Synthesis and anticonvulsant activity of N-3-arylamide substituted 5,5-cyclopropanespirohydantoin derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.09.052

In the present study on the development of new anticonvulsants, twenty new N-3-arylamide substituted 5,5-cyclopropanespirohydantoin derivatives were synthesized and tested for anticonvulsant activity using the maximal electroshock (MES), subcutaneous pentylenetetrazole (scPTZ) screens, which are the most widely employed seizure models for early identification of candidate anticonvulsants. Their neurotoxicity was determined applying the rotorod test. Three compounds 5d, 5j and 5t showed promising anticonvulsant activities in both models employed for anticonvulsant evaluation. The most active compound 5j showed the MES-induced seizures with ED₅₀ value of 9.2 mg/kg and TD₅₀ value of 421.6 mg/kg after intraperitoneally injection to mice, which provided compound 5j with a protective index (TD₅₀/ED ₅₀) of 45.8 in the MES test.

Full text of DOI:10.1016/j.ejmech.2010.09.052

European Journal of Medicinal Chemistry 45 (2010) 5870e5877
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticonvulsant activity of N-3-arylamide substituted 5,
5-cyclopropanespirohydantoin derivatives
Xianran He a, Min Zhong b, Tao Zhang a, Wen Wu a, Zhongyuan Wu a, Jin Yang a, Yuling Xiao a,
,
Yuanhu Pan a, Guofu Qiu a, Xianming Hu a
*
a State Key Laboratory of Virology, College of Pharmacy, Wuhan University, Luojiashan Road, Wuhan 430072, China
b Hubei Research Institute of Chemistry, Wuhan 430074, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study on the development of new anticonvulsants, twenty new N-3-arylamide substituted
5,5-cyclopropanespirohydantoin derivatives were synthesized and tested for anticonvulsant activity using
the maximal electroshock (MES), subcutaneous pentylenetetrazole (scPTZ) screens, which are the most
widely employed seizure models for early identification of candidate anticonvulsants. Their neurotoxicity
was determined applying the rotorod test. Three compounds 5d, 5j and 5t showed promising anticon-
vulsant activities in both models employed for anticonvulsant evaluation. The most active compound 5j
showed the MES-induced seizures with ED50 value of 9.2 mg/kg and TD50 value of 421.6 mg/kg after
intraperitoneally injection to mice, which provided compound 5j with a protective index (TD50/ED50) of
45.8 in the MES test.
Received 27 May 2010
Received in revised form
8 August 2010
Accepted 23 September 2010
Available online 1 October 2010
Keywords:
N-3-arylamide substituted
5,5-cyclopropanespirohydantoin
Anticonvulsant activity
MES test
Crown Copyright Ó 2010 Published by Elsevier Masson SAS. All rights reserved.
scPTZ test
1. Introduction
pharmacological properties, e.g., antihypertensive [9], herbicidal
[10], antitumor [11], anti-HIV [12], antibacterial [13] and antiviral
Epilepsy is a common neurologic affection characterized by
excessive temporary neuronal discharge that affects about 1% of the
world’s population [1]. Despite the availability of many antiepi-
leptic drugs (AEDs), there is still an urgent need for the develop-
ment of more effective and safer AEDs, since about 30% of epileptic
patients are not seizure-free with the existing AEDs [2]. Besides,
many AEDs such as phenobarbital, phenytoin, carbamazepine,
vigabatrin, valproate, felbamate, and lamotrigine, which are effec-
tive toward only 60e80% of patients have some undesirable side
effects, such as headache, nausea, anorexia, ataxia, hepatotoxicity,
drowsiness, gastrointestinal disturbance, gingival hyperplasia, and
hirsutism [3e6]. Thus, there is an enormous need for the devel-
opment of novel AEDs with fewer side effects and more effectives.
The SAR studies of clinically available AEDs and other anticon-
vulsant active compounds showed that most of these compounds
included 5- or 6-member cyclic imides moiety in their molecules
(Fig.1). Hydantoins, a class of cyclic imides, have been demonstrated
to possess good anticonvulsant property [7,8]. Depending on the
nature of substitution on the hydantoin ring, a wide range of other
[14] activities, have also been identified. Spirohydantoin analogs,
such as 5,5-cyclohexanespirohydantoins [15], 5,5-cycloheptanes-
pirohydantoins [15], and 5,5-cyclooctanespirohydantoins [15], have
been synthesized and shown to have anticonvulsant activity.
Previous studies from our laboratory have demonstrated the potent
anticonvulsant activity among the N-3-substituted 5,5-cyclo-
propanespirohydantoin derivatives, from which the compound 1,1-
dimethyl-6-(4-fluorophenyl)-4,6-diazaspiro[2.4]heptane-5,7-dione
showed more potent anticonvulsant activity at the dose of 7.5 mg/kg
than the standard drug phenytoin sodium [16].
In this paper, we report on the synthesis, structural character-
ization, and preliminary evaluation of the anticonvulsant
properties of a number of N-3-arylamide substituted 5,5-cyclo-
propanespirohydantoin derivatives (5aet). Compounds 5d, 5j and
5t which displayed the remarkable activity, were chosen for
quantification of the pharmacological parameters (ED50 and TD50).
It was interesting to see, compound with a small lipophilic group
like fluorine substituents at the para-position of the phenyl ring of
N-3-arylamide (5h) showed promising anticonvulsant activities,
which is in agreement with the reported SAR for spirohydantoins
[17]. It was also noteworthy that, compound with a CF3 group at
position-4 of the phenyl ring (5j) resulted in the most activity of
this series. This observation indicated that CF3 group is recognized
* Corresponding author. Tel.: þ86 27 68753532; fax: þ86 27 68754629.
E-mail address: hexianran@yahoo.com.cn (X. Hu).
0223-5234/$ e see front matter Crown Copyright Ó 2010 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.052

X. He et al. / European Journal of Medicinal Chemistry 45 (2010) 5870e5877
5871
O
O
O
O
H
N
NH
NH
NH
NH
O
O
O
NH
NH
O
O
pimidone
Phenobarbital
Ethosuximide
Phenytoin
Fig. 1. Structure of clinically available AEDs include cyclic imides moiety.
as pharmacophore and plays a significant role in new drug design,
including anticonvulsant active molecules [18]. The pharmacolog-
ical activity of various N-3-arylamide substituted 5,5-cyclopro-
pane-spirohydantoin derivatives can help better establish their
structure-activity relationship (SAR).
1H-NMR, MS and elemental analysis techniques. The detailed
physical and analytical data are listed in Section 5.
All of these compounds were prepared as racemic mixtures and
no attempt was made to resolve the enantiomers.
3. Pharmacology results and discussion
2. Chemistry
The anticonvulsant activity and neurotoxicity of the synthe-
sized compounds were evaluated following the standard proce-
dures proposed by the NIH anticonvulsant drug development
(ADD) program, via the anticonvulsant screening project (ASP).
The initial evaluation (Phase I) included the maximal electroshock
seizure (MES), the subcutaneous pentylenetetrazole (scPTZ) and
neurotoxicity.
The compounds 5aet were administrated intraperitoneally (ip)
into the mice using dose of 30, 100 and 300 mg/kg and the obser-
vations were taken at two different time intervals (0.5 h and 4.0 h).
Neurotoxicity was measured by the rotorod test. The calculated
Log P (C log p) values were calculated using the software in ACD
Labs 8.0 version. The results are shown in Table 1.
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 5p, 5q, 5r and 5s, indicative of their
ability to prevent seizure spread. Compounds which were active at
100 mg/kg after 0.5 h in MES test included 5b, 5c, 5e, 5h, 5i and 5l
The synthesis of the compounds 5aet were accomplished
according to the reaction sequence illustrated in Scheme 1. Diethyl
2,2-dimethylcyclopropane-1,1-dicarboxylate 1 was synthesized by
a Michael initiated ring closure (MIRC) reaction according to our
previous studies [19,20]. Then monoester 2 was obtained after
monosaponification in a 1 N NaOH/ethanol (1.1 equiv) solution at
room temperature for 12 h [20,21]. This was then converted to
corresponding acyl azide by using ethyl chloroformate in the
presence of N-methyl morpholine (NMM) followed by reaction
with sodium azide in a one-pot synthesis. a-Carboethoxy isocya-
nate 3 was successfully generated by a Curtius reaction in situ on
heating the acyl azide in toluene solution at 75 ꢀC. Isocyanate 3 was
allowed to react directly with various arylacethydrazide without
isolation. The desired semicarbazides 4 were readily obtained.
Finally, those semicarbazides (4aet) cyclized on treatment with
NaOH (1 equiv) in EtOH and provided N-3-arylamide substituted
spirohydantoins 5 with a cyclopropane ring in good yields (yield
ꢁ80%). Their chemical structures were characterized using
1)EtOCOCl
1N NaOH
NMM,NaN3
NCO
COOC2H5
COOC2H5
COOC2H5
COOH
COOC2H5
2)toluene,750C
EtOH
1
2
3
H
H
N
O
O
O
N
O
RCONHNH2
NHCNHNHCR
COOC2H5
Na,EtOH
r.t.
+
N
O
N
R
HN
O
O
R
4a-t
5t
5a-s
R
R
R=C6H5
R=4-CH3C6H4
R=3-CH3C6H4
a:
b:
c:
k: R=4-NO2C6H4
l:
R=4-CH3OC6H4
m:
R=3,4-diCH3OC6H4
n:
d:
e:
f:
g:
h:
i:
R=4-(CH3)3CC6H4
R=4-ClC6H4
R=2-ClC6H4
R=4-BrC6H4
R=4-FC6H4
R=2-FC6H4
R=3,4,5-triCH3OC6H4
R=4-(CH3)2NC6H4
R=2-C4H3O
o:
p:
q:
R=2-C4H3S
r: R=4-C5H4N
s:
R=3-C5H4N
R=4-CF3C6H4
j:
t: R=C6H4(CO)2N
Scheme 1. General method for the synthesis of compounds 5aet.

5872
X. He et al. / European Journal of Medicinal Chemistry 45 (2010) 5870e5877
Table 1
Anticonvulsant activity and neurotoxocity of compounds 5aet administered intraperitoneally to mice.
Compounds
Intraperitoneal injection in micea
MESc
C log Pb
scPTZd
Neurotoxocitye
0.5 h
0.5 h
4.0 h
0.5 h
4.0 h
4.0 h
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
300
100
100
30
100
300
300
100
100
30
300
100
300
300
300
e
e
300
e
e
300
e
e
1.15 ꢂ 0.69
1.61 ꢂ 0.69
1.61 ꢂ 0.69
2.84 ꢂ 0.70
1.91 ꢂ 0.70
1.32 ꢂ 0.70
2.09 ꢂ 0.73
1.37 ꢂ 0.73
0.78 ꢂ 0.74
2.12 ꢂ 0.72
1.11 ꢂ 0.70
1.32 ꢂ 0.70
1.39 ꢂ 0.70
1.34 ꢂ 0.70
1.57 ꢂ 0.70
ꢃ0.06 ꢂ 0.71
0.75 ꢂ 0.70
ꢃ0.11 ꢂ 0.69
0.08 ꢂ 0.70
0.68 ꢂ 0.82
2.52 ꢂ 0.38
0.38 ꢂ 0.46
e
300
e
300
e
e
e
e
100
300
e
100
300
300
e
e
e
e
e
e
e
e
e
300
300
300
300
e
300
300
e
300
e
300
e
300
e
e
e
30
300
e
300
300
e
300
e
e
e
e
e
e
300
300
300
e
300
e
e
300
e
e
e
100
e
300
300
300
300
300
100
30
100
e
e
e
100
100
100
100
e
300
300
300
300
e
5q
5r
5s
5t
e
e
e
e
e
e
e
e
e
30
30
300
e
300
e
Phenytoinf
Ethosuximideg
100
e
100
e
e
e
100
300
a
30,100, and 300 mg/kg of doses were administered ip. The figures in the table indicate the minimal dose whereby bioactivity was demonstrated in half or more of the mice.
The animals were examined at 0.5 h and 4.0 h after injection were administered. A dash indicates an absence of activity at maximum dose administered (300 mg/kg).
b
Clog P was calculated using software ACD Labs 8.0 version.
Maximal electroshock test.
Subcutaneous pentylenetetrazole test.
Neurotoxocity screening(rotorod test).
Data from Ref. [31].
Data from Ref. [32].
c
d
e
f
g
indicative of their good ability to protect from seizure spread at
a higher dose. Among these compounds, 5e and 5h 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 5d, 5j and 5t showed anti-MES activity at the
dose of 30 mg/kg at time periods 0.5 h, the most active compound 5j
were active in the MES test both at 0.5 h and 4.0 h that was equiv-
alent to phenytoin used as reference anticonvulsant drug.
The scPTZ screen showed that compounds 5a, 5b, 5d, 5e, 5f, 5g,
5h, 5j, 5k, 5m, 5o and 5t were found to be active after 0.5 h or/and
4.0 h, the other derivatives devoid of anticonvulsant activity.
Compounds 5b, 5g, 5j, 5m and 5t were active after 4.0 h at the
dose of 300 mg/kg, the other compounds showed no activity.
In the neurotoxicity screen, Compounds 5c, 5d, 5e, 5j, 5k, 5o and
5t did not show any neurotoxicity in the maximum dose admin-
istered (300 mg/kg). Compounds 5n, 5p, 5q, 5r and 5s revealed
neurotoxicity at a dose of 100 mg/kg. The majority of these
compounds exhibited less neurotoxic than phenytoin.
Compounds 5d, 5j and 5t were selected for quantification of the
pharmacological parameters (ED50 and TD50). Results of the quan-
titative 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 5j gave
an ED50 of 62.1 mg/kg and a TD50 of 421.6 mg/kg, resulting in a high
protection index (PI), that is, TD50/ED50, of 6.7 when compared to
phenobarbital and valproate.
i.e., the hydantoin nucleus and phenyl ring provided the basic struc-
tural requirement for anticonvulsant activity [22]. Previous studies
from our laboratory have demonstrated a good anticonvulsant activity
of N-3-substituted 5,5-cyclopropane-spirohydantoin derivatives, the
cyclopropanespirohydantoin may provided to be the pharmacophoric
group of anticonvulsant property [16].
In the present studies, we have synthesized a library of
compounds with cyclopropanespirohydantoin as a core fragment
and at the position-3 of hydantoin ring we have introduced
different arylamide substituents. The results of bioevaluation led to
an understanding of the structure-activity relationship (SAR) of
these compounds. The phenyl ring substituted with Cl or Br in para-
position (5e, 5g) lead to compounds with more anticonvulsant
activity than ortho-position (5f, 5i). On the other hand, the 4-fluoro
substituent derivative (5h) was more potent and less neurotoxic
than chloro, bromo and methyl derivatives. It may be proved that
substitution of a small lipophilic group like fluorine at the para-
position of the phenyl ring of this type of compounds resulted in
increased activity. The inclusion of more methoxy groups to phenyl
ring (5l, 5m, 5n) resulted in compounds have long duration of
action at relatively higher dose (300 mg/kg). Furthermore,
comparison of results obtained previously for the N-3-substituted
5,5-cyclopropanespirohydantoins and compounds described
herein, which may be showed that introduction of amide between
cyclopropanespirohydantoin nucleus and the aromatic ring did not
influence the anticonvulsant activity. It was noteworthy that the
introduction of an aryl group other than phenyl in the 5,5-cyclo-
propanespirohydantoin structure had caused a significant reduc-
tion or the complete loss of activity (compounds 5p, 5q, 5r, 5s), it
seems that phenyl ring play a fundamental role in anti-MES
protection [23]. It was also interested to note that compound 5j
with CF3 group in para-position of phenyl ring exhibited the most
The results of the preliminary anticonvulsant screening revealed
that N-3-arylamide substituted 5,5-cyclopropanespirohydantoin
derivatives exhibit a remarkable anticonvulsant activity. The structure
of this series fulfilled all the pharmacophoric structural requirements,

X. He et al. / European Journal of Medicinal Chemistry 45 (2010) 5870e5877
5873
Table 2
Phase-II quantitative anticonvulsant evaluation in mice.(test drug administered i.p.).
a
b
Compound
ED50
MES
TD50
PIc
scPTZ
MES scPTZ
5d
5j
5t
25.3 (22.8e29.5)d
9.2 (5.4e12.3)
13.7 (10.1e15.7)
9.5 (8.1e10.4)
8.8 (5.5e14.1)
21.8 (21.8e25.5)
272 (247e338)
208.3 (182.8e232.9)
62.1 (43.7e92.3)
136.6 (113.5e155.1)
>300
>100
13.2 (5.8e15.9)
149 (123e177)
382.3 (363.9e417.3)
421.6 (398.3e454.5)
387.2 (364.3e405.2)
65.5 (52.5e72.9)
71.6 (45.9e135)
69 (62.8e72.9)
15.5
45.8
17.8
6.9
8.1
3.2
1.8
6.7
2.8
<0.22
<0.22
5.2
Phenytoine
Carbamazepinee
Phenobarbitale
Valproatee
426 (369e450)
1.6
2.9
Number of animals used: 10; solvent used: polyethylene glycol (0.1 mL, i.p.).
a
Dose in milligrams per kilogram body mass.
Minimal toxicity which was determined by rotorod test 30 min after the test drug was administered.
Protection index (TD50/ED50).
Data in parentheses are the 95% confidence limits.
b
c
d
e
Data from Ref. [33].
activity in this series, the C log P value of 5j was also near 2.0, which
is considered to be the optimum lipophilicity for the congeners that
act on the central nervous system [24]. This observation indicated
the importance of lipophilicity as well as electronic properties of
the substituents on the activity of N-3-arylamide substituted 5,5-
cyclopropanespirohydantoin derivatives. Another noteworthy
observation was that compound 5t exhibited marked anticonvul-
sant activity in both MES and scPTZ tests. The structure of 5t
contains the two dicarboximide functions (CONRCO), which may
contribute to increase activity.
over Na2SO4 and evaporated to give product 2 (3.75 g, 90%) as
a colorless oil: 1H-NMR (400 MHz, CDCl3):
1.25 (s, 3H, CH3),1.33 (t,
d
3H, J ¼ 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 ¼ 7.20 Hz, CH2), 11.28 (br s, 1H, OH). 13C NMR
(150 MHz, CDCl3):
171.30, 181.29.
d 13.92, 20.75, 21.66, 26.94, 33.50, 38.54, 62.08,
5.1.2. General procedure for the synthesis of compounds 4aet
Compound 2 (10 mmol) was dissolved in dry THF (30 mL) and
cooled to ꢃ15 ꢀC. After the addition of EtOCOCl (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 ꢃ10 ꢀC. 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 crude acyl azide. This
crude acyl azide could be further purified by a flash column chro-
matography (PEeEtOAc, 4:1, Rf ¼ 0.7). Purified acyl azide was dis-
solved in toluene (30 mL) and the resulting solution was heated to
75 ꢀC under stirring. After gas evolution had stopped toluene was
4. Conclusions
In summary, the present studies revealed that number of N-3-
arylamide substituted 5,5-cyclopropanespirohydantoin 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 N-
(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)-4-fluo-
robenzamide (5j) which showed ED50 value of 9.2 mg/kg and
a protective index (TD50/ED50) of 45.8 in the MES test in mice. This
compound showed greater ED50 and lower TD50 to the reference
drugs.
removed under reduced pressure to afford
a-carboethoxy isocya-
nate 3 as clear oil. This –carboethoxy isocyanate 3 was directly
a
used in the next step without further purification. Arylhydrazide
(10 mmol) was added to a stirred suspension of isocyanate 3 in
appropriate solvent (40 mL) at r.t. (when highly reactive arylhy-
drazides were used, such as 4-methoxybenzhydrazide, 3,4-di
methoxybenzhydrazide and 3,4,5-tri methoxybenzhydrazide, they
should be dissolved in solvent and added dropwise). The solvent
was removed under reduced pressure when the reaction was
completed (detected by TLC) and the products 4 were purified by
a column chromatography.
5. Experimental protocols
5.1. Chemistry
All the chemicals and solvent were purchased from commercial
sources. Solvents and reagents were dried and purified according to
the literature methods. Melting points were uncorrected and
measured on an XT-4 apparatus. 1H-NMR spectra were obtained on
a Varian Mercury VX400 apparatus in DMSO-d6 and CDCl3 with TMS
as internal 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 oper-
ating at 70 eV.
5.1.2.1. Ethyl 1-(2-benzoylhydrazinecarboxamido)-2,2-dimethylcy-
clopropane carboxylate (4a). Yield: 85%. White solid. Mp:
173e174 ꢀC. 1H-NMR (400 MHz, DMSO-d6):
d 0.80 (d, 1H,
J ¼ 4.92 Hz, Cpr-CH), 1.13 (s, 6H, 2CH3), 1.17 (t, 3H, J ¼ 7.08 Hz, CH3),
1.51 (d, 1H, J ¼ 4.92 Hz, Cpr-CH), 4.06 (q, 2H, J ¼ 7.08 Hz, CH2), 7.10
(br, 1H, NH), 7.47e7.54 (m, 2H, C2,6-ArH), 7.55e7.57 (m, 1H, C4-ArH),
7.88e7.90 (m, 2H, C3,5-ArH), 7.98 (br, 1H, NH), 10.17 (br, 1H, NH).
ESI-MS: 320.2 ([M þ H]þ). Anal. calc. for C16H21N3O4: C 60.17, H
6.63, N 13.61; found: C 60.32, H 6.48, N 13.44.
5.1.1. Synthesis of 1-(ethoxycarbonyl)-2,2-
dimethylcyclopropanecarboxylic acid 2
To a solution of diethyl 2,2-dimethylcyclopropane-1,1-dicar-
boxylate 1 (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 ꢄ 30 mL). The combined extracts were dried
5.1.2.2. Ethyl 2,2-dimethyl-1-(2-(4-methylbenzoyl)hydrazinecarbox-
amido) cyclopropanecarboxylate (4b). Yield: 85%. White solid. Mp:
150e151 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.79 (d, 1H, J ¼ 4.00 Hz,
Cpr-CH), 1.13 (s, 6H, 2CH3), 1.18 (t, 3H, J ¼ 7.04 Hz, CH3), 1.51 (d, 1H,
J ¼ 4.00 Hz, Cpr-CH), 2.36 (s, 3H, CH3), 4.05 (q, 2H, J ¼ 7.04 Hz, CH2),
7.08 (brs, 1H, NH), 7.28 (d, 2H, J ¼ 8.00 Hz, C3,5-ArH), 7.79 (d, 2H,
J ¼ 8.00 Hz, C2,6-ArH), 7.94 (brs, 1H, NH), 10.09 (brs, 1H, NH). ESI-

5874
X. He et al. / European Journal of Medicinal Chemistry 45 (2010) 5870e5877
MS: 334.2 ([M þ H]þ). Anal. calc. for C17H23N3O4: C 61.25, H 6.95, N
Cpr-CH), 1.13 (s, 6H, 2CH3), 1.19 (t, 3H, J ¼ 7.04 Hz, CH3), 1.51 (d, 1H,
J ¼ 4.88 Hz, Cpr-CH), 4.06 (q, 2H, J ¼ 7.04 Hz, CH2), 7.01 (brs, 1H,
NH), 7.27e7.32 (m, 2H, ArH), 7.53e7.54 (m, 1H, ArH), 7.65e7.66 (m,
1H, ArH), 8.10 (brs, 1H, NH), 9.99 (brs, 1H, NH). ESI-MS: 338.2
([M þ H]þ). Anal. calc. for C16H20FN3O4: C 56.97, H 5.98, N 12.46;
found: C 57.12, H 6.11, N 12.23.
12.60; found: C 61.17, H 6.76, N 12.83.
5.1.2.3. Ethyl 2,2-dimethyl-1-(2-(3-methylbenzoyl)hydrazinecarbox-
amido)cyclopropanecarboxylate (4c). Yield: 85%. White solid. Mp:
87e88 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.78 (d, 1H, J ¼ 4.80 Hz,
Cpr-CH), 1.12 (s, 6H, 2CH3), 1.18 (t, 3H, J ¼ 7.04 Hz, CH3), 1.50 (d, 1H,
J ¼ 4.80 Hz, Cpr-H), 2.35 (s, 3H, CH3), 4.05 (q, 2H, J ¼ 7.04 Hz, CH2),
7.07 (brs, 1H, NH), 7.32e7.36 (m, 2H, C4,5-ArH), 7.66e7.67 (m, 1H, C6-
ArH), 7.70 (s, 1H, C2-ArH), 7.96 (brs, 1H, NH), 10.10 (brs, 1H, NH). ESI-
MS: 334.2 ([M þ H]þ). Anal. calc. for C17H23N3O4: C 61.25, H 6.95, N
12.60; found: C 61.12, H 6.77, N 12.85.
5.1.2.10. Ethyl 2,2-dimethyl-1-(2-(4-(trifluoromethyl)benzoyl)hydra-
zinecarboxamido) cyclopropanecarboxylate (4j). Yield: 80%. White
solid. Mp: 138e139 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.86 (d, 1H,
J ¼ 4.84 Hz, Cpr-CH), 1.13 (s, 6H, 2CH3), 1.18 (t, 3H, J ¼ 7.20 Hz, CH3),
1.51 (d, 1H, J ¼ 4.84 Hz, Cpr-CH), 4.05 (q, 2H, J ¼ 7.20 Hz, CH2), 7.21
(brs, 1H, NH), 7.87 (d, 2H, J ¼ 8.28 Hz, C3,5-ArH), 8.07 (d, 2H,
J ¼ 8.28 Hz, C2,6-ArH), 8.10 (brs, 1H, NH), 10.41 (brs, 1H, NH). ESI-
MS: 388.2 ([M þ H]þ). Anal. calc. for C17H20F3N3O4 C 52.71, H 5.20, N
10.85; found : C 52.72, H 5.31, N 10.81.
5.1.2.4. Ethyl 1-(2-(4-tert-butylbenzoyl)hydrazinecarboxamido)-2,2-
dimethylcyclopropanecarboxylate (4d). Yield: 80%. White solid. Mp:
115e116 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.79 (d, 3H, J ¼ 4.88 Hz,
Cpr-CH), 1.13 (s, 6H, 2CH3), 1.18 (t, 3H, J ¼ 7.04 Hz, CH3), 1.30 (s, 9H,
3CH3), 1.51 (d, 1H, J ¼ 4.88 Hz, Cpr-CH), 4.06 (q, 2H, J ¼ 7.04 Hz,
CH2), 7.08 (brs, 1H, NH), 7.48 (d, 2H, J ¼ 8.52 Hz, C3,5-ArH), 7.82 (d,
2H, J ¼ 8.44 Hz, C2,6-ArH), 7.96 (brs,1H, NH),10.10 (brs,1H, NH). ESI-
MS: 376.4 ([M þ H]þ). Anal. calc. for C20H29N3O4: C 63.98, H 7.79, N
11.19; found: C 63.81, H 7.87, N 11.43.
5.1.2.11. Ethyl 2,2-dimethyl-1-(2-(4-nitrobenzoyl)hydrazinecarbox-
amido) cyclopropanecarboxylate (4k). Yield: 75%. Yellow solid. Mp:
155e156 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.79 (d, 1H, J ¼ 4.88 Hz,
Cpr-CH), 1.13 (s, 6H, 2CH3), 1.19 (t, 3H, J ¼ 7.04 Hz, CH3), 1.51 (d, 1H,
J ¼ 4.88 Hz, Cpr-CH), 4.06 (q, 2H, J ¼ 7.04 Hz, CH2), 7.23 (brs, 1H,
NH), 8.10 (brs, 1H, NH), 8.12 (d, 2H, J ¼ 8.72 Hz, C2,6-ArH), 8.33 (d,
2H, J ¼ 8.72 Hz, C3,5-ArH), 10.51 (brs, 1H, NH). ESI-MS: 365.3
([M þ H]þ). Anal. calc. for C16H20N4O6 : C 52.74, H 5.53, N 15.38;
found : C 52.66, H 5.37, N 15.46.
5.1.2.5. Ethyl 1-(2-(4-chlorobenzoyl)hydrazinecarboxamido)-2,2-dim-
ethylcyclopropanecarboxylate (4e). Yield: 80%. White solid. Mp:
95e96 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.78 (d, 1H, J ¼ 4.88 Hz,
Cpr-CH), 1.12 (s, 6H, 2CH3), 1.17 (t, 3H, J ¼ 7.08 Hz, CH3), 1.49 (d, 1H,
J ¼ 4.88 Hz, Cpr-CH), 4.05 (q, 2H, J ¼ 7.08 Hz, CH2), 7.15 (brs, 1H, NH),
7.56 (d, 2H, J ¼ 8.48 Hz, C3,5-ArH), 7.89 (d, 2H, J ¼ 8.48 Hz, C2,6-ArH),
8.00 (brs,1H, NH),10.25 (brs,1H, NH). ESI-MS: 354.2 ([M þ H]þ). Anal.
calc. for C16H20ClN3O4: C 54.32, H 5.70, N 11.88; found: C 54.53, H 5.61,
N 11.95.
5.1.2.12. Ethyl 1-(2-(4-methoxybenzoyl)hydrazinecarboxamido)-2,2-
dimethyl cyclopropanecarboxylate (4l). Yield: 85%. White solid. Mp:
153e154 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.79 (d, 1H,
J ¼ 4.92 Hz, Cpr-CH), 1.13 (s, 6H, 2CH3), 1.19 (t, 3H, J ¼ 7.06 Hz,
CH3), 1.49 (d, 1H, J ¼ 4.92 Hz, Cpr-CH), 3.81 (s, 3H, OCH3), 4.02 (q,
2H, J ¼ 7.06 Hz, CH2), 5.37 (brs, 1H, NH), 7.00 (d, 2H, J ¼ 8.88 Hz,
C3,5-ArH), 7.86 (d, 2H, J ¼ 8.88 Hz, C2,6-ArH), 10.03 (brs, 1H, NH),
11.72 (brs, 1H, NH). ESI-MS: 350.1 ([M þ H]þ). Anal. calc. for
C17H23N3O5 : C 58.44, H 6.64, N 12.03; found : C 58.62, H 6.52, N
12.22.
5.1.2.6. Ethyl 1-(2-(2-chlorobenzoyl)hydrazinecarboxamido)-2,2-dim-
ethyl cyclopropanecarboxylate (4f). Yield: 85%. White solid. Mp:
113e114 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.80 (d, 1H, J ¼ 4.92 Hz,
Cpr-CH), 1.13 (s, 3H, 2CH3), 1.17 (t, 3H, J ¼ 7.04 Hz, CH3), 1.52 (d, 1H,
J ¼ 4.92 Hz, Cpr-CH), 4.06 (q, 2H, J ¼ 7.04 Hz), 6.95 (brs, 1H, NH),
7.39e7.47 (m, 5H, ArH), 8.13 (brs, 1H, NH), 10.07 (brs, 1H, NH). ESI-
MS: 354.2 ([M þ H]þ). Anal. calc. for C16H20ClN3O4: C 54.32, H 5.70,
N 11.88; found: C 54.42, H 5.51, N 11.93.
5.1.2.13. Ethyl 1-(2-(3,4-dimethoxybenzoyl)hydrazinecarboxamido)-
2,2- dimethylcyclopropanecarboxylate (4m). Yield: 85%. White solid.
Mp: 134e135 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.78 (d, 1H,
J ¼ 4.80 Hz, Cpr-CH), 1.16 (s, 6H, 2CH3), 1.18 (t, 3H, J ¼ 7.00 Hz, CH3),
1.50 (d, 1H, J ¼ 4.80 Hz, Cpr-CH), 3.80 (s, 6H, 2OMe), 4.05 (q, 2H,
J ¼ 7.00 Hz, CH2), 7.01e7.07 (m, 2H, C4,5-ArH), 7.47 (brs, 1H, NH),
7.49e7.51 (m, 1H, C2-ArH), 7.92 (brs, 1H, NH), 10.04 (brs, 1H, NH).
ESI-MS: 380.1 ([M þ H]þ). Anal. calc. for C18H25N3O6 : C 56.98, H
6.64, N 11.08; found : C 56.82, H 6.42, N 11.12.
5.1.2.7. Ethyl 1-(2-(4-bromobenzoyl)hydrazinecarboxamido)-2,2-dim-
ethyl cyclopropanecarboxylate (4g). Yield: 82%. White solid. M.p.
101e102 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.77 (d, 1H, J ¼ 4.84 Hz,
Cpr-CH), 1.12 (s, 6H, 2CH3), 1.17 (t, 3H, J ¼ 7.12 Hz, CH3), 1.49 (d, 1H,
J ¼ 4.84 Hz, Cpr-CH), 4.05 (q, 2H, J ¼ 7.12 Hz, CH2), 7.15 (brs, 1H, NH),
7.69 (d, 2H, J ¼ 8.44 Hz, C3,5-ArH), 7.82 (d, 2H, J ¼ 8.44 Hz, C2,6-ArH),
8.00 (brs, 1H, NH), 10.25 (brs, 1H, NH). ESI-MS: 398.1 ([M þ H]þ).
Anal. calc. for C16H20BrN3O4: C 48.25, H 5.06, N 10.55; found: C
48.32, H 5.23, N 10.32.
5.1.2.14. Ethyl 1-(2-(3,4,5-trimethoxybenzoyl)hydrazinecarboxamido)-
2,2-dimethylcyclopropanecarboxylate (4n). Yield: 85%. White solid.
Mp: 187e188 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.78 (d, 1H,
J ¼ 4.96 Hz, Cpr-CH), 1.13 (s, 6H, 2CH3), 1.19 (t, 3H, J ¼ 7.08 Hz, CH3),
1.51 (d, 1H, J ¼ 4.96 Hz, Cpr-CH), 3.71 (s, 3H, C4-ArOCH3), 3.82 (s, 6H,
C3,5-ArOCH3), 4.06 (q, 2H, J ¼ 7.08 Hz, CH2), 7.11 (brs, 1H, NH), 7.22 (s,
2H, C2,6-ArH), 7.97 (brs, 1H, NH), 10.12 (brs, 1H, NH). ESI-MS: 410.1
([M þ H]þ). Anal. calc. for C19H27N3O7 : C 55.74, H 6.65, N 10.26;
found : C 55.43, H 6.43, N 10.37.
5.1.2.8. Ethyl 1-(2-(4-fluorobenzoyl)hydrazinecarboxamido)-2,2-dim-
ethyl cyclopropanecarboxylate (4h). Yield: 83%. White solid. Mp:
169e170 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.78 (d, 1H, J ¼ 4.68 Hz,
Cpr-CH), 1.12 (s, 6H, 2CH3), 1.17 (t, 3H, J ¼ 7.08 Hz, CH3), 1.50 (d, 1H,
J ¼ 4.68 Hz, Cpr-CH), 4.05 (q, 2H, J ¼ 7.08 Hz), 7.13 (brs, 1H, NH),
7.29e7.33 (m, 2H, C3,5-ArH), 7.93e7.97 (m, 2H, C2,6-ArH), 7.99 (brs,
1H, NH),10.18 (brs,1H, NH). ESI-MS: 338.3 ([M þ H]þ). Anal. calc. for
C16H20FN3O4: C 56.97, H 5.98, N 12.46; found: C 57.02, H 6.13, N
12.38.
5.1.2.15. Ethyl
1-(2-(4-(dimethylamino)benzoyl)hydrazinecarbox-
amido)-2,2-dimethylcyclopropanecarboxylate
(4o). Yield:
77%.
White solid. Mp: 186e187 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.78
(d, 1H, J ¼ 4.92 Hz, Cpr-CH), 1.12 (s, 6H, 2CH3), 1.18 (t, 3H, J ¼ 7.04 Hz,
CH3), 1.50 (d, J ¼ 4.92 Hz, Cpr-CH), 2.97 (s, 6H, 2CH3), 6.70 (d, 2H,
5.1.2.9. Ethyl 1-(2-(2-fluorobenzoyl)hydrazinecarboxamido)-2,2-dim-
ethyl cyclopropanecarboxylate (4i). Yield: 88%. White solid. Mp:
117e118 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.79 (d, 1H, J ¼ 4.88 Hz,
J ¼ 8.92 Hz, C3,5-2H), 6.99 (brs, 1H, NH), 7.75 (d, 2H, J ¼ 8.80 Hz, C2,6
-
2H), 7.82 (brs, 1H, NH), 9.81 (brs, 1H, NH). ESI-MS: 363.4 ([M þ H]þ).

X. He et al. / European Journal of Medicinal Chemistry 45 (2010) 5870e5877
5875
Anal. calc. for C18H26N4O4: C 59.65, H 7.23, N 15.46; found : C 59.42,
H 7.12, N 15.29.
(m, 2H, C3,5-ArH), 9.76 (br, 1H, NH). ESI-MS: 274.1 ([M þ H]þ). Anal.
calc. for C14H15N3O3 : C 61.53, H 5.53, N 15.38; found : C 61.62, H
5.42, N 15.58.
5.1.2.16. Ethyl 1-(2-(furan-2-carbonyl)hydrazinecarboxamido)-2,2-
dimethylcyclopropane carboxylate (4p). Yield: 80%. White solid. Mp:
120e121 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.79 (d, 1H, J ¼ 4.96 Hz,
Cpr-CH), 1.13 (s, 6H, 2CH3), 1.17 (q, 3H, J ¼ 7.08 Hz, Me), 1.49 (d, 1H,
J ¼ 4.96 Hz, Cpr-CH), 4.05 (q, 2H, J ¼ 7.08 Hz, CH2), 6.64 (m, 1H, C4-
ArH), 7.04 (brs, 1H, NH), 7.21 (brs, 1H, NH), 7.87e7.88 (m, 1H, C3-
ArH), 7.89e7.90 (m, 1H, C5-ArH), 10.03 (brs, 1H, NH). ESI-MS: 310.2
([M þ H]þ). Anal. calc. for C14H19N3O5 : C 54.36, H 6.19, N 13.58;
found : C 54.61, H 6.27, N 13.69.
5.1.3.2. N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)-
4-methylbenzamide(5b). Yield: 88%. White solid. Mp: 132e133 ꢀC.
1H-NMR (400 MHz, DMSO-d6): 1.18 (d, 1H, J ¼ 4.84 Hz, Cpr-CH), 1.12
(s, 3H, CH3), 1.31 (s, 3H, CH3), 1.41 (d, 1H, J ¼ 4.84 Hz, Cpr-CH), 2.36
(s, 3H, Ar-CH3), 7.34 (d, 2H, J ¼ 8.02 Hz, C3,5-ArH), 7.88 (d, 2H,
J ¼ 8.02 Hz, C2,6-ArH), 8.66 (brs, 1H, NH), 10.42 (brs, 1H, NH). ESI-
MS: 288.1 ([M þ H]þ). Anal. calc. for C15H17N3O3 : C 62.71, H 5.96, N
14.63; found : C 62.92, H 5.82, N 14.43.
5.1.2.17. Ethyl 2,2-dimethyl-1-(2-(thiophene-2-carbonyl)hydrazine-
carboxamido)-cyclopropanecarboxylate (4q). Yield: 77%. White
solid. Mp: 180e181 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.79 (d, 1H,
J ¼ 4.96 Hz, Cpr-CH), 1.13 (s, 6H, 2CH3), 1.18 (t, 3H, J ¼ 7.04 Hz, CH3),
1.50 (d, 1H, J ¼ 4.96 Hz, Cpr-CH), 4.03 (q, 2H, J ¼ 7.04 Hz, CH2), 7.08
5.1.3.3. N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)-
3-methylbenzamide (5c). Yield: 90%. White solid. Mp: 115e116 ꢀC.
1H-NMR (400 MHz, DMSO-d6): 1.17 (d,1H, J ¼ 4.44 Hz, Cpr-CH),1.25
(s, 3H, CH3), 1.30 (s, 3H, CH3), 1.38 (d, 1H, J ¼ 4.44 Hz, Cpr-CH), 2.34
(s, 3H, CH3), 7.41e7.47 (m, 2H, ArH), 7.66e7.71 (m, 2H, ArH), 8.78
(brs, 1H, NH), 10.94 (brs, 1H, NH). 1ESI-MS: 288.1 ([M þ H]þ). Anal.
calc. for C15H17N3O3 : C 62.71, H 5.96, N 14.63; found : C 62.86, H
5.72, N 14.54.
(brs, 1H, NH), 7.17e7.19 (m, 1H, C4-ArH), 7.81e7.82 (m, 2H, C2,5
-
ArH), 8.00 (brs, 1H, NH), 10.19 (brs, 1H, NH). ESI-MS: 326.2
([M þ H]þ). Anal. calc. for C14H19N3O4 S: C 51.68, H 5.89, N 12.91;
found : C 51.43, H 6.07, N 13.14.
5.1.3.4. 4-tert-butyl-N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]
heptan-6-yl)-benzamide (5d). Yield: 84%. White solid. Mp:
169e170 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 1.16 (d, 1H, J ¼ 4.92 Hz,
Cpr-CH), 1.26 (s, 3H, CH3), 1.30 (s, 3H, CH3), 1.32 (s, 9H, 3CH3), 7.48
(d, 2H, J ¼ 8.52 Hz, C3,5-ArH), 7.81 (d, 2H, J ¼ 8.52 Hz, C2,6 ¼ ArH),
8.76 (brs, 1H, NH), 10.08 (brs, 1H, NH). ESI-MS: 330.2 ([M þ H]þ).
Anal. calc. for C18H23N3O3 : C 65.63, H 7.04, N 12.76; found : C 65.45,
H 6.99, N 12.97.
5.1.2.18. Ethyl 1-(2-isonicotinoylhydrazinecarboxamido)-2,2-dime-
thylcyclopropane carboxylate (4r). Yield: 81%. White solid. Mp:
188e189 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.79 (d, 1H, J ¼ 4.96 Hz,
Cpr-CH), 1.13 (s, 6H, 2CH3), 1.17 (t, 3H, J ¼ 7.04 Hz, CH3), 1.50 (d, 1H,
J ¼ 4.96 Hz, Cpr-CH), 4.06 (q, 2H, J ¼ 7.04 Hz, CH2), 7.22 (brs, 1H,
NH), 7.78 (d, 2H, J ¼ 6.04 Hz, C3,5-ArH), 8.10 (brs, 1H, NH), 8.74 (d,
2H, J ¼ 6.04 Hz, C2,6-ArH), 10.47 (brs, 1H, NH). ESI-MS: 321.3
([M þ H]þ). Anal. calc. for C15H20N4O4 : C 56.24, H 6.29, N 17.49;
found : C 56.47, H 6.37, N 17.42.
5.1.3.5. 4-Chloro-N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]hep-
tan-6-yl)benzamide (5e). Yield: 90%. White solid. Mp: 97e98 ꢀC.
1H-NMR (400 MHz, DMSO-d6): 1.18 (d,1H, J ¼ 4.96 Hz, Cpr-CH),1.23
(s, 3H, CH3), 1.30 (s, 3H, CH3), 1.37 (d, 1H, J ¼ 4.96 Hz, Cpr-CH), 7.65
(d, 2H, J ¼ 8.56 Hz, C3,5-ArH), 7.93 (d, 2H, J ¼ 8.56 Hz, C2,6-ArH), 8.81
(brs, 1H, NH), 11.12 (brs, 1H, NH). ESI-MS: 308.3 ([M þ H]þ). Anal.
calc. for C14H14ClN3O3 : C 54.64, H 4.59, N 11.52; found : C 54.53, H
4.64, N 11.77.
5.1.2.19. Ethyl 2,2-dimethyl-1-(2-nicotinoylhydrazinecarboxamido)
cyclopropane carboxylate (4s). Yield: 79%. White solid. Mp:
178e179 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.79 (d, 1H, J ¼ 4.84 Hz,
Cpr-CH), 1.13 (s, 6H, 2CH3), 1.18 (t, 3H, J ¼ 7.04 Hz, CH3), 1.51 (d, 1H,
J ¼ 4.84 Hz, Cpr-CH), 4.06 (q, 2H, J ¼ 7.04 Hz, CH2), 7.22 (brs, 1H,
NH), 7.51e7.56 (m, 1H, ArH), 8.06 (s, 1H, ArH), 8.21e8.23 (m, 1H,
ArH), 8.72e8.73 (m, 1H, ArH), 9.03 (brs,1H, NH), 10.36 (brs,1H, NH).
ESI-MS: 321.3 ([M þ H]þ). Anal. calc. for C15H20N4O4 : C 56.24, H
6.29, N 17.49; found : C 56.44, H 6.34, N 17.62.
5.1.3.6. 2-Chloro-N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]hep-
tan-6-yl)benzamide (5f). Yield: 85%. White solid. Mp: 110e111 ꢀC.
1H-NMR (400 MHz, DMSO-d6): 1.15 (d,1H, J ¼ 4.92 Hz, Cpr-CH),1.23
(s, 3H, CH3), 1.31 (s, 3H, CH3), 1.36 (d, 1H, J ¼ 4.92 Hz, Cpr-CH),
7.44e7.58 (m, 5H, ArH), 8.80 (brs, 1H, NH), 10.94 (brs, 1H, NH). ESI-
MS: 308.3 ([M þ H]þ). Anal. calc. for C14H14ClN3O3 : C 54.64, H 4.59,
N 11.52; found : C 54.41, H 4.79, N 11.75.
5.1.2.20. Ethyl 1-(3-(1,3-dioxoisoindolin-2-yl)ureido)-2,2-dimethylcy-
clopropane carboxylate (4t). Yield: 82%. White solid. Mp:
188e189 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 0.81 (d, J ¼ 4.72 Hz, Cpr-
CH), 1.12 (s, 6H, 2CH3), 1.21 (t, 3H, J ¼ 7.06 Hz, CH3), 1.48 (d, 1H,
J ¼ 4.72 Hz, Cpr-CH), 4.06 (q, 2H, J ¼ 7.06 Hz, CH2), 7.62 (brs, 1H, NH),
7.81e7.91 (m, 4H, ArH), 8.58 (brs, 1H, NH). ESI-MS: 346.3 ([M þ H]þ).
Anal. calc. for C17H19N3O5: C 59.12, H 5.55, N 12.17; found : C 59.34, H
5.77, N 12.08.
5.1.3.7. 4-Bromo-N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]hep-
tan-6-yl)benzamide (5g). Yield: 85%. White solid. Mp: 101e102 ꢀC.
1H-NMR (400 MHz, DMSO-d6): 0.91 (d, 1H, J ¼ 4.88 Hz, Cpr-CH),
1.12 (s, 3H, CH3), 1.17 (s, 3H, CH3), 1.38 (d, 1H, J ¼ 4.88 Hz, Cpr-CH),
7.69 (d, 2H, J ¼ 8.52 Hz, C3,5-ArH), 7.78 (d, 2H, J ¼ 8.52 Hz, C2,6-ArH),
8.80 (brs, 1H, NH), 10.41 (brs, 1H, NH). ESI-MS: 352.2 ([M þ H]þ).
Anal. calc. for C14H14BrN3O3 : C 47.74, H 4.01, N 11.93; found : C
47.55, H 3.98, N 12.13.
5.1.3. General procedure for the synthesis of compounds 5aet
To a solution of compound 4 (1 mmol) and EtOH (10 mL) was
added sodium (1.2 mmol), and the reaction mixture was stirred at
room temperature for 2 h. EtOH was removed under reduced
pressure. Ethyl acetate and water were added to the residue. The
ethyl acetate layer was dried over anhydrous Na2SO4, concentrated,
and recrystallized to give compound 5.
5.1.3.8. N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)-
4-fluorobenzamide (5h). Yield: 88%. White solid. Mp: 109e110 ꢀC.
1H-NMR (400 MHz, DMSO-d6): 1.16 (d,1H, J ¼ 5.56 Hz, Cpr-CH),1.25
(s, 3H, CH3), 1.30 (s, 3H, CH3), 1.37 (d, 1H, J ¼ 5.56 Hz, Cpr-CH),
7.39e7.43 (m, 2H, C3,5-ArH), 7.98e8.01 (m, 2H, C2,6-ArH), 8.80 (brs,
1H, NH), 11.05 (brs, 1H, NH). ESI-MS: 292.3 ([M þ H]þ). Anal. calc.
for C14H14FN3O3 : C 57.73, H 4.84, N 14.43; found : C 57.65, H 4.62, N
14.63.
5.1.3.1. N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)
benzamide (5a). Yield: 91%. White solid. Mp: 125e126 ꢀC. 1H-NMR
(400 MHz, CDCl3): 1.19 (d, 1H, J ¼ 3.12 Hz, Cpr-H), 1.23 (s, 3H, CH3),
1.27 (s, 3H, CH3), 1.43 (d, 1H, J ¼ 3.12 Hz, Cpr-H), 7.28e7.35 (m, 2H,
C2,6-ArH), 7.43e7.47 (m, 1H, C4-ArH), 7.75 (br, 1H, NH), 7.80e7.82

5876
X. He et al. / European Journal of Medicinal Chemistry 45 (2010) 5870e5877
5.1.3.9. N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)-
2-fluorobenzamide (5i). Yield: 85%. White solid. Mp: 83e84 ꢀC. 1H-
NMR (400 MHz, DMSO-d6): 1.15 (d, 1H, J ¼ 5.52 Hz, Cpr-CH), 1.24 (s,
3H, CH3),1.31 (s, 3H, CH3),1.36 (d,1H, J ¼ 5.52 Hz, Cpr-CH), 7.41e7.43
(m, 2H, ArH), 7.99e8.02 (m, 2H, ArH), 8.83 (brs, 1H, NH), 11.12 (brs,
1H, NH). ESI-MS: 292.3 ([M þ H]þ). Anal. calc. for C14H14FN3O3 : C
57.73, H 4.84, N 14.43; found : C 57.81, H 4.74, N 14.55.
1H-NMR (400 MHz, DMSO-d6): 1.14 (d, 1H, J ¼ 4.82 Hz, Cpr-CH), 1.13
(s, 3H, CH3), 1.31 (s, 3H, CH3), 1.36 (d, 1H, J ¼ 4.82 Hz, Cpr-CH),
6.67e6.68 (m, 1H, ArH), 7.86e7.87 (m, 1H, ArH), 7.95e7.06 (m, 1H,
ArH), 8.89 (brs, 1H, NH), 11.12 (brs, 1H, NH). ESI-MS: 264.2
([M þ H]þ). Anal. calc. for C12H13N3O4 : C 54.75, H 4.98, N 15.96;
found : C 54.92, H 5.24, N 16.13.
5.1.3.17. N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)
thiophene-2-carboxamide (5q). Yield: 80%. White solid. Mp:
213e214 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 1.18 (d, 1H, J ¼ 4.96 Hz,
Cpr-CH), 1.25 (s, 3H, CH3), 1.30 (s, 3H, CH3), 1.37 (d, 1H, J ¼ 4.96 Hz,
Cpr-CH), 7.25e7.27 (m, 1H, ArH), 7.89e7.90 (m, 1H, ArH), 7.94e7.95
(m, 1H, ArH), 8.80 (brs, 1H, NH), 11.03 (brs, 1H, NH). ESI-MS: 280.2
([M þ H]þ). Anal. calc. for C12H13N3O3S : C 51.60, H 4.69, N 15.04;
found : C 51.51, H 4.88, N 15.24.
5.1.3.10. N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)-
4-(trifluoromethyl)benzamide (5j). Yield: 88%. White solid. Mp:
81e82 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 1.13 (d, 1H, J ¼ 5.02 Hz,
Cpr-CH), 1.15 (s, 3H, CH3), 1.32 (s, 3H, CH3), 1.34 (d, 1H, J ¼ 5.02 Hz,
Cpr-CH), 7.88e7.90 (d, 2H, J ¼ 8.24 Hz, C3,5-ArH), 8.01e8.03 (d, 2H,
J ¼ 8.24 Hz, C2,6-ArH), 8.68 (brs, 1H, NH), 11.22 (brs, 1H, NH). ESI-
MS: 342.1 ([M þ H]þ). Anal. calc. for C15H14F3N3O3 : C 52.79, H 4.13,
N 12.31; found : C 52.83, H 4.24, N 12.44.
5.1.3.11. N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)-
4-nitrobenzamide (5k). Yield: 92%. Brown solid. Mp: 131e132 ꢀC.
1H-NMR (400 MHz, DMSO-d6): 0.91 (d,1H, J ¼ 4.76 Hz, Cpr-CH),1.14
(s, 3H, CH3), 1.16 (s, 3H, CH3), 1.38 (d, 1H, Cpr-H), 8.10 (d, 2H,
J ¼ 8.80 Hz, C2,6-ArH), 8.18 (d, 2H, J ¼ 8.80 Hz, C3,5-ArH), 8.22 (brs,
1H, NH),10.62 (brs,1H, NH). ESI-MS: 319.1 ([M þ H]þ). Anal. calc. for
C14H14N4O5 : C 52.83, H 4.43, N 17.60; found : C 52.87, H 4.34, N
17.82.
5.1.3.18. N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)
isonicotinamide (5r). Yield: 80%. White solid. Mp: 191e192 ꢀC. 1H-
NMR (400 MHz, DMSO-d6): 1.15 (d, 1H, J ¼ 4.88 Hz, Cpr-CH), 1.23 (s,
3H, CH3), 1.29 (s, 3H, CH3), 1.33 (d, 1H, J ¼ 4.88 Hz, Cpr-CH), 7.76 (d,
J ¼ 6.06 Hz, C2,6-ArH), 8.77 (d, J ¼ 6.06 Hz, C3,5-ArH), 8.89 (brs, 1H,
NH), 10.88 (brs, 1H, NH). ESI-MS: 275.1 ([M þ H]þ). Anal. calc. for
C13H14N4O3 : C 56.93, H 5.14, N 20.43; found : C 56.96, H 5.30, N
20.61.
5.1.3.12. N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)-
4-methoxybenzamide (5l). Yield: 88%. White solid. Mp: 141e142 ꢀC.
1H-NMR (400 MHz, DMSO-d6): 1.16 (d,1H, J ¼ 4.00 Hz, Cpr-CH),1.25
(s, 3H, CH3), 1.30 (s, 3H, CH3), 1.37 (d, 1H, J ¼ 4.00 Hz, Cpr-CH), 3.84
(s, 3H, OCH3), 7.09 (d, 2H, J ¼ 8.80 Hz, C3,5-ArH), 7.90 (d, 2H,
J ¼ 8.80 Hz, C2,6-ArH), 8.76 (brs, 1H, NH), 10.85 (brs, 1H, NH). ESI-
MS: 304.1 ([M þ H]þ). Anal. calc. for C15H17N3O4 : C 59.40, H 5.65, N
13.85; found : C 59.55, H 5.51, N 13.92.
5.1.3.19. N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)
nicotinamide(5s). Yield: 82%. White solid. Mp: 73e74 ꢀC. 1H-NMR
(400 MHz, DMSO-d6): 1.16 (d, 1H, J ¼ 4.82 Hz, Cpr-CH), 1.24 (s, 3H,
CH3), 1.32 (s, 3H, CH3), 1.51 (d, 1H, J ¼ 4.82 Hz, Cpr-CH), 7.52e7.56
(m, 1H, ArH), 8.04 (s, 1H, ArH), 8.22e8.24 (m, 1H, ArH), 8.73e8.75
(m, 1H, ArH), 8.78 (brs, 1H, NH), 10.92 (brs, 1H, NH). ESI-MS: 275.1
([M þ H]þ). Anal. calc. for C13H14N4O3 : C 56.93, H 5.14, N 20.43;
found : C 56.94, H 5.21, N 20.52.
5.1.3.13. N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)-
3,4-dimethoxybenzamide (5m). Yield: 80%. White solid. Mp:
95e96 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 1.17 (d, 1H, J ¼ 4.98 Hz,
Cpr-CH), 1.26 (s, 3H, CH3), 1.28 (s, 3H, CH3), 1.37 (d, 1H, J ¼ 4.98 Hz,
Cpr-CH), 3.82 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 7.11 (d, 1H,
J ¼ 8.48 Hz, C5-ArH), 7.48 (s, 1H, C2-ArH), 7.57 (d, 1H, J ¼ 8.48 Hz, C6-
ArH), 8.77 (brs, 1H, NH), 10.85 (brs, 1H, NH). ESI-MS: 334.1
([M þ H]þ). Anal. calc. for C16H19N3O5 : C 57.65, H 5.75, N 12.61;
found : C 57.77, H 5.64, N 12.77.
5.1.3.20. 2-(1,1-Dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)
isoindoline-1,3-dione (5t). Yield: 80%. White solid. Mp: 97e98 ꢀC.
1H-NMR (400 MHz, DMSO-d6): 1.15 (d, J ¼ 4.94 Hz, Cpr-CH), 1.22 (s,
3H, CH3), 1.33 (s, 3H, CH3), 1.52 (d, 1H, J ¼ 4.94 Hz, Cpr-CH),
7.83e8.01 (m, 4H, ArH), 8.88 (brs, 1H, NH). ESI-MS: 300.1
([M þ H]þ). Anal. calc. for C17H18N4O4 : C 59.64, H 5.30, N 16.37;
found : C 59.72, H 5.43, N 16.55.
5.1.3.14. N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)-
3,4,5-trimethoxybenzamide (5n). Yield: 80%. White solid. Mp:
221e222 ꢀC. 1H-NMR (400 MHz, DMSO-d6): 1.16 (d, 1H, J ¼ 4.86 Hz,
Cpr-CH), 1.26 (s, 3H, CH3), 1.30 (s, 3H, CH3), 1.36 (d, 1H, J ¼ 4.86 Hz,
Cpr-CH), 3.74 (s, 3H, OCH3), 3.85 (s, 6H, 2OCH3), 7.25 (s, 2H, ArH),
8.80 (brs, 1H, NH), 10.93 (brs, 1H, NH).ESI-MS: 364.1 ([M þ H]þ).
Anal. calc. for C17H21N3O6 : C 56.19, H 5.83, N 11.56; found : C 56.27,
H 5.63, N 11.72.
5.2. Anticonvulsant screening
Anticonvulsant activity assays were carried out by the Antiepi-
leptic Drug Development Program, Epilepsy Branch, National
Institute of Neurological and Communicative Disorders and Stroke,
National Institute of Health in Bethesda, USA [25]. Male Kunming
mice (20 ꢂ 2.0 g) and male Kunming rats (250 ꢂ 5.0 g) were used as
experimental animals in this study. Animals of the same age and
weight have been selected in order to minimize biological. The
animals were approved by the Animal Care Committee of Wuhan
University. All the animals were purchased from Wuhan University
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 anticonvulsant activity of the tested compounds were
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 [26].
5.1.3.15. N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)-
4-(dimethylamino)-benzamide (5o). Yield: 80%. White solid. Mp:
133e134 ꢀC. 1H-NMR (400 MHz, CDCl3): 1.22 (d, 1H, J ¼ 4.78 Hz,
Cpr-CH), 1.24 (s, 3H, CH3), 1.30 (s, 3H, CH3), 1.35 (d, 1H,, J ¼ 4.78 Hz,
Cpr-CH), 3.00 (s, 6H, 2CH3), 6.75 (d, 2H, J ¼ 8.80 Hz, C3,5-ArH), 7.77
(d, 2H, J ¼ 8.80 Hz, C2,4-ArH), 8.71 (brs, 1H, NH), 10.54 (brs, 1H, NH).
ESI-MS: 317.1 ([M þ H]þ).Anal. calc. for C16H20N4O3 : C 60.75, H 6.37,
N 17.71; found: C 60.54, H 6.54, N 17.63.
5.1.3.16. N-(1,1-dimethyl-5,7-dioxo-4,6-diazaspiro[2.4]heptan-6-yl)
furan-2-carboxamide (5p). Yield: 88%. White solid. Mp: 97e98 ꢀC.

X. He et al. / European Journal of Medicinal Chemistry 45 (2010) 5870e5877
5877
5.2.1. MES-maximal electroshock seizure pattern test
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electrodes. Absence of tonic extension suggests that the tested
compound was considered as positive criteria.
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Acknowledgments
This investigation was made possible through the financial
support of Key Project of Science & Technology of HuBei Province,
China (Grant NO. 2008CDA 057) and the Important National
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Science
& Technology Specific Projects, China (Grant No.
2009ZX09301-14).