Synthesis and evaluation of anticonvulsant and antidepressant activities of 7-alkyl-7H-tetrazolo[1,5-g]purine derivatives

Article abstract of DOI:10.1007/s00044-014-1030-0

Seventeen 7-alkyl-7H-tetrazolo[1,5-g]purine derivatives were synthesized, and their anticonvulsant and antidepressant activities were evaluated in a mouse model. The anticonvulsant effect and neurotoxicity of the compounds were evaluated with a maximal electroshock test and a rotated test in mice, respectively. Most of the compounds had anticonvulsant activity; among the compounds studied, 7-(3-chlorobenzyl)-7H-tetrazolo[1,5-g]purine (3h) was found to be the most potent compound with a median effective dose (ED₅₀) value of 28.9 mg/kg and a protective index value of 15.8, possessing better anticonvulsant activity and higher safety than the marketed drug carbamazepine. To explain the possible mechanism of anticonvulsant activity, compound 3h was tested in pentylenetetrazole-induced seizures tests, and the results suggest that compound 3h exerts anticonvulsant activity through a GABA-mediated mechanism. Forced swimming test showed that at a dose of 40 mg/kg, five compounds have significant antidepressant activity, the most active compound was 7-(2-chlorobenzyl)-7H-tetrazolo[1,5-g]purine (3g), which decreased immobility time by 56 %.

Full text of DOI:10.1007/s00044-014-1030-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-1030-0
ORIGINAL RESEARCH
Synthesis and evaluation of anticonvulsant and antidepressant
activities of 7-alkyl-7H-tetrazolo[1,5-g]purine derivatives
•
Shi-Ben Wang Xian-Qing Deng Da-Chuan Liu
•
•
•
Hong-Jian Zhang Zhe-Shan Quan
Received: 7 December 2013 / Accepted: 7 May 2014
Ó Springer Science+Business Media New York 2014
Abstract Seventeen 7-alkyl-7H-tetrazolo[1,5-g]purine
derivatives were synthesized, and their anticonvulsant and
antidepressant activities were evaluated in a mouse model.
The anticonvulsant effect and neurotoxicity of the com-
pounds were evaluated with a maximal electroshock test
and a rotated test in mice, respectively. Most of the com-
pounds had anticonvulsant activity; among the compounds
studied, 7-(3-chlorobenzyl)-7H-tetrazolo[1,5-g]purine (3h)
was found to be the most potent compound with a median
effective dose (ED50) value of 28.9 mg/kg and a protective
index value of 15.8, possessing better anticonvulsant
activity and higher safety than the marketed drug car-
bamazepine. To explain the possible mechanism of anti-
convulsant activity, compound 3h was tested in
pentylenetetrazole-induced seizures tests, and the results
suggest that compound 3h exerts anticonvulsant activity
through a GABA-mediated mechanism. Forced swimming
test showed that at a dose of 40 mg/kg, five compounds
have significant antidepressant activity, the most active
compound was 7-(2-chlorobenzyl)-7H-tetrazolo[1,5-g]pur-
ine (3g), which decreased immobility time by 56 %.
Keywords Tetrazole ꢀ Anticonvulsant ꢀ Antidepressant ꢀ
Maximal electroshock test ꢀ Forced swimming test ꢀ
PTZ
Introduction
The purine nucleus is one of the most important and widely
exploited heterocyclic ring for the development of bioac-
tive molecules. The literature contains many examples of
the synthesis of purine derivatives, with these compounds
exhibiting a range of biological properties, including anti-
bacterial (Hirokawa et al., 2009; Braendvang et al., 2009),
antifungal (Bordon-Pallier et al., 2004), antimalarial
´
(Mallari et al., 2009), anticancer (Zhou, 2011; Lopez-Cara
´
et al., 2011; Conejo-Garcıa et al., 2011), and anti-inflam-
matory (Wang et al., 2010) activities.
The tetrazole nucleus is another heterocycle present in a
variety of therapeutically important agents, including those
with anticonvulsant (Sun et al., 2010a, b; Alhano et al.,
2006) and antidepressant activities (Deng et al., 2010;
Wang et al., 2009; Beghi et al., 2004). In our previous
study (Wang et al., 2009), we reported that 5-alkoxytet-
razolo[1,5-a]quinazolines (I) had anticonvulsant properties
(Fig. 1). We also found that most of these compounds had
significant antidepressant activities. The best of these,
5-(hexyloxy)tetrazolo[1,5-a]quinazoline and 5-(4-meth-
oxy-phenoxy)tetrazolo[1,5-a]quinazoline, decreased immo-
bility times by 62 and 52 %, respectively.
S.-B. Wang ꢀ D.-C. Liu ꢀ H.-J. Zhang ꢀ Z.-S. Quan (&)
College of Pharmacy, Yanbian University, No. 977,
Park Road, Yanji 133002, Jilin, China
Given these findings, we targeted a series of 7-alkyl-7H-
tetrazolo[1,5-g]purines (3a–3q) and examined their anti-
convulsant and antidepressant activities in a mouse model,
via the maximal electroshock seizure (MES) test and
forced swimming test (FST), respectively. Their structures
e-mail: zsquan@ybu.edu.cn
X.-Q. Deng
Medical College, Jinggangshan University, Ji’an 343009,
JiangXi, China
1
were characterized using IR, H NMR, MS, and 13C NMR
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Med Chem Res
obtained were sufficiently pure for use in the next step
without further purification.
OR
R
N
N
N
N
General procedure for synthesis of 7-alkyl-7H-
N
N
N
N
N
N
tetrazolo[1,5-g]purines (3a–3q)
N
N
3a-3q
l
The appropriate compound (2a–2q, 2.0 mmol) was dis-
solved in of 30 % HCl (20 mL), and the resulting mixture
cooled to 0 °C in an ice bath. A solution of NaNO2
(2.1 mmol) in water (5 mL) was slowly (dropwise) added,
with care taken to maintain the reaction temperature at
below 5 °C. The resulting mixture was then stirred at room
temperature for 2 h. After the reaction was completed
(TLC), the precipitate was filtered and washed with water
to give 7-alkyl-7H-tetrazolo[1,5-g]purines (3a–3q). The
yield, melting point, and spectral data of each compound
are given below.
Fig. 1 Structures of compounds I and target compounds 3a–3q
techniques. PTZ-induced seizure tests were performed on
the most active compound (3h) to determine the possible
mechanism of action.
Materials and methods
Chemistry
7-Hexyl-7H-tetrazolo[1,5-g]purine (3a)
Melting points were determined in open capillary tubes and
were uncorrected. IR spectra were recorded (in KBr) on
White solid; mp: 66–68 °C. IR (KBr) cm-1: 1656 (C=N).
1H NMR (CDCl3, 300 MHz) d: 0.86 (t, 3H, J = 6.79 Hz,
–CH3), 1.35–2.12 (m, 8H, –CH2–), 4.63 (t, 2H, J = 7.20 Hz,
–CH2–), 8.22 (s, 1H, Imidazole-H), 9.56 (s, 1H, Pyrimi-
dine-H). 13C NMR (CDCl3) d: 13.86 (C-11), 22.36 (C-10),
26.00 (C-9), 30.31 (C-8), 31.03 (C-7), 48.55 (C-6), 111.59
(C-1), 133.45 (C-5), 142.38 (C-4), 144.34 (C-2), 151.46
(C-3). MS-EI m/z 246 (M?1). Anal. Calcd. for C11H15N7:
C 53.86, H 6.16, N 39.97 %. Found: C 53.79, H 6.19, N
40.01 %.
1
IRPrestige-21. H NMR and 13C NMR spectra were mea-
sured on an AV-300(Bruker, Switzerland), and all chemical
shifts were given in ppm relative to tetramethysilane. Mass
spectra were measured on an HP1100LC (Agilent Tech-
nologies, USA). The major chemicals were purchased from
Aldrich Chemical Corporation.
General procedure for synthesis of 6-chloro-9-alkyl-9H-
purines (1a–1q)
6-Chloro-9H-purine (2 g, 12.9 mmol), K2CO3 (2.2 g,
14.2 mmol), and the appropriate alkyl bromide or benzyl
chloride derivative (14.2 mmol) were combined in DMF
(25 mL) in a 100-mL round-bottomed flask, and the resulting
mixture was stirred for 24 h at room temperature. After the
reaction was completed (TLC), the mixture was poured into
water (50 mL), and then extracted with CH2Cl2. The com-
bined organic extracts were washed with water, dried
(MgSO4), and concentrated to dryness in vacuo. The residue
was purified by chromatography on silica gel, eluting with
CH2Cl2, to give compounds 1a–1q as white solids.
7-Heptyl-7H-tetrazolo[1,5-g]purine (3b)
White solid; mp: 78–79 °C. IR (KBr) cm-1: 1656 (C=N).
1H NMR (CDCl3, 300 MHz) d: 0.87 (t, 3H, J = 6.00 Hz,
–CH3), 1.27–2.12 (m, 10H, –CH2–), 4.65 (t, 2H,
J = 6.00 Hz, –CH2–), 8.25 (s, 1H, Imidazole-H), 9.58 (s,
1H, Pyrimidine-H). 13C NMR (CDCl3) d: 13.99 (C-12),
22.48 (C-11), 26.33 (C-10), 28.60 (C-9), 30.37 (C-8), 31.52
(C-7), 48.73 (C-6), 111.64 (C-1), 133.69 (C-5), 142.37 (C-
4), 144.23 (C-2), 150.97 (C-3). MS-EI m/z 260 (M?1).
Anal. Calcd. for C12H17N7: C 55.58, H 6.61, N 37.81 %.
Found: C 55.51, H 6.53, N 37.71 %.
General procedure for synthesis of 1-(9-alkyl-9H-purin-6-
yl)hydrazines (2a–2q)
7-Octyl-7H-tetrazolo[1,5-g]purine (3c)
White solid; mp: 71–72 °C. IR (KBr) cm-1: 1656 (C=N).
1H NMR (CDCl3, 300 MHz) d: 0.85 (t, 3H, J = 6.79 Hz,
–CH3), 1.24–2.12 (m, 12H, –CH2–), 4.63 (t, 2H,
J = 7.18 Hz, –CH2–), 8.22(s, 1H, Imidazole-H), 9.57 (s,
1H, Pyrimidine-H). 13C NMR (CDCl3) d: 14.00 (C-13),
22.52 (C-12), 26.34 (C-11), 28.88 (C-10), 28.98 (C-9),
30.35 (C-8), 31.61 (C-7), 48.57 (C-6), 111.61 (C-1), 133.46
A solution of the appropriate (1a–1q, 5.0 mmol) in meth-
anol (15 mL) was added dropwise to a solution of hydra-
zine hydrate (0.6 mL) in methanol (10 mL) at room
temperature. The resulting mixture was stirred at 50 °C for
1 h. The reaction mixture was then cooled to room tem-
perature, and the resulting precipitate was collected by
filtration, washed with methanol. The products (2a–2q)
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Med Chem Res
7-(3-Chlorobenzyl)-7H-tetrazolo[1,5-g]purine (3h)
(C-5), 142.38 (C-4), 144.32 (C-2), 151.46 (C-3). MS-EI m/
z 274 (M?1). Anal. Calcd. for C13H19N7: C 57.12, H 7.01,
N 35.87 %. Found: C 57.09, H 6.86, N 35.77 %.
White solid; mp: 134–135 °C. IR (KBr) cm-1: 1660
(C=N). 1H NMR (CDCl3, 300 MHz) d: 5.80 (s, 2H, –CH2–),
7.31–7.43 (m, 4H, Ar–H), 8.31 (s, 1H, Imidazole-H), 9.56 (s,
1H, Pyrimidine-H). 13C NMR (CDCl3) d: 51.22 (C-6),
111.69 (C-10), 126.47 (C-12), 128.26 (C-1), 129.50 (C-8),
130.72 (C-11), 133.79 (C-9), 135.24 (C-7), 135.88 (C-5),
142.33 (C-4), 144.10 (C-2), 151.48 (C-3). MS-EI m/z 286
(M?1). Anal. Calcd. for C12H8ClN7: C 50.45, H 2.82, N
34.32 %. Found: C 50.40, H 2.90, N 34.26 %.
7-(2-Fluorobenzyl)-7H-tetrazolo[1,5-g]purine (3d)
White solid; mp: 150–152 °C. IR (KBr) cm-1: 1662
(C=N). 1H NMR (CDCl3, 300 MHz) d: 5.86 (s, 2H, –CH2–),
7.08–7.81 (m, 4H, Ar–H), 8.34 (s, 1H, Imidazole-H), 9.54 (s,
1H, Pyrimidine-H). 13C NMR (CDCl3) d: 45.93 (C-6),
111.63 (C-9), 116.08 (C-11), 121.43 (C-7), 125.04 (C-10),
131.37 (C-1), 131.66 (C-12), 133.59 (C-5), 142.37 (C-4),
144.70 (C-2), 151.27 (C-3), 162.68 (C-8). MS-EI m/z 270
(M?1). Anal. Calcd. for C12H8FN7: C 53.53, H 2.99, N
36.42 %. Found: C 53.39, H 3.06, N 36.51 %.
7-(4-Chlorobenzyl)-7H-tetrazolo[1,5-g]purine (3i)
White solid; mp: 152–154 °C. IR (KBr) cm-1: 1660
(C=N). 1H NMR (CDCl3, 300 MHz) d: 5.79 (s, 2H, –CH2–),
7.34–7.47 (dd, 4H, Ar–H), 8.29 (s, 1H, Imidazole-H), 9.56
(s, 1H, Pyrimidine-H). 13C NMR (CDCl3) d: 51.26 (C-6),
111.63 (C-1), 129.63 (C-9, C-11), 129.76 (C-8, C-12),
132.38 (C-10), 133.75 (C-7), 135.44 (C-5). 142.35 (C-4),
143.95 (C-2), 151.53 (C-3). MS-EI m/z 286 (M?1). Anal.
Calcd. for C12H8ClN7: C 50.45, H 2.82, N 34.32 %. Found:
C 50.51, H 2.72, N 34.39 %.
7-(3-Fluorobenzyl)-7H-tetrazolo[1,5-g]purine (3e)
White solid; mp: 126–128 °C. IR (KBr) cm-1: 1662
(C=N). 1H NMR (CDCl3, 300 MHz) d: 5.82 (s, 2H, –CH2–),
7.04–7.36 (m, 4H, Ar–H), 8.31 (s, 1H, Imidazole-H), 9.56 (s,
1H, Pyrimidine-H). 13C NMR (CDCl3) d: 51.30 (C-6),
111.72 (C-10), 115.42 (C-8), 123.86 (C-12), 131.11 (C-1),
131.22 (C-11), 133.77 (C-7), 136.28 (C-5), 142.35 (C-4),
144.03 (C-2), 151.49 (C-3), 164.70 (C-9). MS-EI m/z 270
(M?1). Anal. Calcd. for C12H8FN7: C 53.53, H 2.99, N
36.42 %. Found: C 53.42, H 3.08, N 36.49 %.
7-(2-Benzonitrile)-7H-tetrazolo[1,5-g]purine (3j)
White solid; mp: 198–200 °C. IR (KBr) cm-1: 1662
(C=N). 1H NMR (CDCl3, 300 MHz) d: 6.02 (s, 2H, –CH2–),
7.31–8.03 (m, 4H, Ar–H), 8.55 (s, 1H, Imidazole-H), 9.58 (s,
1H, Pyrimidine-H). 13C NMR (CDCl3) d: 54.29 (C-6),
116.67 (C-8), 121.89 (–CN), 127.92 (C-10), 134.45 (C-1),
134.61 (C-12), 134.81 (C-9), 138.29 (C-11), 138.59 (C-7),
139.15 (C-5), 142.51 (C-4), 147.04 (C-2), 150.19 (C-3). MS-
EI m/z 277 (M?1). Anal. Calcd. for C13H8N8: C 56.52, H
2.92, N 40.56 %. Found: C 56.36, H 2.97, N 40.47 %.
7-(4-Fluorobenzyl)-7H-tetrazolo[1,5-g]purine (3f)
White solid; mp: 158–160 °C. IR (KBr) cm-1: 1662
(C=N). 1H NMR (CDCl3, 300 MHz) d: 5.78 (s, 2H, –CH2–),
7.04–7.54 (dd, 4H, Ar–H), 8.27 (s, 1H, Imidazole-H), 9.56
(s, 1H, Pyrimidine-H). 13C NMR (CDCl3) d: 50.78 (C-6),
111.59 (C-8, C-12), 116.61 (C-7), 130.36 (C-1), 130.48
(C-9, C-11), 133.74 (C-5), 142.38 (C-4), 143.93 (C-2),
151.50 (C-3), 164.74 (C-10). MS-EI m/z 270 (M?1). Anal.
Calcd. for C12H8FN7: C 53.53, H 2.99, N 36.42 %. Found: C
53.40, H 2.89, N 36.51 %.
7-(4-Benzonitrile)-7H-tetrazolo[1,5-g]purine (3k)
White solid; mp: 208–209 °C. IR (KBr) cm-1: 1662
(C=N). 1H NMR (CDCl3, 300 MHz) d: 5.87 (s, 2H, –CH2–),
7.59–7.72 (dd, 4H, Ar–H), 8.32 (s, 1H, Imidazole-H), 9.59
(s, 1H, Pyrimidine-H). 13C NMR (CDCl3) d: 55.57 (C-6),
116.16 (C-10), 117.36 (–CN), 122.90 (C-1), 133.84 (C-8,
C-12), 137.61 (C-9, C-11), 138.94 (C-7), 144.72 (C-5),
147.01 (C-4), 149.96 (C-2), 156.38 (C-3). MS-EI m/z 277
(M ? 1). Anal. Calcd for C13H8N8: C 56.52, H 2.92, N
40.56 %. Found: C 56.41, H 2.88, N 40.51 %.
7-(2-Chlorobenzyl)-7H-tetrazolo[1,5-g]purine (3g)
White solid; mp: 188–189 °C. IR (KBr) cm-1: 1660
(C=N). 1H NMR (CDCl3, 300 MHz) d: 5.94 (s, 2H, –CH2–),
7.30–7.79 (m, 4H, Ar–H), 8.37 (s, 1H, Imidazole-H), 9.57 (s,
1H, Pyrimidine-H). 13C NMR (CDCl3) d: 49.45 (C-6),
111.80 (C-11), 127.88 (C-10), 130.28 (C-1), 131.04 (C-9),
131.53 (C-12), 131.59 (C-8), 133.64 (C-7), 134.07 (C-5),
142.43 (C-4), 144.72 (C-2), 151.28 (C-3). MS-EI m/z 286
(M?1). Anal. Calcd. for C12H8ClN7: C 50.45, H 2.82, N
34.32 %. Found: C 50.36, H 2.91, N 34.27 %.
7-Benzyl-7H-tetrazolo[1,5-g]purine (3l)
White solid; mp: 156–157 °C. IR (KBr) cm-1: 1660 (C=N).
1H NMR (CDCl3, 300 MHz) d: 5.82 (s, 2H, –CH2–),
7.37–7.51 (m, 5H, Ar–H), 8.27 (s, 1H, Imidazole-H), 9.56 (s,
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Med Chem Res
1H, Pyrimidine-H). 13C NMR (CDCl3) d: 51.12 (C-6),
112.69 (C-10), 126.37 (C-1), 129.50 (C-9, C-11), 133.79
(C-8, C-12), 135.24 (C-7), 135.88 (C-5), 142.33 (C-4),
144.10 (C-2), 151.68 (C-3). MS-EI m/z 252 (M?1). Anal.
Calcd. for C12H9N7: C 57.37, H 3.61, N 39.02 %. Found:
C 57.30, H 3.70, N 38.93 %.
(C-1), 114.71 (C-9, C-11), 125.84 (C-7), 130.01 (C-8,
C-12), 133.52 (C-10), 138.66 (C-5), 143.92 (C-4), 143.92
(C-2), 160.26 (C-3). MS-EI m/z 282 (M?1). Anal. Calcd.
for C13H11N7O: C 55.51, H 3.94, N 34.86 %. Found:
C 55.39, H 4.05, N 34.77 %.
7-(4-Nitrobenzyl)-7H-tetrazolo[1,5-g]purine (3q)
7-(2,6-Dichlorobenzyl)-7H-tetrazolo[1,5-g]purine (3m)
White solid; mp: 191–193 °C. IR (KBr) cm-1: 1660
(C=N). 1H NMR (CDCl3, 300 MHz) d: 5.92 (s, 2H, –CH2–),
7.66–8.28 (dd, 4H, Ar–H), 8.34 (s, 1H, Imidazole-H), 9.59
(s, 1H, Pyrimidine-H). 13C NMR (CDCl3) d: 55.19 (–CH2–),
116.14 (C-1), 128.92 (C-9, C-11), 134.19 (C-8, C-12),
139.19 (C-7), 146.94 (C-5), 147.03 (C-4), 150.23 (C-10),
152.70 (C-2), 156.43 (C-3). MS-EI m/z 291 (M?1). Anal.
Calcd. for C12H8N8O2: C 48.65, H 2.72, N 37.82 %. Found:
C 48.57, H 2.82, N 37.76 %.
White solid; mp: 182–184 °C. IR (KBr) cm-1: 1662
(C=N). 1H NMR (CDCl3, 300 MHz) d: 6.19 (s, 2H, –CH2–),
7.41–7.52 (m, 3H, Ar–H), 7.97 (s, 1H, Imidazole-H), 9.60 (s,
1H, Pyrimidine-H). 13C NMR (CDCl3) d: 46.97 (C-6),
112.39 (C-1), 129.17 (C-9, C-11), 129.40 (C-10), 131.86
(C-7), 133.62 (C-8, C-12), 136.89 (C-5), 143.26 (C-4),
143.26 (C-2), 153.31 (C-3). MS-EI m/z 320 (M?1). Anal.
Calcd. for C12H7Cl2N7: C 45.02, H 2.20, N 30.63 %. Found:
C 45.09, H 2.12, N 30.54 %.
Pharmacology
7-(2,4-Dichlorobenzyl)-7H-tetrazolo[1,5-g]purine (3n)
Anticonvulsant activity was assessed by the most widely
used animal model assay, the MES and neurotoxicity test
(Krall et al., 1978; Poter et al., 1984). Antidepressant
activities were evaluated using forced swimming test
(Porsolt et al., 1977). Studies were performed in Kunming
mice (weighing 20–24 g), which were purchased from the
Laboratory of Animal Research, College of Pharmacy,
Yanbian University, China. The animals had free access to
feed and water, except during the actual testing period.
Compounds were administered as suspensions in aqueous
Tween 80 (3 % v/v, 0.9 % NaCl).
White solid; mp: 171–172 °C. IR (KBr) cm-1: 1662
(C=N). 1H NMR (CDCl3, 300 MHz) d: 5.89 (s, 2H, –CH2–),
7.27–7.81 (m, 3H, Ar–H), 8.38 (s, 1H, Imidazole-H), 9.57 (s,
1H, Pyrimidine-H). 13C NMR (CDCl3) d: 48.85 (C-6),
111.66 (C-1), 128.19 (C-11), 130.12 (C-9), 130.16 (C-12),
132.46 (C-10), 133.75 (C-8), 134.79 (C-7), 136.45 (C-5),
142.33 (C-4), 144.72 (C-2), 151.35 (C-3). MS-EI m/z 320
(M?1). Anal. Calcd. for C12H7Cl2N7: C 45.02, H 2.20, N
30.63 %. Found: C 45.10, H 2.14, N 30.57 %.
7-(4-Methylbenzyl)-7H-tetrazolo[1,5-g]purine (3o)
Anticonvulsant effects in the MES
White solid; mp: 172–173 °C. IR (KBr) cm-1: 1658
(C=N). 1H NMR (CDCl3, 300 MHz) d: 2.34 (s, 3H, –CH3),
5.77 (s, 2H, –CH2–), 7.18–7.40 (dd, 4H, Ar–H), 8.23 (s,
1H, Imidazole-H), 9.55 (s, 1H, Pyrimidine-H). 13C NMR
(CDCl3) d: 21.16 (–CH3), 51.82 (C-6), 111.72 (C-1),
128.40 (C-10), 130.05, 130.05 (C-9, C-11), 133.54 (C-8,
C-12), 139.36 (C-5), 142.46 (C-4), 144.00 (C-2), 151.47
(C-3). MS-EI m/z 266 (M?1). Anal. Calcd. for C13H11N7:
C 58.86, H 4.18, N 36.96 %. Found: C 58.78, H 4.12,
N 36.88 %.
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. 30 min after the administration of the compounds,
the activities were evaluated in MES test.
Neurotoxicity (NT) screening
7-(4-Methoxybenzyl)-7H-tetrazolo[1,5-g]purine (3p)
The neurotoxicity of the compounds was measured in mice
by the rotarod test. The mice were trained to stay on an
accelerating rotarod of diameter 3.2 cm that rotates at
10 rpm. Trained animals were given i.p. injection of the
test compounds. 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.
White solid; mp: 164–166 °C. IR (KBr) cm-1: 1658
(C=N). 1H NMR (CDCl3, 300 MHz) d: 3.79 (s, 3H,
–OCH3), 5.74 (s, 2H, –CH2–), 6.88–7.47 (dd, 4H, Ar–H),
8.23 (s, 1H, Imidazole-H), 9.55 (s, 1H, Pyrimidine-H).
13C NMR (CDCl3) d: 51.58 (C-6), 55.33 (–OCH3), 111.63
123

Med Chem Res
Scheme 1 Synthesis of
compounds 3a–3q (i) K2CO3,
RX, DMF, rt, 24 h; (ii)
9
8
7
10
11
R''
R
N
R
N
6
NH2NH2ꢀH2O, CH3OH, 50 °C,
1 h; (iii) NaNO2, 0–5 °C, 2 h;
R0 3a: C6H13, 3b: C7H14, 3c:
C8H17; R’’ 3d: o-F, 3e: m-F, 3f:
p-F, 3g: o-Cl, 3h: m-Cl, 3i: p-Cl,
3j: o-CN, 3k: p-CN, 3l: H, 3m:
2,6-Cl, 3n: 2,4-Cl, 3o: p-CH3,
3p: p-OCH3, 3q: p-NO2
12
H
N
N
N
N
2
1
N
N
(i)
(ii)
(iii)
3
5
N
N
N
N
N
N
N
N
N
4
Cl
NHNH2
2a-2q
Cl
N
N
3d-3q
1a-1q
R'
N
2
N
(iii)
3
5
N
N
1
N
4
N
N
3a-3c
scPTZ-induced seizures test
(Scheme 1). 6-chloro-9-alkyl-9H-purines (1a–1q) were
obtained by reacting with the appropriate alkyl bromide or
benzyl chloride derivatives in a solution of DMF under
basic conditions (Kim et al., 2004). Treatment of 1a–1q
with hydrazine hydrate in methanol afforded 1-(9-alkyl-
9H-purin-6-yl)hydrazines 2a–2q in high yield (Sun et al.,
2009). Finally, diazotization of 2a–2q yielded the desired
purine derivatives 3a–3q (Reisinger et al., 2004). The
chemical structures of the compounds synthesized were
elucidated on the basis of IR, 1H NMR, 13C NMR, and MS.
At 0.5 h after the administration of the test compound,
85 mg/kg PTZ dissolved in saline was administered sc. The
animals (10 mice in one group) placed in individual cages
and observed for 0.5 h. The numbers of clonic seizure
(range from exaggerated twitches of the limbs to violent
shaking or vibrating of the stiffened extremities) and tonic
seizure (the extremities pull toward the body or rigidly
push away from it, usually maximal extension of the hind
leg) as well as the number of deaths were noted.
Pharmacology
Forced swimming test (FST)
Anticonvulsant activity
Male Kunming mice (20–24 g) were used in the FST. On
the test day, mice were assigned to different groups (n = 8
for each group). The synthesized compounds and the
standard drug fluoxetine were administered as intraperito-
neal injections. Control animals received a 3 % aqueous
solution of Tween 80. After 30 min, the mice were dropped
one at a time into a Plexiglas cylinder (height 25 cm,
diameter 10 cm, containing water to a height of 10 cm at
23–25 °C) and observed for 6 min. After the first 2 min of
vigorous struggling, the animals were immobile. A mouse
was judged immobile if it floated in the water in an upright
position and made only slight movements to prevent
sinking. The total duration of immobility was recorded
during the last 4 min of the 6 min test.
The choice of an appropriate animal model for the initial
compound screening process represents a very important
step in antiepileptic drug discovery. There are currently
three models available for the in vivo evaluation of a drug
candidate’s anticonvulsant activity, namely the MES,
subcutaneous pentylenetetrazole (scPTZ), and kindling
models, which are routinely used by most AED discovery
programs. Of these tests, the MES and scPTZ seizure
models represent the two animal seizure models which are
most widely used in the search for new AEDs (White,
2003; Levy et al., 1995). The MES test is believed to
provide an effective prediction of drugs that will be
effective against generalized seizures of the tonic–clonic
(grand mal) type, whereas the scPTZ test is used to find
drugs that are effective against the generalized seizures of
the petit mal (absence) type. In the current study, the MES
seizure model was used for screening the anticonvulsant
activity of target compounds.
Results and discussion
Chemistry
The MES test was used for the preliminary screening of
3a–3q. Compounds were administered intraperitoneally
(ip) into mice as aqueous suspensions, at three dose levels
For the preparation of all compounds described in this
paper, 6-chloro-9H-purine was used as the starting material
123

Med Chem Res
Table 1 Anticonvulsant activities of compounds 3a–3q in MES tests
Table 2 Quantitative anticonvulsant data in mice (i.p.)
Compound
ED50 (MES)a
TD50 (NT)b
PIc
R
N
N
3d
44.0 (28.1–50.8)d
31.7 (27.4–36.6)
28.9 (22.1–37.8)
31.7 (27.5–36.6)
11.8 (8.5–16.4)
448.7 (256.2–667.7)
387.2 (320.0–468.5)
458.3 (416.6–504.1)
294.6 (257.7–336.7)
76.1 (55.8–103.7)
10.2
12.2
15.8
9.3
3e
N
N
N
3h
N
N
3j
Carbamazepine
6.4
3a-3q
a
ED50 median effective dose affording anticonvulsant protection in
50 % of animals, the dose is measured in mg/kg
Compound
R
MES (mg/kg)a
b
TD50 median toxic dose eliciting minimal neurological toxicity in
50 % of animals, the dose is measured in mg/kg
300
100
30
c
PI protective index (TD50/ED50
)
3/3b
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
1/3
2/3
3/3
3/3
2/3
0/3
2/3
1/3
2/3
1/3
3/3
3/3
2/3
1/3
3/3
1/3
3/3
0/3
1/3
2/3
3/3
1/3
0/3
1/3
0/3
0/3
0/3
1/3
1/3
0/3
0/3
2/3
0/3
1/3
d
3a
3b
3c
3d
3e
3f
–C6H13
–C7H15
–C8H17
95 % confidence intervals given in parentheses
The PI value gave bigger margin of safe compared to car-
bamazepine, which has a PI value of 6.4.
–CH2C6H4 (o-F)
–CH2C6H4 (m-F)
–CH2C6H4 (p-F)
–CH2C6H4 (o-Cl)
–CH2C6H4 (m-Cl)
–CH2C6H4 (p-Cl)
–CH2C6H4 (o-CN)
–CH2C6H4 (p-CN)
–CH2C6H5
An analysis of the anticonvulsant activities of com-
pounds 3a–3q showed the following structure–activity
relationships. Among the benzyl group-substituted deriva-
tives, the position and electronic nature of the substituent
on the benzene ring significantly influenced the MES test
results of a given compound. For derivatives with electron-
withdrawing substituents, the anticonvulsant activity order
was m-F [ o-F [ p-F, m-Cl [ 2,4-Cl [ 2,6-Cl [ o-Cl,
p-Cl, o-CN [ p-CN. For derivatives with electron-donat-
ing substituents, the activity order was p-CH3 [ p-OCH3.
To explain the possible mechanism for anticonvulsant
action, compound 3h was subjected to PTZ, which induced
seizure test. Compound 3h and reference drug carbamaz-
epine were administered into mice at a dose of 50 mg/kg
(i.p.). In the scPTZ model (Table 3), compound 3h and
carbamazepine showed the clonic seizures, tonic seizures,
and lethality at the rates of 100, 0, 20, 90, 10, and 50 %,
induced by scPTZ. The data showed that compound 3h and
carbamazepine can protect the mice from tonic seizures
and death, which revealed that compound 3h possessed
activity against sc-PTZ. It has been reported that convul-
sant induced seizure by inhibiting r-aminobutyric acid
(GABA) neurotransmission (such as PTZ) and GABAA
antagonist (Okada et al., 1989; Olsen, 1981). Inhibition of
GABA ergic neurotransmission or activity has been shown
to promote and facilitate seizures (Gale, 1992). The find-
ings of the present study suggest that the newly synthesized
compound 3h might inhibit or attenuate PTZ-induced sei-
zures in mice by enhancing GABAergic neurotransmission.
3g
3h
3i
3j
c
3k
3l
-
0/3
0/3
0/3
0/3
–
3m
3n
3o
3p
3q
a
–CH2C6H3 (2,6-Cl)
–CH2C6H3 (2,4-Cl)
–CH2C6H4 (p-CH3)
–CH2C6H4 (p-OCH3)
–CH2C6H4 (p-NO3)
0/3
Maximal electroshock: doses of 30, 100, and 300 mg/kg were
administrated intraperitoneally in mice
The animals were examined 0.5 h after administration
b
n1/n2: the animals protected/the animals tested
c
Not test
(30, 100, and 300 mg/kg), and observations were made at
0.5 h. As shown in Table 1, most compounds were active,
as shown by their ability to prevent seizure spread. Spe-
cifically, four compounds (3d, 3e, 3h, 3j) showed protec-
tion against MES at the dose of 30 mg/kg, while at
100 mg/kg, all except compounds 3 k and 3p were seizure
protective, with 3d–3e, 3 h, 3j, and 3n protective in 3/3
animals. At a dose of 300 mg/kg, all except 3p were
protective.
In the second stage of the pharmacological research,
ED50, TD50, and PI values for compounds 3d, 3e, 3h, and 3j
were determined (Table 2). Among the tested compounds,
7-(3-chlorobenzyl)-7H-tetrazolo[1,5-g]purine (3h) was the
most promising compound with relatively high activity and
the lowest toxicity. In the anti-MES potency test, it showed
ED50 of 28.9 mg/kg, a TD50 of 458.3 mg/kg, and a PIof 15.8.
Antidepressant activity
In this study, the results are expressed as the mean SEM;
n represents the number of animals. Data obtained from
pharmacological experiments were analyzed using one-
way analysis of variance, followed by Dunnett’s post hoc
123

Med Chem Res
Table 3 Effects of compound 3h on chemical-induced seizures in mice (i.p.)
Chemical substances
Pentylenetetrazol
Compound
Doses (mg/kg)
Test time (h)a
Clonic seizures (%)b
Tonic seizures (%)
Lethality (%)
DMSO
–
0.5
0.5
0.5
100
100
90
100
0
100
20
Carbamazepine
3h
50
50
10
50
a
At 0.5 h after the administration of the test compound, chemical substances were administered in mice
b
The animals clonic sezures, tonic seizures and deaths, respectively / the animals tested 9 100 %
(p \ 0.01). The most active compound, 7-(2-chloroben-
zyl)-7H-tetrazolo[1,5-g]purine (3g), displayed an antide-
pressant activity nearly equal to that of fluoxetine
(p \ 0.01). The other compounds (3e and 3q) exhibited
weak antidepressant activity compared with fluoxetine and
with a control (0.01 \ p \ 0.05).
Table 4 Antidepressant activities of compounds 3a–3q in FST
Compound
R
Antidepressant activitiesa
Duration of
immobility(s)
(mean SEM)b
Change from
control (%)
3a
–C6H13
157.1 13.95
130.0 11.09
103.6 19.91
135.0 21.35
94.88 15.49*
153.6 15.50
73.63 9.58**
128.8 9.87
122.0 22.84
125.8 18.83
6.76
3b
–C7H15
22.85
38.51
19.88
43.69
8.84
3c
–C8H17
Conclusion
3d
–CH2C6H4 (o-F)
–CH2C6H4 (m-F)
–CH2C6H4 (p-F)
–CH2C6H4 (o-Cl)
–CH2C6H4 (m-Cl)
–CH2C6H4 (p-Cl)
–CH2C6H4 (o-CN)
–CH2C6H4 (p-CN)
–CH2C6H5
3e
In conclusion, a series of purine derivatives were synthe-
sized, and their anticonvulsant and antidepressant activities
were evaluated. Pharmacological testing showed that at a
dose of 100 mg/kg, most of the compounds had anticon-
vulsant activity. Among the compounds studied, 7-(3-
chlorobenzyl)-7H-tetrazolo[1,5-g]purine (3h) was found to
be the most potent compound with a median effective dose
(ED50) value of 28.9 mg/kg and a protective index (PI)
value of 15.8, possessing better anticonvulsant activity and
higher safety than the marketed drug carbamazepine. To
explain the possible mechanism of anticonvulsant activity,
compound 3h was tested in pentylenetetrazole PTZ-
induced seizures tests. The results suggest that compound
3h exerts anticonvulsant activity through a GABA-medi-
ated mechanism. Five compounds have significant antide-
pressant activity during the forced swimming test. The
most active compound was 7-(2-chlorobenzyl)-7H-tetraz-
olo[1,5-g]purine (3g), which decreased immobility time by
56 %.
3f
3g
56.30
23.56
27.59
25.34
3h
3i
3j
3k
76.50 17.98** 54.59
3l
126.3 20.45
129.5 23.05
138.3 21.17
96.25 22.12
25.04
23.14
17.92
42.87
3m
–CH2C6H3 (2,6-Cl)
–CH2C6H3 (2,4-Cl)
–CH2C6H4 (p-CH3)
3n
3o
3p
–CH2C6H4 (p-OCH3) 75.13 23.06** 55.41
3q
–CH2C6H4 (p-NO3)
85.13 15.53*
67.0 9.34**
168.5 12.14
49.47
60.23
–
Fluoxetine
Control
a
–
–
Compounds prepared were administered at 40 mg/kg. Fluoxetine
was administered at 40 mg/kg
b
Values represent the mean SEM. (n = 8)
* Significantly compared to control (0.01 \ p\0.05)
** Very significantly compared to control (p \ 0.01)
Acknowledgments This work was supported by the National Nat-
ural Science Foundation of China (No. 81160382) and National
Science and Technology Major Project of China (No. 2011ZX09102-
003-03).
test, using Pharmacologic Calculation System Version 4.1
(Microcomputer Specialists). A P value of less than 0.05
was considered statistically significant.
Compounds 3a–3q were screened for antidepressant
activity using the FST, which effectively predict the
activities of a wide variety of antidepressants, including
monoamine oxidase (MAO) inhibitors and selective sero-
tonin-reuptake inhibitors (SSRIs) such as fluoxetine. The
results are shown in Table 4, together with those of flu-
oxetine, for comparison (all doses were 40 mg/kg).
Three compounds (3g, 3k, and 3p) significantly reduced
immobility times during the FST compared with a control
Conflict of interest We declare that we have no conflict of interest
with respect to this study.
References
Alhano C, Cupello A, Mainardi P, Scarrone S, Favale E (2006)
Successful treatment of epilepsy with serotonin reuptake inhib-
itors: proposed mechanism. Neurochem Res 31:509–514
123

Med Chem Res
Beghi E, Roncolato M, Visona G (2004) Depression and altered
quality of life in women with epilepsy of childbearing age.
Epilepsia 45:64–70
Mallari JP, Guiguemde WA, Guy RK (2009) Antimalarial activity of
thiosemicarbazones and purine derived nitriles. Bioorg Med
Chem Lett 19:3546–3549
Bordon-Pallier F, Jullian N, Ferrari P, Girard AM, Bocquel MT, Biton
J, Bouquin N, Haesslein JL (2004) Inhibitors of Civ1 kinase
belonging to 6-aminoaromatic-2-cyclohexyldiamino purine ser-
ies as potent anti-fungal compounds. Biochim Biophys Acta
1697:211–223
Okada R, Negishi N, Nagaya H (1989) The role of the nigrotegmental
GABAergic pathway in the propagation of pentylenetetrazol-
induced seizures. Brain Res 480:383–387
Olsen RW (1981) GABA-benzodiazepine-barbiturate receptor inter-
actions. J Neuro chem 37:1–13
Braendvang M, Charnock C, Gundersen LL (2009) Synthesis and
antimycobacterial activity of 5-formylaminopyrimidines; ana-
logs of antibacterial purines. Bioorg Med Chem Lett 19:3297–
3299
Porsolt RD, Bertin A, Jalfre M (1977) Behavioral despair in mice: a
primary screening test for antidepressants. Arch Int Pharmaco-
dyn Ther 229:327–336
Poter RJ, Cereghino JJ, Gladding GD, Hessie BJ, Kupferberg HJ,
Scoville B (1984) Antiepileptic drug development program.
Clevel Clinic Q 51:293–305
Reisinger A, Bernhardt PV, Wentrup C (2004) Synthesis of 1,3-
diazepines and ring contraction to cyanopyrroles. Org Biomol
Chem 2:246–256
´
´
´
Conejo-Garcıa A, Garcıa-Rubin˜o ME, Marchal JA, Nun˜ez MC,
´
´
´
´
Ramırez A, Cimino S, Garcıa MA, Aranega A, Gallo MA,
Campos JM (2011) Synthesis and anticancer activity of (RS)-9-
(2,3-dihydro-1,4-benzoxaheteroin-2-ylmethyl)-9H-purines.
Eur J Med Chem 46:3795–3801
Deng XQ, Wei CX, Song MX, Chai KY, Sun ZG, Quan ZS (2010)
Synthesis and studies on anticonvulsant and antidepressant
activities of 5-alkoxy-tetrazolo[1,5-a]quinolines. Bulletin Kor-
ean Chem Soc 31:447–452
Sun XY, Guan LP, Zhang L, Wei CX, Piao HR, Quan ZS (2009)
Design, synthesis and anticonvulsant activity evaluation of
7-substituted-4H-[1,2,4]triazino[3,4-a]phthalazin-4-one deriva-
tives. J Braz Chem Soc 20:826–831
Gale K (1992) GABA and epilepsy, basic concepts from preclinical
research. Epilepsia 33:S3–S12
Sun XY, Wei CX, Deng XQ, Sun ZG, Quan ZS (2010a) Evaluation of
the anticonvulsant activity of 6-(4-chlorophenyoxy)-tetrazol-
o[5,1-a]phthalazine in various experimental seizure models in
mice. Pharmacol Rep 62:273–277
Sun XY, Wei CX, Deng XQ, Sun ZG, Quan ZS (2010b) Synthesis
and primary anticonvulsant activity evaluation of 6-alkyoxyl-
tetrazolo[5,1-a]phthalazine derivatives. Arzneim-Forsch 60:
289–292
Wang HJ, Wei CX, Deng XQ, Li FN, Quan ZS (2009) Synthesis and
evaluation on anticonvulsant and antidepressant activities of
5-alkoxy-tetrazolo[1,5-a]quinazolines. Arch Pharm Chem Life
Sci 342:671–675
Wang Y, Yang X, Zheng X, Li J, Ye C, Song X (2010) Theacrine, a
purine alkaloid with anti-inflammatory and analgesic activities.
Fitoterapia 81:627–631
Hirokawa Y, Kinoshita H, Tanaka T, Nakamura T, Fujimoto K,
Kashimoto S, Kojima T, Kato S (2009) Pleuromutilin derivatives
having a purine ring. Part 2: influence of the central spacer on the
antibacterial activity against Gram-positive pathogens. Bioorg
Med Chem Lett 19:170–174
Kim BY, Ahn JB, Lee HW, Kang SK, Lee JH, Shin JS, Ahn SK,
Hong CI, Yoon SS (2004) Synthesis and biological activity of
novel substituted pyridines and purines containing 2,4-thiazo-
lidinedione. Eur J Med Chem 39:433–447
Krall RJ, Penry JK, White BG, Kupferberg HJ (1978) Antiepileptic
drug development: II. Anticonvulsant drug screening. Epilepsia
19:409–428
Levy RH, Mattson RH, Meldrum BS (eds) (1995) Antiepileptic drugs,
4th edn. Raven Press, New York, pp 99–110
White HS (2003) Preclinical development of antiepileptic drugs: past,
present, and future directions. Epilepsia 44:2–8
´
Lopez-Cara LC, Conejo-Garcıa A, Marchal JA, Macchione G, Cruz-
´
´
´
´
´
Lopez O, Boulaiz H, Garcıa MA, Rodrıguez-Serrano F, Ramırez
A, Cativiela C, Jimenez AI, Garcıa-Ruiz JM, Choquesillo-
Zhou LX (2011) Computational study on the mechanisms of action of
the potential anticancer drug trans-isopropylaminedimethylami-
nedichloroplatinum (trans-IPADMADP) and its cis isomer with
DNA purine bases. Inorg Chim Acta 376:44–56
´
´
´
Lazarte D, Aranega A, Campos JM (2011) New (RS)-ben-
zoxazepin-purines with antitumour activity: the chiral switch
from (RS)-2,6-dichloro-9-[1-(p-nitrobenzenesulfonyl)-1,2,3,5-
tetrahydro-4,1-benzoxazepin-3-yl]-9H-purine. Eur J Med Chem
46:249–258
123