Synthesis and anticonvulsant activity of ethyl 2,2-dimethyl-1-(2- substitutedhydrazinecarboxamido) cyclopropanecarboxylate derivatives

Article abstract of DOI:10.1111/cbdd.12310

In this study on the development of new anticonvulsants, fourteen ethyl 2,2-dimethyl-1-(2-substitutedhydrazinecarboxamido) cyclopropanecarboxylate derivatives were synthesized and tested for anticonvulsant activity using the maximal electroshock, subcutan

Full text of DOI:10.1111/cbdd.12310

Chem Biol Drug Des 2014
Research Article
Synthesis and Anticonvulsant Activity of Ethyl
2,2-dimethyl-1-(2-substitutedhydrazinecarboxamido)
Cyclopropanecarboxylate Derivatives
Min Zhong1, Yongmin Zhang2,3 and Xianran He2,*
AEDs, as about 30% of epileptic patients are not seizure-
free with the existing AEDs (4,5). Besides, patients often
1Wuhan Economic and Technological Development Zone,
School of Chemical and Environmental Engineering,
Jianghan University, Wuhan 430056, China
suffer side-effects during therapy process, such as head-
ache, nausea, anorexia, ataxia, hepatotoxicity, drowsiness,
gastrointestinal disturbance, gingival hyperplasia, and
hirsutism (6–8).
2Wuhan Economic and Technological Development Zone,
Institute for Interdisciplinary Research, Jianghan University,
Wuhan 430056, China
3
The SAR studies of clinically available AEDs and other anti-
convulsant active compounds showed that most of these
compounds included a general model consist of three
essential fragments for anticonvulsant activity in their mole-
cules: (i) amides group as hydrogen-binding domain; (ii) an
electron donor group; (iii) an aryl group (Figure 1) (9,10).
Taking into consideration of the above, our laboratory has
demonstrated the potent anticonvulsant activity among the
urea derivatives, from which compound I with ED50 value
of 14.3 mg/kg, compound II with ED50 value of 9.8
mg/kg and a protective index (TD50/ED50) of 33.9 in the
MES test in mice (11,12) (Figure 2).
ꢀ
Institut Parisien de Chimie Moleculaire, CNRS UMR 7201,
Universite Pierre et Marie Curie-Paris 6, 4 Place Jussieu,
ꢀ
75005 Paris, France
*Corresponding author: Xianran He, hexianran@163.com
In this study on the development of new anticonvulsants,
fourteen ethyl 2,2-dimethyl-1-(2-substitutedhydrazine-
carboxamido) cyclopropanecarboxylate derivatives
were synthesized and tested for anticonvulsant activity
using the maximal electroshock, subcutaneous penty-
lenetetrazole screens, which are the most widely
employed seizure models for early identification of can-
didate anticonvulsants. Their neurotoxicity was deter-
mined applying the rotorod test. Two compounds 6f and
6k showed promising anticonvulsant activities in both
models employed for anticonvulsant evaluation. The
most active compound 6k showed the maximal electro-
shock-induced seizures with ED50 value of 9.2 mg/kg
and TD50 value of 387.5 mg/kg after intraperitoneally
injection to mice, which provided compound 6k with a
protective index (TD50/ED50) of 42.1 in the maximal elec-
troshock test.
Above these facts and in continuation of our research pro-
gram on design and synthesis of new anticonvulsant agents,
the urea pharmacophore has been attached to an alkyl and
aryl amine group and to a cyclopropane unit in order to
develop new potent and safe AEDs (Figure 3). We synthe-
sized and comparatively evaluated the anticonvulsant activity
and neurotoxicity of a number of ethyl 2,2-dimethyl-1-(2-aryl-
hydrazinecarboxamido) cyclopropanecarboxylate derivatives
(6a–n). Compounds 6f and 6k, which displayed the remark-
able activity, were chosen for quantification of the pharmaco-
logical parameters (ED50 and TD50).
Key words: anticonvulsant activity, Ethyl 2,2-dimethyl-1-(2-substi-
tutedhydrazinecarboxamido) cyclopropanecarboxylate, MES test,
scPTZ test
All of these compounds were prepared as racemic mix-
tures, and no attempt was made to resolve the enantio-
mers.
Received 24 November 2013, revised 31 December 2013
and accepted for publication 7 February 2014
Epilepsy is a chronic neurological disorder characterized
by the onset of spontaneous convulsant and non-convul-
sant seizures that result from neuronal hyperexcitability
and hypersynchronous neuronal firing (1). Over 1% of the
world’s population are affected by epilepsy, and many of
them suffer from epilepsy over their lifetime (2,3). Despite
several new antiepileptic drugs (AEDs) have been marked
during the last two decades, there is still a substantial
need for the development of more effective and safer
Experimental Procedures
All chemicals and solvents were purchased from Aldrich or
Fluka. Solvents and reagents were dried and purified
according to the literature methods. Melting points were
uncorrected and measured on an XT-4 apparatus.1H,
and 13C NMR spectra were obtained on a Bruker AV400
apparatus in DMSO-d6 and CDCl3 with TMS as internal
ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12310
1

Zhong et al.
Figure 1: Structure of anticonvulsant drugs with their vital structural fragments. (A) aryl group, (B) amides group as hydrogen-binding
domain, (C) electron donor group.
(11 mmol) and NMM (12 mmol), the mixture was stirred
for 20 min. A solution of NaN3 (25 mmol) in H2O was
added and stirred for 1 h at À10 °C. The solution was
then diluted with H2O and extracted with EtOAc. The
organic layers were washed with brine, dried over Na2SO4,
Figure 2: Structure of compounds I and II.
and concentrated under reduced pressure to give crude
acyl azide. This crude acyl azide could be further purified
by
a flash column chromatography (PE-EtOAc, 4:1,
Rf = 0.7). Purified acyl azide was dissolved in toluene
(30 mL), and the resulting solution was heated to 75 °C
under stirring. After gas evolution had stopped, toluene
was removed under reduced pressure to afford (ethoxy-
carbonyl)isocyanate 5 as clear oil. This alfa-carboethoxy
isocyanate 5 was directly used in the next step without
further purification. Alkyl or aryl hydrazine (10 mmol) was
added to a stirred suspension of (ethoxycarbonyl)isocya-
nate 5 in appropriate solvent (40 mL) at r.t. The solvent
was removed under reduced pressure when the reaction
was completed (detected by TLC), and the products 6
were purified by a column chromatography.
Figure 3: Pharmacophoric
compounds. (A) aryl group, (B) amides group as hydrogen-binding
domain, (C) electron donor group.
structural
fragments
of
title
standard. The elemental analysis (C, H, N) data were
obtained from a VarioEL III (German) elemental analyzer.
The mass spectra (MS) were recorded on AMD-604 mass
spectrometer operating at 70 eV.
Ethyl 1-(hydrazinecarboxamido)-2,2-
dimethylcyclopropanecarboxylate (6a)
Synthesis of 1-(ethoxycarbonyl)-2,2-
dimethylcyclopropanecarboxylic acid 4
Yield: 83%. White solid. Mp: 125–127 °C. IR (KBr, cmÀ1):
3376 (N-H), 1712 (C=O), 1664 (C=O). 1H-NMR (400 MHz,
CDCl3): d 0.80 (d, 1H, J = 4.92 Hz, Cpr-CH), 1.14(s, 6H,
2CH3), 1.16(t, 3H, J = 7.04 Hz, CH3), 1.51 (d, 1H,
J = 4.92 Hz, Cpr-CH), 4.06 (q, 2H, J = 7.04 Hz, CH2),
4.96-5.23(m, 2H, NH2), 7.18(br, 1H, NH), 8.31(br, 1H,
NH). ESI-MS: 216.2 ([M+H]+). Anal. calc. for C9H17N3O3:
C 50.22, H 7.96, N 19.52; found: C 50.33, H 7.99, N
19.48.
To a solution of diethyl 2,2-dimethylcyclopropane-1,1-di-
carboxylate 3 (4.8 g, 22 mmol) in EtOH (25 mL) was added
1 N sodium hydroxide (25 mL, 1.1 equiv, 25 mmol), and
the resulting mixture was stirred at room temperature for
12 h. EtOH was removed under reduced pressure, water
was added to the residue, and the mixture was acidified by
means of a saturated KHSO4 solution and extracted with
ethyl acetate (3 9 30 mL). The combined extracts were
dried over Na2SO4 and evaporated to give product 4
(3.75 g, 90%) as a colorless oil without purified: 1H-NMR
(400 MHz, CDCl3): d 1.25 (s, 3H, CH3), 1.33 (t, 3H,
J = 7.20 Hz, CH3), 1.38 (s, 3H, CH3), 1.78 (s, 1H, Cpr-H),
1.85 (s, 1H, Cpr-H), 4.29 (q, 2H, J = 7.20 Hz, CH2), 11.28
(br s, 1H, OH); 13C-NMR (150 MHz, CDCl3): d 13.92,
20.75, 21.66, 26.94, 33.50, 38.54, 62.08, 171.30, 181.29.
Ethyl 2,2-dimethyl-1-(2-
methylhydrazinecarboxamido)
cyclopropanecarboxylate (6b)
Yield: 85%. White solid. Mp: 130–131 °C. IR (KBr, cmÀ1):
3352 (N-H), 1729 (C=O), 1672 (C=O). 1H-NMR (400 MHz,
CDCl3): 0.79 (d, 1H, J = 4.88 Hz, Cpr-CH), 1.13 (s, 6H,
2CH3), 1.18 (t, 3H, J = 7.04 Hz, CH3), 1.50 (d, 1H,
J = 4.88 Hz, Cpr-CH), 2.38 (s, 3H, CH3), 4.05 (q, 2H,
J = 7.04 Hz, CH2), 7.14 (brs, 1H, NH), 8.04 (brs, 1H, NH),
8.89 (brs, 1H, NH). ESI-MS: 230.2 ([M+H]+). Anal. calc. for
C10H19N3O4: C 52.39, H 8.35, N 18.33; found: C 52.43,
H 8.41, N 18.43.
General procedure for the synthesis of compounds
6a-t
Compound 4 (10 mmol) was dissolved in dry THF (30 mL)
and cooled to À15 °C. After the addition of EtOCOCl
2
Chem Biol Drug Des 2014

Synthesis and Anticonvulsant Activity of Cyclopropanecarboxylate Derivatives
Ethyl 2,2-dimethyl-1-(2-
phenylhydrazinecarboxamido)
cyclopropanecarboxylate (6c)
CDCl3): 0.78 (d, 1H, J = 4.88 Hz, Cpr-CH), 1.12 (s, 6H,
2CH3), 1.18 (t, 3H, J = 7.08 Hz, CH3), 1.46 (d, 1H,
J = 4.88 Hz, Cpr-CH), 4.08 (q, 2H, J = 7.08 Hz, CH2),
Yield: 85%. White solid. Mp: 101–102 °C. IR (KBr, cmÀ1):
3359 (N-H), 1703 (C=O), 1668 (C=O). 1H-NMR (400 MHz,
CDCl3): 0.79 (d, 1H, J = 4.80 Hz, Cpr-CH), 1.14 (s, 6H,
2CH3), 1.18 (t, 3H, J = 7.04 Hz, CH3), 1.52 (d, 1H,
J = 4.80 Hz, Cpr-H), 4.05 (q, 2H, J = 7.04 Hz, CH2), 7.22
(brs, 1H, NH), 7.44–7.54 (m, 2H, C2,6-ArH), 7.56–7.58 (m,
1H, C4-ArH), 7.89–8.00 (m, 2H, C3,5-ArH), 8.86 (brs, 1H,
NH), 9.89 (brs, 1H, NH). ESI-MS: 292.2 ([M+H]+). Anal.
calc. for C15H21N3O3: C 61.84, H 7.27, N 14.42; found: C
61.91, H 7.37, N 14.35.
7.10-7.14(m, 4H, C2,4,5,6-ArH), 7.25 (brs, 1H, NH), 8.40
(brs, 1H, NH), 9.25 (brs, 1H, NH). ESI-MS: 325.2 ([M+H]+).
Anal. calc. for C15H20ClN3O3: C 55.30, H 6.19, N 12.90;
found: C 55.44, H 6.39, N 12.84.
Ethyl 1-(2-(2-chlorobenzoyl)
hydrazinecarboxamido)-2,2-dimethyl
cyclopropanecarboxylate (6h)
Yield: 82%. White solid. Mp: 101–102 °C. IR (KBr, cmÀ1):
3374 (N-H), 1712 (C=O), 1677 (C=O). 1H-NMR (400 MHz,
CDCl3): 0.80 (d, 1H, J = 4.92 Hz, Cpr-CH), 1.15 (s, 3H,
2CH3), 1.17 (t, 3H, J = 7.04 Hz, CH3), 1.52 (d, 1H,
J = 4.92 Hz, Cpr-CH), 4.06 (q, 2H, J = 7.04 Hz), 6.99
(brs, 1H, NH), 7.41–7.52 (m, 5H, ArH), 8.56 (brs, 1H, NH),
9.27 (brs, 1H, NH). ESI-MS: 325.2 ([M+H]+). Anal. calc. for
Ethyl 1-(2-(4-chlorophenyl)hydrazinecarboxamido)-
2,2-dimethylcyclopropanecarboxylate (6d)
Yield: 88%. White solid. Mp: 87–89 °C. IR (KBr, cmÀ1): 3346
1
(N-H), 1692 (C=O), 1652 (C=O). H-NMR (400 MHz, CDCl3):
0.79 (d, 1H, J = 4.12 Hz, Cpr-CH), 1.14 (s, 6H, 2CH3), 1.19
(t, 3H, J = 7.12 Hz, CH3), 1.54 (d, 1H, J = 4.12 Hz, Cpr-H),
4.04 (q, 2H, J = 7.12 Hz, CH2), 7.13 (brs, 1H, NH), 7.55 (d,
C
15H20ClN3O3: C 55.30, H 6.19, N 12.90; found: C 55.44,
H 6.38, N 12.88.
2H, J = 8.48 Hz, C3,5-ArH), 7.87 (d, 2H, J = 8.48 Hz, C2,6
-
ArH), 8.45 (brs, 1H, NH), 9.56 (brs, 1H, NH). ESI-MS: 325.2
([M+H]+). Anal. calc. for C15H20ClN3O3: C 55.30, H 6.19, N
12.90; found: C 55.41, H 6.37, N 12.83.
Ethyl 1-(2-(3-bromophenyl)hydrazinecarboxamido)-
2,2-dimethyl cyclopropanecarboxylate (6i)
Yield: 80%. White solid. Mp: 87–88 °C. IR (KBr, cmÀ1):
3371 (N-H), 1708 (C=O), 1669 (C=O). 1H-NMR (400 MHz,
CDCl3): 0.77 (d, 1H, J = 4.88 Hz, Cpr-CH), 1.13 (s, 6H,
2CH3), 1.18 (t, 3H, J = 7.14 Hz, CH3), 1.49 (d, 1H,
J = 4.88 Hz, Cpr-CH), 4.06 (q, 2H, J = 7.14 Hz, CH2),
7.18 (brs, 1H, NH), 7.25–7.30(m, 4H, C2,4,5,6-ArH), 8.15
(brs, 1H, NH), 9.35 (brs, 1H, NH). ESI-MS: 370.1 ([M+H]+).
Anal. calc. for C15H20BrN3O3: C 48.66, H 5.44, N 11.35;
found: C 48.82, H 5.39, N 11.43.
Ethyl 1-(2-(4-bromophenyl)hydrazinecarboxamido)-
2,2-dimethylcyclopropanecarboxylate (6e)
Yield: 85%. White solid. Mp: 92–93 °C. IR (KBr, cmÀ1):
3359 (N-H), 1703 (C=O), 1668 (C=O). 1H-NMR (400 MHz,
CDCl3): 0.78 (d, 1H, J = 4.42 Hz, Cpr-CH), 1.12 (s, 6H,
2CH3), 1.18 (t, 3H, J = 7.14 Hz, CH3), 1.52 (d, 1H,
J = 4.42 Hz, Cpr-H), 4.06 (q, 2H, J = 7.16 Hz, CH2), 7.22
(brs, 1H, NH), 7.65 (d, 2H, J = 8.44 Hz, C3,5-ArH), 7.85
(d, 2H, J = 8.44 Hz, C2,6-ArH), 8.45 (brs, 1H, NH), 9.88
(brs, 1H, NH). ESI-MS: 370.1 ([M+H]+). Anal. calc. for
C15H20BrN3O3: C 48.66, H 5.44, N 11.35; found: C
48.82, H 5.39, N 11.43.
Ethyl 1-(2-(3,5-dichlorophenyl)
hydrazinecarboxamido)-2,2-dimethyl
cyclopropanecarboxylate (6j)
Yield: 88%. White solid. M.p. 104–106 °C. IR (KBr, cmÀ1):
3356 (N-H), 1712 (C=O), 1678 (C=O). 1H-NMR (400 MHz,
CDCl3): 0.77 (d, 1H, J = 4.74 Hz, Cpr-CH), 1.14 (s, 6H,
2CH3), 1.20 (t, 3H, J = 7.22 Hz, CH3), 1.50 (d, 1H,
J = 4.74 Hz, Cpr-CH), 4.09 (q, 2H, J = 7.22 Hz, CH2),
6.85 (brs, 1H, NH), 7.92–7.18 (m, 3H), 8.58 (brs, 1H, NH),
9.35 (brs, 1H, NH). ESI-MS: 361.2 ([M+H]+). Anal. calc. for
C15H19Cl2N3O3: C 50.01, H 5.32, N 11.66; found: C
50.23, H 5.30, N 11.54.
Ethyl 1-(2-(4-fluorophenyl)hydrazinecarboxamido)-
2,2-dimethylcyclopropanecarboxylate (6f)
Yield: 80%. White solid. Mp: 132–133 °C. IR (KBr, cmÀ1):
3377 (N-H), 1743 (C=O), 1672 (C=O).1H-NMR (400 MHz,
CDCl3): 0.78 (d, 3H, J = 4.86 Hz, Cpr-CH), 1.12 (s, 6H,
2CH3), 1.19 (t, 3H, J = 7.04 Hz, CH3), 1.51 (d, 1H,
J = 4.86 Hz, Cpr-CH), 4.06 (q, 2H, J = 7.04 Hz, CH2),
7.04 (brs, 1H, NH), 7.27–7.33 (m, 2H, C3,5-ArH), 7.92–7.96
(m, 2H, C2,6-ArH), 8.88 (brs, 1H, NH), 9.56 (brs, 1H, NH).
ESI-MS: 310.2 ([M+H]+). Anal. calc. for C15H20FN3O3: C
58.24, H 6.52, N 13.58; found: C 58.31, H 6.67, N 13.43.
Ethyl 1-(2-(3-chloro-4-fluorophenyl)
hydrazinecarboxamido)-2,2-dimethyl
cyclopropanecarboxylate (6k)
Yield: 83%. White solid. Mp: 129–130 °C. IR (KBr, cmÀ1):
3376 (N-H), 1745 (C=O), 1690 (C=O). 1H-NMR (400 MHz,
CDCl3): 0.78 (d, 1H, J = 4.88 Hz, Cpr-CH), 1.12 (s, 6H,
2CH3), 1.18 (t, 3H, J = 7.18 Hz, CH3), 1.51 (d, 1H,
J = 4.88 Hz, Cpr-CH), 4.09 (q, 2H, J = 7.18 Hz),
Ethyl 1-(2-(3-chlorophenyl)hydrazinecarboxamido)-
2,2-dimethylcyclopropanecarboxylate (6g)
Yield: 80%. White solid. Mp: 95–96 °C. IR (KBr, cmÀ1):
3376 (N-H), 1721 (C=O), 1668 (C=O). 1H-NMR (400 MHz,
Chem Biol Drug Des 2014
3

Zhong et al.
6.56-6.72 (m, 3H, C2,5,6-ArH), 7.35 (brs, 1H, NH), 8.29
(brs, 1H, NH), 9.47 (brs, 1H, NH). ESI-MS: 344.1 ([M+H]+).
Anal. calc. for C15H19ClFN3O3: C 52.41, H 5.57, N 12.22;
found: C 52.38, H 5.63, N 12.37.
age and weight have been selected to minimize biological.
The animals were approved by the Animal Care Commit-
tee of Wuhan University and purchased from Wuhan Uni-
versity Laboratory Animal Center (Wuhan, China). The
tested compounds were injected intraperitoneally to mice
as a suspension in 0.5% methylcellulose at dose of 30,
100, and 300 mg/kg to one to four mice. The anticonvul-
sant activity of the tested compounds was evaluated by
two models, namely MES and scPTZ models. Phenytoin
and ethosuximide were used as the standard drugs for the
comparison. The neurological toxicity was determined in
the rotorod test. Procedures employed for evaluation of
anticonvulsant activity and neurotoxicity were described
elsewhere (14).
Ethyl 2,2-dimethyl-1-(2-p-
tolylhydrazinecarboxamido)
cyclopropanecarboxylate (6l)
Yield: 88%. White solid. Mp: 122–123 °C. IR (KBr,
cmÀ1): 3374 (N-H), 1743 (C=O), 1688 (C=O). 1H-NMR
(400 MHz, CDCl3): 0.80 (d, 1H, J = 4.88 Hz, Cpr-CH),
1.15 (s, 6H, 2CH3), 1.21 (t, 3H, J = 7.14 Hz, CH3), 1.52
(d, 1H, J = 4.88 Hz, Cpr-CH), 2.89(s, 3H, Ar-CH3), 4.08
(q, 2H, J = 7.14 Hz, CH2), 7.41 (brs, 1H, NH), 7.29 (d,
2H, J = 8.00 Hz, C3,5-ArH), 7.82 (d, 2H, J = 8.00 Hz,
C2,6-ArH), 8.47 (brs, 1H, NH), 9.72 (brs, 1H, NH). ESI-
MS: 306.2 ([M+H]+). Anal. calc. for C16H23N3O3: C 62.93,
H 7.59, N 13.76; found: C 62.87, H 7.61, N 13.83.
MES-maximal electroshock seizure pattern test
This activity was tested according to the method of Swin-
yard et al. (15). In experiments with mice, a 60-Hz current
of 50-mA intensity was applied through corneal electrodes
for a 0.25-second duration; the procedures caused imme-
diate hindlimb tonic extension. After 0.5 and 4.0 h of drug
administration, electroshocks were via corneal electrodes.
Absence of tonic extension suggests that the tested com-
pound was considered as positive criteria.
Ethyl 2,2-dimethyl-1-(2-(4-nitrophenyl)
hydrazinecarboxamido) cyclopropanecarboxylate
(6m)
Yield: 80%. Yellow solid. Mp: 138–139 °C. IR (KBr, cmÀ1):
3368 (N-H), 1721 (C=O), 1682 (C=O). 1H-NMR (400 MHz,
CDCl3): 0.86 (d, 1H, J = 4.88 Hz, Cpr-CH), 1.16 (s, 6H,
2CH3), 1.22 (t, 3H, J = 7.20 Hz, CH3), 1.53 (d, 1H,
J = 4.88 Hz, Cpr-CH), 4.12 (q, 2H, J = 7.20 Hz, CH2),
7.27 (brs, 1H, NH), 7.87 (d, 2H, J = 8.28 Hz, C3,5-ArH),
8.07 (d, 2H, J = 8.28 Hz, C2,6-ArH), 8.54 (brs, 1H, NH),
9.43 (brs, 1H, NH). ESI-MS: 337.2 ([M+H]+). Anal. calc. for
C15H20N4O5: C 53.56, H 5.99, N 16.66; found: C 53.62,
H 5.81, N 16.83.
Pentylenetetrazole (PTZ) induced seizure test
For the chemically induced convulsant test according to
the method of Vamecq et al. (16), pentylenetetrazole was
dissolved in sufficient 0.9% saline to allow subcutaneous
injections to mice or rat. Standard drug in this model was
ethosuximide. After 0.5 and 4.0 h of drug administration,
the failure to observe even a threshold seizure (a single
episode of clonic spasms of at least 5-second duration) is
defined as protection.
Ethyl 1-(2-(2,4-dinitrophenyl)
hydrazinecarboxamido)-2,2-
dimethylcyclopropanecarboxylate (6n)
Neurotoxicity screening
Yield: 75%. Yellow solid. Mp: 157–158 °C. IR (KBr, cmÀ1):
3388 (N-H), 1766 (C=O), 1682 (C=O). 1H-NMR (400 MHz,
CDCl3): 0.80 (d, 1H, J = 4.92 Hz, Cpr-CH), 1.16 (s, 6H,
2CH3), 1.22 (t, 3H, J = 7.14 Hz, CH3), 1.54 (d, 1H,
J = 4.92 Hz, Cpr-CH), 4.09 (q, 2H, J = 7.14 Hz, CH2),
7.28 (dd, 1H, J1 = 8.84 Hz, J2 = 2.52 Hz), 7.33 (brs, 1H,
NH), 7.46 (d, 1H, J = 2.52 Hz), 7.58 (d, 1H, J = 8.84 Hz),
8.15 (brs, 1H, NH), 9.36 (brs, 1H, NH). ESI-MS: 382.1
([M+H]+). Anal. calc. for C15H19N5O7: C 47.24, H 5.02, N
18.37; found: C 47.36, H 5.17, N 18.45.
Minimal motor impairment was measured in mice or rats
using standardized rotorod test (17). The mouse was
placed on a 1 in. diameter knurled plastic rod rotating at
6 rpm. Trained animals were given an ip injection of the
test compounds in doses of 30, 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 four trials.
Quantification studies
Anticonvulsant activity was expressed in terms of the med-
ian effective dose (ED50), that is, the dose of drug required
to produce the biological responses in 50% of animals, and
neurotoxicity was expressed as the median toxic dose
(TD50). For determination of the ED50 and TD50, groups of
10 mice were given a range of ip doses of the test drug until
at least three points were established in the range of 10–
90% seizure protection or minimal observed neurotoxicity
Pharmacology
Anticonvulsant activity assays were carried out by the
Antiepileptic Drug Development Program, Epilepsy Branch,
National Institute of Neurological and Communicative Dis-
orders and Stroke, National Institute of Health in Bethesda,
USA (13). Male Kunming mice (20 Æ 2.0 g) were used as
experimental animals in this study. Animals of the same
4
Chem Biol Drug Des 2014

Synthesis and Anticonvulsant Activity of Cyclopropanecarboxylate Derivatives
(18). From the plot of these data, the respective ED50 and
The compounds 6a-n were administrated intraperitoneally
(ip) into the mice using dose of 30, 100, and 300 mg/kg,
and the observations were taken at two different time
intervals (0.5 and 4.0 h). Neurotoxicity was measured by
the rotorod test. The calculated LogP (Clogp) values were
calculated using the software in ACD Labs 8.0 version
(Advanced Chemistry Development, Inc., Toronto, ON,
Canada). The results are shown in Table 1.
TD50 values, 95% confidence intervals, slope of the
regression line, and standard error of the slope were
calculated by means of a computer program written at
NINDS, NIH.
Results and Discussion
The synthesis of the compounds 6a-n was accomplished
according to the reaction sequence illustrated in Scheme 1.
Diethyl 2-(2-methylpropylidene)malonate 1 was obtained by
reacted isobutyraldehyde with diethyl malonate in the pres-
ence of piperidine and acetic acid. Compound 2 was
obtained after bromination of compound 2 with NBS and
AIBN in CCl4. Diethyl 2,2-dimethylcyclopropane-1,1-dicarb-
oxylate 3 was synthesized by a Michael-initiated ring clo-
The initial anticonvulsant evaluation indicated that all the
compounds were effective in ip MES and/or scPTZ
screens. In the MES test, all of the compounds showed
protection in half or more of the tested mice after 0.5 h
except 6a and 6b, indicative of their ability to prevent sei-
zure spread. Compounds which were active at 100 mg/kg
after 0.5 h in MES test included 6c, 6d, 6e, 6 h, 6i, and
6j indicative of their good ability to protect from seizure
spread at a higher dose. Among these compounds, 6e,
6 h, and 6j were also active at the same dose after 4.0 h.
This showed that these compounds have quick onset and
long duration of action at relatively higher dose. From
these series 6f, 6 g, and 6k showed anti-MES activity at
the dose of 30 mg/kg at time periods 0.5 h, the most
active compound 6k was active in the MES test both at
0.5 and 4.0 h in same dose, that was equivalent to phe-
nytoin used as reference anticonvulsant drug.
ꢀ
sure (MIRC) reaction according to R. Verhe (19–22). Then,
monoester 4 was obtained after monosaponification in a
1 N NaOH/ethanol (1.1 equiv) solution at room temperature
for 12 h (23,24). This was then converted to corresponding
acyl azide using ethyl chloroformate in the presence of N-
methylmorpholine (NMM) followed by reacting with sodium
azide in a one-pot synthesis. a-Carboethoxy isocyanate 5
was successfully generated by a Curtius reaction in situ on
heating the acyl azide in toluene solution at 75 °C. The
desired compounds 6a-n were readily obtained by reacting
isocyanate 5 with various hydrazines. Their chemical struc-
tures were characterized using 1H-NMR, MS, and elemental
analysis techniques.
The scPTZ screen showed that compounds 6c, 6e, 6f, 6 g,
6 h, 6i, and 6k were found to be active after 0.5 h or/and
4.0 h, the other derivatives devoid of anticonvulsant activity.
Compounds 6c and 6 g were active after 4.0 h at the dose
of 300 mg/kg, the other compounds showed no activity.
The anticonvulsant activity and neurotoxicity of the synthe-
sized compounds were evaluated following the standard
procedures proposed by the NIH anticonvulsant drug
development (ADD) program, via the anticonvulsant
screening project (ASP). The initial evaluation (Phase Ι )
included the maximal electroshock seizure (MES), the sub-
cutaneous pentylenetetrazole (scPTZ), and neurotoxicity.
In the neurotoxicity screen, compounds 6d, 6e, 6f, 6j, 6k,
and 6 m did not show any neurotoxicity in the maximum
dose administered (300 mg/kg). Compounds 6n revealed
neurotoxicity at a dose of 100 mg/kg. The majority of these
compounds exhibited less neurotoxic than phenytoin.
COOC2H5
COOC2H5
AcOH, piperdine, benzene
NBS, AIBN, CCl4
reflux, 4 h, 95%
COOEt
COOEt
+
CHO
COOC2H5
COOC2H5
reflux, 4 h, 80%
Br
1
2
1)EtOCOCl,NMM,NaN3,0oC
2)toluene,75oC,
NaBH4, EtOH
0oC, 1 h, 77%
1N NaOH, EtOH
r.t. 12 h, 95%
COOC2H5
COOC2H5
COOH
NCO
COOC2H5
COOC2H5
70%
3
4
5
O
RNHNH2, THF
r.t. 2 h, 80-88%
NH-C-NH-NHR
COOC2H5
R
R
6a-n
a: R=H
b:
h: R=2-ClC6H4
i: R=3-BrC6H4
R=CH3
c: R=C6H5
d: R=4-ClC6H4
e: R=4-BrC6H4
R=4-FC6H4
R=3-ClC6H4
j:
k:
R=3,5diClC6H3
R=3-Cl-4-FC6H3
l: R=4-CH
3
C6H4
R=4-NO2C6H4
R=2,4-(NO2)2C6H3
f:
g:
m:
n:
Scheme 1: General method for the synthesis of compounds 6a-n.
Chem Biol Drug Des 2014
5

Zhong et al.
Table 1: Anticonvulsant activity and neurotoxicity of compounds 6a-n administered intraperitoneally to mice
Intraperitoneal injection in micea
MESc
0.5 h
scPTZd
Neurotoxicitye
0.5 h
Compounds
4.0 h
0.5 h
4.0 h
4.0 h
Clog Pb
6a
6b
–
–
100
100
100
30
–
–
–
–
300
100
300
300
–
300
30
–
–
–
–
–
100
100
–
300
–
300
300
–
–
–
300
–
–
300
300
–
–
–
300
–
–
–
–
300
–
–
–
–
–
–
–
–
–
–
–
–
100
100
–
–
–0.64 Æ 0.28
0.42 Æ 0.58
2.17 Æ 0.58
3.16 Æ 0.59
3.34 Æ 0.62
2.62 Æ 0.62
3.20 Æ 0.59
2.68 Æ 0.58
3.38 Æ 0.62
4.25 Æ 0.60
3.76 Æ 0.63
2.63 Æ 0.58
2.63 Æ 0.59
2.82 Æ 0.60
2.52 Æ 0.38
0.38 Æ 0.46
300
300
–
–
–
300
300
300
–
–
300
–
6c
6d
6e
6f
6g
30
6h
6i
100
100
100
30
300
300
300
30
6j
6k
6l
6m
300
–
–
–
–
100
6n
Phenytoinf
Ethosuximideg
300
100
–
30
–
–
300
a30, 100, and 300 mg/kg of doses were administered ip. The figures in the table indicate the minimal dose whereby bioactivity was dem-
onstrated in half or more of the mice. The animals were examined at 0.5 and 4.0 h after injection were administered. A dash indicates an
absence of activity at maximum dose administered (300 mg/kg).
bClog P was calculated using software ACD Labs 8.0 version.
cMaximal electroshock test.
dSubcutaneous pentylenetetrazole test.
eNeurotoxicity screening (rotorod test).
fData from Ref. (29).
gData from Ref. (30).
Table 2: Phase-II quantitative anticonvulsant evaluation in mice.(test drug administered i.p.)
PIc
a
ED50
MES
b
Compound
scPTZ
TD50
MES
scPTZ
6f
22.5 (20.8–24.5)d
9.2 (5.3–11.9)
9.5 (8.1–10.4)
8.8 (5.5–14.1)
21.8 (21.8–25.5)
272 (247–338)
173.3 (161.8–198.3)
58.1 (41.4–87.1)
>300
>100
13.2 (5.8–15.9)
149 (123–177)
342.7 (319.3–366.2)
387.5 (368.2–404.5)
65.5 (52.5–72.9)
71.6 (45.9–135)
69 (62.8–72.9)
15.2
42.1
6.9
8.1
3.2
2.0
6.7
<0.22
<0.22
5.2
6k
Phenytoine
Carbamazepinee
Phenobarbitale
Valproatee
426 (369–450)
1.6
2.9
Number of animals used: 10; solvent used: polyethylene glycol (0.1 mL, i.p.).
aDose in milligrams per kilogram body mass.
bMinimal toxicity which was determined by rotorod test 30 min after the test drug was administered.
cProtection index (TD50/ED50).
dDates in parentheses are the 95% confidence limits.
eDate from references (31).
Compounds 6f and 6k were selected for quantification
of the pharmacological parameters (ED50 and TD50).
Results of the quantitative test for these compounds,
along with the data on the standard drugs (phenytoin,
carbamazepine, phenobarbital, and valproate), are
reported in Table 2. In the mice MES screen, the tested
compounds showed a higher protective index (PI) than
all the standard drugs. In the mice ip scPTZ screen,
compound 6k gave an ED50 of 58.1 mg/kg and a TD50
of 387.5 mg/kg, resulting in a high protection index (PI),
that is, TD50/ED50, of 6.7 when compared to phenobar-
bital and valproate.
The results of the preliminary anticonvulsant screening
revealed that ethyl 2,2-dimethyl-1-(2-substitutedhydrazine-
carboxamido) cyclopropanecarboxylate derivatives exhibit
a remarkable anticonvulsant activity. The structure of this
series fulfilled the pharmacophoric structural requirements,
6
Chem Biol Drug Des 2014

Synthesis and Anticonvulsant Activity of Cyclopropanecarboxylate Derivatives
that is, the hydrazinecarboxamide fragment and phenyl
ring provided the basic structural requirement for anticon-
vulsant activity (25).
for anti-epileptic drugs.
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In the present studies, we have synthesized a library of
compounds with hydrazinecarboxamide as a core frag-
ment, and at the position-N’ of urea, we have introduced
different amine substituents. Compounds 6f and 6k
showed apparent activity in MES and scPTZ screens. It is
noteworthy that the introduction of an alkyl group other
than phenyl in the hydrazinecarboxamide structure caused
a complete loss of activity (compounds 6a, 6b), and it
seems that phenyl ring plays a fundamental role in anti-
MES protection (26). The ethyl 2,2-dimethyl-1-(2-substit-
uedhydrazinecarboxamido) cyclopropanecarboxylate deriv-
atives could be regarded chemically as bioisosteres of the
cyclic acylurea moiety of phenytoin. The inhibition of elec-
trically induced seizures that is characteristic for sulfonylu-
reas and thioureas may indicate the influence of
compounds on Na+ 2 HCI- K+ cotransporter as the most
plausible mechanism of anticonvulsant action (12,27,28).
The anticonvulsant properties of ethyl 2,2-dimethyl-1-(2-
substituedhydrazinecarboxamido) cyclopropanecarboxylate
derivatives could perhaps be mediated by a similar path-
way. Further experiments in binding and electrophysiology
are clearly necessary to elucidate the mechanism of action
of these novel anticonvulsants.
11. He X., Zhong M., Zhang T., Wu W., Wu Z., Xiao Y.,
Hu X. (2012) Synthesis and anticonvulsant activity of
ethyl 1-(2-arylhydrazinecarboxamido)-2,2-dimethylcy-
clopropanecarboxylate derivatives. Eur J Med Chem;
45:542–548.
12. He X., Zhong M., Yang J., Wu Z., Xiao Y., Guo H., Hu
X. (2012) Synthesis and anticonvulsant activity of 1-(2-
(8-(benzyloxy)quinolin-2-yl)-1-butyrycyclopropyl)3-
substituted urea derivatives. Chem Biol Drug
Des;79:771–779.
13. He X., Zhong M., Zhang T., Wu W., Wu Z., Yang J.,
Xiao Y., Pang Y., Qiu G., Hu X. (2010) Synthesis and
anticonvulsant activity of N-3-arylamide substituted
5,5-cyclopropanespirohydantion derivatives. Eur J Med
Chem;45:5870–5877.
In summary, the present studies revealed that numbers of
ethyl 2,2-dimethyl -1-(2-substituedhydrazinecarboxamido)
cyclopropanecarboxylate derivatives were effective in the
MES and/or scPTZ screens. The anticonvulsant activity
depended on the kind and position of substituents at the
phenyl moiety. In the neurotoxicity studies, some of the
active compounds were devoid of toxicity. The most active
was ethyl 1-(2-(3-chloro-4-fluorophenyl)hydrazinecarbox-
amido)-2,2-dimethylcyclopropanecarboxylate (6k), which
showed ED50 value of 9.2 mg/kg and a protective index
(TD50/ED50) of 42.1 in the MES test in mice. This com-
pound showed greater ED50 and lower TD50 to standard
drugs.
14. He X., Zhong M., Zhang T., Yang J., Wu Z., Xiao Y.,
Guo H., Qiu G., Hu X. (2012) Synthesis and anticon-
vulsant activity of 1-(8-(benzyloxy)quinolin-2-yl)-6-
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Acknowledgments
This investigation was made possible through the financial
support of National Natural Science Foundation of China
(Grant No: 21302065). The authors gratefully acknowledge
the animal experiment with Associate Professor Yuling
Xiao, School of Pharmacy, Wuhan University.
16. Vamecq J., Lambert D., Poupaert J.H., Masereel B.,
Stables J.P. (1998) Anticonvulsant activity and interac-
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