Synthesis and potential anticonvulsant activity of new 5,5-cyclopropanespirohydantoin derivatives

Article abstract of DOI:10.1007/s00044-015-1465-y

In this study, sixteen new 5,5-cyclopropanespirohydantoin derivatives were synthesized and tested for anticonvulsant activity using maximal electroshock (MES), subcutaneous pentylenetetrazole screens. Their neurotoxicity was determined by the rotarod test

Full text of DOI:10.1007/s00044-015-1465-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1465-y
ORIGINAL RESEARCH
Synthesis and potential anticonvulsant activity of new
5,5-cyclopropanespirohydantoin derivatives
1
,2
1,2
1,2
•
Yousong Nie
1
Fei Ding
•
Min Zhong
1,2
•
Yangguang Gao
3
,2
1,2
•
Hang Li
•
Yongmin Zhang
•
Xianran He
Received: 24 March 2015 / Accepted: 4 September 2015
Ó Springer Science+Business Media New York 2015
Abstract In this study, sixteen new 5,5-cyclopropanespiro-
hydantoin derivatives were synthesized and tested for anti-
convulsant activity using maximal electroshock (MES),
subcutaneous pentylenetetrazole screens. Their neurotoxicity
was determined by the rotarod test. Two compounds 7f and 7n
showed promising anticonvulsant activities in both models
employed for anticonvulsant evaluation. The most active
compound 7f showed the MES-induced seizures with ED50
value of 8.5 mg/kg and TD50 value of 381.7 mg/kg after
intraperitoneally injection to mice, which provided compound
firing (Fisher et al., 2005). Over 50 million people world-
wide are affected by epilepsy, and many of patients suffer
from epilepsy over their lifetime (Brown and Holmes,
2001; Yogeeswari et al., 2007). Despite the development of
several new antiepileptic drugs (AEDs), the treatment of
epilepsy remains still inadequate and the patients suffer
from many side effects such as headache, ataxia, hepato-
toxicity, gingival hyperplasia and nausea (Perucca et al.,
2007; Smith et al., 2007; Kwan and sander, 2004; James
and Lesley, 2005; Zaccara et al., 2007; Emilio, 1999).
The commonly used classic AEDs showed that most of
these compounds included three essential fragments for
anticonvulsant activity, namely an amide function, an
electron-donating group and an aryl group (Bojic et al.,
7
f with a protective index (TD /ED ) of 44.9 in the MES test.
50 50
Keywords 5,5-Cyclopropanespirohydantoin Á MES Á
scPTZ Á Neurotoxicity
1996; Bialer, 2006) (Fig. 1). Using the above data, our
laboratory has focused on 5,5-cyclopropanespirohydantoin
as a new anticonvulsant pharmacophore, and the previous
work exhibited some of these compounds including 5,5-
cyclopropanespirohydantoin fragment showed more potent
anticonvulsant activity than standard drug phenytoin (He
et al., 2010a, b, 2012a, b, c) (Fig. 2).
Introduction
Epilepsy is a group of long-term neurological chronic
disorders characterized by the onset of spontaneous con-
vulsant and nonconvulsant seizures that result from neu-
ronal hyperexcitability and hypersynchronous neuronal
Above these facts and in continuation of our research
program on design and synthesis of new antiepileptic agents,
the new 5,5-cyclopropanespirohydantoin derivatives have
been obtained in order to search for safer and more effective
AEDs (Fig. 3). The obtained results showed that the most
potent compounds were 7f and 7n, which were chosen for
further pharmacological screening (ED50 and TD50).
&
Xianran He
hexianran@163.com
1
Institute for Interdisciplinary Research, Jianghan University,
Wuhan Economic and Technological Development Zone,
Wuhan 430056, People’s Republic of China
2
3
Key Laboratory of Optoelectronic Chemical Materials and
Devices of Ministry of Education, Jianghan University,
Wuhan Economic and Technological Development Zone,
Wuhan 430056, People’s Republic of China
Results and discussion
The synthetic strategy for target compounds (7a–p) was
executed as outlined in Scheme 1. Compound 1 was
obtained by reacting isobutyraldehyde with diethyl
Institut Parisien de Chimie Mol e´ culaire, CNRS UMR 7201,
Universit e´ Pierre et Marie Curie-Paris 6, 4 Place Jussieu,
7
5005 Paris, France
123

Med Chem Res
Fig. 1 Structure of anticonvulsant drugs with vital structural fragments. A aryl group, B amide function, C electron-donating group
O
Me
O
N
F
F
N
H
F
O
H
N
O
OH
N
N
N
O
O
HN
S
N
O
OCH Ph
O
2
O
H
I
II
III
ED = 8.6 mg/kg
ED = 12.5 mg/kg
ED50 = 9.2 mg/kg
(MES test)
50
5
0
(
MES test)
(
MES test)
Fig. 2 Structure of compounds I, II and III
Fig. 3 Structure of title
compounds
Finally, those compounds 6a–p cyclized on treatment with
Na (1 equiv) in EtOH and provided N-3-amino-substituted
spirohydantoins 7a–p with cyclopropane ring in good
yields (yield C80 %). The compounds were checked by
1
13
TLC, H NMR, C NMR and MS.
All the experimental protocols were carried out fol-
lowing the standard procedures proposed by the NIH
anticonvulsant drug development (ADD) program, via the
anticonvulsant screening project (ASP). The initial evalu-
ation (phase I) included the maximal electroshock seizure
(MES), the subcutaneous pentylenetetrazole (scPTZ) and
neurotoxicity.
malonate in the presence of acetic acid and piperidine.
Then, bromination of compound 1 with azobisisobuty-
ronitrile (AIBN) and N-bromosuccinimide (NBS) in CCl4
afforded the desired bromo compound 2. Diethyl 2,2-
dimethylcyclopropane-1,1-dicarboxylate 3 was synthesized
by a Michael initiated ring closure (MIRC) reaction
´
according to the previous report (VERHE et al., 1978).
The compounds 7a–p were administrated intraperi-
toneally (ip) into the mice using dose of 30, 100 and
300 mg/kg, and the observations were taken at two dif-
ferent time intervals (0.5 and 4.0 h). Neurotoxicity was
measured by the rotarod test. The calculated logP (Clogp)
values were calculated using the software in ACD Labs 8.0
version. The results are shown in Table 1.
Then, monoester 4 was synthesized after monosaponifica-
tion in a 1-N NaOH/ethanol (1.1 equiv) solution at room
temperature for 12 h (He et al., 2012a, b, c). 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. The isocyanate 5 was successfully generated
by a Curtius reaction in situ on heating the acyl azide in
toluene solution at 75 °C. Isocyanate 5 was allowed to
react directly with various hydrazines without isolation.
The desired compounds 6a–p were readily obtained.
The initial anticonvulsant evaluation indicated that all the
compoundswereeffectiveinipMES and/orscPTZ screens. In
the MES test, all of the compounds showed protection in half
ormore ofthe tested mice after 0.5 h except7a, 7b, 7o and 7p,
indicative of their ability to prevent seizure spread.
1
23

Med Chem Res
Scheme 1 Steps for the synthesis of compounds 7a–p
Compounds that were active at 100 mg/kg after 0.5 h in MES
test included 7c, 7d, 7e and 7k indicative of their good ability
to protect from seizure spread at a higher dose. Among these
compounds, 7e and 7k were also active at the same dose after
In the neurotoxicity screen, compounds 7b, 7d, 7e, 7f, 7j,
7m and 7n did not show any neurotoxicity in the maximum
dose administered (300 mg/kg). Compounds 7p revealed
neurotoxicity at a dose of 100 mg/kg. The majority of these
compounds exhibited less neurotoxic than phenytoin.
Compounds 7f and 7n were selected for the quantifi-
cation of the pharmacological parameters (ED50 and
TD ). Results of the quantitative test for these com-
4
.0 h. This showed that these compounds have quick onset
and long duration of action at relatively higher dose. From
these series, 7f and 7n showedanti-MES activityatthe doseof
3
0 mg/kgattimeperiods0.5 h,andthemostactivecompound
5
0
7
n was active in the MES test at both 0.5 and 4.0 h in the same
pounds, 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 7f gave an ED50 of 61.8 mg/kg and a TD50 of
381.7 mg/kg, resulting in a high protection index (PI),
that is, TD /ED , of 5.8 when compared to phenobar-
dose that was equivalent to phenytoin used as reference anti-
convulsant drug.
The scPTZ screen showed that compounds 7c, 7e, 7f, 7g,
h, 7k, 7m and 7n were found to be active after 0.5 h and/or
7
4.0 h, and the other derivatives devoid of anticonvulsant
activity. Compounds 7f and 7n were active after 4.0 h at the
dose of 300 mg/kg, and the other compounds showed no
activity.
5
0
50
bital and valproate.
123

Med Chem Res
Table 1 Anticonvulsant activity and neurotoxicity of compounds 7a–n administered intraperitoneally to mice
a
b
Compounds
Intraperitoneal injection in mice
ClogP
c
d
e
MES
scPTZ
Neurotoxicity
0.5 h
0
.5 h
4.0 h
0.5 h
4.0 h
4.0 h
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
–
–
300
300
–
–
–
300
–
–
-0.55 ± 0.67
-0.22 ± 0.67
1.53 ± 0.67
2.52 ± 0.68
2.70 ± 0.70
1.98 ± 0.70
2.56 ± 0.68
2.04 ± 0.68
2.74 ± 0.70
3.61 ± 0.68
3.12 ± 0.72
1.99 ± 0.67
1.99 ± 0.68
2.18 ± 0.69
1.37 ± 0.69
1.88 ± 0.69
2.52 ± 0.38
0.38 ± 0.46
–
–
–
100
100
100
30
–
300
–
–
300
–
–
–
–
300
100
300
–
100
100
–
–
–
–
300
300
–
–
–
g
h
i
300
300
300
300
100
300
300
30
–
300
300
300
–
300
–
–
–
–
–
j
–
–
–
–
k
l
300
–
–
300
–
–
–
–
–
300
–
m
n
o
p
300
30
–
300
300
–
–
–
300
–
–
–
–
300
300
100
–
300
300
100
–
–
–
–
–
f
Phenytoin
30
30
–
–
–
g
Ethosuximide
–
100
300
a
3
0, 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 and 4.0 h after injection was administered. A dash indicates an absence of activity
at maximum dose administered (300 mg/kg)
b
ClogP was calculated using software ACD Labs 8.0 version
c
Maximal electroshock test
d
Subcutaneous pentylenetetrazole test
e
Neurotoxicity screening (rotarod test)
f
Data from Ref. (Dimmock et al., 1995)
g
Data from Ref. (Rajak et al., 2009)
Conclusion
purified according to the literature methods. Melting points
1
were uncorrected and measured on an XT-4 apparatus. H-
1
3
In summary, the present studies revealed that numbers of
NMR and C-NMR spectra were obtained on a Bruker
AV400 apparatus in DMSO-d and CDCl with TMS as
3
-amino-substituted
5,5-cyclopropanespirohydantoin
6
3
derivatives were effective in the MES and/or scPTZ
screens. The most active was 6-(4-fluorophenylamino)-1,1-
dimethyl-4,6-diazaspiro[2.4]heptane-5,7-dione (7f) which
showed ED50 value of 8.5 mg/kg and a protective index
internal standard. The mass spectra (MS) were recorded on
AMD-604 mass spectrometer operating at 70 eV.
Synthesis of 1-(ethoxycarbonyl)-2,2-
(
TD50/ED50) of 44.9 in the MES test in mice. This com-
dimethylcyclopropanecarboxylic acid (4)
pound showed greater ED50 and lower TD50 to standard
drugs.
Compound 4 was synthesized as described previously in
our laboratory (He et al., 2010a, b).
To a solution of compound 3 (10 mmol) in anhydrous
ethanol (50 mL) was added 1 N sodium hydroxide (25 mL,
Experimental
Chemistry
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
All chemicals and solvents were purchased from com-
mercial sources. Solvents and reagents were dried and
mixture was acidified by means of a saturated KHSO
and
4
extracted with ethyl acetate (3 9 30 mL). The combined
1
23

Med Chem Res
Table 2 Phase II quantitative anticonvulsant evaluation in mice (test drug administered ip)
a
b
c
PI
Compounds
ED50
TD50
MES
scPTZ
MES
scPTZ
d
7
f
8.5 (5.8–14.1)
61.8 (41.8–96.2)
126.1 (101.2–153.2)
[300
381.7 (369.3–416.2)
441.5 (401.2–474.5)
65.5 (52.5–72.9)
71.6 (45.9–135)
69 (62.8–72.9)
44.9
30.8
6.9
5.8
3.5
7
n
14.3 (10.1–11.9)
9.5 (8.1–10.4)
8.8 (5.5–14.1)
21.8 (21.8–25.5)
272 (247–338)
e
Phenytoin
\0.22
\0.22
5.2
e
Carbamazepine
e
Phenobarbital
e
Valproate
[100
8.1
13.2 (5.8–15.9)
149 (123–177)
3.2
426 (369–450)
1.6
2.9
Number of animals used: 10; solvent used: polyethylene glycol (0.1 mL, ip)
a
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
d
e
Protection index (TD50/ED50
)
Data in parentheses are the 95 % confidence limits
Data from references (Ucar et al., 1998)
extracts were dried over Na SO and evaporated to give
2
4.06 (q, 2H, J = 7.04 Hz, CH
7.18(br, 1H, NH), 8.31(brs, 1H, NH). C-NMR (CDCl
00 Hz): d 13.1, 18.3, 19.8, 28.4, 30.4, 43.5, 62.3, 163.0,
2 2
), 4.96–5.23(m, 2H, NH ),
4
1
3
products 4 without further purification.
,
3
1
?
General method for the synthesis of compounds 6a–p
172.1. ESI-MS: 216.2 ([M ? H] ).
Ethyl 2,2-dimethyl-1-(2-methylhydrazinecarboxamido) cyclo-
Compounds 6a–p were synthesized as described previously
in our laboratory (He et al., 2012a, b, c).
propanecarboxylate (6b) Yield: 85 %. White solid. Mp:
-
1
1
30–131 °C. IR (KBr, cm ): 3352 (N–H), 1729 (C=O),
Compound 4 (10 mmol) was dissolved in dry THF
30 mL) and cooled to -15 °C. After the addition of
1
672 (C=O). H-NMR (CDCl 400 MHz): 0.79 (d, 1H,
1
3
,
(
J = 4.88 Hz, Cpr-CH), 1.13 (s, 6H, 2CH ), 1.18 (t, 3H,
3
ClCOOC H (11 mmol) and N-methylmorpholine (NMM)
2
5
J = 7.04 Hz, CH ), 1.50 (d, 1H, J = 4.88 Hz, Cpr-CH),
3
(
12 mmol), the mixture was stirred for 20 min. A solution
2
.38 (s, 3H, NCH ), 4.05 (q, 2H, J = 7.04 Hz, CH ), 7.14
3 2
of NaN (25 mmol) in H O was added and stirred for 1 h at
3
2
13
brs, 1H, NH), 8.04 (brs, 1H, NH), 8.89 (brs, 1H, NH). C-
(
-
10 °C. The solution was then diluted with H O and
2
NMR (CDCl , 100 Hz): d 13.2, 18.4, 19.8, 27.9, 31.2, 43.2,
3
extracted with EtOAc. The organic layers were washed
?
6.3, 61.1, 162.8, 171.4. ESI-MS: 230.2 ([M ? H] ).
4
with brine, dried over Na SO and concentrated under
2
4
reduced pressure to give crude acyl azide. This crude acyl
azide could be further purified by a flash column chro-
Ethyl 2,2-dimethyl-1-(2-phenylhydrazinecarboxamido)cy-
clopropanecarboxylate (6c) Yield: 85 %. White solid.
-
1
matography (PE-EtOAc, 4:1, R = 0.6). Purified acyl azide
f
Mp: 101–102 °C. IR (KBr, cm ): 3359 (N–H), 1703
(C=O), 1668 (C=O). H-NMR (CDCl3, 400 MHz): 0.79 (d,
1
was dissolved in toluene (30 mL), and the resulting solu-
tion was heated to 75 °C under stirring. After gas evolution
had stopped, toluene was removed under reduced pressure
to afford isocyanate 5 as clear oil. This isocyanate 5 was
directly used in the next step without further purification.
Hydrazine (10 mmol) was added to a stirred suspension of
isocyanate 5 in dry THF (40 mL) at room temperature for
1H, J = 4.80 Hz, Cpr-CH), 1.14 (s, 6H, 2CH
3H, J = 7.04 Hz, CH ), 1.52 (d, 1H, J = 4.80 Hz, Cpr-H),
4.05 (q, 2H, J = 7.04 Hz, CH ), 7.22 (brs, 1H, NH),
7.44–7.54 (m, 2H, C2,6-ArH), 7.56–7.58 (m, 1H, C -ArH),
7.89–8.00 (m, 2H, C3,5-ArH), 8.86 (brs, 1H, NH), 9.89 (brs,
), 1.18 (t,
3
3
2
4
1
3
1H, NH). C-NMR (CDCl , 100 Hz): d 13.2, 18.4, 19.8,
3
1
h. The solvent was removed under reduced pressure to
27.9, 31.2, 43.2, 61.1, 126.4, 128.4, 129.3, 132.1, 162.8,
?
171.4. ESI-MS: 292.2 ([M ? H] ).
give products 6 and were recrystallized as white solid.
Ethyl 1-(hydrazinecarboxamido)-2,2-dimethylcyclo-
propanecarboxylate (6a) Yield: 83 %. White solid. Mp:
Ethyl 1-(2-(4-chlorophenyl)hydrazinecarboxamido)-2,2-
dimethylcyclopropanecarboxylate (6d) Yield: 88 %.
-1
-
1
1
1
25–127 °C. IR (KBr, cm ): 3376 (N–H), 1712 (C=O),
White solid. Mp: 87–89 °C. IR (KBr, cm ): 3346 (N–H),
1692 (C=O), 1652 (C=O). H-NMR (CDCl3, 400 MHz):
1
1
664 (C=O). H-NMR (CDCl 400 MHz): d 0.80 (d, 1H,
3
,
J = 4.92 Hz, Cpr-CH), 1.14(s, 6H, 2CH ), 1.16(t, 3H,
3
0.79 (d, 1H, J = 4.12 Hz, Cpr-CH), 1.14 (s, 6H, 2CH
),
3
J = 7.04 Hz, CH ), 1.51 (d, 1H, J = 4.92 Hz, Cpr-CH),
3
1.19 (t, 3H, J = 7.12 Hz, CH ), 1.54 (d, 1H, J = 4.12 Hz,
3
123

Med Chem Res
Cpr-H), 4.04 (q, 2H, J = 7.12 Hz, CH ), 7.13 (brs, 1H,
2
17.9, 20.0, 28.4, 33.9, 41.9, 65.2, 123.1, 126.8, 128.5,
129.0, 131.4, 146.1, 163.9, 171.8. ESI-MS: 325.2
NH), 7.55 (d, 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,
?
([M ? H] ).
1
3
NH). C-NMR (CDCl , 100 Hz): d 13.2, 18.4, 19.8, 27.9,
3
Ethyl 1-(2-(3-bromophenyl)hydrazinecarboxamido)-2,2-
3
1.2, 43.2, 61.1, 126.4, 128.4, 129.3, 132.1, 162.8, 171.4.
dimethyl cyclopropanecarboxylate (6i) Yield: 80 %.
-
? 2?
ESI-MS: 325.2 ([M ? H] ), 327.2([M ? 2H] ).
1
White solid. Mp: 87–88 °C. IR (KBr, cm ): 3371 (N–H),
1
1708 (C=O), 1669 (C=O). H-NMR (CDCl , 400 MHz):
Ethyl 1-(2-(4-bromophenyl)hydrazinecarboxamido)-2,2-
3
dimethylcyclopropanecarboxylate (6e) Yield: 85 %.
-
0.77 (d, 1H, J = 4.88 Hz, Cpr-CH), 1.13 (s, 6H, 2CH3),
1
White solid. Mp: 92–93 °C. IR (KBr, cm ): 3359 (N–H),
1.18 (t, 3H, J = 7.14 Hz, CH ), 1.49 (d, 1H, J = 4.88 Hz,
3
1
703 (C=O), 1668 (C=O). H-NMR (CDCl3, 400 MHz):
1
0
1
Cpr-CH), 4.06 (q, 2H, J = 7.14 Hz, CH ), 7.18 (brs, 1H,
2
.78 (d, 1H, J = 4.42 Hz, Cpr-CH), 1.12 (s, 6H, 2CH3),
NH), 7.25–7.30(m, 4H, C2,4,5,6-ArH), 8.15 (brs, 1H, NH),
1
3
.18 (t, 3H, J = 7.14 Hz, CH ), 1.52 (d, 1H, J = 4.42 Hz,
3
9.35 (brs, 1H, NH). C-NMR (CDCl , 100 Hz): d 12.6,
3
Cpr-H), 4.06 (q, 2H, J = 7.16 Hz, CH ), 7.22 (brs, 1H,
2
17.9, 20.0, 28.4, 33.9, 41.9, 65.2, 123.1, 126.8, 128.5,
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,
129.0, 131.4, 146.1, 163.9, 171.8. ESI-MS: 370.1
?
2?
([M ? H] ), 372.1 ([M ? 2H] ).
Ethyl 1-(2-(3,5-dichlorophenyl)hydrazinecarboxamido)-
,2-dimethyl cyclopropanecarboxylate (6j) Yield: 88 %.
1
3
NH). C-NMR (CDCl , 100 Hz): d 13.4, 18.1, 20.2, 28.1,
3
3
3.2, 41.4, 64.2, 122.7, 129.4, 131.2, 134.7, 163.3, 172.2.
2
?
2?
ESI-MS: 370.1 ([M ? H] ), 372.1([M ? 2H] ).
Ethyl 1-(2-(4-fluorophenyl)hydrazinecarboxamido)-2,2-
dimethylcyclopropanecarboxylate (6f) Yield: 80 %.
-
1
White solid. Mp: 104–106 °C. IR (KBr, cm ): 3356 (N–
H), 1712 (C=O), 1678 (C=O). H-NMR (CDCl3,
1
400 MHz): 0.77 (d, 1H, J = 4.74 Hz, Cpr-CH), 1.14 (s,
6H, 2CH ), 1.20 (t, 3H, J = 7.22 Hz, CH ), 1.50 (d, 1H,
-
1
White solid. Mp: 132–133 °C. IR (KBr, cm ): 3377 (N–
3
3
1
H), 1743 (C=O), 1672 (C=O). H-NMR (CDCl 400 MHz):
J = 4.74 Hz, Cpr-CH), 4.09 (q, 2H, J = 7.22 Hz, CH2),
3
,
0
.78 (d, 3H, J = 4.86 Hz, Cpr-CH), 1.12 (s, 6H, 2CH3),
6.85 (brs, 1H, NH), 7.92–7.18 (m, 3H), 8.58 (brs, 1H, NH),
1
3
1
.19 (t, 3H, J = 7.04 Hz, CH ), 1.51 (d, 1H, J = 4.86 Hz,
3
9.35 (brs, 1H, NH). C-NMR (CDCl , 100 Hz): d 12.9,
3
Cpr-CH), 4.06 (q, 2H, J = 7.04 Hz, CH ), 7.04 (brs, 1H,
2
18.2, 20.3, 27.9, 34.2, 42.5, 66.7, 117.14, 122.82, 135.13,
?
140.72, 164.2, 171.4. ESI-MS: 361.2 ([M ? H] ), 363.2
2?
([M ? 2H] ).
NH), 7.27–7.33 (m, 2H, C3,5-ArH), 7.92–7.96 (m, 2H, C2,6-
1
3
ArH), 8.88 (brs, 1H, NH), 9.56 (brs, 1H, NH). C-NMR
(
CDCl , 100 Hz): d 13.1, 18.4, 19.7, 28.3, 33.6, 42.1, 63.7,
16.2, 128.0, 157.3, 161.6, 163.7, 171.8. ESI-MS: 310.2
?
[M ? H] ).
3
Ethyl 1-(2-(3-chloro-4-fluorophenyl)hydrazinecarboxam-
1
ido)-2,2-dimethyl cyclopropanecarboxylate (6k) Yield:
1
(
-
8
3 %. White solid. Mp: 129–130 °C. IR (KBr, cm ): 3376
1
Ethyl 1-(2-(3-chlorophenyl)hydrazinecarboxamido)-2,2-
(N–H), 1745 (C=O), 1690 (C=O). H-NMR (CDCl3,
400 MHz): 0.78 (d, 1H, J = 4.88 Hz, Cpr-CH), 1.12 (s,
6H, 2CH ), 1.18 (t, 3H, J = 7.18 Hz, CH ), 1.51 (d, 1H,
dimethylcyclopropanecarboxylate (6g) Yield: 80 %.
-
White solid. Mp: 95–96 °C. IR (KBr, cm ): 3376 (N–H),
1
0
1
1
3
3
1
721 (C=O), 1668 (C=O). H-NMR (CDCl , 400 MHz):
J = 4.88 Hz, Cpr-CH), 4.09 (q, 2H, J = 7.18 Hz),
3
.78 (d, 1H, J = 4.88 Hz, Cpr-CH), 1.12 (s, 6H, 2CH3),
6.56–6.72 (m, 3H, C2,5,6-ArH), 7.35 (brs, 1H, NH), 8.29
1
3
.18 (t, 3H, J = 7.08 Hz, CH ), 1.46 (d, 1H, J = 4.88 Hz,
3
(brs, 1H, NH), 9.47 (brs, 1H, NH). C-NMR (CDCl3,
100 Hz): d 13.3, 18.8, 20.5, 28.3, 34.8, 41.9, 66.9, 123.1,
126.8, 128.5, 129.0, 131.4, 146.1, 163.5, 172.1. ESI-MS:
Cpr-CH), 4.08 (q, 2H, J = 7.08 Hz, CH ), 7.10–7.14(m,
2
4
9
1
1
H, C2,4,5,6-ArH), 7.25 (brs, 1H, NH), 8.40 (brs, 1H, NH),
1
3
?
2?
.25 (brs, 1H, NH). C-NMR (CDCl , 100 Hz): d 13.2,
344.1 ([M ? H] ). 346.1 ([M ? 2H] ).
3
8.6, 20.2, 28.9, 34.2, 42.5, 64.9, 119.6, 122.1, 128.5,
Ethyl
2,2-dimethyl-1-(2-p-tolylhydrazinecarboxamido)
31.1, 135.7, 150.3, 164.1, 172.2. ESI-MS: 325.2
?
cyclopropanecarboxylate (6l) Yield: 88 %. White solid.
-
2?
(
[M ? H] ), 327.2[M ? 2H] .
1
Mp: 122–123 °C. IR (KBr, cm ): 3374 (N–H), 1743
1
(C=O), 1688 (C=O). H-NMR (CDCl , 400 MHz): 0.80 (d,
Ethyl 1-(2-(2-chlorobenzoyl)hydrazinecarboxamido)-2,2-
3
dimethyl cyclopropanecarboxylate (6h) Yield: 82 %.
-
1H, J = 4.88 Hz, Cpr-CH), 1.15 (s, 6H, 2CH ), 1.21 (t,
3
1
White solid. Mp: 101–102 °C. IR (KBr, cm ): 3374 (N–
H), 1712 (C=O), 1677 (C=O). H-NMR (CDCl3,
3H, J = 7.14 Hz, CH ), 1.52 (d, 1H, J = 4.88 Hz, Cpr-
3
1
CH), 2.89(s, 3H, Ar-CH ), 4.08 (q, 2H, J = 7.14 Hz, CH ),
3
2
4
6
00 MHz): 0.80 (d, 1H, J = 4.92 Hz, Cpr-CH), 1.15 (s,
H, 2CH ), 1.17 (t, 3H, J = 7.04 Hz, CH ), 1.52 (d, 1H,
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),
3
3
1
3
J = 4.92 Hz, Cpr-CH), 4.06 (q, 2H, J = 7.04 Hz), 6.99
9.72 (brs, 1H, NH). C-NMR (CDCl , 100 Hz): d 14.5,
3
(
brs, 1H, NH), 7.41–7.52 (m, 4H, ArH), 8.56 (brs, 1H, NH),
1
20.1.8, 21.5, 22.8, 29.1, 35.9, 42.5, 67.4, 120.8, 130.0,
?
133.5, 136.1, 156.9, 172.4. ESI-MS: 306.2 ([M ? H] ).
3
9
.27 (brs, 1H, NH). C-NMR (CDCl , 100 Hz): d 12.6,
3
1
23

Med Chem Res
Ethyl 2,2-dimethyl-1-(2-(4-nitrophenyl)hydrazinecarbox-
General method for the synthesis of compounds 7a–p
amido) cyclopropanecarboxylate (6m) Yield: 80 %.
-
1
Yellow solid. Mp: 138–139 °C. IR (KBr, cm ): 3368 (N–
H), 1721 (C=O), 1682 (C=O). H-NMR (CDCl3,
Compounds 7a–p were synthesized as described previously
in our laboratory (He et al., 2012a, b, c)
1
4
6
00 MHz): 0.86 (d, 1H, J = 4.88 Hz, Cpr-CH), 1.16 (s,
H, 2CH ), 1.22 (t, 3H, J = 7.20 Hz, CH ), 1.53 (d, 1H,
6
-Amino-1,1-dimethyl-4,6-diazaspiro[2.4]heptane-5,7-dione
1
3
3
(
7a) Yield: 83 %. White solid. Mp: 112–113 °C. H-
J = 4.88 Hz, Cpr-CH), 4.12 (q, 2H, J = 7.20 Hz, CH2),
NMR (DMSO-d , 400 MHz): 1.19 (d, 1H, J = 4.22 Hz,
6
7
8
9
2
1
.27 (brs, 1H, NH), 7.87 (d, 2H, J = 8.28 Hz, C3,5-ArH),
Cpr-H), 1.25 (s, 3H, CH ), 1.29 (s, 3H, CH ), 1.44 (d, 1H,
3
3
.07 (d, 2H, J = 8.28 Hz, C2,6-ArH), 8.54 (brs, 1H, NH),
1
.43 (brs, 1H, NH). C-NMR (CDCl , 100 Hz): d 14.9,
J = 4.22 Hz, Cpr-H), 4.33 (br, 2H, NH ), 7.75 (br, 1H,
2
3
3
13
NH). C-NMR (DMSO-d , 100 Hz): d 18.8, 22.3, 26.1,
6
0.2, 22.7, 27.4, 29.1, 44.5, 61.4, 117.6, 125.8, 141.3,
?
47.4, 155.6, 171.2. ESI-MS: 337.2 ([M ? H] ).
?
6.4, 49.8, 157.7, 175.5. ESI-MS: 169.3 ([M ? H] ).
2
1
,1-Dimethyl-6-(methylamino)-4,6-diazaspiro[2.4]heptane-5,7-
Ethyl 1-(2-(2,4-dinitrophenyl)hydrazinecarboxamido)-2,2-
dione (7b) Yield: 85 %. White solid. Mp: 133–134 °C.
H-NMR (DMSO-d6, 400 MHz): 1.17 (d, 1H,
dimethylcyclopropanecarboxylate (6n) Yield: 75 %.
-
1
1
Yellow solid. Mp: 157–158 °C. IR (KBr, cm ): 3388 (N–
H), 1766 (C=O), 1682 (C=O). H-NMR (CDCl3,
J = 4.08 Hz, Cpr-H), 1.23 (s, 3H, CH ), 1.30 (s, 3H, CH ),
3
1
3
1
.44 (d, 1H, J = 4.08 Hz, Cpr-H), 3.01(s, 3H, NCH ), 4.21
3
4
6
00 MHz): 0.80 (d, 1H, J = 4.92 Hz, Cpr-CH), 1.16 (s,
H, 2CH ), 1.22 (t, 3H, J = 7.14 Hz, CH ), 1.54 (d, 1H,
1
br, 1H, NH), 7.88 (br, 1H, NH). C-NMR (DMSO-d6,
3
(
3
3
1
00 Hz): d 18.4, 22.8, 26.2, 26.4, 48.5, 49.3, 155.6, 176.1.
J = 4.92 Hz, Cpr-CH), 4.09 (q, 2H, J = 7.14 Hz, CH2),
.28 (dd, 1H, J = 8.84 Hz, J = 2.52 Hz), 7.33 (brs, 1H,
?
ESI-MS: 184.2 ([M ? H] ).
7
1
2
NH), 7.46 (d, 1H, J = 2.52 Hz), 7.58 (d, 1H,
1,1-Dimethyl-6-(phenylamino)-4,6-diazaspiro[2.4]heptane-5,7-
1
J = 8.84 Hz), 8.15 (brs, 1H, NH), 9.36 (brs, 1H, NH). C-
3
dione (7c) Yield: 88 %. White solid. Mp: 115–116 °C.
1
NMR (CDCl , 100 Hz): d 14.2, 21.4, 22.9, 28.3, 30.1, 42.1,
H-NMR (DMSO-d , 400 MHz): 1.18(d, 1H, J = 3.44 Hz,
3
6
6
3.2, 118.3, 125.2, 129.3, 131.5, 133.6, 149.9, 157.6,
Cpr-CH), 1.25(s, 3H, CH ), 1.28(s, 3H, CH ), 1.42(d, 1H,
3
3
1
72.1. ESI-MS: 382.1 ([M ? H] ?).
J = 3.44 Hz, Cpr-CH), 7.28–7.35(m, 2H, C2,6-ArH),
7
.45–7.48(m, 1H, C -ArH), 7.55(brs, 1H, NH),
4
Ethyl 1-(2-(2-furan-2-yl)hydrazinecarboxamido)-2,2-dimethyl-
1
.79–7.83(m, 2H, C3,5-ArH), 9.88(brs, 1H, NH). C-NMR
3
7
cyclopropanecarboxylate (6o) Yield: 70 %. White solid.
-
(DMSO-d , 100 Hz): d 18.5, 22.6, 27.4, 28.2, 48.5, 126.4,
1
6
Mp: 92–93 °C. IR (KBr, cm ): 3287 (N–H), 1736 (C=O),
1
28.3, 129.5, 132.5, 160.6, 172.1. ESI-MS: 246.3
1
698 (C=O). H-NMR (CDCl , 400 MHz): 0.78 (d, 1H,
1
3
?
[M ? H] ).
(
J = 4.26 Hz, Cpr-CH), 1.17 (s, 6H, 2CH ), 1.22 (t, 3H,
3
J = 7.20 Hz, CH ), 1.59 (d, 1H, J = 4.26 Hz, Cpr-CH),
3
6-(4-Chlorophenylamino)-1,1-dimethyl-4,6-diazaspiro[2.4]hep-
4
.13 (q, 2H, J = 7.20 Hz, CH ), 7.16–7.24 (m, 3H, ArH),
2
tane-5,7-dione (7d) Yield: 80 %. White solid. Mp:
1
7
.72 (brs, 1H, NH), 8.56 (brs, 1H, NH), 9.67 (brs, 1H, NH).
152–153 °C. H-NMR (DMSO-d , 400 MHz): 1.18(d, 1H,
6
1
3
C-NMR (CDCl , 100 Hz): d 14.1, 20.7, 22.6, 29.1, 31.5,
J = 4.92 Hz, Cpr-CH), 1.27(s, 3H, CH ), 1.30(s, 3H,
3
3
4
4.5, 65.7, 111.4, 143.5, 149.7, 149.9, 155.3, 170.9. ESI-
CH ), 1.33(d, 1H, J = 4.92 Hz, Cpr-CH), 7.62(d, 2H,
3
MS: 282.3 ([M ? H] ?).
J = 8.54 Hz, C3,5-ArH), 7.66(brs, 1H, NH), 7.91(d, 2H,
1
J = 8.52 Hz, C2,6 = ArH), 10.08(brs, 1H, NH). C-NMR
3
Ethyl
1-(2-(2,-thiophen-2-yl)hydrazinecarboxamido)-2,2-
(
DMSO-d , 100 Hz): d 18.2, 22.9, 26.9, 28.1, 49.3, 124.2,
6
dimethylcyclopropanecarboxylate (6p) Yield: 75 %. White
-
1
30.3, 131.2, 152.7, 161.3, 171.5. ESI-MS: 280.7
1
solid. Mp: 104–105 °C. IR (KBr, cm ): 3372 (N–H),
? 2?
[M ? H] ). 282.1 ([M ? 2H] ).
(
1
788(C=O), 1690(C=O). H-NMR (CDCl , 400 MHz):
1
0
1
3
.81 (d, 1H, J = 4.76 Hz, Cpr-CH), 1.14 (s, 6H, 2CH3),
6-(4-Bromophenylamino)-1,1-dimethyl-4,6-diazaspiro[2.4]hep-
.25 (t, 3H, J = 7.24 Hz, CH ), 1.54 (d, 1H, J = 4.76 Hz,
3
tane-5,7-dione (7e) Yield: 80 %. White solid. Mp:
1
Cpr-CH), 4.06 (q, 2H, J = 7.24 Hz, CH ), 7.28–7.45 (m,
2
158–159 °C. H-NMR (DMSO-d , 400 MHz): 1.08(d, 1H,
6
3
H, ArH), 7.65 (brs, 1H, NH), 8.85 (brs, 1H, NH), 9.76
1
J = 4.96 Hz, Cpr-CH), 1.17(s, 3H, CH ), 1.26(s, 3H,
3
3
(
brs, 1H, NH). C-NMR (CDCl , 100 Hz): d 14.4, 21.2,
CH ), 1.38(d, 1H, J = 4.96 Hz, Cpr-CH), 7.65(d, 2H,
3
3
2
2.3, 28.3, 30.9, 42.7, 66.3, 109.2, 139.5, 147.2, 148.1,
J = 8.52 Hz, C3,5-ArH), 7.73 (brs, 1H, NH), 7.88 (d, 2H,
1
J = 8.52 Hz, C2,6-ArH), 10.12 (brs, 1H, NH). C-NMR
3
1
54.7, 169.5. ESI-MS: 298.4 ([M ? H] ?).
123

Med Chem Res
?
159.9, 177.1. ESI-MS: 315.1 ([M ? H] ), 317.1
(
DMSO-d , 100 Hz): d 18.8, 22.4, 27.4, 28.6, 49.6, 121.3,
6
2
([M ? 2H] ).
?
1
32.1, 132.5, 154.9, 162.6, 173.1. ESI-MS: 325.2
?
2?
(
[M ? H] ), 327.2 ([M ? 2H] ).
-(4-Fluorophenylamino)-1,1-dimethyl-4,6-diazaspiro[2.4]hep-
tane-5,7-dione (7f) Yield: 85 %. White solid. Mp:
6-(3-Chloro-4-fluorophenylamino)-1,1-dimethyl-4,6-diaza-
6
spiro[2.4]heptane-5,7-dione (7k) Yield: 80 %. White
1
solid. Mp: 136–137 °C. H-NMR (DMSO-d , 400 MHz):
6
1
1
62–163 °C. H-NMR (DMSO-d , 400 MHz): 1.16 (d, 1H,
1.09 (d, 1H, J = 4.78 Hz, Cpr-CH), 1.14 (s, 3H, CH3),
6
J = 5.58 Hz, Cpr-CH), 1.23(s, 3H, CH ), 1.30 (s, 3H,
1.27 (s, 3H, CH ), 1.38 (d, 1H, J = 4.78 Hz, Cpr-H), 7.66
3
3
CH ), 1.36 (d, 1H, J = 4.92 Hz, Cpr-CH), 7.44–7.58 (m,
3
(s, 1H, C -ArH), 8.18 (m, 2H, C4,5-ArH), 8.35 (brs, 1H,
2
1
3
H, ArH), 8.80 (brs, 1H, NH), 10.94 (brs, 1H, NH). C-
13
NH), 10.67 (brs, 1H, NH). C-NMR (DMSO-d , 100 Hz):
4
6
NMR (DMSO-d , 100 Hz): d 18.5, 22.6, 27.8, 28.2, 48.5,
d 21.4, 23.3, 27.1, 28.5, 51.2, 118.2, 125.4, 129.1, 131.8,
133.8, 150.2, 163.2, 172.0. ESI-MS: 319.1 ([M ? H] ),
6
?
1
16.3, 128.1, 128.3, 157.2, 161.3, 172.4. ESI-MS: 264.3
?
2?
321.1 ([M ? 2H] ).
(
[M ? H] ).
-(3-Chlorophenylamino)-1,1-dimethyl-4,6-diazaspiro[2.4]hep-
tane-5,7-dione (7g) Yield: 80 %. White solid. Mp:
6
1,1-Dimethyl-6-(p-tolylamino)-4,6-diazaspiro[2.4]heptane-5,7-
1
dione (7l) Yield: 90 %. White solid. Mp: 103–104 °C. H-
1
1
42–143 °C. H-NMR (DMSO-d , 400 MHz): 1.12 (d, 1H,
NMR (DMSO-d , 400 MHz): 1.16 (d, 1H, J = 4.00 Hz,
6
6
J = 4.46 Hz, Cpr-CH), 1.16 (s, 3H, CH ), 1.19 (s, 3H,
Cpr-CH), 1.24 (s, 3H, CH ), 1.30 (s, 3H, CH ), 1.37 (d, 1H,
3
3
3
CH ), 1.39 (d, 1H, J = 4.46 Hz, Cpr-CH), 7.41–7.47 (m,
3
J = 4.00 Hz, Cpr-CH), 2.41(s, 3H, Ar-CH ), 7.32 (d, 2H,
3
2
1
1
1
2
H, ArH), 7.66–7.71(m, 2H, ArH), 8.88 (brs, 1H, NH),
1
J = 8.80 Hz, C3,5-ArH), 7.91 (d, 2H, J = 8.80 Hz, C2,6-
13
ArH), 8.77 (brs, 1H, NH), 10.88 (brs, 1H, NH). C-NMR
3
0.42 (brs, 1H, NH). C-NMR (DMSO-d , 100 Hz): d
6
8.7, 22.2, 27.1, 28.4, 49.5, 121.0, 122.8, 125.9, 131.3,
?
35.4, 155.0, 164.4, 172.3. ESI-MS: 280.7 ([M ? H] ),
(DMSO-d , 100 Hz): d 18.5, 22.6, 28.1, 48.5, 55.7, 114.7,
6
124.6, 127.9, 157.6, 159.4, 172.7. ESI-MS: 260.3
?
([M ? H] ).
2
?
82.7 ([M ? 2H] ).
-(2-Chlorophenylamino)-1,1-dimethyl-4,6-diazaspiro[2.4]hep-
tane-5,7-dione (7h) Yield: 83 %. White solid. Mp:
6
1,1-Dimethyl-6-(4-nitrophenylamino)-4,6-diazaspiro[2.4]hep-
tane-5,7-dione (7m) Yield: 80 %. Yellow solid. Mp:
1
1
1
54–155 °C. H-NMR (DMSO-d , 400 MHz): 1.16 (d, 1H,
128–129 °C. H-NMR (DMSO-d , 400 MHz): 1.16 (d, 1H,
6
6
J = 5.52 Hz, Cpr-CH), 1.24(s, 3H, CH ), 1.31(s, 3H,
J = 4.12 Hz, Cpr-CH), 1.25 (s, 3H, CH ), 1.32 (s, 3H,
3
3
CH ), 1.37 (d, 1H, J = 5.52 Hz, Cpr-CH), 7.39–7.56 (m,
3
CH ), 1.37 (d, 1H, J = 4.12 Hz, Cpr-CH), 7.69 (d, 2H,
3
1
3
H, ArH), 8.84 (brs, 1H, NH), 11.15 (brs, 1H, NH). C-
4
J = 8.88 Hz, C3,5-ArH), 8.02 (d, 2H, J = 8.88 Hz, C2,6-
1
ArH), 8.90 (brs, 1H, NH), 10.88 (brs, 1H, NH). C-NMR
3
NMR (DMSO-d , 100 Hz): d 18.4, 22.6, 27.1, 28.3, 49.6,
6
1
24.0, 126.8, 127.8, 128.7, 131.3, 150.1, 164.4, 171.0. ESI-
(DMSO-d , 100 Hz): d 19.0, 22.2, 27.4, 27.7, 48.4, 124.7,
6
?
MS: 280.7 ([M ? H] ).
-(3-Bromophenylamino)-1,1-dimethyl-4,6-diazaspiro[2.4]hep-
tane-5,7-dione (7i) Yield: 82 %. White solid. Mp:
127.4, 139.0, 145.3, 155.1, 172.3. ESI-MS: 291.2
?
[M ? H] ).
(
6
6-(2,4-Dinitrophenylamino)-1,1-dimethyl-4,6-diaza-
spiro[2.4]heptane-5,7-dione (7n) Yield: 80 %. Yel-
1
1
09–110 °C. H-NMR (DMSO-d , 400 MHz): 1.13(d, 1H,
6
1
J = 4.46 Hz, Cpr-CH), 1.22 (s, 3H, CH ), 1.28 (s, 3H,
low solid. Mp: 144–156 °C. H-NMR (DMSO-d6,
400 MHz): 1.13 (d, 1H, J = 4.12 Hz, Cpr-CH), 1.22 (s,
3
CH ), 1.34 (d, 1H, J = 4.46 Hz, Cpr-CH), 7.41–7.46(m,
3
2
1
1
1
3
H, ArH), 7.99–8.12(m, 2H, ArH), 8.45 (brs, 1H, NH),
3
3H, CH ), 1.28 (s, 3H, CH ), 1.33 (d, 1H, J = 4.12 Hz,
3
3
1
1.72 (brs, 1H, NH). C-NMR (DMSO-d , 100 Hz): d
Cpr-CH), 7.46 (s, 1H, C -ArH), 7.91–8.02 (m, 2H, C5,6-
3
6
1
ArH), 8.53 (brs, 1H, NH), 11.82 (brs, 1H, NH). C-NMR
3
8.8, 22.3, 26.7, 28.1, 48.8, 121.3, 122.4, 125.6, 132.1,
?
35.2, 157.1, 166.2, 171.4. ESI-MS: 325.2 ([M ? H] ),
(DMSO-d , 100 Hz): d 19.1, 23.1, 27.6, 27.9, 48.3, 123.9,
6
2
?
27.2 ([M ? 2H] ).
125.5, 126.7, 138.8, 142.2, 144.9, 156.3, 171.6. ESI-MS:
?
35.1 ([M ? H] ).
3
6
-(3,5-Dichlorophenylamino)-1,1-dimethyl-4,6-diaza-
spiro[2.4]heptane-5,7-dione (7j) Yield: 85 %. White
1
6-(Furan-2-ylamino)-1,1-dimethyl-4,6-diazaspiro[2.4]hep-
solid. Mp: 129–130 °C. H-NMR (DMSO-d , 400 MHz):
tane-5,7-dione (7o) Yield: 82 %. White solid. Mp:
1
6
1
1
7
.13 (d, 1H, J = 5.22 Hz, Cpr-CH), 1.16 (s, 3H, CH3),
102–103 °C. H-NMR (DMSO-d , 400 MHz): 1.12 (d, 1H,
6
.30 (s, 3H, CH ), 1.38 (d, 1H, J = 5.22 Hz, Cpr-CH),
3
J = 4.52 Hz, Cpr-CH), 1.22 (s, 3H, CH ), 1.28 (s, 3H,
3
.54 (s, 2H, C2,6-ArH), 7.66 (s, 1H, C -ArH), 8.68 (brs, 1H,
4
CH ), 1.34 (d, 1H, J = 4.52 Hz, Cpr-CH), 6.68–6.69 (m,
3
1
3
NH), 11.22 (brs, 1H, NH). C-NMR (DMSO-d , 100 Hz):
6
1H, ArH), 7.86–7.87 (m, 1H, ArH), 7.95–8.03 (m, 1H,
1
ArH), 8.89 (brs, 1H, NH), 10.84 (brs, 1H, NH). C-NMR
3
d 23.7, 27.0, 32.1, 32.8, 51.3, 130.7, 132.5, 139.1, 140.1,
1
23

Med Chem Res
(
DMSO-d , 100 Hz): d 19.1, 20.3, 27.9, 28.5, 48.4, 108.6,
activity and neurotoxicity assessment at 0.5 and 4 h after
administration.
6
1
11.1, 142.2, 148.9, 163.7, 169.7. ESI-MS: 236.2
?
(
[M ? H] ).
,1-Dimethyl-6-(thiophen-2-ylamino)-4,6-diazaspiro[2.4]hep-
tane-5,7-dione (7p) Yield: 82 %. White solid. Mp:
Neurotoxicity screening
1
Minimal motor impairment was measured in mice by using
standardized rotarod test (Dunham et al., 1957). 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 on a
rod for at least 1 min.
1
1
09–110 °C. H-NMR (DMSO-d , 400 MHz): 1.15 (d, 1H,
6
J = 4.32 Hz, Cpr-CH), 1.26 (s, 3H, CH ), 1.32 (s, 3H,
3
CH ), 1.36 (d, 1H, J = 4.32 Hz, Cpr-CH), 7.12–7.14 (m,
3
1
H, ArH), 7.89–7.91 (m, 1H, ArH), 8.01–8.03 (m, 1H,
1
ArH), 8.33 (brs, 1H, NH), 11.54 (brs, 1H, NH). C-NMR
3
(
DMSO-d , 100 Hz): d 18.9, 22.4, 27.1, 29.6, 49.0, 107.3,
6
1
09.2, 137.4, 147.6, 165.4, 170.7. ESI-MS: 252.3
Quantification studies
?
[M ? H] ).
(
All quantitative in vivo anticonvulsant and neurotoxicity
studies were conducted in terms of the median effective
dose (ED ) and the median toxic dose (TD ). Groups of
Pharmacology
5
0
50
Anticonvulsant tests were established by the Antiepileptic
Drug Development Program, Epilepsy Branch, National
Institute of Neurological and Communicative Disorders
and Stroke, National Institute of Health in Bethesda, USA
at least eight mice were given with various doses of the
candidate drug until at least two points were established
between the limits of 100 % protection or minimal toxicity
and 0 % protection or minimal toxicity (White et al.,
(
Swinyard et al., 1952). Male Kunming mice (20 ± 2.0 g)
2002). The dose of drug required to produce the desired
were used as experimental animals. All the animals were
purchased from Wuhan University Laboratory Animal
Center. The animals were approved by the Animal Care
Committee of Wuhan University. The tested compounds
were injected intraperitoneally to mice as a suspension in
end point in 50 % of animals in each test, the 95 % con-
fidence interval, the slope of the regression line and stan-
dard error of the slope was then calculated by a computer
program based on the method described by Finney (Finney,
1971).
0
.5 % methylcellulose at the dose of 30, 100 and 300 mg/
kg to one to four mice. The anticonvulsant activity of the
tested compounds was detected by both electrical and
chemoconvulsant seizure tests. Phenytoin and ethosux-
imide were chosen as the standard drugs for the compari-
son. The neurological toxicity was determined in the
rotarod test (Krall et al., 1978).
Acknowledgments This investigation was supported by National
Science Foundation of China (NSFC) (Grant No: 21302065), Scien-
tific Research Foundation for the Returned Overseas Chinese Schol-
ars, State Education Ministry of China and Technology Foundation
for Selected Overseas Chinese Scholar, Ministry of Personnel of
China.
MES—maximal electroshock seizure pattern test
References
For the MES test according to the method of Swinyard
Bialer M (2006) New antiepileptic drugs that are second generation to
existing antiepileptic drugs. Expert Opin Investig Drugs
(
Swinyard, 1969). In the MES screen, an electrical stimulus
1
5:637–647
of 0.25 s in duration (50 mA in mice) is delivered via
corneal electrodes primed with an electrolyte solution
containing an anesthetic agent.
Bojic U, Elmazar MMA, Hauck RS, Nau H (1996) Further branching
of valproate related carboxylic acids reduces the teratogenic
activity, but not the anticonvulsant effect. Chem Res Toxicol
9
:866–870
Brown TR, Holmes GI (2001) Primary care: epilepsy. N Engl J Med
344:1145–1151
PTZ-induced seizure test
Dimmock JR, Pandey SN, Quail JW, Pugazhenthi U, Allen TM, Kao
GY, Balzarini J, DeClercq E (1995) Evaluation of the semicar-
bazones, thiosemicarbazones and bis-carbohydrazones of some
aryl alicyclic ketones for anticonvulsant and other biological
properties. Eur J Med Chem 30:303–314
This screen utilizes a dose of pentylenetetrazole that pro-
duces clonic seizures lasting for a period of at least 5 s in
9
7 % (CD97) of animals tested (Vamecq et al., 1998), and
Dunham NW, Miya TA, Edwards LD (1957) The pharmacological
activity of a series of basic esters of mono- and dialkylmalonic
acids. J Am Pharm Assoc 46:64–66
Emilio P (1999) The clinical pharmacokinetics of the new antiepilep-
tic drugs. Epilepsia 40(Suppl. 9):7–13
pentylenetetrazole was dissolved in sufficient 0.9 % saline
to allow subcutaneous injections to mice. All the com-
pounds were injected intraperitoneally into mice at the dose
levels of 30, 100 and 300 mg/kg with anticonvulsant
123

Med Chem Res
Finney DJ (1971) Statistical logic in the monitoring of reactions to
therapeutic drugs. Methods Inf Med 10:237–245
Rajak H, Deshmukh R, Aggarwal N, Kashaw S, Kharya MD, Mishra
P (2009) Synthesis of novel 2,5-disubstituted 1,3,4-thiadiazoles
for their potential anticonvulsant activity: pharmacophoric
model studies. Arch Pharm 342:453–461
Smith M, Wilcox KS, White HS (2007) Discovery of antiepileptic
drugs. Neurotherapeutics 4:12–17
Fisher RS, Emde Boas W, Blume W, Elger C, Genton P, Lee P, Engel
J Jr (2005) Epileptic seizures and epilepsy: definitions proposed
by the International League against epilepsy (ILAE) and the
International Bureau for epilepsy (IBE). Epilepsia 46:470–472
He X, Qiu G, Yang J, Xiao Y, Wu Z, Qiu G, Hu X (2010a) Synthesis
and anticonvulsant activity of new 6-methyl-1-substituted-4,6-
Swinyard EA (1969) Laboratory evaluation of antiepileptic drugs.
Epilepsia 10:107–119
diazaspiro[2.4]heptane-5,7-diones.
5:3818–3830
He X, Zhong M, Zhang T, Wu W, Wu Z, Yang J, Xiao Y, Pan Y, Qiu
G, Hu X (2010b) Synthesis and anticonvulsant activity of N-3-
arylamide substituted 5,5-cyclopropanespirohydantion deriva-
tives. Eur J Med Chem 45:5870–5877
Eur
J
Med
Chem
Swinyard EA, Brown WC, Goodman LS (1952) Comparative assays
of antiepileptic drugs in mice and rats. J Pharmacol Exp Ther
106:319–330
Ucar H, Van derpoorten K, Cacciaguerra S, Spampinato S, Stables JP,
Depovere P, Isa M, Masereel B, Delarge J, Poupaert JH (1998)
Synthesis and anticonvulsant activity of 2 (3H)-benzoxazolone
4
He X, Zhong M, Zhang T, Yang J, Wu Z, Xiao Y, Guo H, Qiu G, Hu
X (2012a) Synthesis and anticonvulsant activity of 1-(8-(benzy
loxy)quinolin-2-yl)-6-substituted-4,6-diazaspiro[2,4]heptane-5,7-di
ones. Eur J Med Chem 48:338–346
He X, Zhong M, Zhang T, Wu W, Wu Z, Xiao Y, Hu X (2012b)
Synthesis and anticonvulsant activity of ethyl 1-(2-arylhy-
drazinecarboxamido)-2,2-dimethylcyclopropanecarboxylate deri
vatives. Eur J Med Chem 54:542–548
and
41:1138–1145
2
(3H)-benzothiazolone derivatives.
J
Med Chem
Vamecq J, Lambert D, Poupaert JH, Masereel B, Stables JP (1998)
Anticonvulsant activity and interactions with neuronal voltage-
dependent sodium channel of analogs of ameltolide. J Med
Chem 41:3307–3313
Verh e´ R, De Kimpe N, De Buyck L, Courtheyn D, Schamp N (1978)
Preparation of 2,2-dialkylcyclopropanes geminally substituted
with electron-withdrawing groups. Synthesis 1978:530–532
White HS, Woodhead JH, Wilcox KS, Stables JP, Kupferberg HJ,
Wolf HH, Levy RH, Mattson RH, Meldrum BS, Perucca E
(2002) Antiepileptic drugs. Lippincott Williams & Wilkins
Publishers, New York, pp 36–48
He X, Zhong M, Yang J, Wu Z, Xiao Y, Guo H, Hu X (2012c)
Synthesis and anticonvulsant activity of 1-(2-(8-(benzy-
loxy)quinolin-2-yl)-1-butyrycyclopropyl)3-substituted
derivatives. Chem Biol Drug Des 79:771–779
James ES, Lesley J (2005) Zonisamide. CNS Drugs 19:347–367
urea
Krall RL, Penry JK, White BG, Kupferberg HJ, Swinyard EA (1978)
Antiepileptic drug development: II. Anticonvulsant drug screen-
ing. Epilepsia 19:409–428
Yogeeswari P, Ragavendran JV, Sriram D (2007) Neuropathic pain:
strategies in drug discovery and treatment. Expert Opion Drug
Discover 2:169–184
Kwan P, Sander JW (2004) The natural history of epilepsy: an
epidemiological view.
5:1376–1381
Zaccara G, Franciotta D, Perucca E (2007) Idiosyncratic adverse
reactions to antiepileptic drugs. Epilepsia 48:1223–1244
J
Neurol Neurosurg Psychiatry
7
Perucca E, French J, Bialer M (2007) Development of new
antiepileptic drugs: challenges, incentives, and recent advances.
Lancet Neurol 6:793–804
1
23