Design, synthesis and pharmacological evaluation of novel N-(2-(1, 1-dimethyl-5, 7-dioxo-4, 6-diazaspiro[2.4]heptan-6-yl)ethyl) sulfonamide derivatives as potential anticonvulsant agents

Article abstract of DOI:10.1016/j.ejmech.2015.01.008

A series of new N-(2-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)ethyl) sulfonamide derivatives (8aei) and ethyl 2,2-dimethyl-1-(3-(2-(sulfonamido)ethyl)ureido) cyclopropanecarbox-ylate derivatives (9aei) were designed, synthesized and evaluate

Full text of DOI:10.1016/j.ejmech.2015.01.008

European Journal of Medicinal Chemistry 92 (2015) 370e376
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and pharmacological evaluation of novel N-(2-(1, 1-
dimethyl-5, 7-dioxo-4, 6-diazaspiro[2.4]heptan-6-yl)ethyl)
sulfonamide derivatives as potential anticonvulsant agents
Jinping Li, Jun Lou, Zhiming Wang, Ting Wang, Yuling Xiao, Xianming Hu, Peng Liu,
Xuechuan Hong*
State Key Laboratory of Virology, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, Wuhan University School of
Pharmaceutical Sciences, 430071, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new N-(2-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)ethyl) sulfonamide de-
rivatives (8aei) and ethyl 2,2-dimethyl-1-(3-(2-(sulfonamido)ethyl)ureido) cyclopropanecarbox-ylate
derivatives (9aei) were designed, synthesized and evaluated for their anticonvulsant activities using
maximal electroshock shock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure models in mice.
N-(2-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)ethyl)-4-fluoroben- zenesulfonamide (8f)
and N-(2-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)ethyl)-4- methylbenzenesulfonamide
(8e) have shown promising anticonvulsant activities in MES model. The most active compound 8f has
shown the MES-induced seizures with ED50 value of 28.05 mg/kg and TD50 value of 561 mg/kg after
intraperitoneal injection to mice, which provided compound 8f with a protective index (TD50/ED50) of 20
in the MES test. Further, rotarod toxicity method was used to study the acute neurotoxicity profile of
selected compounds.
Received 24 May 2014
Received in revised form
4 January 2015
Accepted 6 January 2015
Available online 7 January 2015
Keywords:
Anticonvulsant
MES test
scPTZ test
Sulfonamide
© 2015 Published by Elsevier Masson SAS.
1. Introduction
Cyclopropanespirohydantoins have also been synthesized and
demonstrated to have good anticonvulsant activities with less
Epilepsy is a chronic brain disorder characterized by excessive
temporary neuronal discharge and affects about 1% of the world's
population [1]. A number of anti-epileptic drugs (AEDs) such as
topiramate [2], lamotrigine [3], tiagabine [4], felbamate [5], vig-
abatrin [6], and zonisamide [7] have been introduced to treat epi-
lepsy diseases. However, 20e30% of patients are failed to control
seizures by current medications. Substantial increases in drug
resistance and undesirable side effects such as neurotoxicity of
current AEDs make it necessary to develop more efficacious anti-
epileptic and anti-epileptogenic drug along with less side effects.
Over last decade, anticonvulsant drug development has become
one of the most active research areas in medicinal chemistry.
Hydantoins show various types of biological activities [8,9].
Phenytoin, a hydantion derivative has been known for anticon-
vulsant activity since 1938. It is still prescribed as one of most
toxicity than that of phenytoin [10e12]. Those interesting biological
activities have drawn us more attention on the development of the
novel 5, 5-cyclopropanespirohydantoin as the core structure of
potential anticonvulsant drugs (Fig. 1). Acetazolamide (Fig. 1), an
old AED, containing a sulfonamide group, has displayed excellent
anticonvulsant activity [13]. It was found that N- (2, 2, 3, 3-
tetramethylcyclopropanecarboxamide)-p-phenylsulfonamide
(compound I) exhibits high potency and a wide protective index
(PI ¼ TD50/ED50) during the rat-MES seizure test [14]. Our research
mainly focuses on the synthesis and biological studies of compound
II containing a sulfone moiety [11]. As an extension, we set out to
introduce
a
sulfonamide
group
to
5,
5-
cyclo-
propanespirohydantoin, in order to enhance biological activity.
Therefore, a series of compounds (type IeIV) are designed and
synthesized as listed in Fig. 1. Unfortunately, type I and type II
compounds are not stable enough (The synthetic route of type I and
type II compounds was shown in the supporting information).
Herein, type III and type IV compounds are synthesized and their
pharmacological activities as potential anticonvulsant agents are
common
drugs
for
treating
grand
mal.
5,
5-
* Corresponding author.
E-mail address: xhy78@whu.edu.cn (X. Hong).
http://dx.doi.org/10.1016/j.ejmech.2015.01.008
0223-5234/© 2015 Published by Elsevier Masson SAS.

J. Li et al. / European Journal of Medicinal Chemistry 92 (2015) 370e376
371
Fig. 1. Structure of designed compounds and model AEDs.
evaluated in this paper.
administrated intraperitoneally (ip.) into the Kunming mice
(18e22 g) using dose of 300, 100 and 30 mg/kg and the observa-
tions were taken at 0.5 h and 4.0 h after injection and the results are
summarized in Table 1.
2. Results and discussion
As shown in Table 1, compounds 8e and 8f, which showed
protection at a dose of 30 mg/kg after 0.5 h, were found to be the
most active in the mice ip. MES screen. It's worth noting that
compound 8f protected the tested mice from seizures at a dose of
30 mg/kg after 4 h while compound 8e was active at the same time
but at a higher dose of 100 mg/kg. The data suggest that compound
8f has a long duration of anticonvulsant activity. Compounds 8b,
8d, 8h, 8i, 9b, 9e, 9f and 9i were active at 100 mg/kg after 0.5 h in
MES test. Among those compounds, 8b and 9f kept active at the
same dose after 4 h. In the scPTZ test, all of the compounds were
ineffective to protect the tested mice except 8e and 8f which were
active at a dose of 300 mg/kg after 0.5 h. Given R1 ¼ R2, it is self-
evident that N-(2-(1, 1-dimethyl-5, 7 -dioxo-4, 6-diazaspiro [2.4]
heptan- 6-yl) ethyl) sulfonamide derivatives (8aei) were more
active than that of their ring-open derivatives (9aei). For example,
compound 8f showed protection at a dose of 30 mg/kg following
0.5 h while compound 9f exhibited activity at a higher dose of
100 mg/kg. Furthermore, introduction of methyl or fluorine group
at para position of the phenyl ring gave the best anticonvulsant
activity. However, the activity was decreased when nitro substitu-
ent was on phenyl ring. In the neurotoxicity test, compounds 8e, 8h
and 9d showed neurotoxicity at the maximum dose. Compound 8d
was neurotoxic at a dose of 100 mg/kg after 0.5 h and it was also
found to reveal neurotoxicity after 4 h at a dose of 300 mg/kg. The
rest compounds did not cause any motor impairment at all.
Crossing the bloodebrain barrier (BBB) is an important factor
influencing anticonvulsant activity. It is believed that log P values
between 1 and 2 is sufficient value for crossing BBB. The optimum
compound show favorable anticonvulsant activity [21]. The Clog P
of the tested compounds is summarized in Table 1. Compounds 8a
and 8c with low ClogP values (ꢀ1.015 and ꢀ1.159 respectively)
showed no anticonvulsant activity at the max dose (300 mg/kg).
The most active compounds 8e and 8f showed optimal ClogP values
of 1.273 and 1.2194 respectively.
2.1. Chemistry
The synthetic route of compounds 8aei and 9aei was shown in
Scheme 1. Compound 2 was prepared by condensation of diethyl
malonate with isobutyraldehyde in the presence of piperidine and
acetic acid. The reaction of compound 2 with NBS and AIBN in
chloroform afforded the desired bromo compound 3. The resulting
compound 3 underwent cyclopropane formation by Michael initi-
ated ring closure (MIRC) reaction to afford compound 4. Then
monoester 5 was obtained after monohydrolysation in a 1 N NaOH/
ethanol-water solution at room temperature for 12 h. It was then
converted to corresponding acyl azide by using ethyl chloroformate
with triethylamine followed by reaction with sodium azide in a
one-pot synthesis. Compound 6 was successfully made by a Curtius
reaction. Then it was treated with hydrazine in THF to provide
compound 7. The subsequent sulfonylation of compound 7 with a
variety of different sulfonylating reagents gave the target com-
pounds 8aei. Reactions between compound 6 and a variety of
sulfonyl ethylenediamines resulted in the formation of target
compounds 9aei.
The chemical structures of the titled compounds were eluci-
dated on the basis of 1H NMR, 13C NMR and mass spectra. The
detailed physical and analytical data are listed in Section 4. All of
these compounds were prepared as racemic mixtures and no
attempt was made to resolve the enantiomers.
2.2. Anticonvulsant activity
It is highly unlikely that any animal model will ever successfully
predict the full therapeutic potential of an investigational AED due
to its highly heterogenous nature of seizure disorders in humans
and the complexity of the seizure phenotypes [15]. A number of
animal models have been used in the search for more efficacious
AEDs. For example, maximal electroshock (MES) and subcutaneous
pentylenetetrazole (scPTZ) tests are reported to detect the effects
on generalized tonic-clonic seizures and generalized absence sei-
zures, respectively [16,17].
In the present study, maximal electroshock seizure (MES),
subcutaneous pentylenetetrazole (scPTZ) were chosen in the early
stages of testing because they are recognized as a gold standard
[18]. Neurotoxicity is also determined in the minimal motor
impairment-rotarod screening. All the tested compounds were
In view of the performance of those tested compounds in initial
evaluation, compounds 8e and 8f with the highest anticonvulsant
potencies are selected for the quantification of TD50 and ED50 pa-
rameters. The estimated time of peak effect (TPE) is evaluated after
administrated intraperitoneally (ip.) into mice. The results are lis-
ted in Table 2. It is seen that compounds 8e and 8f showed a higher
protective index (PI ¼ TD50/ED50) than that of standard drug
phenytoin. All freshly synthesized cyclopropanespirohydantoins

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J. Li et al. / European Journal of Medicinal Chemistry 92 (2015) 370e376
Scheme 1. Synthesis of compounds 8aei and 9aei. Reagents and conditions: (a) Isobutyraldehyde, Piperidine, Acetic acid, Cyclohexane; (b) NBS, AIBN, Chloroform; (c) NaBH4; (d)
NaOH; (e) i: Et3N, Ethyl chloroformate, NaN3, THF; ii: Toluene; (f) Ethylenediamine.
showed much higher TD50 and ED50 than that of phenytoin. The
TD50 value of compounds 8f and 8e is 561 mg/kg and 487.6 mg/kg
respectively while the TD50 value of phenytoin is 65.5 mg/kg.
However, compounds 8e and 8f have lower TPE value than that of
phenytoin.
gel plates (E. Merck), 1H NMR spectra are obtained on a Varian
Mercury VX400 apparatus in DMSO-d6 or CDCl3 or CD3OD with
TMS as internal standard. . The liquid chromatography/mass spec-
trometry (LC/MS) spectra for the titled compounds are obtained on
Applied Biosystem/MDS-SCIEX API 2000 with Agilent HPLCs.
The general mechanisms of action of the established anticon-
vulsant agents include (1) effects on neurotransmitter action, (2)
effects on repetitive neuronal firing mechanisms, (3) effects on
neuronal networks, and (4) effects on neuronal ionic transport. The
possible mechanism related to Naþ channels for compounds 8e and
8f is tentatively proposed since all these compounds have a similar
hydantoin core as phenytoin [29,30]. Further experiments are
necessary to elucidate the mechanisms of action of novel synthe-
sized compounds in the future.
4.1.1. Synthesis of diethyl 2, 2-dimethylcyclopropane-1,1-
dicarboxylate (4)
The synthesis of compound 4 use a more environment-friendly
method developed from our previously work [23]. To a stirred so-
lution of diethyl malonate 1 (16 g, 0.1 mol) in cyclohexane (100 mL)
was added isobutyraldehyde (10.8 g, 1.5 equiv), piperidine and
acetic acid. The resulting mixture was heated refluxed for 12 h and
an oil-water separator was used to move the water produced in this
Knoevenagel condensation. The reaction was completed and
monitored by TLC. Then the mixture was cooled to room temper-
ature and was washed with water and saturated brine. The organic
phase was dried over sodium sulfate and concentrated. The ob-
tained oil (used without purification) was dissolved in 200 ml
chloroform, then 20 g N-bromo succinimide (NBS) and catalytic
amount AIBN were added to the solution. The mixture was heated
to reflux for 4 h and then cooled to room temperature and washed
with water and saturated brine. The organic phase was dried over
sodium sulfate and concentrated. The obtained oil did not need to
be purified. The ethanol solution of this oil was added to the so-
lution of 8 g NaBH4 in 250 ml EtOH in an ice bath, The resulting
mixture was stirred for 12 h, 6 N hydrochloric acid solution was
added until the pH reached at 3. The solid was removed by filtra-
tion, then the liquid was evaporated to remove ethanol. The residue
was extracted with ethyl acetate, the combine organic layer was
dried over sodium sulfate and concentrated. The crude product was
further purified by column chromatography (PE: EA, 4:1, Rf ¼ 0.9) to
give 9.2 g (yield 43% for three steps) product 4 as a colorless oil. 1H
NMR (400 M, CDCl3): 1.26 (s, 6H, eCH3), 1.29 (t, 6H, eCH2CH3), 1.43
(s, 2H, eCH2), 4.21 (q, 4H, eCH2CH3).
3. Conclusion
In conclusion, we have accomplished the design, synthesis and
pharmacological evaluation of novel N-(2-(1, 1-dimethyl-5, 7-
dioxo-4, 6-diazaspiro[2.4]heptan-6-yl)ethyl) sulfonamide de-
rivatives 8aei and its ring-open analogues 9aei as potential anti-
convulsant agents. The nature of groups in sulfonyl moiety is very
important for anticonvulsant activity. Among them, compounds 8f
and 8e have shown promising anticonvulsant activities in MES
model. The most active compound 8f has shown the MES-induced
seizures with ED50 value of 28.05 mg/kg and TD50 value of 561 mg/
kg after intraperitoneal injection to mice, which provided com-
pound 8f with a protective index (TD50/ED50) of 20 in the MES test
higher than that of standard drug phenytoin (TD50/ED50). Further
investigations regarding the precise mechanism and pharmacoki-
netic parameters in progress, and results will be reported in due
course.
4. Experimental
4.1. Chemistry
4.1.2. Synthesis of 1-(ethoxycarbonyl)-2,2-
All the chemicals and solvents are purchased from commercial
sources. Solvents and reagents are dried and purified according to
the literature methods. Melting points are uncorrected and
measured on an XT-4 apparatus. All reactions are monitored by
thin-layer chromatography (TLC) using 0.25 mm pre-coated silica
dimethylcyclopropanecarboxylic acid (5)
1 N sodium hydroxide (25 mL, 1.1 equiv, 25 mmol) was added
drop wise to a solution of diethyl 2,2-dimethylcyclopropane-1,1-
dicar-boxylate1 (4.8 g, 22 mmol) in EtOH (25 mL), and the result-
ing mixture was stirred at room temperature for 12 h. EtOH was

J. Li et al. / European Journal of Medicinal Chemistry 92 (2015) 370e376
373
Table 1
Anticonvulsant activity and neurotoxicity of compounds 8aei and 9aei administered intraperitoneally.
Compounds
Intraperitioneal injection in micea
MESc
ClogPb
scPTZd
Neurotoxocitye
0.5 h
0.5 h
4 h
0.5 h
4 h
4 h
8a
8b
8c
8d
8e
8f
8g
8h
8i
9a
9b
9c
9d
9e
9f
9g
ef
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
300
e
e
ꢀ1.015
0.572
ꢀ1.159
0.6885
1.273
1.2194
1.165
1.165
5.5055
1.3084
2.8954
1.3984
2.8169
3.4014
3.3478
3.2934
3.2934
7.1834
100
e
100
e
e
e
e
e
e
e
100
30
30
300
100
100
e
300
100
30
e
e
100
300
e
300
e
300
300
e
e
e
e
e
e
300
e
e
300
e
e
e
e
e
e
100
e
e
e
e
e
e
e
e
e
300
100
100
e
e
e
300
e
e
300
100
e
e
e
e
e
e
e
e
e
9h
9i
e
e
e
e
e
300
30
e
e
e
e
Phenytoinh
Ethosuximideg
30
e
e
100
e
100
e
e
100
a
30, 100, and 300 mg/kg of doses were administered ip. The data in the table indicate the minimal dose whereby bioactivity was demonstrated. The animals were examined
at 0.5 h and 4.0 h after injection was administered. A dash indicates an absence of activity at maximum dose administered (300 mg/kg).
b
Clog P was calculated using software ChemDraw Ultra 6.0.1.
Maximal electroshock test.
Subcutaneous pentylenetetrazole test.
Neurotoxocity screening (rotarod test).
A dash indicates the absence of anticonvulsant activity and neurotoxicity at the maximum dose administered (300 mg/kg).
Data from Ref. [19].
Data from Ref. [20].
c
d
e
f
g
h
removed under reduced pressure, water (10 mL) was added to the
residue and extracted with ethyl acetate (3 ꢁ 30 mL), and the
aqueous residue was acidified by means of a saturated KHSO4 so-
lution and extracted with ethyl acetate (3 ꢁ 30 mL). The combined
extracts were dried over Na2SO4 and evaporated to give crude
product 5 (3.75 g, 90%), The product was used directly in the next
step without further purification or purified by column chroma-
tography (PE: EA, 4:1) to give pure compound 5 as colorless oil: 1H
acarboethoxy isocyanate 6 in dry THF, the reaction mixture was
stirred for 2 h at room temperature. The solvent was then evapo-
rated under vacuum and the residue was dissolved in ethyl acetate
and washed with 1N hydrochloric acid solution and brine. The
organic phase was dried over Na2SO4 and evaporated to give an oil.
The oil was chromatographed on a silica-gel column to give 9aei
respectively.
NMR (400 MHz, CDCl3)
d
:1.25 (s, 3H, CH3),1.33 (t, 3H, J ¼ 7.20 Hz,
4.1.4.1. Ethyl 2,2-dimethyl-1-(3-(2-(methylsulfonamido)ethyl)ureido)
CH3), 1.38 (s, 3H, CH3), 1.78 (s, 1H, Cpr-H),1.85 (s, 1H, Cpr-H), 4.29 (q,
cyclopropanecarboxylate (9a). Yield: 83% (white solid), mp:
2H, J ¼ 7.20 Hz, CH2), 11.28 (br s, 1H, OH).13C NMR (101 MHz, CDCl3)
120e122 ꢂC; 1H NMR (400 MHz, CDCl3)
d 5.82 (s, 1H, NH), 5.69 (s,
d:13.9, 20.7, 21.6, 26.9, 33.5, 38.5, 62.1171.3, 181.3.
1H, NH), 5.55 (s, 1H, NH), 4.28e4.10 (q, J ¼ 7.1, 2H, eCH2CH3),
3.53e3.30 (m, 2H, eCH2CH2e), 3.24 (d, J ¼ 5.3 Hz, 2H, eCH2CH2e),
2.96 (s, 3H，eCH3), 1.73 (d, J ¼ 5.0 Hz, 1H, Cpr-CH), 1.28 (s, 3H, Cpr-
CH), 1.25 (t, J ¼ 7.1 Hz, 3H, eCH2CH3), 1.21 (s, 3H, eCH3), 0.97 (t,
4.1.3. Synthesis of ethyl 1-isocyanato-2, 2-
dimethylcyclopropanecarboxylate (6)
Compound 5 (1.86 10 mmol) was dissolved in dry THF (30 mL)
and cooled to ꢀ15 ꢂC. After the addition of EtOCOCl (11 mmol) and
NMM (12 mmol), the mixture was stirred for 20 min NaN3 (1.6 g)
was added and stirred for 1 h, The solution was then diluted with
H2O and extracted with EtOAc. The organic layers were washed
with brine, dried over Na2SO4 and concentrated under reduced
pressure to give the crude acyl azide. This crude product was pu-
rified by a flash column chromatography (PE: EA, 4:1, Rf 0.5) to give
1.4 g acyl azide. Then it was dissolved in toluene (30 mL) and the
solution was heated to 80 ꢂC under stirring. After gas evolution had
stopped, toluene was removed under reduced pressure to afford
acarboethoxy isocyanate 6 as a clear oil. This acarboethoxy isocy-
anate 6 was used directly in the next step without further
purification.
J ¼ 7.4 Hz, 1H, Cpr-CH). 13C NMR (101 MHz, CDCl3)
d: 172.6, 159.4,
61.5, 43.8, 43.2, 40.0, 28.3, 21.9, 19.9, 14.3. ESI-MS: 323 ([M þ H]þ).
4.1.4.2. Ethyl 1-(3-(2-(butylsulfonamido)ethyl)ureido)-2,2-
dimethylcyclopropanecarboxylate (9b). Yield: 85% (white solid),
mp: 52e54 ꢂC; 1H NMR (400 MHz, CDCl3)
d: 5.77 (s, 2H, NH), 5.60
(s, 1H, NH), 4.18 (q, J ¼ 7.1, 2H, eCH2CH3), 3.50e3.26 (m, 2H,
eCH2CH2e), 3.21 (dd, J ¼ 11.1, 5.5 Hz, 2H，eCH2e), 3.08e2.91 (m,
2H，eCH2e), 1.83e1.74 (m, 2H, eCH2e), 1.73 (d, J ¼ 4.9 Hz, 1H, Cpr-
CH), 1.44 (dq, J ¼ 14.8, 7.4 Hz, 2H, eCH2e), 1.27 (s, 3H, eCH3), 1.25 (t,
J ¼ 7.1 Hz, 3H, eCH2CH3), 1.21 (s, 3H，eCH3), 0.99 (d, J ¼ 5.0 Hz, 1H,
Cpr-CH), 0.94 (t, J ¼ 7.3 Hz, 3H，eCH3). 13C NMR (101 MHz, CDCl3)
d: 172.5, 159.4, 61.5, 52.2, 43.7, 43.2, 40.4, 28.4, 25.5, 21.9, 21.5, 19.9,
14.3, 13.6. ESI-MS: 365 ([M þ H]þ).
4.1.4. General produce for the synthesis of compounds (9aei)
A suitable N-(2-aminoethyl) sulfonamide (1.2eq) dissolved in
4.1.4.3. Ethyl 1-(3-(2-(N,N-dimethylsulfamoylamino)ethyl)ureido)-
2,2-dimethylcyclopropaneca-rboxylate (9c). Yield: 88% (white
solid), mp: 128e130 ꢂC; 1H NMR (400 MHz, CDCl3) 5.34 (s, 1H, NH),
anhydrous THF was slowly added to
a stirred solution of

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J. Li et al. / European Journal of Medicinal Chemistry 92 (2015) 370e376
J ¼ 7.1 Hz, 3H, eCH2CH3), 0.91 (d, J ¼ 4.9 Hz, 1H, Cpr-CH). 13C NMR
Table 2
Quantitative anticonvulsant date (Anti-MES) in mice dosed intraperitoneally.
(101 MHz, CDCl3) d: 173.0, 159.9, 153.7, 137.6, 137.6, 126.5, 110.8,
Compounds
TPE
ED50a
TD50b
PIc
61.6, 45.1, 43.3, 39.4, 28.1, 21.7, 19.9, 14.2. ESI-MS: 432 ([M þ H]þ).
8f
8e
0.5 h
0.5 h
2.0 h
28.05 (23.88e32.94)d
40.65 (37.9e43.58)
9.2 (8.1e10.4)
561 (516.2e611.2)
487.6 (425.0e559.5)
65.5 (52.5e72.9)
20
12
6.9
4.1.4.8. Ethyl 2,2-dimethyl-1-(3-(2-(3-nitrophenylsulfonamido)ethyl)
Phenytoine
ureido)cyclopropanecarbo-xylate (9h). Yield: 73% (white solid), mp:
125e127 ꢂC; 1H NMR (400 MHz, CDCl3)
d 8.70 (s, 1H, C2-ArH), 8.41
Number of animals used: 10; solvent used: polyethylene glycol (0.1 mL, ip.).
a
(d, J ¼ 8.1 Hz, 1H, C4-ArH), 8.21 (d, J ¼ 7.8 Hz, 1H, C6-ArH), 7.73 (t,
J ¼ 8.0 Hz, 1H, C5-ArH), 6.68 (s, 1H, NH), 5.57 (s, 1H, NH), 5.39 (s, 1H,
NH), 4.28e4.06 (q, J ¼ 7.1 Hz, 2H, eCH2CH3), 3.49e3.22 (m, 2H,
eCH2CH2e), 3.11 (d, J ¼ 5.0 Hz, 2H, eCH2CH2e), 1.73 (d, J ¼ 4.8 Hz,
1H, Cpr-CH),1.27 (s, 3H, eCH3),1.24 (t, J ¼ 7.1 Hz, 3H, eCH2CH3),1.21
(s, 3H, eCH3), 0.94 (t, J ¼ 4.9 Hz, 1H, Cpr-CH). 13C NMR (101 MHz,
Dose in milligrams per kilogram body mass.
b
Minimal toxicity which was determined by rotarod test 30 min after the test
drug was administered.
c
Protection index (TD50/ED50).
Data in parentheses are the 95% confidence limits.
Data from Ref. [22].
d
e
CDCl3) d: 172.4, 159.6, 148.3, 142.4, 132.7, 130.5, 126.9, 122.2, 61.7,
5.28 (s, 1H, NH), 5.26 (s, 1H, NH), 4.20e4.07 (q, J ¼ 5.7 Hz, 2H,
eCH2CH3), 3.36e3.15 (m, 2H, eCH2CH2e), 3.01 (m, 2H, eCH2CH2e),
2.71 (s, 6H), 1.71 (d, J ¼ 5.0 Hz, 1H, Cpr-CH), 1.26 (s, 3H), 1.23 (t,
J ¼ 5.7 Hz, 3H, eCH3), 1.19 (s, 3H, eCH3), 0.97 (d, J ¼ 5.0 Hz, 1H, Cpr-
44.0, 43.2, 39.9, 28.3, 21.8, 20.0, 14.3. ESI-MS: 432 ([M þ H]þ).
4 . 1 . 4 . 9 . E t h y l 2 , 2 - d i m e t h y l - 1 - ( 3 - ( 2 - ( 2 , 4 , 6 -
triisopropylphenylsulfonamido)ethyl)ureido)cyclopr-opanecarbox-
ylate (9i). Yield: 77% (white solid), mp: 128e130 ꢂC; 1H NMR
CH). 13C NMR (101 MHz, CDCl3)
d: 172.3159.2, 130.7, 129.3, 128.8,
128.7, 77.4, 77.0, 76.7, 61.5, 58.9, 44.2, 43.1, 40.6, 28.3, 21.8, 19.9, 14.3.
ESI-MS: 352 ([M þ H]þ).
(400 MHz, CDCl3)
d 7.15 (s, 2H, C3,5-ArH), 5.55 (s, 1H, NH), 5.36 (s,
1H, NH), 5.31 (s, 1H, NH), 4.14 (m, 4H, eCH2CH2e, eCH2CH3),
3.45e3.25 (m, 2H, eCH2CH2e), 3.08 (m, 5.4 Hz, 2H, eCH(CH3)2),
2.98e2.79 (m, 1H, eCH(CH3)2), 1.72 (d, J ¼ 5.0 Hz, 1H, Cpr-CH),
1.36e1.13 (m, 27H, eCH3), 0.97 (d, J ¼ 5.0 Hz, 1H, Cpr-CH). 13C
4.1.4.4. Ethyl 2,2-dimethyl-1-(3-(2-(thiophene-2-sulfonamido)ethyl)
ureido)cyclopropanecarboxy-late (9d). Yield: 85% (white solid), mp:
94e95 ꢂC; 1H NMR (400 MHz, CDCl3)
d
7.60 (dd, J ¼ 3.7, 1.3 Hz, 1H,
NMR (101 MHz, CDCl3) d: 173.7, 159.4, 152.6, 150.2, 123.8, 61.5, 43.4,
C5-ArH), 7.57 (dd, J ¼ 5.0, 1.3 Hz, 1H, C4-ArH), 7.08 (dd, J ¼ 5.0,
3.8 Hz, 1H, C3-ArH), 6.23 (s, 1H, NH), 5.58 (s, 1H, NH), 5.44 (s, 1H,
NH), 4.25e4.14 (q, J ¼ 7.1 Hz, 2H, eCH2CH3), 3.43e3.28 (m, 2H,
eCH2CH2e), 3.13 (m, 2H, eCH2CH2e), 1.69 (d, J ¼ 5.0 Hz, 1H, Cpr-
CH), 1.27 (s, 3H, eCH3), 1.24 (t, J ¼ 7.1 Hz, 3H, eCH2CH3), 1.20 (s,
3H, eCH3), 0.96 (d, J ¼ 5.0 Hz, 1H, Cpr-CH). 13C NMR (101 MHz,
43.2, 40.1, 34.1, 29.6, 24.9, 23.6, 21.8, 19.9, 14.3. ESI-MS: 501
([M þ H]þ).
4.1.5. Synthesis of 6-(2-aminoethyl)-1,1-dimethyl-4,6-diazaspiro
[2.4]heptane-5,7-dione (7)
1.8 g isocyanate was added to a stirred solution of 2 g 1,2-
ethylenediamine in 50 ml THF for 3 h at room temperature. The
solvent was removed under reduced pressure when the reaction
was completed (detected by TLC) and the products 7 (1 g) were
purified by a column chromatography. Yield: 50% (white solid).
CDCl3) d: 172.7, 159.4, 140.9, 132.0, 131.7, 127.4, 61.7, 44.2, 43.2, 39.8,
28.2, 21.8, 19.9, 14.3. ESI-MS: 391 ([M þ H]þ).
4.1.4.5. Ethyl2,2-dimethyl-1-(3-(2-(4-methylphenylsulfonamido)
1H NMR (400 MHz, CDCl3)
d
3.61 (t, J ¼ 6.2 Hz, 2H), 2.93 (t,
ethyl)ureido)cyclopropanecarbo-xylate (9e). Yield: 84% (white
solid), mp: 136e138 ꢂC; 1H NMR (400 MHz, CDCl3)
d 7.74 (d,
J ¼ 6.1 Hz, 2H), 1.46 (d, J ¼ 5.5 Hz, 1H), 1.40 (s, 3H), 1.28 (s, 3H), 1.19
(d, J ¼ 5.5 Hz, 1H). 13C NMR (101 MHz, CDCl3)
d: 168.6, 153.2, 43.2,
J ¼ 8.2 Hz, 2H, C2,6-ArH), 7.29 (d, J ¼ 8.1 Hz, 2H, C3,5-ArH), 5.79 (s,
1H, NH), 5.41 (s, 1H, NH), 5.35 (s, 1H, NH), 4.18 (q, J ¼ 7.1, 2H,
eCH2CH3), 3.32 (m, 2H, eCH2CH2e), 3.03 (m, 2H, eCH2CH2e), 2.42
(s, 3H, ArCH3), 1.72 (d, J ¼ 5.0 Hz, 1H, Cpr-CH), 1.26 (s, 3H, CH3), 1.23
(t, J ¼ 7.1 Hz, 3H, eCH2CH3), 1.20 (s, 3H, CH3), 0.95 (d, J ¼ 5.0 Hz, 1H,
36.2, 35.1, 22.2, 21.7, 16.9, 13.1.
4.1.6. General produce for the synthesis of compounds (8aei)
A sulfonyl chloride (1.2eq) was dissolved in anhydrous THF, then
this solution was slowly added to a stirred solution of the 6-(2-
aminoethyl)-1,1-dimethyl-4,6-diazaspiro[2.4]heptane-5,7 -dione 7
and triethylamine in dry dichloromethane, the reaction mixture
was stirred for 6 h at room temperature. The mixture was washed
with water and saturated brine. After drying over Na2SO4, the sol-
vent was removed under reduced pressure to give the crude
product as an oil, The oil was chromatographed on a silica-gel
column to give 8aei respectively.
Cpr-CH). 13C NMR (101 MHz, CDCl3)
d: 172.3, 159.3, 143.3, 136.7,
129.5, 126.8, 61.4, 43.8, 43.0, 39.7, 28.0, 21.7, 21.5, 19.8, 14.2. ESI-MS:
399 ([M þ H]þ).
4.1.4.6. Ethyl 1-(3-(2-(4-fluorophenylsulfonamido)ethyl)ureido)-2,2-
dimethylcyclopropanecarbo-xylate (9f). Yield: 75% (white solid),
mp: 107e109 ꢂC; 1H NMR (400 MHz, CDCl3)
d
7.88 (dd, J ¼ 8.7,
5.1 Hz, 2H, C2,6-ArH), 7.18 (t, J ¼ 8.5 Hz, 2H, C3,5-ArH), 6.03 (s, 1H,
NH), 5.40 (s, 1H, NH), 5.33 (s, 1H, NH), 4.27e4.12 (q, J ¼ 7.1, 2H,
eCH2CH3), 3.41e3.23 (m, 2H, eCH2CH2e), 3.14e2.97 (m, 2H,
eCH2CH2e), 1.73 (d, J ¼ 4.9 Hz, 1H, Cpr-CH), 1.27 (s, 3H, CH3), 1.24 (t,
J ¼ 7.1 Hz, 3H, eCH2CH3), 1.21 (s, 3H), 0.95 (d, J ¼ 5.0 Hz, 1H, Cpr-
4.1.6.1. N-(2-(1, 1-dimethyl-5, 7-dioxo-4, 6-diazaspiro[2.4]heptan-6-
yl)ethyl)methanesulfonamide (8a). Yield: 70% (White solid), mp:
138e140 ꢂC; 1H NMR (400 MHz, CDCl3)
d 6.85 (s, 1H, NH), 5.61 (s,
CH). 13C NMR (101 MHz, CDCl3)
d: 172.6166.2, 163.7, 159.4, 129.8,
1H, NH), 3.74 (d, J ¼ 5.1 Hz, 2H, eCH2CH2e), 3.41 (d, J ¼ 5.1 Hz, 2H,
129.7, 116.4, 116.2, 61.6, 43.9, 43.2, 39.8, 28.2, 21.8, 19.9, 14.3. ESI-
MS: 403 ([M þ H]þ).
eCH2CH2e), 2.95 (s, 3H, eCH3), 1.50 (d, J ¼ 5.5 Hz, 1H, Cpr-CH), 1.37
(s, 3H, eCH3), 1.27 (s, 3H, eCH3), 1.21 (d, J ¼ 5.5 Hz, 1H, Cpr-CH). 13
C
NMR (101 MHz, CDCl3) d: 173.8, 158.2, 48.6, 41.6, 40.7, 38.7, 27.8,
4.1.4.7. Ethyl 2,2-dimethyl-1-(3-(2-(4-nitrophenylsulfonamido)ethyl)
ureido)cyclopropanecarbo-xylate (9g). Yield: 60% (white solid), mp:
27.3, 22.3, 18.3. ESI-MS: 276 ([M þ H]þ).
136e138 ꢂC; 1H NMR (400 MHz, CDCl3)
d
8.06 (d, J ¼ 9.1 Hz,2H, C3,5
-
4.1.6.2. N-(2-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-
ArH), 6.52 (d, J ¼ 9.1 Hz, 2H, C2,6-ArH), 5.88 (s, 1H, NH), 5.26 (s, 1H,
NH), 5.24 (s, 1H, NH), 4.19e4.01 (q, J ¼ 7.1 Hz, 2H, eCH2CH3),
3.60e3.42 (m, 2H, eCH2CH2e), 3.29 (m, 2H, eCH2CH2e), 1.71 (d,
J ¼ 4.8 Hz, 1H, Cpr-CH), 1.26 (s, 3H, CH3), 1.19 (s, 3H, eCH3), 1.16 (t,
yl)ethyl)butane-1-sulfonamide (8b). Yield: 90% (White solid), mp:
127e129 ꢂC; 1H NMR (400 MHz, CDCl3)
d 6.72 (s, 1H, NH), 5.38 (s,
1H, NH), 3.80e3.64 (m, 2H, eCH2CH2e), 3.39 (m, 2H, eCH2CH2e),
3.04e2.93 (m, 2H, eCH2CH2CH2CH3), 1.82e1.70 (m, 2H,

J. Li et al. / European Journal of Medicinal Chemistry 92 (2015) 370e376
375
eCH2CH2CH2CH3), 1.50 (d, J ¼ 5.7 Hz, 1H, Cpr-CH), 1.44 (dd, J ¼ 14.9,
7.5 Hz, 2H, eCH2CH2CH2CH3), 1.37 (s, 3H, eCH3), 1.27 (s, 3H, eCH3),
1.20 (d, 5.7 Hz, 1H, Cpr-CH), 0.94 (t, 7.3 Hz, 3H,
eCH2CH2CH2CH3). 13C NMR (101 MHz, CDCl3)
: 173.7, 158.1, 38.9,
([M þ H]þ).
J
¼
J
¼
4.1.6.9. N-(2-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-
d
yl)ethyl)-2,4,6-triisopropylbe-nzenesulfonamide (8i). yield: 80%
27.8, 27.3, 25.6, 22.3, 21.5, 18.3, 13.6. ESI-MS: 318 ([M þ H]þ).
(White solid), mp: 180e182 ꢂC; 1H NMR (400 MHz, CDCl3)
d 7.15 (s,
2H, C3,5-ArH), 6.95 (s, 1H, NH), 5.53 (t, J ¼ 5.5 Hz, 1H, NH), 4.13 (dt,
J ¼ 13.5, 6.7 Hz, 2H, eCH(CH3)2), 3.73 (dd, J ¼ 11.0, 5.7 Hz, 2H,
eCH2CH2e), 3.27e3.14 (m, 2H, eCH2CH2e), 2.97e2.74 (m, 1H,
eCH(CH3)2), 1.49 (d, J ¼ 5.7 Hz, 1H, Cpr-CH), 1.36 (s, 3H, eCH3), 1.27
(s, 3H, eCH3), 1.25 (d, J ¼ 6.7 Hz,18H, eCH(CH3)2), 1.20 (d, J ¼ 5.7 Hz,
4.1.6.3. N-(2-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-
yl)ethyl) N,N-dimethylsulfona-mide (8c). Yield: 80% (White solid);
mp: 141e143 ꢂC; 1H NMR (400 MHz, CDCl3)
d 6.89 (s, 1H, NH), 5.27
(t, J ¼ 5.7 Hz, 1H, NH), 3.78e3.65 (m, 2H, eCH2CH2e), 3.33 (dd,
J ¼ 11.1, 5.7 Hz, 2H, eCH2CH2e), 2.78 (s, 6H, eCH3), 1.50 (d,
J ¼ 5.7 Hz,1H, Cpr-CH), 1.37 (s, 3H, eCH3), 1.28 (s, 3H, eCH3), 1.20 (d,
1H, Cpr-CH). 13C NMR (101 MHz, CDCl3)
d: 173.7, 158.4, 152.5, 150.4,
132.4, 123.7, 48.6, 41.9, 38.3, 34.1, 29.6, 27.9, 27.4, 24.9, 23.6, 22.3,
18.3. ESI-MS: 464 ([M þ H]þ).
J ¼ 5.7 Hz, 1H, Cpr-CH). 13C NMR (101 MHz, CDCl3)
d
: 173.8, 158.3,
48.6, 42.4, 38.5, 38.0, 27.8, 27.3, 22.3, 18.3. ESI-MS: 305 ([M þ H]þ).
4.2. Anticonvulsant screening
4.1.6.4. N-(2-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-
yl)ethyl)thiophene-2-sulfona-mide (8d). Yield: 80% (White sol-
The initial Anticonvulsant activity assays were carried out by the
Antiepileptic Drug Development (ADD) Program in Epilepsy
Branch, National Institute of Neurological and Communicative
Disorders and Stroke, National Institute of Health in Bethesda, USA
[25]. Male Kunming mice (18e22 g) were used as experimental
animals in this study. Animals of the same age and weight have
been selected in order to minimize biological difference. All the
animals were purchased from Wuhan University Laboratory Ani-
mal Center (Wuhan, China). The mice were housed in metabolic
case and allowed free access to food and water, The tested com-
pounds were injected intraperitoneally to mice as a suspension in
0.5% methylcellulose at dose of 300, 100, and 30 mg/kg to mice. The
anticonvulsant activity of the tested compounds were evaluated by
two models, namely, MES and scPTZ models. Ethosuximide and
Phenytoin were used as the standard drugs for the comparison. The
neurological toxicity was determined in the rotarod test. Pro-
cedures employed for evaluation of anticonvulsant activity and
neurotoxicity were described in Ref. [24].
id),mp: 160e162 ꢂC; 1H NMR (400 MHz, CDCl3)
d 7.58 (m, 2H, C4,5-
ArH), 7.08 (dd, J ¼ 4.8, 3.9 Hz, 1H, C3-ArH), 6.79 (s, 1H, NH), 5.87 (t,
J ¼ 5.3 Hz,1H, NH), 3.82e3.60 (m, 2H, eCH2CH2e), 3.31 (dd, J ¼ 10.7,
5.4 Hz, 2H, eCH2CH2e), 1.49 (d, J ¼ 5.7 Hz, 1H, Cpr-CH), 1.36 (s, 3H,
eCH3), 1.29 (s, 3H, eCH3), 1.20 (d, J ¼ 5.7 Hz, 1H, Cpr-CH). 13C NMR
(101 MHz, CDCl3) d: 173.6, 158.2, 140.9, 132.1, 131.8, 127.4, 48.6, 42.4,
38.1, 27.9, 27.4, 22.3, 18.3. ESI-MS: 344 ([M þ H]þ).
4.1.6.5. N-(2-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4] heptan-6-
yl)ethyl)-4-methylbenzenesulfo -namide (8e). Yield: 83% (White
solid), mp: 162e163 ꢂC; 1H NMR (400 MHz, CDCl3)
d 7.72 (d,
J ¼ 8.2 Hz, 2H, C2,6-ArH), 7.29 (d, J ¼ 8.1 Hz, 2H, 2H, C3,5-ArH), 6.83
(s, 1H, NH), 5.72 (t, J ¼ 5.6 Hz, 1H, NH), 3.79e3.58 (m, 2H,
eCH2CH2e), 3.20 (m, 2H, eCH2CH2e), 2.41 (s, 3H, eCH3), 1.46 (d,
J ¼ 5.7 Hz, 1H, Cpr-CH), 1.35 (s, 3H, eCH3), 1.27 (s, 3H, eCH3), 1.18 (d,
J ¼ 5.7 Hz, 1H, Cpr-CH). 13C NMR (101 MHz, CDCl3)
d: 173.6, 158.2,
143.4, 137.0, 129.7, 127.0, 48.6, 42.0, 38.3, 27.8, 27.3, 22.3, 21.5, 18.3.
ESI-MS: 352 ([M þ H]þ).
4.2.1. MES
4.1.6.6. N-(2-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-
In the MES screen, an electrical stimulus of 0.2 s in duration
(50 mA in mice at 60 Hz) is delivered via corneal electrodes primed
with an electrolyte solution. Protection against the spread of MES-
induced seizures was defined as that the abolition of the hind limb
tonic extension spasm [25].
yl)ethyl)-4-fluorobenzenesul-fonamide (8f). Yield: 85% (White
solid), mp: 130e132 ꢂC; 1H NMR (400 MHz, CDCl3)
d 7.86 (dd,
J ¼ 8.8, 5.1 Hz, 2H, C2,6-ArH), 7.17 (t, J ¼ 8.6 Hz, 2H C3,5-ArH), 6.83
(s, 1H, NH), 5.91 (s, 1H, NH), 3.73e3.59 (m, 2H, eCH2CH2e), 3.23 (m,
2H, eCH2CH2e), 1.47 (d, J ¼ 5.7 Hz, 1H, Cpr-CH), 1.36 (s, 3H, eCH3),
1.27 (s, 3H, eCH3), 1.19 (d, J ¼ 5.7 Hz, 1H, Cpr-CH). 13C NMR
4.2.2. Pentylenetetrazole (PTZ) induced seizure test
(101 MHz, CDCl3)
d: 173.5, 166.3, 163.7, 158.2, 129.8, 129.7, 116.5,
For the chemically induced convulsant test according to the
method described in Refs. [26], pentylenetetrazole was dissolved in
sufficient 0.9% saline to allow subcutaneous injections to mice.
Ethosuximide was chosen as Standard drug in this model. The
failure to observe even a threshold seizure (a single episode of
clonic spasms of at least 5s duration) is defined as protection.
116.2, 48.6, 41.9, 38.2, 27.9, 27.4, 22.3, 18.3. ESI-MS: 356 ([M þ H]þ).
4.1.6.7. N-(2-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-
yl)ethyl)-4-nitrobenzenesulfo-namide (8g). yield: 75% (White solid),
mp: 197e199 ꢂC, 1H NMR (400 MHz, CD3OD)
d
7.67 (d, J ¼ 8.7 Hz,
2H, C3,5-ArH), 7.32 (d, J ¼ 8.7 Hz, 2H, C2,6-ArH), 2.85 (t, J ¼ 5.3 Hz,
2H, eCH2CH2e), 2.44 (t, J ¼ 5.3 Hz, 2H, eCH2CH2e), 0.62 (d,
J ¼ 5.2 Hz, 1H, Cpr-CH), 0.61 (s, 3H, eCH3), 0.51 (s, 3H, eCH3), 0.41
4.2.3. Neurotoxicity screening
Minimal motor impairment was measured in mice by using
standardized rotarod test [27]. The mouse was placed on a 1 in
diameter knurled plastic rod rotating at 6 rpm. The acute motor
impairment can be demonstrated by the inability of the animal to
maintain equilibrium for 1 min.
(d, J ¼ 5.2 Hz, 1H, Cpr-CH). 13C NMR (101 MHz, CD3OD)
d: 175.4,
159.1, 129.3, 125.5, 41.6, 39.5, 28.1, 27.5, 22.2, 18.8. ESI-MS: 383
([M þ H]þ).
4.1.6.8. N-(2-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-
yl)ethyl)-3-nitrobenzenesulfo-namide (8h). yield: 86% (White solid),
4.2.4. Quantification studies
mp: 151e153 ꢂC; 1H NMR (400 MHz, CD3OD)
d
8.05 (s, 1H, C2-ArH),
Anticonvulsant activity was expressed in terms of the median
effective dose (ED50), that is, the dose of drug required to produce
the biological responses in 50% of animals, neurotoxicity was
expressed as the median toxic dose (TD50). For determination of the
ED50and 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 10e90% seizure protection or minimal observed
7.70 (d, J ¼ 8.1 Hz, 1H, C4-ArH), 7.43 (d, J ¼ 7.7 Hz, 1H, C6-ArH), 7.06
(t, J ¼ 8.0 Hz, 1H, C5-ArH), 2.86e2.72 (m, 2H, eCH2CH2e), 2.39 (t,
J ¼ 5.8 Hz, 2H, eCH2CH2e), 0.58 (d, J ¼ 5.5 Hz, 1H, Cpr-CH), 0.57 (s,
3H, eCH3), 0.47 (s, 3H, eCH3), 0.38 (d, J ¼ 5.5 Hz, 1H, Cpr-CH). 13
C
NMR (101 MHz, CD3OD) d: 175.5, 159.1, 149.8, 144.2, 133.6, 132.1,
128.0, 122.9, 41.6, 39.5, 28.1, 27.5, 22.2, 18.8. ESI-MS: 383

376
J. Li et al. / European Journal of Medicinal Chemistry 92 (2015) 370e376
Synthesis and anticonvulsant activity of 1-(8-(benzyloxy)quinolin-2-yl)-6-
substituted-4,6-diazaspiro[2,4]heptane-5,7-diones, Eur. J. Med. Chem. 48
(2012) 338e346.
neurotoxicity [28]. The ED50, TD50 and the 95% confidence interval
were calculated by the Graphpad prism 5. The PIs were calculated
by dividing the TD50 by the ED50.
[13] T.H. Maren, Carbonic anhydrase: chemistry, physiology, and inhibition,
Physiol. Rev. 47 (1967) 595e781.
[14] J.A. Shimshoni, M. Bialer, B. Yagen, Synthesis and anticonvulsant activity of
aromatic tetramethylcyclopropanecarboxamide derivatives, Bioorg. Med.
Chem. 16 (2008) 6297e6305.
Acknowledgment
[15] M.M. Castel-Branco, G.L. Alves, I.V. Figueiredo, A.C. Falcao, M.M. Caramona,
The maximal electroshock seizure (MES) model in the preclinical assessment
of potential new antiepileptic drugs, Methods Find. Exp. Clin. Pharmacol. 31
(2009) 101e106.
[16] M. Smith, K.S. Wilcox, H.S. White, Discovery of antiepileptic drugs, Neuro-
therapeutics 4 (2007) 12e17.
[17] H.S. White, Clinical significance of animal seizure models and mechanism of
action studies of potential antiepileptic drugs, Epilepsia 38 (1997) S9eS17.
[18] H.S. White, Preclinical development of antiepileptic drugs: past, present, and
future directions, Epilepsia 44 (2003) 2e8.
This work was supported by the National Mega Project on Major
Drug Development (2011ZX09401-302), the National Natural Sci-
ence Foundation of China (No. 21102107, 81273363, 81373254,
21390402) and Innovation Seed Fund of Wuhan University School
of Medicine.
Appendix A. Supplementary data
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J. Balzarini, E. DeClercq, Evaluation of the semicarbazones, thio-
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[21] H. Ucar, V.D. Kim, S. Cacciaguerra, S. Spampinato, J.P. Stables, P. Depovere,
M. Isa, B. Masereel, J. Delarge, J.H. Poupaert, Synthesis and anticonvulsant
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J. Med. Chem. 41 (1998) 1138e1145.
[22] N. Hen, M. Bialer, B. Wlodarczyk, R.H. Finnell, B. Yagen, Syntheses and eval-
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of branched aliphatic carboxylic acids with 4-aminobenzensulfonamide,
J. Med. Chem. 53 (2010) 4177e4186.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.01.008.
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