Design, synthesis and evaluation of 5-substituted 1-H-tetrazoles as potent anticonvulsant agents

Article abstract of DOI:10.1007/s12272-016-0881-y

Abstract: A series of 5-substituted 1-H-tetrazoles were designed and synthesized as potent anticonvulsant agents. Their preliminary anticonvulsant activities were evaluated using maximal electroshock and subcutaneous pentylenetetrazole (scPTZ) seizure tests. Neurotoxicity was determined using rotarod test. The results indicated that the compound 2j in scPTZ model exhibited the ED₅₀ values of 83.3?mg/kg, superior to the standard drug ethosuximide with the maximum activity. In addition, compound 2k showed the most potent activity in MES model with ED₅₀ value of 9.6?mg/kg and TD₅₀ value of 189.5?mg/kg after intraperitoneal injection in mice, and displayed a high protective index (TD₅₀/ED₅₀) of 19.7 compared to reference antiepileptic drugs. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s12272-016-0881-y

Arch. Pharm. Res.
DOI 10.1007/s12272-016-0881-y
RESEARCH ARTICLE
Design, synthesis and evaluation of 5-substituted 1-H-tetrazoles
as potent anticonvulsant agents
Ai-Mei Liao1,4 Tiantian Wang2,3 Bangrong Cai1 Yi Jin2,3 Seunghoon Cheon1
•
•
•
•
•
ChangJu Chun1 Zengtao Wang2
•
Received: 4 July 2016 / Accepted: 21 December 2016
Ó The Pharmaceutical Society of Korea 2016
Abstract A series of 5-substituted 1-H-tetrazoles were
designed and synthesized as potent anticonvulsant agents.
Their preliminary anticonvulsant activities were evalu-
ated using maximal electroshock and subcutaneous
pentylenetetrazole (scPTZ) seizure tests. Neurotoxicity was
determined using rotarod test. The results indicated that the
compound 2j in scPTZ model exhibited the ED50 values of
83.3 mg/kg, superior to the standard drug ethosuximide
with the maximum activity. In addition, compound 2k
showed the most potent activity in MES model with ED50
value of 9.6 mg/kg and TD50 value of 189.5 mg/kg after
intraperitoneal injection in mice, and displayed a high
protective index (TD50/ED50) of 19.7 compared to refer-
ence antiepileptic drugs.
Graphical Abstract
Keywords Synthesis Á 5-Substituted 1-H-tetrazoles Á
Anticonvulsant
3
The National Pharmaceutical Engineering Center for Solid
Preparation in Chinese Herbal Medicine, Nanchang 330006,
China
& ChangJu Chun
cchun1130@jnu.ac.kr
& Zengtao Wang
zengtaowang@126.com
4
School of life Science, Hefei Normal University,
Hefei 230601, China
1
Research Institute of Drug Development, College of
Pharmacy, Chonnam National University, 77 Yongbong-ro,
Buk-Gu, Gwangju 61186, Republic of Korea
2
Department of Medicinal Chemistry, College of Pharmacy,
JiangXi University of Traditional Chinese Medicine,
Nanchang 330004, China
123

A.-M. Liao et al.
characteristics of existing clinically effective AEDs and
typical anticonvulsant compounds. Accordingly, this
method has been applied mainly for introducing structural
modifications into existing AEDs to obtain more effica-
cious drugs that can suppress seizures and/or drugs with
minimal or no adverse effects compared to the parental
compounds.
Introduction
Epilepsy defined as a brain disorder is characterized by a
long-term predisposition to epileptic seizures and has
neurobiological, cognitive, psychological, and social con-
sequences (Eadie 1984). As a major neurological disorder,
epilepsy is an emerging medical and socioeconomic threat
that affects more than 50 million people, approximately 1
and 2% of the world’s population. The number is steadily
increasing as the modern lifestyle is faster and more
stressful (Bialer and White 2010). Currently available
antiepileptic drugs (AEDs), the only medical choice of
epilepsy, effectively reduce the severity and number of
seizures in only 60% of patients. Around 40% of patients
suffer from types of seizure which can’t be adequately
controlled by AEDs even are resistant to them, leading to
lower quality of life and higher death ratio (Kwan and
Brodie 2000; French 2007; Hirsch and Arzimanoglou
2004). Moreover, the recent antiepileptic therapy is often
limited by severe adverse effects and occupying with other
dose related toxicities (Greenwood 2000). Consequently,
safe and effective drugs to protect and treat epilepsy are in
great urgent demand.
In previous decades, many approaches have been
employed to identify the structural features that are
essential for anticonvulsant activity in various compounds.
As a result, it was well established that the crucial core
fragment of anticonvulsant agents contains an aromatic
functional group joined to the heterocyclic ring system,
which is considered as ‘drug-like’. This common template
is present in the structures of AEDs such as rufinamide,
lamotrigine, loreclezole, and nafimidone (Fig. 1).
Based on these observations, in order to develop new
and therapeutic anticonvulsants, we designed and synthe-
sized a series of 5-substituted 1-H-tetrazoles based on the
structural characteristics analysis of current AEDs, and
evaluated their anticonvulsant activities in comparison to
those of phenytoin, phenobarbital, and ethosuximide as
reference drugs.
Studies have greatly expanded the discovery of
antiepileptic drugs from screening processes to develop-
ment of drugs. However, design of new compounds with
medical activity is a major challenge in pharmaceutical
industry. Furthermore, due to the incomplete and uncertain
pathogenesis of human epilepsy, also the complex and
variable acting mechanism of AEDs, it is more difficult to
develop new antiepileptic drugs using rational method-
ologies (Barkmeier and Loeb 2009). Molecular modifica-
tion is considered to be an effective source for new
biological active agents (Kraus 1983). Thus, an important
and most useful strategy for developing new anticonvul-
sants is the ligand-based approach that establishes and
utilizes various pharmacophores by analyzing structural
Materials and methods
Chemistry
Melting points were determined in open capillary tubes and
were uncorrected. Infrared spectra (vs = very strong,
s = strong, m = medium, w = weak) were measured on a
PerkinElmer Fourier transform infrared spectroscopy
instrument (Waltham, MA, USA) over a wavelength range
of 4000–450 cm-1, where m = stretching vibrations and
1
d = bending vibrations. H NMR spectra were measured
on
a Bruker Avance 600 MHz NMR spectrometer
Fig. 1 Reported and designed nitrogen hetero atomic system as anticonvulsants
123

Design, synthesis and evaluation of 5-substituted 1-H-tetrazoles as potent anticonvulsant…
(Billerica, MA, USA), with all chemical shifts given in
ppm relative to tetramethylsilane. Chemical shift values are
in hertz. Splitting patterns are indicated as s, singlet; d,
doublet; t, triplet; m, multiplet; dd, doublet of doublets; dt,
doublet of triplets for 1H-NMR data. Thin layer chro-
matography (TLC) was performed using Merck silica gel
GF254 aluminum plates (Darmstadt, Germany). Reagents
were purchased from Xiya Reagent Co., Ltd. (Chengdou,
China).
3004 (w, m(C–H)), 2917 (w, m(CH3)), 2860 (w, m(CH3)),
1900–1600 (w, overtones d(C–H, Ph)), 1575 (s, d(N–H)),
1
1514 (s, m(C=C)), 813 (s, d(C–H, Ph)) cm-1; H NMR
(DMSO-d6, 600 MHz) d: 7.13–7.16 (m, 4H, ArH), 4.23 (s,
2H, CH2), 2.27 (s, 3H, CH3). ESI-HRMS calcd for
C9H10N4 ([M?H]?): 175.0905; found: 175.0980.
5-(4-(Trifluoromethyl)benzyl)-1-H-tetrazole (2d)
White solid (yield 68.9%); mp: 174–176 °C; FT-IR:
3100–2300 (w, m(N–H)), 3008 (w, m(C–H)), 2956 (w,
m(CH2)), 1900–1600 (w, overtones d(C–H, Ph)), 1623 (s,
m(C=C)), 1576 (s, d(N–H)), 1325 (vs, m(C–F)), 844 (s, d(C–
General procedure for preparation of 5-substituted
1-H-tetrazoles (2a–2m)
Nitriles (1 mmol) were added to a solution of NaN3
(3 mmol), PhMe (50 mL), and Et3NÁHCl (3 mmol) in a
round-bottomed flask. The reaction mixture was stirred at
110 °C. After completion of the reaction (as indicated by
TLC), the product was cooled and extracted with water.
Next, 36% HCl was added dropwise to the aqueous layer to
precipitate the tetrazoles. After filtration, the solid was
dried under reduced pressure and recrystallized from
EtOAc/Et2O to yield the 5-substituted-1-H-tetrazoles.
1
H, Ph)) cm-1; H NMR (DMSO-d6, 600 MHz) d: 7.72 (d,
1H, ArH, J = 8.4 Hz), 7.52 (d, 1H, ArH, J = 8.4 Hz), 4.42
(s, 2H, CH2). ESI-HRMS calcd for C9H7F3N4 ([M?H]?):
229.0623; found: 229.0697.
5-(4-Fluorobenzyl)-1-H-tetrazole (2e)
White solid (yield 53%); mp: 147–150 °C; FT-IR:
3100–2300 (w, m(N–H)), 3007 (w, m(C–H)), 2971 (w,
m(CH2)), 1900–1600 (w, overtones d(C–H, Ph)), 1601 (s,
m(C=C)), 1580 (s, d(N–H)), 1508 (s, m(C=C)), 1224 (vs,
5-Benzyl-1-H-tetrazole (2a)
1
White solid (yield 56%); mp: 123–125 °C (123–124 °C
(Cantillo et al. 2011)); FT-IR: 3200–2400 (w, m(N–H)),
3103 (w, m(C–H)), 2951 (w, m(CH2)), 1900–1600 (w,
overtones d(C–H, Ph)), 1603 (s, m(C=C)), 1531 (s, d(N–
H)), 1493 (s, m(C=C)), 1458 (s, d(CH2)), 733 (s, d(C–H,
m(C–F)), 833 (s, d(C–H, Ph)) cm-1; H NMR (DMSO-d6,
600 MHz) d: 7.31–7.33 (m, 2H, ArH), 7.16–7.19 (m, 2H,
ArH), 4.29 (s, 2H, CH2). ESI-HRMS calcd for C8H7FN4
([M?H]?): 179.0655; found: 179.0731.
Ph)), 695 (s, d(C–H, Ph)) cm-1
;
1H NMR (DMSO-d6,
5-(3-Nitrobenzyl)-1-H-tetrazole (2f)
600 MHz) d: 7.33–7.36 (m, 2H, ArH), 7.26–7.29 (m, 2H,
ArH), 4.29 (s, 2H, CH2). ESI-HRMS calcd for C8H8N4
([M?H]?): 161.0749; found: 161.0823.
White solid (yield 61%); mp: 159–161 °C; FT-IR:
3100–2400 (w, m(N–H)), 3007 (w, m(C–H)), 2955 (w,
m(CH2)), 1900–1600 (w, overtones d(C–H, Ph)), 1573 (s,
d(N–H)), 1520 (s, m(C=C)), 1351 (s, m(NO2)), 775 (s, d(C–
H, Ph)), 727 (s, d(C–NO2)), 697 (s, d(C–H, Ph)); 1H-NMR
(DMSO-d6, 600 MHz) d: 8.23 (s, 1H, ArH), 8.16 (d, 1H,
ArH, J = 7.8 Hz), 7.77 (d, 1H, ArH, J = 7.8 Hz), 7.66 (t,
1H, ArH, J = 7.8 Hz), 4.49 (s, 2H, CH2). ESI-HRMS calcd
for C8H7N5O2 ([M?H]?): 206.0600; found: 206.0668.
5-(4-Methoxybenzyl)-1-H-tetrazole (2b)
White solid (yield 43%); mp: 158–161 °C (156–158 °C
(Rama et al. 2011)); FT-IR: 3200–2300 (w, m(N–H)), 3015
(w, m(C–H)), 2970 (w, m(CH3)), 2954 (s, m(CH2)),
1900–1600 (w, overtones d(C–H, Ph)), 1612 (s, m(C=C)),
1555 (s, d(N–H)), 1513 (s, m(C=C)), 1463 (s, d(CH2)), 1246
(s, m(C–O, Ph)), 1026 (s, m(C–O, OCH3)), 835 (s, d(C–H,
Ph)) cm-1; 1H NMR (DMSO-d6, 600 MHz) d: 7.19 (d, 1H,
ArH, J = 8.4 Hz), 6.89 (d, 1H, ArH, J = 8.4 Hz), 4.20 (s,
2H, CH2), 3.72 (s, 3H, CH3). ESI-HRMS calcd for
C9H10N4O ([M?H]?): 191.0855; found: 191.0929.
5-(3-Methylbenzyl)-1-H-tetrazole (2g)
White solid (yield 63%); mp: 130–132 °C; FT-IR: 3120
(w, m(C–H)), 3100–2300 (w, m(N–H)), 2989 (w, m(CH3)),
2921 (s, m(CH2)), 1900–1600 (w, overtones d(C–H, Ph)),
1610 (s, m(C=C)), 157 7(s, d(N–H)), 1493 (s, m(C=C)),
1
758(s, d(C–H, Ph)), 688 (s, d(C–H, Ph)) cm-1; H-NMR
5-(4-Methylbenzyl)-1-H-tetrazole (2c)
(DMSO-d6, 600 MHz) d: 7.22 (t, 1H, ArH, J = 7.8 Hz),
7.08–7.09 (m, 2H, ArH), 7.05–7.06 (m, 1H, ArH), 4.24 (s,
2H, CH2), 2.28 (s, 3H, CH3). ESI-HRMS calcd for
C9H10N4 ([M?H]?): 175.0905; found: 175.0976.
White solid (yield 46%); mp: 158–160 °C (161 °C (Weh-
man and Popov 1966)); FT-IR: 3100–2300 (w, m(N–H)),
123

A.-M. Liao et al.
5-(2-Chlorobenzyl)-1-H-tetrazole (2h)
H)), 2953 (w, m(CH2)), 1900–1600 (w, overtones d(C–H,
Ph)), 1599 (s, m(C=C)), 1539 (s, d(N–H)), 1505 (s,
m(C=C)), 863 (s, d(C–H, Ph)), 813 (s, d(C–H, Ph)), 762 (s,
White solid (yield 62%); mp: 124–126 °C; FT-IR:
3000–2400 (w, m(N–H)), 2993 (w, m(CH2)), 1900–1600 (w,
overtones d(C–H, Ph)), 1581 (s, d(N–H)), 1492 (s,
m(C=C)), 1474 (s, m(C=C)), 755 (s, d(C–H, Ph)) cm-1; 1H-
NMR (DMSO-d6, 600 MHz) d: 7.47–7.50 (m, 1H, ArH),
7.39–7.42 (m, 1H, ArH), 7.34–7.38 (m, 2H, ArH), 4.39 (s,
2H, CH2). ESI-HRMS calcd for C8H7ClN4 ([M?H]?):
195.0359; found: 195.0433.
d(C-H, Ph)), cm-1 1H NMR (DMSO-d6, 600 MHz) d:
;
7.88–7.91 (m, 3H, ArH), 7.80 (s, 1H, ArH), 7.49–7.54 (m,
2H, ArH), 7.42 (dd, 1H, ArH, J = 1.8, 8.4 Hz), 4.47 (s, 2H,
CH2). ESI-HRMS calcd for C12H10N4 ([M?H]?):
211.0905; found: 211.0979.
5-Benzhydryl-1-H-tetrazole (2m)
White solid (yield 70%); mp: 166–168 °C (165–166 °C
(Gutmann et al. 2010)); FT-IR: 3100–2400 (w, m(N–H)),
3090 (w, m(C–H)), 3058 (w, m(C–H)), 3003 (w, m(C–H)),
2949 (w, m(CH2)), 1900–1600 (w, overtones d(C–H, Ph)),
1599 (s, m(C=C)), 1565 (s, d(N–H)), 1496 (s, m(C=C)), 1455
5-(2-Methylbenzyl)-1-H-tetrazole (2i)
White solid (yield 55%); mp: 156–158 °C; FT-IR:
3100–2100 (w, m(N–H)), 2962 (w, m(CH2)), 1900–1600 (w,
overtones d(C–H, Ph)), 1607 (s, m(C=C)), 1562 (s, d(N–
H)), 1485 (s, m(C=C)), 1463 (s, m(C=C)), 744 (s, d(C–H,
(s, d(CH2)), 743 (s, d(C–H, Ph)), 692 (s, d(C–H, Ph)) cm-1
;
1H NMR (DMSO-d6, 600 MHz) d: 7.34–7.37 (m, 4H, ArH),
7.27–7.31 (m, 6H, ArH), 5.96 (s, 1H, CH). ESI-HRMS calcd
for C14H12N4 ([M?H]?): 237.1062; found: 237.1131.
1
Ph)) cm-1; H NMR (DMSO-d6, 600 MHz) d: 7.13–7.21
(m, 4H, ArH), 4.26 (s, 2H, CH2), 2.27 (s, 3H, CH3). ESI-
HRMS calcd for C9H10N4 ([M?H]?): 175.0905; found:
175.0976.
Pharmacology
5-(2-Chlorophenyl)-1-H-tetrazole (2j)
Maximal electroshock seizure test (MES)
White solid (yield 58%); mp: 178–180 °C (176–177 °C
(Obushak et al. 2008); FT-IR: 3200–2300 (w, m(NH)), 3060
(w, m(C–H)), 2971 (w, m(CH2)), 1900–1600 (w, overtones
d(C–H, Ph)), 1601 (s, m(C=C)), 1562 (s, d(N–H)), 1469 (s,
The maximal electroshock seizure test was carried out
according to the standard protocol (Krall et al. 1978). Male
mice (Kunming, China), weighing 18–22 g, were used as
experimental animals purchased from the Laboratory of
Animal Research, JiangXi University of Traditional Chinese
Medicine (Nanchang, JiangXi Province, China). The ani-
mals were allowed to acclimatize with free access to food
and water for a 24 h period before testing except during the
experiment. Abolition of hind limb tonic extension spasm
was recorded as anticonvulsant activity. The test compounds
were dissolved in an aqueous solution of 50% polyethylene
glycol. In preliminary screening, each compound was
administered as an ip injection at three dose levels (30, 100,
300 mg/kg body mass) and the anticonvulsant activity
assessed after 0.5, 1, 2, and 4 h intervals of administration.
1
m(C=C)), 776 (s, m(C–Cl)), 745 (s, d(CH, Ph)) cm-1; H
NMR (DMSO-d6, 600 MHz) d: 7.47–7.50 (m, 1H, ArH),
7.39–7.42 (m, 1H, ArH), 7.34–7.38 (m, 2H, ArH), 4.39 (s,
2H, CH2). ESI-HRMS calcd for C7H5ClN4 ([M?H]?):
181.0203; found: 181.0277.
5-(o-Tolyl)-1-H-tetrazole (2k)
White solid (yield 52%); mp: 153–155 °C (155–156 °C
(Butler and Garvin 1981)). FT-IR: 3200–2300 (w, m(N–
H)), 3029 (w, m(C–H)), 2970 (w, m(CH3)), 1900–1600 (w,
overtones d(C–H, Ph)), 1607 (s, m(C=C)), 1562 (s, d(N–
H)), 1485 (s, m(C=C)), 1463 (s, d(CH3)), 744 (s, d(C–H,
Ph)) cm-1; 1H NMR (DMSO-d6, 600 MHz) d: 7.69 (d, 1H,
ArH, J = 7.8 Hz), 7.49 (td, 1H, ArH, J = 1.2, 7.2 Hz),
7.44 (d, 1H, ArH, J = 7.8 Hz), 7.40 (dt, 1H, ArH, J = 1.2,
7.2 Hz), 2.48 (s, 3H, CH3). ESI-HRMS calcd for C8H8N4
([M?H]?): 161.0749; found: 161.0825.
Subcutaneous pentylenetetrazole seizure test
(scPTZ)
This test involved treating the mice with metrazol
(pentylenetetrazole, 85 mg/kg in mice). This produced
clonic seizures lasting for a period of at least 5 s in 97% of
the animals tested. At the anticipated time of testing, the
convulsant was subcutaneously administered. The test
compounds were intraperitoneally administered in mice
and the animals were observed over a 30 min period. Mice
5-(Naphthalen-2-ylmethyl)-1-H-tetrazole (2l)
White solid (yield 66%); mp: 159–161 °C; FT-IR:
3200–2500 (w, m(N–H)), 3130 (w, m(C–H)), 3051 (w, m(C–
123

Design, synthesis and evaluation of 5-substituted 1-H-tetrazoles as potent anticonvulsant…
were tested 0.5 and 4 h after doses of 100 and 300 mg/kg
of the test compound were administered. The absence of
clonic spasms over the period of observation indicated the
compound’s ability to abolish the effect of pentylenete-
trazol on the seizure threshold.
Results and discussion
The synthetic strategy used to prepare the target com-
pounds 2a–2m is depicted in Scheme 1. Substituted ben-
zonitriles 1a–1m were reacted with sodium azide (NaN3) in
the presence of toluene (PhMe) and triethylamine
hydrochloride (Et3NÁHCl) at a refluxing temperature to
afford 5-substituted 1-H-tetrazoles (Lenda et al. 2005). All
products are known compounds and their structures were
established by FT-IR, 1H NMR, LC–MS and melting point
analysis and confirmed by comparing their physical and
spectra data with those in the literature. The physico-
chemical properties and structures of 2a–2m are summa-
rized in Table 1. Since lipophilicity is important feature of
central nervous system drugs, enabling them to pass the
blood brain barrier; the clog P values of the compounds
were calculated using ChemDrawUltra 12.0 program. As
shown in Table 1, clog P values of compound 2d (clog P:
2.85) is higher than those of other compounds (clog P:1.80
for compound 2b, clog P: 2.42 for compound 2c, clog P:
2.49 for 2h, and clog P: 2.04 for 2k).
Neurotoxicity screening
The rotarod test was used to evaluate neurotoxicity. The
mice were trained to stay on an accelerating rotarod that
rotated at 6 revolutions per minute. The rod diameter was
3.2 cm. Trained animals were given ip injection of the test
compounds in doses of 100 and 300 mg/kg. Neurotoxicity
was indicated by the inability of the animal to maintain
equilibrium on the rod for at least 1 min in each of the
three trials.
Quantification studies
The median effective (ED50) was calculated using the Bliss
method. The ED50 values were presented as the mean with
95% confidence intervals. Neurotoxicity was indicated by
the inability of the animal to maintain equilibrium on the
rod for at least 1 min in each of the three trials (Dunham
et al. 1957). Neurotoxicity was expressed as the median
toxic dose (TD50 in mg/kg) eliciting minimal neurological
toxicity in 50% of animals. The quantitative determination
of ED50 and TD50 values was performed at the previously
estimated time of peak effect (1 h) after ip injection into
mice. Groups of six animals received various doses of
compound 2k until at least three points were established in
the range of 10–90% seizure protection. The results are
shown in Table 3.
The preclinical discovery and development of a new
bioactive chemical entity for the treatment of epilepsy rely
mainly on the use of predictable animal models. The
obtained compounds 2a–2m were submitted to in vivo
evaluation using the methods described in the Antiepileptic
Drug Development Program of the National Institutes of
Health according to previously described testing proce-
dures (Krall et al. 1978; Porter et al. 1984). Generally,
primary studies of anticonvulsants in mice involve maxi-
mal electroshock (MES), and subcutaneous pentylenete-
trazole (scPTZ). MES and scPTZ are regarded as the ‘gold
standard’ of the measurement of new anticonvulsants. The
first test detects compounds possessing the therapeutic
activity for human generalized tonic–clonic seizures
induced by an electrical stimulus. The second test is cap-
able to recognize the agents raising the threshold of
chemically-induced generalized absence seizures (Farghaly
and El-Kashef 2005). In the study, antiepileptic property of
synthesized compounds was detected by these methods.
Further, their neurotoxicity profiles was primarily evalu-
ated by Rotarod screening. For the most promising
derivatives, ED50 and TD50 values were further
determined.
Calculation of drug-likeness and ADME properties
A computational study of titled compounds was performed
to predict ADME properties. Polar surface area (TPSA)
(Ertl et al. 2000), number of rotatable bonds, molecular
volume, number of hydrogen donor and acceptor atoms,
and violations of Lipinski’s rule of five (Lipinski et al.
2001) were calculated using the Molinspiration online
property calculation toolkit (Molinspiration Cheminfor-
matics 2010). Absorption (%ABS) was calculated as:
%ABS = 109 - (0.345 9 TPSA) (Zhao et al. 2002).
The test compounds were administered intraperitoneally
(ip) into mice at doses of 30, 100, and 300 mg/kg (at 0.5, 1,
Scheme 1 Synthetic protocol
of the target compounds 2a–2m
123

A.-M. Liao et al.
phenobarbital were selected as standard drugs for the MES
and minimal motor impairment test, whereas ethosuximide
was used for scPTZ screening. The screening results for
anticonvulsant activity and neurotoxocity studies are
summarized in Table 2.
Table 1 Physicochemical properties and structures of all synthesized
compounds 2a–2m
In this test, the activities of the synthesized compounds
in the MES screening in mice were highest for ortho-
substituted derivatives. 2-Chloro derivative 2h displayed
high protection at doses of 30 mg/kg for 1 h, but with
300 mg/kg for 0.5 h. The possible reason is that 2h with an
unfavorable lipophilicity enabled the longer time of peak
concentration. Replacement of the Cl of 2h with a CH3
group gave 2i, which, interestingly, showed protective
effect at a dose of 30 mg/kg with markedly slow absorption
at 4 h. A similar trend was observed in meta-substituted
compounds 2f and 2g, of which only the 3-CH3 derivative
2g revealed notable activity at a dose of 30 mg/kg at 4 h in
the MES test. Another nitro-derivative 2f was moderately
active at a dose of 100 mg/kg and 1 h of testing. Except for
2b and 2c at doses of 100 mg/kg (at 4 and 2 h post-ad-
ministration, respectively), the other para-substituted
compounds 2d and 2e were low or inactive in the prelim-
inary MES screening, which showed a similar potency as
unsubstituted derivative 2a. High hydrophobic naphthalene
derivative 2l showed moderate activity at a dose of
100 mg/kg (at 1 and 2 h post-administration), which had
lower activity than diphenylmethane derivative 2m (at a
dose of 30 mg/kg at 1 h post-administration).
Compounds
R
H
N
1
Structures
Clog Pa
1.93
2a
2b
2c
2d
4-OCH3
4-CH3
4-CF3
1
1
1
1.80
2.42
2.85
2e
2f
4-F
1
1
2.09
1.96
3-NO2
2g
2h
2i
3-CH3
2-Cl
1
1
1
2.42
2.49
2.42
Increased clog P values of compound 2d resulted in
decreased and lost MES activity, respectively, when com-
pared to compounds (2b and 2c) which are more active
with lower clog P values. Similar trend was observed when
comparison to 2h and 2k. The reason for this appearance
was the fact that increased lipophilicity does not neces-
sarily enhance anticonvulsant properties; conversely, it
may sometimes lead to reduced or lost activity. Therefore,
lipophilicity is not the only important parameter for anti-
convulsant ability, but some additional properties and
action mechanisms of the compounds also account for the
anticonvulsant activity.
2-CH3
2j
2-Cl
0
0
2.11
2.04
2k
2-CH3
2l
1
1
2.93
3.60
Preliminary structure–activity relationship (SAR) stud-
ies indicated that ortho-substituted derivatives 2h and 2i
displayed remarkable activity. Consequently, further
structural modifications of 2h and 2i are necessary. We
prepared compounds 2j and 2k with one less carbon atom
chain than that in 2h and 2i. Interestingly, these prepared
compounds showed significantly increased anticonvulsant
activity. Compound 2k showed the highest activity in this
series according to the MES screening results. Addition-
ally, 2j exhibited high activity in the scPTZ model at a dose
of 100 mg/kg at 0.5 and 4 h. Pentylenetetrazole destroys
the balance between glutamic acid and c-aminobutyric
acid, which are an excitatory neurotransmitter and an
2m
a
Calculated using Chemdraw Ultra 12.0 program (Cambridge
Software)
2, and 4 h) for MES screening, whereas 100 and 300 mg/
kg were used for scPTZ screening (at 0.5 and 4 h) and
neurotoxicity testing (at 0.5, 1, 2, and 4 h). Phenytoin and
123

Design, synthesis and evaluation of 5-substituted 1-H-tetrazoles as potent anticonvulsant…
Table 2 Anticonvulsant
Compound
Intraperitoneal injection in micea
MESb
activity and neurotoxocity of
compounds 2a–2m
scPTZc
0.5 h
NTd
administered intraperitoneally
to mice
0.5 h
1 h
2 h
4 h
4 h
0.5 h
1 h
2 h
4 h
2a
300
300
300
–
300
300
–
300
300
100
300
–
300
100
300
–
–
–
300
300
300
300
300
300
300
300
300
300
300
300
300
100
100
–
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
300
100
300
–
2b
300
300
300
–
300
–
2c
2d
300
–
300
–
2e
300
–
–
2f
100
100
30
–
300
30
100
30
300
300
–
–
–
2g
–
100
100
–
–
–
2h
300
–
–
–
2i
100
100
30
–
–
2j
100
30
–
100
–
100
–
2k
–
2l
300
300
30
100
30
100
100
–
–
2m
–
–
–
Phenytoin
Phenobarbital
Ethosuximide
a
30
30
–
–
–
100
–
30
100
300
300
–
–
–
–
Doses of 30, 100, and 300 mg/kg were administered in MES screening, whereas doses of 100 and
300 mg/kg were used in scPTZ and NT screening. The animals were examined at 0.5, 1, 2, and 4.0 h after
injection for MES and NT and 0.5 and 4.0 h for scPTZ. The dash (–) indicates the absence of activity at the
maximum dose administered (300 mg/kg)
b
Maximal electroshock test
c
Subcutaneous pentylenetetrazole test
d
Neurotoxicity screening (rotarod test)
Table 3 Quantitative anticonvulsant evaluation in mice (ip)
Compounds
TPE (h)a
ED50 (MES)b
ED50 (scPTZ)b
TD50 (NT)c
PId
2j
1
1
2
1
2
nd
83.3 (51.0–125.4)e
nd
[200
[2.4 (scPTZ)
19.7 (MES)
2k
9.6 (7.5–26.7)
9.5 (8.1–10.4)
21.8 (21.8–25.5)
–
189.5 (137.1–276.5)
65.5 (52.5–72.9)
69 (62.8–72.9)
[500
Phenytoin
Phenobarbital
Ethosuximide
[300
6.9 (MES)
13.2 (5.8–15.9)
167.0 (116.0–237.0)
3.2 (MES) 5.2 (scPTZ)
[3.0 (scPTZ)
a
Time to peak effect
b
ED50 median effective dose affording anticonvulsant protection in 50% of animals, the dose is measured in mg/kg
TD50 median toxic dose eliciting minimal neurological toxicity in 50% of animals, the dose is measured in mg/kg
c
d
e
PI protective index (TD50/ED50
)
95% confidence intervals given in parentheses
nd not determined
inhititory one, respectively, leading to the tonic convulsion
(Baughman and Gilbert 1980). Increase and accumulating
glutamic acid damages the hippocampus which is respon-
sible for sensation, cognition, learning and memory (Wang
et al. 2006). Therefore, these active compounds especially
2j and 2k may prevent CNS excitation or stress, since they
were demonstrated to protect seizures in scPTZ test (Nu-
groho et al. 2013).
Acute neurological toxicity of synthesized compounds
study was carried out by rotarod test (Sun et al. 2006). The
results indicated that all compounds showed neurotoxicity
at the highest dose (300 mg/kg) with a wide range of
safety. The toxicity of compound 2f is highest, which
maybe because of the presence of nitro group.
Based on the results of preliminary screening, 2j and 2k
were further examined in the second phase to quantify their
123

A.-M. Liao et al.
Table 4 Pharmacokinetic
parameters important for good
oral bioavailability of
semicarbazones compounds 2a–
2m
Compounds
MW
HBA
HBD
NVio
N-ROTB
Volume
TPSA
% ABS
Rule
2a
2b
2c
2d
2e
2f
\500
\10
4
\5
1
B1
0
–
2
3
2
3
2
3
2
2
2
1
1
2
3
–
–
–
160.18
190.21
174.21
228.18
178.17
205.18
174.21
194.62
174.21
180.60
160.18
210.24
236.28
144.77
170.32
161.33
176.07
149.70
168.10
161.33
158.31
161.33
141.50
144.53
188.76
216.21
54.47
63.70
54.47
54.47
54.47
100.29
54.47
54.47
54.47
54.47
54.47
54.47
54.47
90.21
87.02
90.21
90.21
90.21
74.40
90.21
90.21
90.21
90.21
90.21
90.21
90.21
5
1
0
4
1
0
4
1
0
4
1
0
7
1
0
2g
2h
2i
4
1
0
4
1
0
4
1
0
2j
4
1
0
2k
2l
4
1
0
4
1
0
2m
4
1
0
MW molecular weight, HBA number of hydrogen bond acceptors, HBD number of hydrogen bond donors,
nVio number of Lipinski’s violations, n-ROTB number of rotatable bonds, volume molecular volume, TPSA
topological polar surface area, %ABS percentage of absorption
anticonvulsant activity (indicated by ED50) and neurotox-
icity (indicated by TD50) in mice. Their protective index
(PI) fixed as the ratio of TD50–ED50 were calculated. For
compound 2j (ED50 = 83.3) in the scPTZ model, although
the ED50 was much lower than that of ethosuximide
(ED50 = 167.0), its PI was lower than that of the standard
due to the lower TD50 value for the drug. Compound 2k
showed an ED50 value of 9.6 mg/kg in the MES model and
a TD50 value of 189.5 mg/kg. Consequently, the PI value
of 2k (19.7) was higher than those of the reference drugs,
6.9 for phenytoin and 3.2 for phenobarbital (Table 3). 2k
was the most potent anticonvulsants of all tested com-
pounds in this study since the PI value is considered to be
an index representing the margin of safety and tolerability
between anticonvulsant doses and doses of anticonvulsant
drugs exerting acute adverse effects (Loscher and Nolting
1991).
Conclusion
In summary, we designed and synthesized a series of
5-substituted 1-H-tetrazoles as anticonvulsant agents. The
anticonvulsant activity and neurotoxicity of all synthesized
compounds were evaluated using MES- and scPTZ-in-
duced seizure testing and rotarod test. The SARs and
ADME predictions of these compounds were also covered.
The anticonvulsant potency of these compounds probably
attributed to the favorable structural combination of ortho-
substituted derivatives. In current study, the most potent
anticonvulsant agent in scPTZ screen was 2j with an ED50
value of 83.3 mg/kg, which is much lower than that of the
standard drug ethosuximide, showing maximum activity. In
addition, the compound 2k showed the highest activity in
MES model, with an ED50 value of 9.6 mg/kg and TD50
value of 189.5 mg/kg, after ip injection in mice, and dis-
played a higher PI value of 19.7 compared to reference
antiepileptic drugs. This research shed light on the useful
information for developing new anticonvulsant agents with
high activity and low toxicity and further investigation on
the precise mechanism of action of novel anticonvulsants
in future.
A computational study to predict the absorption, distri-
bution, metabolism and excretion (ADME) properties of the
compounds was performed. Topological polar surface area
(TPSA) is a descriptor that is strongly correlated with passive
molecular transport through membranes, and accordingly
predicts the transport properties of drugs in the intestines and
blood brain barrier crossing (Ertl et al. 2000). The percentage
of absorption (%ABS) was calculated using TPSA. Based on
these parameters, all titled compounds exhibited a high
%ABS ranging from 74.4 to 90.21% (Table 4). None of the
compounds violated Lipinski’s parameters, indicating that
they are promising agents for use in epilepsy therapy.
Acknowledgements This work was supported by the Science and
Technology Research Projects of Jiangxi Provincial Education
Department (No. GJJ150845), Traditional Chinese Medicine
Research Project of Jiangxi Provincial Health and Family Planning
Commission (No. 2016A035) and PhD Start-up Fund of Jiangxi
University of Traditional Chinese Medicine (No. 2014BS016).
123

Design, synthesis and evaluation of 5-substituted 1-H-tetrazoles as potent anticonvulsant…
gamma-aminobutyrate system. Pharmacol Res Commun
15:183–189
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