NMR (d ppm, CDCl3): 8.42 (1H, s, –CH), 8.27
(2H, d, J = 7.5 Hz, Ar–H), 7.88 (1H, d, J = 8.4 Hz, Ar–H), 7.25–7.63 (9H, m, Ar–
H); IR (KBr) cm-1: 3098, 1498, 1176; MS m/z 373 (M ? 1), Anal. Calcd. for
C21H13ClN4O: C, 67.66; H, 3.51; N, 15.03; Found: C, 67.48; H, 3.37; N, 1531.
6-(3-(3-Methoxyphenyl)-4H-1,2,4-triazol-4-yl)-2-phenylbenzo[d]oxazole (3n)
Yield: 66%; m.p. = 165–167°C; 1H-NMR (d ppm, CDCl3): 8.41 (1H, s, –CH), 8.27
(2H, d, J = 7.8 Hz, Ar–H), 6.91–7.88 (10H, m, Ar–H), 3.74 (3H, s, –OCH3); IR
(KBr) cm-1: 3121, 1514, 1042; MS m/z 369 (M ? 1), Anal. Calcd. for C22H16N4O2:
C, 71.73; H, 4.38; N, 15.21; Found: C, 71.45; H, 4.56; N, 15.54.
6-(3-(4-Methoxyphenyl)-4H-1,2,4-triazol-4-yl)-2-phenylbenzo[d]oxazole (3o)
Yield: 69%; m.p. = 247°C dec; 1H-NMR (d ppm, CDCl3): 8.38 (1H, s, –CH), 8.26
(2H, d, J = 7.8 Hz, Ar–H), 6.83–7.88 (10H, m, Ar–H), 3.83 (3H, s, –OCH3); IR
(KBr) cm-1: 3126, 1545, 1004; MS m/z 369 (M ? 1), Anal. Calcd. for C22H16N4O2:
C, 71.73; H, 4.38; N, 15.21; Found: C, 71.45; H, 4.55; N, 15.52.
2-Phenyl-6-(3-p-tolyl-4H-1,2,4-triazol-4-yl)benzo[d]oxazole (3p)
Yield: 69%; m.p. = 147–150°C; 1H-NMR (d ppm, CDCl3): 8.55 (1H, s, –CH), 8.28
(2H, d d, J = 7.5 Hz, J = 1.5 Hz, Ar–H), 7.40–7.93 (10H, m, Ar–H), 1.73 (3H, s,
CH3); IR (KBr) cm-1: 3111, 1518, 1076; MS m/z 353 (M ? 1), Anal. Calcd. for
C22H16N4O: C, 74.98; H, 4.58; N, 15.90; Found: C, 74.87; H, 4.74; N, 15.79.
6-(3-(4-Nitrophenyl)-4H-1,2,4-triazol-4-yl)-2-phenylbenzo[d]oxazole (3q)
Yield: 66%; m.p. = 262°C dec; 1H-NMR (d ppm, CDCl3): 8.55 (1H, s, –CH), 8.27
(2H, d, J = 7.6 Hz, Ar–H), 7.85 (1H, d, J = 8.1 Hz, Ar–H), 7.28–7.59 (8H, m, Ar–
H), 6.58 (2H, d, J = 8.5 Hz, Ar–H); IR (KBr) cm-1: 3091, 1519, 1111; MS m/z 384
(M ? 1), Anal. Calcd. for C21H13N5O3: C, 65.79; H, 3.42; N, 18.27; Found: C,
65.98; H, 3.68; N, 18.52.
Pharmacology
The evaluation of the anticonvulsant activity followed the Anticonvulsant Drug
Development (ADD) Program of the National Institutes of Health, following
previously described testing procedures (USA). Adult male and female KunMing
mice (18–25 g) were used as experimental animals. Animals of the same age and
weight were selected in order to minimize biological variability. The animals were
maintained on a 12 h light/dark cycle and allowed free access to food and water,
except during the time they were removed from their cages for testing. The test

Med Chem Res (2010) 19:925–935
931
substance was administered in 30% polyethylene glycol 400 (PEG-400) and 10%
water. The drugs were administered intraperitoneally (i.p.) in mice in a volume of
0.05 ml/20 g body weight.
MES screening
Seizures were elicited with a 60-Hz alternating current of 50-mA intensity in mice.
The current was applied via corneal electrodes for 0.2 s. Protection against the
spread of MES-induced seizures was defined as the abolition of the hind leg and
tonic maximal extension component of the seizure. At 30 min after the adminis-
tration of the compounds, the activities were evaluated in MES test.
Pentylenetetrazole-induced seizure
At 30 min after the administration of the compounds, the Sc dose of pentylenetet-
razole (85 mg/kg) at which 100% of the animals showed clonic seizure was
determined by a dose–percentage effect curve. This dose was then administered to
animals 30 min after different treatments. The doses were calculated which
prevented 50% of the treated animals from clonic convulsions (ED50).
Rotarod test
At 30 min after administration of the compounds, animals were tested on a 1-inch-
diameter, knurled plastic rod rotating at 6 rpm for 1 min. Neurotoxicity was
indicated by inability of an animal to maintain equilibrium in each of three trials.
Results and discussion
Chemistry
Target compounds 3a–q were synthesized according to Scheme 1. Compound 1 was
prepared using 2-amino-5-nitrophenol as starting material, which reacted further
with benzoic acid for 8 h according to the reference method (Sener et al., 2000;
Pinar et al., 2004; Temiz et al., 2005). Compound 1 was reduced by iron powder in
hydrochloride condition to product 2 (Piao et al., 2008). Compounds 3a–q were
obtained through the condensation reaction according to the reference (Stocks et al.,
2004).
Pharmacology
Initial anticonvulsant evaluation of the 6-(3-substituted-4H-1,2,4-triazol-4-yl)-2-
phenylbenzo[d]oxazoles was undertaken by following the Anticonvulsant Drug
Development (ADD) program protocol (Krall et al., 1978; Poter et al., 1984). The
profile of anticonvulsant activity was established after i.p. injections by electrical
and chemical tests. The electrical test employed was the MES pattern test. The

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N
NH2
OH
N
O
Fe/HCl
Ph COOH
Ph
Ph
O
H2N
PPA
O2N
O2N
2
1
OCH3
OCH3
RCONHNH2
N
O
R
N
Ph
N
N
N
3a-3q
Scheme 1 Synthesis of compounds 3a–q
Table 1 Phase I: quantitative anticonvulsant data in mice (test drug administered i.p.)
N
R
Ph
O
N
N
N
3a-3q
Comp.
R
MES (100 mg/kg)
Sc-PTZ (100 mg/kg)
Toxicity (300 mg/kg)
0.5 h
4 h
0.5 h
4 h
0.5 h
4 h
3a
3b
3c
3d
3e
3f
–H
5/5
5/5
3/5
2/5
1/5
0/5
0/5
5/5
3/5
2/5
0/5
2/5
1/5
5/5
2/5
2/5
0/5
5/5
5/5
1/5
0/5
0/5
0/5
0/5
2/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
3/5
3/5
2/5
3/5
1/5
0/5
1/5
1/5
0/5
0/5
1/5
2/5
0/5
0/5
0/5
1/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
1/5
5/5
2/5
1/5
0/5
1/5
5/5
4/5
0/5
0/5
0/5
5/5
0/5
3/5
0/5
5/5
4/5
0/5
3/5
0/5
0/5
0/5
1/5
5/5
1/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
3/5
3/5
–CH3
–C2H5
n-C3H7
n-C4H9
n-C5H11
n-C9H19
–Ph
3g
3h
3i
–Ph(3-F)
–Ph(4-F)
–Ph(2-Cl)
–Ph(3-Cl)
–Ph(4-Cl)
–Ph(3-OCH3)
–Ph(4-OCH3)
–Ph(4-CH3)
–Ph(4-NO2)
3j
3k
3l
3m
3n
3o
3p
3q
chemical test employed was the Sc-PTZ seizure threshold test. The acute
neurological toxicity was determined in the rotorod test (Sun et al., 2006). The
activity and toxicity profile for the tested compounds are summarized in Table 1

Med Chem Res (2010) 19:925–935
933
along with literature data for standard drugs. All the compounds were active in the
MES test, indicative of their ability to prevent seizure spread.
The results of preliminary (phase I) screening of 3a–q are summarized in
Table 1. At 0.5 h time period after drug administration, compounds that showed
100% protection at 100 mg/kg dose of the tested mice were 3a, 3b, 3h, and 3n.
Compounds that exhibited anti-MES activity at both the 0.5 and 4 h time periods
include 3a and 3b. All of the compounds inordinately exhibited anticonvulsant
activity in both the MES and the sc-PTZ screenings. In the acute neurological
toxicity screen, compounds 3e, 3i, 3j, 3l, 3n, and 3p showed no neurotoxicity at the
maximum dose administered (300 mg/kg).
As a result of preliminary screening, compounds 3a, 3b, 3h, and 3n were then
subjected to phase II trials for quantification of their anticonvulsant activity and
neurotoxicity in mice. This phase provides an evaluation of the ED50 and TD50
values. The 95% confidence interval, slope of the regression line, and standard error
(SE) of the slope were then calculated. These data are shown in Table 2, which also
includes comparisons with the marketed antiepileptic drug carbamazepine. No
compounds exhibited better anticonvulsant activity than the reference drug
carbamazepine. Among the synthesized compounds, compound 6-(3-methyl-4H-
1,2,4-triazol-4-yl)-2-phenylbenzo[d]oxazole (3b) exhibited the strongest activity,
with ED50 = 26.5 mg/kg, but it possessed stronger neurotoxicity, with
TD50 = 167 mg/kg and PI = 6.3. Compared with compound 3b, compound 2-
phenyl-6-(4H-1,2,4-triazol-4-yl)-benzo [d]oxazole (3a) could be considered the
potentially most useful and safe therapeutic compound, with ED50 = 29.6 mg/kg,
TD50 = 285 mg/kg, and PI = 9.7, Its neurotoxicity was the lowest among all the
synthesized compounds and was also markedly lower than that of the reference drug
carbamazepine with PI value of 6.4.
Analyzing the activities of the synthesized compounds 3a–q, the following
structure–activity relationship (SAR) was gained. The length of the alkyl chain
Table 2 Phase II: anticonvulsant and toxicity data in mice (i.p.)a
Compound
Numbers
EDb50
ED50
TDc50
PI (TD50/ED50
)
(MES)
(Sc-PTZ)
MES
Sc-PTZ
3a
29.5
26.5
65.4
66.5
11.8
69.8
75.6
285
167
266
249
76.1
9.7
6.3
4.1
3.7
6.4
4.1
2.3
–
3b
d
–
3h
3n
–
–
Carbamazepine
[100
\1.3
a
All tested compounds were dissolved in polyethylene glycol-400
Dose measured in mg/kg
b
c
Minimal neurotoxicity was determined by the rotarod test 30 min after test compounds were
administrated
d
Not tested

934
Med Chem Res (2010) 19:925–935
appeared to have a direct impact on anticonvulsant activity of the 6-(3-alkyl-4H-
1,2,4-triazol-4-yl)-2-phenylbenzo[d]oxazoles derivatives. From compound 3a to 3g,
as the alkyl chain length increased, anticonvulsant activity gradually decreased, with
compound 3a being the most active. The pharmacology results revealed that the
position of the substituent on the phenyl ring greatly influenced the anticonvulsant
activity in 6-(2-aryl-4H-1,2,4-triazol-4-yl)-2-phenylbenzo[d]oxazoles derivatives
3i–q; for example, the activity order of the position of F atom on the phenyl ring
was 3-F [ 4-F; the activity order of the position of Cl atom on the phenyl ring was
3-Cl [ 4-Cl [ 2-Cl. Among the six p-substituted phenyl derivatives, the potency
order was H [ F = CH3O = CH3 [ Cl [ NO2.
Conclusions
A series of 6-(3-substituted-4H-1,2,4-triazol-4-yl)-2-phenylbenzo[d]oxazoles were
synthesized. The anticonvulsant effect and the neurotoxicity of the compounds were
calculated with MES test, Sc-PTZ, and rotarod tests with intraperitoneally injected
mice. The results showed that the series of derivatives exhibit different anticon-
vulsant activities. No compounds exhibited better anticonvulsant activity than the
reference drug carbamazepine. Among the synthesized compounds, compound 6-(3-
methyl-4H-1,2,4-triazol-4-yl)-2-phenylbenzo[d]oxazole (3b) exhibited the strongest
activity with ED50 = 26.5 mg/kg, but it possessed stronger neurotoxicity, with
TD50 = 167 mg/kg and PI = 6.3. Compared with compound 3b, compound 2-
phenyl-6-(4H-1,2,4-triazol-4-yl)benzo[d]oxazole (3a) could be considered the
potentially most useful and safe therapeutic compound, with ED50 = 29.6 mg/kg,
TD50 = 285 mg/kg, and PI = 9.7. Its neurotoxicity was the lowest among all the
synthesized compounds and was also markedly lower than that of the reference drug
carbamazepine with PI value of 6.4.
Acknowledgment This work was supported by the National Natural Science Foundation of China (Nos.
30760290 and 30860340) and Important Item Foundation of Ministry of Education P.R. China (No.
20070422029).
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