Synthesis and anticonvulsant activity of 1-(8-(benzyloxy)quinolin-2-yl)-6- substituted-4,6-diazaspiro[2,4]heptane-5,7-diones

Article abstract of DOI:10.1016/j.ejmech.2011.12.037

In the present study on the development of new anticonvulsants, 16 new1-(8-(benzyloxy)quinolin-2-yl-6-substituted-4,6-diazaspiro[2,4]heptane-5, 7-diones were synthesized and tested for anticonvulsant activity using the maximal electroshock (MES), subcutan

Full text of DOI:10.1016/j.ejmech.2011.12.037

European Journal of Medicinal Chemistry 48 (2012) 338e346
Contents lists available at SciVerse ScienceDirect
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journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticonvulsant activity of 1-(8-(benzyloxy)quinolin-2-yl)-6-
substituted-4,6-diazaspiro[2,4]heptane-5,7-diones
Xianran He a, Min Zhong b, Tao Zhang a, Jin Yang c, Zhongyuan Wu a, Yuling Xiao a, Hao Guo a,
,
Guofu Qiu a, Xianming Hu a
*
a State Key Laboratory of Virology, Ministry of Education Laboratory of Combinatorial Biosynthesis and Drug Discovery, Wuhan University School of Pharmaceutical Science,
Luojiashan Road, Wuhan 430071, China
b Hubei Research Institute of Chemistry, Wuhan 430074, China
c Hubei University for Nationalities School of Medicine, Enshi 445000, 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, 16 new1-(8-(benzyloxy)quinolin-2-yl-
6-substituted-4,6-diazaspiro[2,4]heptane-5,7-diones 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. Two compounds 8e and 8j showed promising
anticonvulsant activities in both models employed for anticonvulsant evaluation. The most active
compound 8e showed the MES-induced seizures with ED50 value of 8.6 mg/kg and TD50 value of
365.3 mg/kg after intraperitoneally injection to mice, which provided compound 8e with a protective
index (TD50/ED50) of 26.8 in the MES test.
Received 19 May 2011
Received in revised form
16 December 2011
Accepted 23 December 2011
Available online 29 December 2011
Keywords:
1-(8-(Benzyloxy)quinolin-2-yl-6-
substituted-4,6-diazaspiro[2,4]heptane-5,7-
diones
Ó 2011 Elsevier Masson SAS. All rights reserved.
Anticonvulsant activity
MES test
scPTZ test
1. Introduction
anticonvulsant [15] activities. On the other hand, many spi-
rohydantoin derivations have been documented as potent anti-
Epilepsy is a chronic disorder of the brain characterized by
recurrent unprovoked seizures that affects about 0.5e1% of the
world’s population [1,2]. In spite of the large therapeutic arsenal of
old and new antiepileptic drugs (AEDs), about 30% of epileptic
patients are not seizure-free [3]. Besides, many AEDs such as
phenobarbital, phenytoin, carbamazepine, vigabatrin, valproate,
felbamate, and lamotrigine, which are effective toward only
60e80% of patients have some undesirable side effects, such as
headache, nausea, anorexia, ataxia, hepatotoxicity, drowsiness,
gastrointestinal disturbance, gingival hyperplasia, and hirsutism
[4e8]. Thus, there is an enormous need for the development of
novel AEDs with fewer side effects and more effectives.
convulsants [16e18]. We have previously reported that the
structure of cyclopropanespirohydantoin exhibit excellent anti-
convulsant activities in various animal seizure models [19e21]. In
our previous study,
a series of derivatives of 6-methyl-1-
substituted-4, 6-diazaspiro[2.4]heptane-5,7-diones were first
found to have anticonvulsant activities, among which 6-methyl-
1-(4-(methylsulfonyl)phenyl)-4,6-diazaspiro[2.4]heptanes-5,7-
dione (Compound I) showed the strongest activity with an ED50
value of 12.5 mg/kg in the maximal electroshock test (MES) and
a TD50 value greater than 300 mg/kg (Fig. 1) [19]. Another
derivatives in the group of N-3-arylamide substituted 5,5-
cyclopropanespirohydantoin,1,1-dimethyl-6-(4-(trifluoromethyl)
phenyl)4,6-diazaspiro[2.4]heptanes-5,7-dione (Compound II)
showed ED50 values of 9.2 mg/kg in the maximal electroshock
test (MES), protective index (PI ¼ TD50/ED50) values of 45.8 and
6.7 in the MES and the pentylenetetrazole tests respectively
(Fig. 1) [20]. As a result, these compounds are potential leads for
further design of more active compounds.
Quinoline derivatives, which show several beneficial heterog-
enous and varied pharmacological properties, for example, anti-
bacterial [9], immunosuppressive [10], analgesic [11],
vasorelaxing [12], antiplasmodial [13], anticancer [14] and
Above these facts and in continuation of our research
program on design and synthesis of new anticonvulsant agents,
* Corresponding author. Tel.: þ86 27 68754621; fax: þ86 27 68754629.
E-mail address: hexianran@yahoo.com.cn (X. Hu).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.037

X. He et al. / European Journal of Medicinal Chemistry 48 (2012) 338e346
339
underwent cyclopropane formation with trimethylsulphox-
oniumylide (TMSOI) to afford compound 4 [24]. Then monoester
compound 5 was obtained after monosaponification in a 1 N NaOH/
ethanol (1.1 equiv) solution at room temperature for 12 h [25]. This
was then converted to acyl azide by using ethyl chloroformate in
the presence of triethylamine (Et3N) followed by reaction with
O
N
F
F
F
N
H
O
O
H
N
N
O
S
sodium azide in a one-pot synthesis. a-Carboethoxy isocyanate 6
O
was successfully generated by a Curtius reaction in situ on heating
the acyl azide in anhydrous toluene solution at 75 ꢀC. Isocyanate 6
was allowed to react directly with various amines without isolation.
N
H
O
O
I
The desired
a-carboethoxy ureas 7aep were readily obtained.
II
Finally, compounds 7aep cyclized on treatment with Na (1.1 equiv)
in EtOH and provided 8aep in good yields (yield ꢁ90%).
The chemical structures of the compounds synthesized were
elucidated on the basis of 1H NMR, 13C NMR, MS and elemental
analysis. The detailed physical and analytical data are listed in
Section 5.
Fig. 1. Structure of compounds I and II.
we introduced quinoline structure to propane ring of cyclo-
propanespirohydantoin with different substituents at N-3 position
of hydantoin ring in order to develop new potent and safe AEDs.
We synthesized and comparatively evaluated the anticonvulsant
activity and neurotoxicity of 16 new derivatives of 1-(8-(benzyloxy)
quinolin-2-yl)-6-substituted-4,6-diazaspiro[2,4] heptane-5,7-di-
ones. Compounds 8e and 8j were quantitatively evaluated for its
anticonvulsant activity (ED50) and neurotoxicity (TD50).
All of these compounds were prepared as racemic mixtures and
no attempt was made to resolve the enantiomers.
3. Pharmacology results and discussion
The anticonvulsant activity and neurotoxicity of the synthesized
compounds were evaluated following the standard procedures
proposed by the NIH anticonvulsant drug development (ADD)
program, via the anticonvulsant screening project (ASP) [26]. The
initial evaluation (Phase I) included the maximal electroshock
seizure (MES), the subcutaneous pentylenetetrazole (scPTZ) and
neurotoxicity.
The MES test is associated with the electrical induction of the
seizure, whereas PTZ test involves a chemical induction to generate
the convulsion. Neurotoxicity is primary determined in the
minimal motor impairment-rotorod screen. The calculated Log P
2. Chemistry
The synthesis of target compounds 8aep was accomplished as
presented in Scheme 1. We have succeeded in a simple and
convenient preparation of compound
methylquinolin-8-ol with benzyl bromide in anhydrous ethanol
in the presence of NaOC2H5 in 90% yield [22]. Compound 1 was
reacted with SeO2 in anhydrous dioxane to afford the compound 2
in high yield (98%) [23]. Then compound 3 was prepared by
condensation of diethyl malonate with Compound 2. Compound 3
1 by reaction of 2-
EtONa
CH2(COOC2H5)2
SeO2
PhCH2Br
N
N
OCH2Ph
1
N
OCH2Ph
2
CHO
OH
COOC2H5
COOH
COOC2H5
COOC2H5
NaH, TMSOI
DMSO
1N, NaOH
N
N
N
COOC2H5
COOC2H5
OCH2Ph
OCH2Ph
OCH2Ph
5
3
4
O
NHCNHR
1)EtOCOCl
NCO
RNH2
r.t.
Na, EtOH
r.t.
Et3N, NaN3
N
N
COOC2H5
COOC2H5
2) toluene, 750C
OCH2Ph
OCH2Ph
7a-p
6
O
R
N
a: R=H
i: R=4-CH3C6H4
j: R=4-OCH3C6H4
k: R=4-NO2C6H4
l: R=4-FC6H4
m: R=4-CF3C6H4
n: R=4-ClC6H4
o: R=4-BrC6H4
p: R=3,5-diClC6H3
N
N
H
O
b: R=CH3
OCH2Ph
c: R=C4H9
d: R=(CH3)2CH
8a-p
e: R=CH3CH2(CH2OH)CH
f: R=C6H11
g: R=C6H5
h: R=CH2C6H5
Scheme 1. General method for the synthesis of compounds 8aep.

340
X. He et al. / European Journal of Medicinal Chemistry 48 (2012) 338e346
(Clog p) values were calculated using the software in ACD Labs 8.0
version.
at the three doses at the two time periods. Compounds 8g and 8p
were less neurotoxic than phenytoin and exhibited motor impair-
ment at the dose of 300 mg/kg at 0.5 h and also 4.0 h (8g).
Compound 8n and 8o revealed neurotoxicity at a dose of 100 mg/kg
at 0.5 h.
Compounds 8e and 8j were selected for quantification of the
pharmacological parameters (ED50 and TD50). Results of the quan-
titative test for the compounds, along with the data on the standard
drugs (phenytoin, carbamazepine, phenobarbital, and valproate),
are reported in Table 2. In the mice ip MES screen, compound 8e
and 8j showed a higher protective index (PI) than all the standard
drugs. In the mice ip scPTZ screen, compound 8e gave an ED50 of
212.3 mg/kg and a TD50 of 365.3 mg/kg, resulting in a high
protective index (PI), that is, TD50/ED50, of 1.7 when compared to
phenobarbital and valproate.
In the present studies, we have synthesized a library of
compounds with 2-quinolincyclopropanespirohydantoin as
a core fragment and at the position-3 of hydantoin ring we have
introduced different substituents. In the preliminary anti-
convulsant screen, all of the alkyl- and aryl-substituted 2-
quinolincyclopropanespirohydantoins showed better protection
against ip MES test but less response toward scPTZ test.
Compounds with long straight chain butyl (8c, 8d) substituents
at N-3 have shown less active than alkyl-substituted derivatives
(8b), the introduction of hydrophilic group at branch of alkyl chain
(8e) resulted in increased anticonvulsant activity. When the alkyl
groups were replaced by a phenyl ring, substitution of an electron-
donating group like methoxy and methyl at the para position of
phenyl ring resulted in increased anticonvulsant activity. For
example, the 4-methyl (8i) and 4-methoxy (8j) substituent deriv-
atives were more potent than 4-nitro (8k) and 4-trifuloro (8m)
derivatives. Compounds 8n, 8o, and 8p did not show apparent
activity in MES and scPTZ screen, and the Clog P of these
compounds were far from 2.0, which is considered to be the
optimum lipophilicity for the congeners that act on the central
nervous system [27]. It may be indicated the importance of lip-
ophilicity as well as electronic properties of the substituents on the
activity of these compounds.
The compounds 8aep 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 results are
shown in Table 1.
The initial anticonvulsant evaluation showed that all
compounds 8aep, with different substituents at N-3 of 2-
quinolincyclopropanespirohydantoin, were effective in ip MES
and/or scPTZ screens.
In the MES test, compounds 8e and 8j were active against MES
test at dose of 30 mg/kg at 0.5 h. The most active compound was 8e,
which presented 50% of protection at dose of 30 mg/kg at 0.5 h.
Interestingly, compound 8e continued to protect from the seizures
at a dose of 30 mg/kg at 4.0 h also. It indicates that 8e have rapid
onset and long duration of anticonvulsant at lower dose.
Compound 8j was active at 4.0 h but at the higher dose of 100 mg/
kg.
Compounds that were active at a dose of 100 mg/kg only at 0.5 h
in MES screen included 8b and 8i, indicating that they have rapid
onset and short duration of anticonvulsant activity. Compound 8p
did not show any activity at two time periods in MES screen.
In the scPTZ screen, compounds that showed protection at 0.5 h
were 8a, 8d, 8e, 8f, 8h, 8j, 8k and 8p. Among these compounds 8d
and 8e showed anti-scPTZ activity at the dose of 100 mg/kg at time
periods 0.5 h. Compounds 8b, 8c and 8m were active only at 4.0 h at
a dose of 300 mg/kg indicative of the long duration of action of
these compounds.
In the neurotoxicity screen, compounds that did not show any
neurotoxicity at the highest dose (300 mg/kg) included 8c, 8d, 8e,
8j and 8k. These compounds did not cause any motor impairment
Table 1
Anticonvulsant activity and neurotoxocity of compounds 8aep administered
intraperitoneally to mice.
Compounds
Intraperitoneal injection in micea
MESc scPTZd Neurotoxocitye
0.5 h 4.0 h 0.5 h 4.0 h 0.5 h
Clog Pb
4. Conclusion
4.0 h
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
300
100
300
300
30
e
300
e
e
300
e
e
1.63 ꢂ 0.41
1.86 ꢂ 0.68
3.45 ꢂ 0.68
2.74 ꢂ 0.68
2.11 ꢂ 0.68
3.92 ꢂ 0.68
4.15 ꢂ 0.61
3.69 ꢂ 0.68
4.61 ꢂ 0.62
3.24 ꢂ 0.64
4.43 ꢂ 0.63
4.44 ꢂ 0.67
5.12 ꢂ 0.66
4.98 ꢂ 0.63
5.16 ꢂ 0.67
6.05 ꢂ 0.72
2.52 ꢂ 0.38
0.38 ꢂ 0.46
e
300
300
e
300
e
In summary, the present studies revealed that number of
1-(8-(benzyloxy)quinolin-2-yl)-6-substituted-4,6-diazaspiro[2,4]
heptane-5,7-diones were effective in the MES and/or scPTZ screens.
In the neurotoxicity studies some of the active compounds were
devoid of toxicity. The most active was 1-(8-(benzyloxy)quinolin-2-
yl)-6-(1-hydroxybutan-2-yl)-4,6-diazaspiro[2,4]heptane-5,7-dione
(8e) which showed ED50 value of 8.6 mg/kg and a protective index
(TD50/ED50) of 26.8 in the MES test in mice. This compound showed
greater ED50 and lower TD50 to standard drugs.
e
e
e
100
30
300
300
300
e
100
100
300
e
e
e
300
e
e
e
300
e
e
300
300
300
300
e
e
300
e
e
300
e
e
100
30
e
e
100
300
e
300
300
e
e
e
e
e
e
e
300
300
300
e
e
e
300
300
300
100
e
300
e
e
300
e
e
e
100
100
300
100
e
5. Experimental protocols
300
e
e
e
e
300
e
e
Phenytoinf
Ethosuximideg
30
e
30
e
e
100
e
5.1. Chemistry
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 adminis-
tered (300 mg/kg).
All chemicals and solvents were purchased from Aldrich, or
Fluka. Solvents and reagents were dried and purified according to
the literature methods. Melting points were uncorrected and
measured on an XT-4 apparatus. 1H and 13C-NMR spectra were
obtained on a Bruker AV400 apparatus in DMSO-d6 and CDCl3 with
TMS as internal standard. The elemental analysis (C, H, N) data were
obtained from a VarioEL III (German) elemental analyzer. The mass
spectra (MS) were recorded on AMD-604 Mass Spectrometer
operating at 70 eV.
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. [34].
Data from Ref. [35].
c
d
e
f
g

X. He et al. / European Journal of Medicinal Chemistry 48 (2012) 338e346
341
Table 2
Phase-II quantitative anticonvulsant evaluation in mice (test drug administered i.p.).
a
b
Compound
ED50
MES
TD50
PIc
scPTZ
MES
scPTZ
8e
8j
8.6 (6.4e12.5)d
212.3 (172.4e222.6)
278.6 (221.5e297.2)
>300
>100
13.2 (5.8e15.9)
149 (123e177)
365.3 (353.7e395.5)
432.6 (378.4e464.8)
65.5 (52.5e72.9)
71.6 (45.9e135)
69 (62.8e72.9)
26.8
22.2
6.9
8.1
3.2
1.7
1.6
<0.22
<0.22
5.2
19.5 (16.3e22.7)
9.5 (8.1e10.4)
8.8 (5.5e14.1)
21.8 (21.8e25.5)
272 (247e338)
Phenytoine
Carbamazepineee
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.
Protective index (TD50/ED50).
Date in parentheses are the 95% confidence limits.
b
c
d
e
Date from Ref. [36].
5.1.1. Synthesis of 8-(benzyloxy)-2-methylquinoline 1
PhCH2O), 6.96 (d, 1H, J ¼ 8.42 Hz), 7.21e7.49 (m, 8H), 7.76 (s, 1H,
CH]C), 8.03 (d, 1H, J ¼ 8.44 Hz). 13C NMR (100 MHz, CDCl3):
To a mixture of 2-methylquinolin-8-ol (8.00 g, 50.3 mmol) and
Na2CO3 (6.30 g, 59.4 mmol) in ethanol (100 ml), a solution of benzyl
bromide (10.1 g, 59.4 mmol) in acetone (20 ml) was added. In the
dark, the reaction mixture was stirred at r.t. for 12 h. After filtration,
the resulting solution was evaporated. Short column chromatog-
raphy on silica gel with chloroform and recrystallization from
PEeETOAc (6:1) gave 1 (11.22 g, 90%) as a white solid: 1H NMR
d
13.99, 14.11, 61.40, 61.76, 71.01, 110.74, 119.71, 123.34, 127.66,
127.95, 128.62, 129.33, 129.76, 136.65, 139.84, 140.63, 150.09,
154.95, 164.11, 166.14.
5.1.4. Synthesis of diethyl 2-(8-(benzyloxy)quinoline-2-yl)
cyclopropane-1,1-dicarboxylate 4
(400 MHz, CDCl3):
d
2.81 (s, 3H, CH3), 5.46 (s, 2H, PhCH2O), 6.98 (d,
To a stirred suspension of sodium hydride (2.12 g, 0.044 mol,
57% in oil dispersion) in dry dimethyl sulfoxide (DMSO) (100 ml)
under nitrogen was added of trimethylsulfoxonium iodide (TMSI)
(9.66 g, 0.044 mol) in a single portion [24]. After frothing ceased,
the grey-white suspension was cooled to 15 ꢀC, and to this mixture
was added with stirring a solution of 16.22 g (0.04 mol) of 3 in 25 ml
of DMSO in one portion. The stirring was continued for 10 min at
15 ꢀC, overnight at room temperature and 4 h at 50e60 ꢀC. After it
was cooled and 400 ml of ice-water was added to it, the mixture
was extracted with ether, and the combined ether fractions were
washed with brine, dried over anhydrous sodium sulfate (Na2SO4),
and evaporated to give a pale yellow oil. The residue was purified
via a chromatography eluting with hexaneseEtOAc (6:1) to give 4
1H, J ¼ 7.60 Hz), 7.26e7.52 (m, 6H), 7.54 (d, 2H, J ¼ 7.20 Hz), 8.03 (d,
1H, J ¼ 8.40 Hz). 13C NMR (100 MHz, CDCl3):
d 24.82, 70.91, 110.83,
118.72, 123.41, 126.45, 126.88, 127.12, 127.95, 128.67, 136.64, 137.27,
139.54, 154.34, 157.36.
5.1.2. Synthesis of 8-(benzyloxy)quinoline-2-carbaldehyde 2
8-(Benzyloxy)-2-methylquinoline (9.20 g, 0.035 mmol) was
added to a dry flask under nitrogen, followed by dry dioxane
(300 ml) and molecular sieves (4 Å, 60 mg). Selenium dioxide
(4.65 g, 0.042 mmol) was added and the reaction was stirred at
95 ꢀC for 12 h and then cooled to room temperature. The reaction
mixture was filtered through celite to remove the black residue and
molecular sieves. The dioxane was removed by rotary evaporation
and the yellow oil was re-dissolved in ethyl acetate (500 ml),
washed with brine (100 ml), water (100 ml), saturated potassium
carbonate solution (100 ml) and dried (MgSO4). Rotary evaporation
of the solvent and recrystallization from PEeETOAc (4:1) give 2
(15.0 g, 65%) as a colorless oil: 1H NMR (400 MHz, CDCl3):
d 0.74 (t,
3H, J ¼ 7.12 Hz, CH3), 1.28 (t, 3H, J ¼ 7.12 Hz, CH3), 1.89e1.93 (m, 1H,
CpreH), 2.68e2.70 (m, 1H, CpreH), 3.30e3.35 (m, 1H, CpreH), 3.83
(q, 2H, J ¼ 7.12 Hz, CH2), 4.23 (q, 2H, J ¼ 7.12 Hz, CH2), 5.34 (s, 1H,
PhCH2O), 7.07 (d, 1H, J ¼ 8.48 Hz), 7.25e7.45 (m, 6H), 7.58 (d, 2H,
J ¼ 7.56 Hz), 8.04 (d, 1H, J ¼ 8.44 Hz). 13C NMR (100 MHz, CDCl3):
(9.02 g, 98%) as a white solid: 1H NMR (400 MHz, CDCl3):
d 5.46 (s,
2H, PhCH2O), 7.16 (d, 1H, J ¼ 8.40 Hz), 7.32e7.56 (m, 6H), 8.07 (d,
d 12.45, 13.01, 19.11, 32.45, 35.94, 59.96, 60.79, 70.05, 110.65, 119.08,
2H, J ¼ 8.44 Hz), 8.28 (d, 1H, J ¼ 8.56 Hz), 10.32 (s, 1H, CHO). 13C
121.35, 125.09, 125.71, 125.98, 126.57, 127.39, 134.96, 136.27, 139.00,
153.17, 153.64, 165.41, 168.80.
NMR (100 MHz, CDCl3):
d 71.32, 111.88, 118.43, 123.32, 126.71,
126.89,127.54,128.33,128.76,136.54,138.43,139.90,154.33,155.78,
193.23.
5.1.5. Synthesis of 2-(8-(benzyloxy)quinolin-2-yl)-1-
(ethoxycarbonyl) cyclopropanecarboxylic acid 5
5.1.3. Synthesis of diethyl 2-((8-(benzyloxy)quinoline-2-yl)
methylene)malonate 3
To a solution of compound 4 (10 mmol) in anhydrous ethanol
(50 ml) was added 1 N sodium hydroxide (25 ml, 1.1 equiv,
25 mmol), and the resulting mixture was stirred art 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 and extracted with ethyl acetate
(3 ꢃ 30 ml). The combined extracts were dried over Na2SO4 and
evaporated to give product 5 as a colorless oil: 1H NMR (400 MHz,
To a solution of compound 2 (10.0 g, 93.0 mmol) and diethyl
malonate (17.94 g, 112.0 mmol), in toluene (200 mL) was added
piperidine (7.95 g, 93 mmol) followed by acetic acid (5.61g,
93 mmol).The reaction mixture was heated to reflux for 20 h and
the distillate was collected in a DeaneStark apparatus. It was
cooled to room temperature and concentrated in vacuo. The dark
brown residue was dissolved in ethyl acetate and washed
successively with NaHCO3 (saturated) and NaCl (saturated), dried
over Na2SO4 and concentrated in vacuo. The residue was purified
via a flash chromatography eluting with hexaneseEtOAc (4:1) to
give 3 (15.0 g, 65%) as a white solid: 1H NMR (400 MHz, CDCl3):
CDCl3):
d
0.51 (t, 3H, J ¼ 7.12 Hz, CH3), 2.02e2.06 (m, 1H, CpreH),
2.43e2.46 (m, 1H, CpreH), 3.67 (q, 2H, J ¼ 7.12 Hz, CH2),
3.74e3.80 (m, 1H, CpreH), 5.32 (s, 1H, PhCH2O), 7.11 (d, 1H,
J ¼ 7.16 Hz), 7.33e7.55 (m, 6H), 7.54 (d, 2H, J ¼ 7.32 Hz), 8.09 (d, 1H,
J ¼ 8.48 Hz). 13C NMR (100 MHz, CDCl3):
d 13.16, 19.02, 31.95, 37.24,
d
1.04 (t, 3H, J ¼ 7.12 Hz, CH3), 1.25 (t, 3H, J ¼ 7.12 Hz, CH3), 4.11 (q,
60.15, 69.95, 111.41, 119.90, 122.65, 126.10, 126.98, 127.47, 127.94,
128.26, 136.00, 137.43, 139.19, 153.55, 154.82, 165.97, 170.63.
2H, J ¼ 7.12 Hz, CH2), 4.23 (q, 2H, J ¼ 7.12 Hz, CH2), 5.27 (s, 1H,

342
X. He et al. / European Journal of Medicinal Chemistry 48 (2012) 338e346
5.1.6. General procedure for the synthesis of compounds 7aep
Compound 5 (10 mmol) was dissolved in dry THF (30 mL) and
cooled to ꢄ15 ꢀC. After the addition of EtOCOCl (11 mmol) and Et3N
(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 ꢄ10 ꢀ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
J1 ¼ 16.04 Hz, J2 ¼ 6.60 Hz, 2ꢃ CH3), 2.26e2.29 (m, 1H, CpreCH),
2.50e2.52 (m, 1H, CpreCH), 3.46e3.50 (m, 1H, CpreCH), 3.72 (q,
2H, J ¼ 7.12 Hz, CH2), 4.13 (m, 1H, CpreCH), 5.52 (br s, 1H, NH), 5.38
(s, 2H, PhCH2O), 5.86 (br s, 1H, NH), 7.07e7.09 (m, 1H), 7.29e7.43
(m, 6H), 7.56 (d, 1H, J ¼ 7.44 Hz), 8.03 (d, 1H, J ¼ 8.48 Hz). 13C
NMR (CDCl3, 100 Hz), d 13.14, 20.11, 22.58, 22.77, 34.94, 38.17, 41.87,
60.86, 70.93, 111.06, 119.92, 122.72, 126.40, 127.09, 127.75, 128.30,
128.49, 136.03, 137.16, 139.87, 154.08, 155.28, 161.95, 170.99. MS m/z
448.5 (Mþ þ 1). Anal. Calcd. ForC26H29N3O4: C 69.78, H 6.53, N 9.39.
Found: C 69.88, H 6.47, N 9.27.
5.1.6.5. Ethyl 2-(8-(benzyloxy)quinolin-2-yl)-1-(3-(1-hydroxybutan-
removed under reduced pressure to afford
a
-carboethoxy isocya-
2-yl)ureido)cyclopropanecarboxylate
(7e). Mp: 148e150 ꢀC,
nate 6 as clear oil. This -carboethoxy isocyanate 6 was directly
a
yield ¼ 85%. 1H NMR (CDCl3, 400 MHz),
d
0.52 (t, 3H, J ¼ 7.12 Hz,
used in the next step without further purification. Arylhydrazide
(10 mmol) was added to a stirred suspension of isocyanate 6 in
appropriate solvent (40 mL) at r.t. The solvent was removed under
reduced pressure when the reaction was completed (detected by
CH3), 1.02 (t, 3H, J ¼ 7.44 Hz, CH3), 1.55e1.71 (m, 2H, CH2),
2.25e2.28 (m, 1H, CpreCH), 2.52e2.55 (m, 1H, CpreCH), 3.32 (br s,
1H, OH), 3.52e3.54 (m, 2H, CH2), 3.58e3.63 (m, 1H, CH), 3.70e3.75
(m, 2H, CH2), 3.90e3.95 (m, 1H, CpreCH), 5.33 (br s, 1H, NH), 5.38
(s, 2H, PhCH2O), 5.89 (br s, 1H, NH), 7.08e7.09 (m, 1H), 7.30e7.44
(m, 6H), 7.55 (d, 1H, J ¼ 7.40 Hz), 8.05 (d, 1H, J ¼ 8.44 Hz). 13C
TLC) and the products
chromatography.
7
were purified by
a
column
NMR (CDCl3, 100 Hz), d 13.13, 13.81, 20.24, 19.69, 31.47, 34.89, 38.41,
5.1.6.1. Ethyl 2-(8-(benzyloxy)quinolin-2-yl)-1-ureidocyclopropane-
39.80, 60.89, 71.02, 111.27, 119.95, 122.71, 126.37, 127.05, 127.71,
128.32, 128.48, 136.00, 137.25, 139.99, 154.16, 155.23, 160.83, 171.08.
MS m/z 478.4 (Mþ þ 1). Anal. Calcd. For C27H31N3O5: C 67.91, H 6.54,
N 8.80. Found: C 67.85, H 6.44, N 8.63.
carboxylate (7a). Mp: 151e153 ꢀC, yield ¼ 85%. 1H NMR (CDCl3,
400 MHz),
d
0.51 (t, 3H, J ¼ 7.12 Hz, CH3), 2.28e2.32 (m, 1H,
CpreCH), 2.58e2.61 (m, 1H, CpreCH), 3.53 (q, 2H, J ¼ 7.12 Hz, CH2),
3.71e3.76 (m, 1H, CpreCH), 5.01e5.13 (m, 1H, NH2), 5.39 (s, 2H,
PhCH2O), 5.66 (br s, 1H, NH), 7.07e7.09 (m, 1H), 7.30e7.44 (m, 6H),
7.56 (d, 2H, J ¼ 7.40 Hz), 8.04 (d, 1H, J ¼ 8.44 Hz). 13C NMR (CDCl3,
5.1.6.6. Ethyl 2-(8-(benzyloxy)quinolin-2-yl)-1-(3-cyclohexylureido)
cyclopropanecarboxylate (7f). Mp: 163e165 ꢀC, yield ¼ 80%. 1H
100 Hz),
d
13.07, 18.34, 33.41, 38.37, 60.41, 69.98, 111.35, 119.86,
NMR (CDCl3, 400 MHz),
d
0.41 (t, 3H, J ¼ 7.12 Hz, CH3), 1.22e1.88 (m,
122.49, 126.16, 127.10, 127.52, 127.89, 128.28, 135.89, 137.40, 139.25,
153.58, 154.99, 159.83, 172.02. MS m/z 406.4 (Mþ þ 1). Anal. Calcd.
For C23H23N3O4: C 68.13, H 5.72, N 10.36. Found: C 68.21, H 5.81, N
10.47.
10H), 2.28e2.31 (m, 1H, CpreCH), 2.51e2.54 (m, 1H, CpreCH), 3.53
(q, 2H, J ¼ 7.12 Hz, CH2), 3.71e3.76 (m, 1H, CpreCH), 3.73e3.82 (m,
1H), 4.89 (br s, 1H, NH), 5.22 (br s, 1H, NH), 5.29 (s, 2H, PhCH2O),
7.07e7.09 (m, 1H), 7.29e7.44 (m, 6H), 7.92 (d, 1H, J ¼ 7.48 Hz), 8.38
(d, 1H, J ¼ 8.44 Hz). 13C NMR (CDCl3, 100 Hz),
d 12.12, 19.15, 23.66,
5.1.6.2. Ethyl
2-(8-(benzyloxy)quinolin-2-yl)-1-(3-methylureido)
24.64, 31.69, 31.91, 33.93, 37.22, 47.54, 52.44, 59.82, 69.99, 110.19,
118.93, 121.67, 125.34, 126.04, 126.68, 127.27, 127.45, 134.95, 136.19,
138.92, 153.11, 154.27, 159.74, 169.98. MS m/z 488.6 (Mþ þ 1). Anal.
Calcd. For C29H33N3O4: C 71.44, H 6.82, N 8.62. Found: C 71.51, H
6.79, N 8.55.
cyclopropanecarboxylate (7b). Mp: 134e136 ꢀC, yield ¼ 83%. 1H
NMR (CDCl3, 400 MHz),
d
0.50 (t, 3H, J ¼ 7.16 Hz, CH3), 2.30e2.33
(m, 1H, CpreCH), 2.52e2.55 (m, 1H, CpreCH), 2.90 (s, 3H, NCH3),
3.51 (q, 2H, J ¼ 7.16 Hz, CH2), 3.71e3.75 (m, 1H, CpreCH), 4.56 (br s,
1H, NH), 5.34 (br s,1H, NH), 5.40 (s, 2H, PhCH2O), 7.06e7.08 (m,1H),
7.30e7.43 (m, 6H,), 7.55 (d, 2H, J ¼ 7.40 Hz), 8.04 (d, 1H, J ¼ 8.44 Hz).
5.1.6.7. Ethyl 2-(8-(benzyloxy)quinolin-2-yl)-1-(3-phenylureido)cyc-
13C NMR (CDCl3, 100 Hz),
d
12.14, 19.05, 25.72, 33.87, 37.49, 59.92,
lopropanecarboxylate (7g). Mp: 117e120 ꢀC, yield ¼ 85%. 1H NMR
70.01, 110.32, 118.94, 121.69, 125.40, 126.06, 126.72, 127.35, 127.48,
135.04, 136.26, 139.00, 153.15, 154.18, 158.63, 171.06. MS m/z 420.2
(Mþ þ 1). Anal. Calcd. For C24H25N3O4: C 68.72, H 6.01, N 10.02.
Found: C 68.81, H 6.07, N 10.07.
(CDCl3, 400 MHz),
d
0.40 (t, 3H, J ¼ 7.16 Hz, CH3), 2.31e2.34 (m, 1H,
CpreCH), 2.51e2.53 (m, 1H, CpreCH), 3.47 (q, 2H, J ¼ 7.16 Hz, CH2),
3.62e3.68 (m, 1H, CpreCH), 5.27 (s, 2H, PhCH2O), 6.01 (br s, 1H,
NH), 6.62 (br s, 1H, NH), 6.96e7.01 (m, 2H), 7.19e7.29 (m, 7H), 7.33
(d,1H, J ¼ 8.44 Hz), 7.46 (d, 2H, J ¼ 7.44 Hz), 7.53 (d, 2H, J ¼ 8.00 Hz),
5.1.6.3. Ethyl 2-(8-(benzyloxy)quinolin-2-yl)-1-(3-butylureido)cyclo-
7.93 (d, 1H, J ¼ 8.44 Hz). 13C NMR (CDCl3, 100 Hz),
d 12.06, 19.48,
propanecarboxylate (7c). Mp: 143e145 ꢀC, yield ¼ 78%. 1H NMR
34.15, 38.51, 60.19, 69.90, 110.13, 118.88, 119.04, 121.60, 123.05,
125.51, 126.05, 126.71, 127.30, 127.45, 127.85, 135.12, 136.08, 137.16,
138.87, 153.06, 153.75, 158.06, 171.23. MS m/z 482.6 (Mþ þ 1). Anal.
Calcd. For C29H27N3O4: C 72.33, H 5.65, N 8.73. Found: C 72.45, H
5.54, N 8.66.
(CDCl3, 400 MHz),
d
0.51 (t, 3H, J ¼ 7.16 Hz, CH3), 0.95 (t, 3H,
J ¼ 7.32 Hz, CH3), 1.25e1.43 (m, 2H, CH2), 1.53e1.58 (m, 2H, CH2),
2.28e2.31 (m, 1H, CpreCH), 2.51e2.54 (m, 1H, CpreCH), 3.29e3.40
(m, 2H, CH2), 3.53 (q, 2H, J ¼ 7.16 Hz, CH2), 3.71e3.76 (m, 1H,
CpreCH), 4.62 (br s, 1H, NH), 4.99 (br s, 1H, NH), 5.39 (s, 2H,
PhCH2O), 7.07e7.09 (m, 1H), 7.29e7.44 (m, 6H), 7.56 (d, 1H,
J ¼ 7.48 Hz), 8.04 (d, 1H, J ¼ 8.44 Hz). 13C NMR (CDCl3, 100 Hz),
5.1.6.8. Ethyl 2-(8-(benzyloxy)quinolin-2-yl)-1-(3-benzylureido)cyc-
lopropanecarboxylate (7h). Mp: 101e103 ꢀC, yield ¼ 88%. 1H NMR
d
12.48, 13.16, 19.55, 29.03, 30.79, 34.26, 37.68, 39.13, 60.23, 70.33,
(CDCl3, 400 MHz),
d
0.33 (t, 3H, J ¼ 7.16 Hz, CH3), 2.21e2.25 (m, 1H,
110.52, 119.27, 122.05, 125.72, 126.42, 127.07, 127.65, 127.83, 135.36,
136.54,139.27,153.46,154.57,157.18,170.38. MS m/z 462.3 (Mþ þ 1).
Anal. Calcd. For C27H31N3O4: C 70.26, H 6.77, N 9.10. Found: C 70.33,
H 6.57, N 9.17.
CpreCH), 2.43e2.46 (m, 1H, CpreCH), 3.39 (q, 2H, J ¼ 7.16 Hz,
CH2), 3.52e3.60 (m, 1H, CpreCH), 4.42 (s, 2H, PhCH2), 5.23 (s, 2H,
PhCH2O), 6.44 (br s, 1H, NH), 6.91e6.95 (m, 1H), 7.06e7.24 (m,
11H), 7.41 (d, 2H, J ¼ 7.24 Hz), 7.87 (d, 1H, J ¼ 8.48 Hz), 8.41 (br s,
1H, NH). 13C NMR (CDCl3, 100 Hz),
d 13.14, 20.38, 34.91, 38.68,
5.1.6.4. Ethyl 2-(8-(benzyloxy)quinolin-2-yl)-1-(3-isopropylureido)
44.15, 60.98, 70.93, 111.16, 119.96, 122.72, 126.50, 127.06, 127.34,
127.77, 128.35, 128.45, 128.52, 128.69, 136.11, 137.19, 138.35,
139.94, 154.12, 155.09, 158.12, 170.90. MS m/z 496.6 (Mþ þ 1). Anal.
cyclopropanecarboxylate (7d). Mp: 123e125 ꢀC, yield ¼ 80%. 1H
NMR (CDCl3, 400 MHz),
d
0.52 (t, 3H, J ¼ 7.12 Hz, CH3), 1.22 (dd, 6H,

X. He et al. / European Journal of Medicinal Chemistry 48 (2012) 338e346
343
Calcd. For C30H29N3O4: C 72.71, H 5.90, N 8.48. Found: C 72.85, H
5.97, N 8.53.
124.34, 126.76, 127.73, 127.79, 127.84, 128.36, 128.66, 129.34, 133.50,
135.87, 136.80, 137.34, 139.98, 154.22, 154.98, 165.45, 171.71. MS m/z
550.5 (Mþ þ 1). Anal. Calcd. For C30H26F3N3O4: C 65.57, H 4.77, N 7.65.
Found: C 65.42, H 4.67, N 7.43.
5.1.6.9. Ethyl
2-(8-(benzyloxy)quinolin-2-yl)-1-(3-p-tolylureido)
cyclopropanecarboxylate (7i). Mp: 159e160 ꢀC, yield ¼ 80%. 1H
NMR (CDCl3, 400 MHz),
d
0.52 (t, 3H, J ¼ 7.16 Hz, CH3), 2.32 (s, 3H,
5.1.6.14. Ethyl 2-(8-(benzyloxy)quinolin-2-yl)-1-(3-(4-chlorophenyl)
CH3), 2.40e2.44 (m, 1H, CpreCH), 2.61e2.64 (m, 1H, CpreCH), 3.57
(q, 2H, J ¼ 7.16 Hz, CH2), 3.75e3.79 (m, 1H, CpreCH), 5.39 (s, 2H,
PhCH2O), 7.07e7.14 (m, 2H), 6.15 (s,1H, NH), 7.30e7.39 (m, 6H), 7.45
(d, 1H, J ¼ 8.44 Hz), 7.50 (d, 2H, J ¼ 8.16 Hz), 7.56 (d, 2H, J ¼ 7.56 Hz),
8.05 (d, 1H, J ¼ 8.48 Hz), 8.27 (s, 1H, NH). 13C NMR (CDCl3, 100 Hz),
ureido)cyclopropanecarboxylate (7n). Mp: 118e120 ꢀC, yield ¼ 80%.
1H NMR (CDCl3, 400 MHz),
d
0.53 (t, 3H, J ¼ 7.12 Hz, CH3), 2.39e2.42
(m, 1H, CpreCH), 2.58e2.61 (m, 1H, CpreCH), 3.58 (q, 2H,
J ¼ 7.12 Hz, CH2), 3.76e3.80 (m, 1H, CpreCH), 5.39 (s, 2H, PhCH2O),
6.71 (br s, 1H, NH), 7.09e7.11 (m, 2H), 7.26e7.58 (m, 11H), 8.08 (d,
d
11.48, 20.21, 20.93, 35.40, 39.23, 61.25, 70.98, 111.17, 115.25, 119.98,
1H, J ¼ 8.44 Hz), 8.55 (br s,1H, NH). 13C NMR (CDCl3,100 Hz),
d 13.11,
126.60, 127.23, 127.42, 127.84, 128.39, 128.56, 129.44, 133.60, 135.82,
136.22, 137.14, 139.91, 154.12, 154.93, 155.97, 171.22. MS m/z 496.6
(Mþ þ 1). Anal. Calcd. For C30H29N3O4: C 72.71, H 5.90, N 8.48.
Found: C 72.62, H 6.04, N 8.55.
20.64, 20.93, 35.11, 39.69, 61.33, 70.97, 111.11, 116.21, 119.92, 121.31,
122.64, 126.63, 127.18, 127.83, 128.39, 128.94, 136.24, 136.83, 137.08,
139.89,144.96,154.10,154.73,156.71,171.30. MS m/z 516.5 (Mþ þ 1).
Anal. Calcd. For C29H26ClN3O4: C 67.50, H 5.08, N 8.41. Found: C
67.62, H 5.16, N 8.55.
5.1.6.10. Ethyl
2-(8-(benzyloxy)quinolin-2-yl)-1-(3-(4-methoxy-
phenyl)ureido)cyclopropanecarboxylate (7j). Mp: 124e126 ꢀC,
5.1.6.15. Ethyl 2-(8-(benzyloxy)quinolin-2-yl)-1-(3-(4-bromophenyl)
yield ¼ 78.7%. 1H NMR (CDCl3, 400 MHz),
d
0.45 (t, 3H, J ¼ 7.16 Hz,
ureido)cyclopropanecarboxylate (7o). Mp: 105e107 ꢀC, yield ¼ 88%.
CH3), 2.33e2.37 (m, 1H, CpreCH), 2.54e2.56 (m, 1H, CpreCH), 3.50
(q, 2H, J ¼ 7.16 Hz, CH2), 3.68e3.73 (m, 1H, CpreCH), 3.75 (s, 3H,
OCH3), 5.33 (s, 2H, PhCH2O), 6.72 (br s, 1H, NH), 6.80 (d, 2H,
J ¼ 8.96 Hz), 7.00e7.50 (m, 11H), 7.99 (d, 1H, J ¼ 8.44 Hz), 8.66 (br s,
1H NMR (CDCl3, 400 MHz),
d
0.42 (t, 3H, J ¼ 7.16 Hz, CH3), 2.20e2.46
(m, 1H, CpreCH), 2.31e2.49 (m, 1H, CpreCH), 3.46 (q, 2H,
J ¼ 7.16 Hz, CH2), 3.63e3.69 (m, 1H, CpreCH), 5.26 (s, 2H, PhCH2O),
6.26 (br s, 1H, NH), 6.39e6.41 (m, 2H), 6.89e7.46 (m, 11H), 7.96 (d,
1H, J ¼ 8.46 Hz), 8.74 (br s, 1H, NH). 13C NMR (CDCl3, 100 Hz),
1H, NH). 13C NMR (CDCl3, 100 Hz),
d 11.00, 18.42, 32.91, 37.40, 53.36,
59.09, 68.92, 109.19, 111.94, 117.83, 119.64, 120.60, 124.40, 124.94,
125.62, 126.26, 126.39, 129.33, 134.01, 135.10, 137.89, 152.07, 152.81,
154.12, 159.74, 171.32. MS m/z 482.6 (Mþ þ 1). Anal. Calcd. For
C30H29N3O4: C 72.71, H 5.90, N 8.48. Found: C 72.62, H 6.04, N 8.53.
d 13.70, 18.12, 34.21, 38.39, 60.31, 69.88, 109.94, 115.63, 118.85,
120.60, 121.57, 125.62, 126.77, 127.79, 129.90, 130.77, 135.25, 135.93,
136.30, 138.72, 144.48, 152.98, 153.68, 156.66, 170.10. MS m/z 561.4
(Mþ þ 1). Anal. Calcd. For C30H29N3O4: C 62.15, H 4.68, N 7.50.
Found: C 62.23, H 4.82, N 7.38.
5.1.6.11. Ethyl 2-(8-(benzyloxy)quinolin-2-yl)-1-(3-(4-nitrophenyl)
ureido)cyclopropanecarboxylate (7k). Mp: 114e116 ꢀC, yield ¼ 85%.
5.1.6.16. Ethyl
2-(8-(benzyloxy)quinolin-2-yl)-1-(3-(3,5-dichloro-
1H NMR (CDCl3, 400 MHz),
d
0.62 (t, 3H, J ¼ 7.12 Hz, CH3), 1.90e1.94
phenyl)ureido)cyclopropanecarboxylate (7p). Mp: 131e133 ꢀC,
(m, 1H, CH), 2.73e2.76 (m, 1H, CH), 3.29e3.33 (q, 2H, J ¼ 7.16 Hz,
CH2), 3.75e3.79 (m, 1H, CH), 5.23 (s, 2H, PhCH2O), 6.33 (br s, 1H,
NH), 7.07e7.14 (m, 2H), 7.30e7.39 (m, 6H), 7.45 (d, 1H, J ¼ 8.44 Hz),
7.50 (d, 2H, J ¼ 8.16 Hz), 7.56 (d, 2H, J ¼ 7.56 Hz), 8.05 (d, 1H,
yield ¼ 82%. 1H NMR (CDCl3, 400 MHz),
d
0.44 (t, 3H, J ¼ 7.12 Hz,
CH3), 2.18e2.34 (m, 1H, CpreCH), 2.33e2.51 (m, 1H, CpreCH), 3.48
(q, 2H, J ¼ 7.12 Hz, CH2), 3.72e3.75 (m, 1H, CpreCH), 5.23 (s, 2H,
PhCH2O), 6.05 (br s,1H, NH), 6.43e6.51 (m, 2H), 6.84e7.55 (m,10H),
8.05 (d, 1H, J ¼ 8.56 Hz), 8.43 (br s, 1H, NH). 13C NMR (CDCl3,
J ¼ 8.48 Hz), 8.64 (br s, 1H, NH). 13C NMR (CDCl3, 100 Hz),
d 12.10,
19.35, 31.83, 37.66, 60.04, 69.88, 109.45, 112.43, 117.76, 119.43,
121.64, 124.88, 124.96, 125.82, 126.72,126.98,130.43,134.87,136.32,
137.92, 143.44, 152.34, 152.98, 156.72, 172.33. MS m/z 527.6
(Mþ þ 1). Anal. Calcd. For C29H26N4O6: C 66.15, H 4.98, N 10.64.
Found: C 66.22, H 4.87, N 10.78.
100 Hz), d 13.88, 20.56, 21.03, 35.87, 39.41, 61.44, 70.98, 110.91,
115.46, 120.28, 126.74, 127.53, 127.76, 127.82, 128.43, 128.76, 129.42,
133.61, 134.84, 136.76, 137.24, 140.94, 154.33, 154.87, 166.91, 171.62.
MS m/z 551.4 (Mþ þ 1). Anal. Calcd. For C29H25Cl2N3O4: C 63.28, H
4.58, N 7.63. Found: C 63.36, H 4.74, N 7.47.
5.1.6.12. Ethyl 2-(8-(benzyloxy)quinolin-2-yl)-1-(3-(4-fluorophenyl)
ureido)cyclopropanecarboxylate (7l). Mp: 159e160 ꢀC, yield ¼ 82%.
5.1.7. General procedure for the synthesis of compounds 8aep
To a solution of compound 7 (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 8.
1H NMR (CDCl3, 400 MHz),
d
0.53 (t, 3H, J ¼ 7.12 Hz, CH3), 2.39e2.42
(m, 1H, CpreCH), 2.58e2.61 (m, 1H, CpreCH), 3.56 (q, 2H,
J ¼ 7.12 Hz, CH2), 3.74e3.80 (m, 1H, CpreCH), 5.23 (s, 2H, PhCH2O),
7.03e7.11 (m, 3H), 6.55 (br s, 1H, NH), 7.30e7.59 (m, 10H), 8.08 (d,
1H, J ¼ 8.48 Hz), 8.27 (br s, 1H, NH). 13C NMR (CDCl3, 100 Hz),
d
11.98, 18.65, 31.88, 37.44, 59.34, 70.68, 108.48, 113.66, 117.88,
119.33, 122.34, 124.76, 125.43, 125.88, 126.74, 126.88, 131.56, 133.46,
136.77, 137.99, 144.46, 153.41, 156.66, 161.54, 172.56. MS m/z 500.6
(Mþ þ 1). Anal. Calcd. For C29H26FN3O4: C 69.73, H 5.25, N 8.41.
Found: C 69.93, H 5.14, N 8.55.
5.1.7.1. 1-(8-(Benzyloxy)quinolin-2-yl)-4,6-diazaspiro[2.4]heptane-
5,7-dione (8a). Mp: 159e160 ꢀC, yield ¼ 92%. 1H NMR (DMSO-d6,
400 MHz),
d 1.72e1.75 (m, 1H, CpreH), 2.05e2.05 (m, 1H, CpreH),
2.83e2.85 (m, 1H, CpreH), 5.32 (s, 2H, PhCH2O), 7.13 (d, 1H,
J ¼ 6.92 Hz), 7.33e7.54 (m, 8H), 8.05 (d, 1H, J ¼ 8.32 Hz), 8.23 (br s,
5.1.6.13. Ethyl
2-(8-(benzyloxy)quinolin-2-yl)-1-(3-(4-(trifluorome-
1H, NH), 10.54 (br s, 1H, NH). 13C NMR (DMSO-d6, 100 Hz),
d 19.12,
thyl)phenyl)ureido)cyclopropanecarboxylate (7m). Mp: 133e135 ꢀC,
36.23, 37.76, 69.54, 110.12, 119.83, 121.45, 125.37, 127.65, 127.71,
127.76, 128.34, 134.69, 137.43, 139.06, 153.51, 157.05, 158.3, 171.12.
MS m/z 360.4 (Mþ þ 1). Anal. Calcd. For C21H17N3O3: C 70.18, H 4.77,
N 11.69. Found: C 69.91, H 4.82, N 11.43.
yield ¼ 78%. 1H NMR (CDCl3, 400 MHz),
d
0.54 (t, 3H, J ¼ 7.12 Hz,
CH3),1.88e1.92 (m,1H, CpreCH), 2.75e2.78 (m,1H, CpreCH), 3.38(q,
2H, J ¼ 7.12 Hz, CH2), 3.75e3.79 (m, 1H, CpreCH), 5.20 (s, 2H,
PhCH2O), 6.43 (br s, 1H, NH), 7.08e7.12 (m, 2H), 7.11e7.55 (m, 11H),
8.05 (d,1H, J ¼ 8.56 Hz), 8.62 (br s,1H, NH). 13C NMR (CDCl3, 100 Hz),
5.1.7.2. 1-(8-(Benzyloxy)quinolin-2-yl)-6-methyl-4,6-diazaspiro[2,4]
d
13.22, 20.35, 20.96, 35.38, 39.43, 61.62, 71.04,111.74,115.89, 119.88,
heptane-5,7-dione (8b). Mp: 142e144 ꢀC, yield ¼ 95%. 1H NMR

344
X. He et al. / European Journal of Medicinal Chemistry 48 (2012) 338e346
(DMSO-d6, 400 MHz),
d
1.68e1.71 (m, 1H, CpreH), 2.06e2.08 (m,
(d, 1H, J ¼ 8.56 Hz), 12.33 (br s, 1H, NH). 13C NMR (DMSO-d6,
1H, CpreH), 2.79e2.82 (m,1H, CpreH), 3.03 (s, 3H, CH3), 5.35 (s, 2H,
PhCH2O), 7.08 (d, 1H, J ¼ 7.12 Hz), 7.36e7.58 (m, 8H), 8.12 (d, 1H,
J ¼ 8.24 Hz), 8.46 (br s, 1H, NH), 10.21 (br s, 1H, NH). 13C NMR
100 Hz), d 19.40, 36.14, 37.89, 69.99, 110.88, 119.21, 119.71, 122.40,
123.13, 126.05, 127.48, 127.63, 127.89, 128.35, 128.76, 135.24, 137.25,
138.95, 139.05, 153.43, 156.13, 158.98, 170.55. MS m/z 436.3
(Mþ þ 1). Anal. Calcd. For C27H21N3O3: C 74.47, H 4.86, N 9.65.
Found: C 74.58, H 4.93, N 9.46.
(DMSO-d6,100 Hz), d 19.56, 24.35, 36.87, 37.16, 70.44,110.23, 118.32,
121.73, 125.97, 127.32, 127.61, 127.78, 129.05, 134.27, 137.81, 139.60,
154.42,157.15,157.74,171.45. MS m/z 374.1 (Mþ þ 1). Anal. Calcd. For
C22H19N3O3: C 70.76, H 5.13, N 11.25. Found: C 70.51, H 4.97, N 11.04.
5.1.7.8. 6-Benzyl-1-(8-(benzyloxy)quinolin-2-yl)-4,6-diazaspiro[2.4]
heptane-5,7-dione (8h). Mp: 141e143 ꢀC, yield ¼ 92%. 1H NMR
5.1.7.3. 1-(8-(Benzyloxy)quinolin-2-yl)-6-butyl-4,6-diazaspiro[2,4]
(DMSO-d6, 400 MHz), d 1.74e1.77 (m, 1H, CpreH), 2.13e2.14 (m, 1H,
heptane-5,7-dione (8c). Mp: 151e153 ꢀC, yield ¼ 90%. 1H NMR
CpreH), 2.88e2.92 (m, 1H, CpreH), 4.37 (s, 2H, PhCH2), 5.34 (s, 2H,
PhCH2O), 7.16 (d, 1H, J ¼ 7.24 Hz), 7.24e7.55 (m, 13H), 8.04 (d, 1H,
J ¼ 8.60 Hz), 11.48 (br s, 1H, NH). 13C NMR (DMSO-d6, 100 Hz),
(DMSO-d6, 400 MHz),
d
0.89 (t, 3H, J ¼ 7.28 Hz, CH3), 1.31e1.46 (m,
4H), 1.71e1.73 (m, 1H, CpreH), 2.09e2.11 (m, 1H, CpreH),
2.85e2.89 (m, 1H, CpreH), 3.09e3.22 (m, 2H, CH2), 5.34 (s, 2H,
PhCH2O), 7.15 (d, 1H, J ¼ 7.12 Hz), 7.32e7.55 (m, 8H), 8.05 (d, 1H,
J ¼ 8.44 Hz), 8.76 (br s, 1H, NH), 10.77 (br s, 1H, NH). 13C NMR
d
19.10, 36.27, 37.48, 42.53, 69.94, 110.25, 119.56, 122.04, 125.59,
126.69, 127.14, 127.21, 127.67, 127.70, 127.76, 128.22, 128.30, 134.44,
137.23, 137.36, 139.04, 139.59, 153.45, 159.57, 171.34. MS m/z 450.6
(Mþ þ 1). Anal. Calcd. For C28H23N3O3: C 74.82, H 5.16, N 9.35.
Found: C 74.93, H 5.37, N 9.44.
(DMSO-d6, 100 Hz),
d 13.22, 19.14, 19.35, 29,87, 36.28, 36.71, 37.82,
69.44, 111.72, 118.53, 122.04, 125.47, 127.26, 127.53, 127.80, 129.33,
134.71, 136.88, 140.50, 155.22, 157.55, 158.76, 170.33. MS m/z 416.1
(Mþ þ 1). Anal. Calcd. For C25H25N3O3: C 72.27, H 6.06, N 10.11.
Found: C 72.34, H 6.22, N 10.08.
5.1.7.9. 1-(8-(Benzyloxy)quinolin-2-yl)-6-p-tolyl-4,6-diazaspiro[2,4]
heptane-5,7-dione (8i). Mp: 171e173 ꢀC, yield ¼ 95%. 1H NMR
(DMSO-d6, 400 MHz), d 1.88e1.90 (m, 1H, CpreH), 2.26 (s, 3H, CH3),
5.1.7.4. 1-(8-(Benzyloxy)quinolin-2-yl)-6-isopropyl-4,6-diazaspiro
2.30e2.32 (m, 1H, CpreH), 3.16e3.20 (m, 1H, CpreH), 5.36 (s, 2H,
PhCH2O), 7.12 (d, 1H, J ¼ 8.28 Hz), 7.13e7.55 (m, 12H), 8.16 (d, 1H,
J ¼ 8.56 Hz), 12.27 (br s, 1H, NH). 13C NMR (DMSO-d6, 100 Hz),
[2,4]heptane-5,7-dione (8d). Mp: 137e139 ꢀC, yield ¼ 90%. 1H NMR
(DMSO-d6, 400 MHz),
d
1.10 (d, 6H, J ¼ 7.28 Hz, J ¼ 17.32 Hz, 2ꢃ
CH3), 1.70e1.72 (m, 1H, CpreH), 2.07e2.09 (m, 1H, CpreH),
2.85e2.88 (m, 1H, CpreH), 3.86e3.89 (m, 1H, CH), 5.34 (s, 2H,
PhCH2O), 7.15 (d, 1H, J ¼ 7.12 Hz), 7.32e7.55 (m, 8H), 8.03 (d, 1H,
J ¼ 8.44 Hz), 8.69 (br s, 1H, NH), 10.71 (br s, 1H, NH). 13C NMR
d 19.35, 20.41, 36.05, 37.79, 70.01, 110.88, 119.13, 119.71, 122.42,
125.97, 127.47, 127.61, 127.88, 128.34, 129.13, 131.86, 135.12, 136.56,
137.30, 139.09, 153.49, 156.30, 159.87, 170.28. MS m⁄z 450.5
(Mþ þ 1). Anal. Calcd. For C28H23N3O3: C 74.82, H 5.16, N 9.35.
Found: C 74.79, H 5.23, N 9.29.
(DMSO-d6, 100 Hz), d 19.06, 22.68, 22.83, 35.93, 37.55, 40.06, 69.90,
110.28, 119.54, 121.98, 125.50, 127.53, 127.63, 127.74, 128.33, 134.34,
137.32,139.05, 153.46,157.80,158.77,170.16. MS m/z 402.1 (Mþ þ 1).
Anal. Calcd. For C24H23N3O3: C 71.80, H 5.77, N 10.47. Found: C 71.66,
H 5.80, N 10.29.
5.1.7.10. 1-(8-(Benzyloxy)quinolin-2-yl)-6-(4-methoxyphenyl)-4,6-
diazaspiro[2.4]heptane-5,7-dione (8j). Mp: 137e139 ꢀC, yield ¼ 90%.
1H NMR (DMSO-d6, 400 MHz),
d 1.88e1.91 (m, 1H, CpreH),
2.30e2.33 (m, 1H, CpreH), 3.18e3.22 (m, 1H, CpreH), 3.72 (s, 3H,
OCH3), 5.36 (s, 2H, PhCH2O), 6.89 (d, 2H, J ¼ 8.96 Hz), 7.18e7.55 (m,
11H), 8.16 (d, 1H, J ¼ 8.56 Hz), 12.04 (br s, 1H, NH). 13C NMR (DMSO-
5.1.7.5. 1-(8-(Benzyloxy)quinolin-2-yl)-6-(1-hydroxybutan-2-yl)-4,6-
diazaspiro[2,4]heptane-5,7-dione (8e). Mp: 175e177 ꢀC, yield ¼ 90%.
1H NMR (DMSO-d6, 400 MHz),
d
0.90 (m, 3H, CH3), 1.75e1.78 (m,1H,
d6, 100 Hz), d 19.31, 35.99, 37.77, 55.08, 70.00, 110.86, 113.82, 119.71,
CpreCH), 1.88e1.92 (m, 2H, CH2), 2.12e2.15 (m, 1H, CpreCH),
2.88e2.30 (m, 1H, CpreCH), 3.75e3.92 (m, 3H, CH2, CH), 4.06 (br
s, 1H, OH), 5.34 (s, 2H, PhCH2O), 7.13 (d, 1H, J ¼ 7.88 Hz), 7.42e7.67
(m, 8H), 8.13 (d, 1H, J ¼ 8.42 Hz). 13C NMR (DMSO-d6, 100 Hz),
120.70, 122.38, 126.01, 127.48, 127.63, 127.88, 128.35, 132.22, 135.19,
137.27, 139.08, 153.46, 155.05, 156.22, 158.79, 170.62. MS m/z 466.5
(Mþ þ 1). Anal. Calcd. For C28H23N3O4: C 72.24, H 4.98, N 9.03.
Found: C 72.39, H 5.02, N 9.17.
d
11.23, 19.72, 21.24, 36.65, 37.81, 56.34, 63.27, 71.26, 110.27, 119.99,
122.72, 126.34, 127.05, 127.71, 128.32, 128.48, 136.00, 137.25, 139.99,
154.16, 155.23, 159.83, 174.08. MS m/z 478.4 (Mþ þ 1). Anal. Calcd.
For C27H31N3O5: C 67.91, H 6.54, N 8.80. Found: C 67.85, H 6.44, N
8.63.
5.1.7.11. 1-(8-(Benzyloxy)quinolin-2-yl)-6-(4-nitrophenyl)-4,6-diaza-
spiro[2,4]heptane-5,7-dione (8k). Mp: 125e127 ꢀC, yield ¼ 90%. 1H
NMR (DMSO-d6, 400 MHz),
d 1.90e1.92 (m, 1H, CpreH), 2.31e2.33
(m, 1H, CpreH), 3.19e3.21 (m, 1H, CpreH), 5.34 (s, 2H, PhCH2O),
7.09 (d, 1H, J ¼ 8.82 Hz), 7.18e7.55 (m, 12H), 8.16 (d, 1H, J ¼ 8.56 Hz),
5.1.7.6. 1-(8-(Benzyloxy)quinolin-2-yl)-6-cyclohexyl-4,6-diazaspiro
12.22 (br s, 1H, NH). 13C NMR (DMSO-d6, 100 Hz),
d 19.26, 36.95,
[2,4]heptane-5,7-dione (8f). Mp: 168e170 ꢀC, yield ¼ 90%. 1H NMR
37.82, 69.98, 110.84, 113.73, 119.53, 121.73, 124.66, 126.06, 127.38,
127.45, 127.79, 128.48, 132.41, 135.21, 139.95, 146.34, 153.46, 155.25,
156.53, 159.32, 171.43. MS m⁄z 481.3 (Mþ þ 1). Anal. Calcd. For
C27H20N4O5: C 67.49, H 4.20, N 11.66. Found: C 67.61, H 4.13, N
11.59.
(DMSO-d6, 400 MHz),
d 1.15e1.72 (m, 10H), 2.08e2.11 (m, 1H,
CpreH), 2.82e2.84 (m, 1H, CpreH), 3.41e3.43 (m, 1H, CH),
3.63e3.65 (m, 1H, CpreH), 5.35 (s, 2H, PhCH2O), 7.16 (d, 1H,
J ¼ 7.68 Hz), 7.33e7.55 (m, 8H), 8.05 (d, 1H, J ¼ 8.56 Hz). 13C NMR
(DMSO-d6, 100 Hz),
d 13.66, 19.69, 24.16, 31.40, 32.57, 36.25, 38.43,
69.92, 110.31, 119.54, 121.98, 125.59, 127.51, 127.77, 127.85, 128.33,
134.47, 137.29, 139.08, 153.47, 157.46, 158.88, 171.04. MS m/z 442.1
(Mþ þ 1). Anal. Calcd. For C27H27N3O3: C 73.45, H 6.16, N 9.52.
Found: C 73.57, H 6.31, N 9.68.
5.1.7.12. 1-(8-(Benzyloxy)quinolin-2-yl)-6-(4-fluorophenyl)-4,6-diaza-
spiro[2,4]heptane-5,7-dione (8l). Mp: 165e167 ꢀC, yield ¼ 93%. 1H
NMR (DMSO-d6, 400 MHz),
d 1.91e1.93 (m, 1H, CpreH), 2.30e2.32
(m, 1H, CpreH), 3.20e3.22 (m, 1H, CpreH), 5.35 (s, 2H, PhCH2O),
7.07 (d, 1H, J ¼ 8.84 Hz), 7.21e7.57 (m, 12H), 8.14 (d, 1H, J ¼ 8.44 Hz),
5.1.7.7. 1-(8-(Benzyloxy)quinolin-2-yl)-6-phenyl-4,6-diazaspiro[2,4]
11.68 (br s, 1H, NH). 13C NMR (DMSO-d6, 100 Hz),
d 20.03, 36.62,
heptane-5,7-dione (8g). Mp: 130e132 ꢀC, yield ¼ 90%. 1H NMR
38.38, 71.05, 111.08, 116.07, 119.72, 121.68, 122.44, 126.89, 127.36,
127.94, 128.47, 128.52, 134.40, 136.32, 137.62, 139.72, 154.23, 154.22,
158.50, 160.64, 171.95. MS m/z 482.6 (Mþ þ 1). Anal. Calcd. For
C27H20FN3O3: C 71.51, H 4.45, N 9.27. Found: C 71.43, H 4.54, N 9.39.
(DMSO-d6, 400 MHz),
d 1.90e1.92 (m, 1H, CpreH), 2.32e2.34 (m,
1H, CpreH), 3.19e3.23 (m, 1H, CpreH), 5.36 (s, 2H, PhCH2O),
7.03e7.07 (m, 1H), 7.19 (d, 1H, J ¼ 7.24 Hz), 7.30e7.63 (m, 12H), 8.17

X. He et al. / European Journal of Medicinal Chemistry 48 (2012) 338e346
345
5.1.7.13. 1-(8-(Benzyloxy)quinolin-2-yl)-6-(4-(trifluoromethyl)phenyl)-
5.2.1. MES-maximal electroshock seizure pattern test
4,6-diazaspiro[2,4]heptane-5,7-dione
(8m). Mp:
165e167
ꢀC,
This activity was tested according to the method of Swinyard
[30]. In experiments with mice, a 60-Hz current of 50-mA intensity
was applied through corneal electrodes for a 0.25 s duration; with
rats, seizure activity was induced by delivery of a 150-mA current
(0.2 s) to the cornea. Both procedures caused immediate hindlimb
tonic extension. After 0.5 h and 4.0 h of drug administration,
electroshocks were via corneal electrodes. Absence of tonic
extension suggests that the tested compound was considered as
positive criteria.
yield ¼ 93%. 1H NMR (DMSO-d6, 400 MHz),
d 1.89e1.91 (m, 1H,
CpreH), 2.28e2.30 (m, 1H, CpreH), 3.21e3.23 (m, 1H, CpreH), 5.36
(s, 2H, PhCH2O), 7.17 (d, 1H, J ¼ 8.82 Hz), 7.14e7.49 (m, 12H), 8.16 (d,
1H, J ¼ 8.36 Hz), 11.77 (br s, 1H, NH). 13C NMR (DMSO-d6, 100 Hz),
d
19.33, 37.12, 37.89, 70.87, 110.45, 113.74, 119.60, 120.96, 122.23,
126.34, 127.57, 127.14, 127.43, 128.13, 132.25, 135.18, 137.64, 139.34,
153.19, 155.05, 156.82, 158.09, 170.22. MS m⁄z 482.6 (Mþ þ 1). Anal.
Calcd. For C28H20F3N3O3: C 66.80, H 4.00, N 8.35. Found: C 66.73, H
4.14, N 8.47.
5.2.2. Pentylenetetrazole (PTZ) induced seizure test
5.1.7.14. 1-(8-(Benzyloxy)quinolin-2-yl)-6-(4-chlorophenyl)-4,6-diaza-
For the chemically induced convulsant test according to the
method of Vamecq et al. [31], pentylenetetrazole was dissolved in
sufficient 0.9% saline to allow subcutaneous injections to mice or
rat. Standard drug in this model was ethosuximide. After 0.5 h and
4.0 h of drug administration the failure to observe even a threshold
seizure (a single episode of clonic spasms of at least 5 s duration) is
defined as protection.
spiro[2,4]heptane-5,7-dione (8n). Mp: 133e135 ꢀC, yield ¼ 93%. 1H
NMR (DMSO-d6, 400 MHz),
d 1.90e1.93 (m, 1H, CpreH), 2.30e2.32
(m, 1H, CpreH), 3.24e3.26 (m, 1H, CpreH), 5.34 (s, 2H, PhCH2O),
7.21 (d, 1H, J ¼ 8.84 Hz), 7.26e7.57 (m, 12H), 8.22 (d, 1H, J ¼ 8.56 Hz),
11.43 (br s,1H, NH). 13C NMR (DMSO-d6, 100 Hz),
d 19.14, 35.41, 37.80,
70.35, 110.98, 112.42, 119.65, 120.32, 122.78, 126.66, 127.65, 127.83,
127.89, 128.14, 132.12, 135.67, 137.31, 139.88, 153.65, 154.12, 156.44,
157.70, 170.21. MS m⁄z 470.4 (Mþ þ 1). Anal. Calcd. For C27H20ClN3O3:
C 69.01, H 4.29, N 8.94. Found: C 69.23, H 4.18, N 9.02.
5.2.3. Neurotoxicity screening
Minimal motor impairment was measured in mice or rats by
using standardized rotorod test [32]. The mouse was placed on a 1
in. diameter knurled plastic rod rotating at 6 rpm. Trained animals
were given an ip injection of the test compounds in doses of 30,100,
and 300 mg/kg. Neurotoxicity was indicated by the inability of the
animal to maintain equilibrium on the rod for at least 1 min in each
of the four trials.
5.1.7.15. 1-(8-(Benzyloxy)quinolin-2-yl)-6-(4-bromophenyl)-4,6-diaza-
spiro[2,4]heptane-5,7-dione (8o). Mp: 128e130 ꢀC, yield ¼ 90%. 1H
NMR (DMSO-d6, 400 MHz),
d 1.89e1.91 (m, 1H, CpreH), 2.28e2.30
(m, 1H, CpreH), 3.22e3.24 (m, 1H, CpreH), 5.33 (s, 2H, PhCH2O),
7.24 (d, 1H, J ¼ 8.88 Hz), 7.28e7.59 (m, 12H), 8.24 (d, 1H,
J ¼ 8.72 Hz), 11.21 (br s, 1H, NH). 13C NMR (DMSO-d6, 100 Hz),
d
19.39, 35.05, 38.70, 69.87, 110.11, 115.81, 118.92, 120.83, 121.89,
5.2.4. Quantification studies
125.76, 126.48, 127.44, 127.90, 130.49, 135.68, 135.01, 136.48,
138.78, 144.11, 152.11, 153.85, 157.62, 170.87. MS m⁄z 515.4 (Mþ þ 1).
Anal. Calcd. For C27H20BrN3O3: C 63.05, H 3.92, N 8.17. Found: C
63.12, H 4.13, N 8.21.
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
ED50 and TD50, groups of 10 mice were given a range of ip doses of
the test drug until at least three points were established in the
range of 10e90% seizure protection or minimal observed neuro-
toxicity [33]. From the plot of these data, the respective ED50 and
TD50 values, 95% confidence intervals, slope of the regression line,
and standard error of the slope were calculated by means of
a computer program written at NINDS, NIH.
5.1.7.16. 1-(8-(Benzyloxy)quinolin-2-yl)-6-(3,5-dimethylphenyl)-4,6-
diazaspiro[2,4]heptane-5,7-dione
(8p). Mp:
114e116
ꢀC,
yield ¼ 90%. 1H NMR (DMSO-d6, 400 MHz),
d 1.88e1.90 (m, 1H,
CpreH), 2.26e2.28 (m, 1H, CpreH), 3.21e3.23 (m, 1H, CpreH), 5.36
(s, 2H, PhCH2O), 7.28 (d,1H, J ¼ 8.68 Hz), 7.31e7.60 (m,12H), 8.28 (d,
1H, J ¼ 8.56 Hz), 11.13 (br s, 1H, NH). 13C NMR (DMSO-d6, 100 Hz),
d
20.58, 35.45, 39.33, 70.25, 111.36, 116.81, 119.11, 121.92, 122.44,
126.32, 127.79, 127.97, 128.84, 129.30, 136.57, 136.82, 137.72, 139.27,
144.10, 154.69, 154.35, 157.32, 170.30. MS m⁄z 505.3 (Mþ þ 1). Anal.
Calcd. For C27H19Cl2N3O3: C 64.30, H 3.80, N 8.33. Found: C 64.42, H
3.76, N 8.24.
Acknowledgments
This investigation was made possible through the financial
support of the Important National Science & Technology Specific
Projects, China (Grant No. 2009ZX09301-14-1) and Key Project of
Science
& Technology of HuBei Province, China (Grant NO.
5.2. Anticonvulsant screening
2008CDA057).
Male Kunming mice (20 ꢂ 2.0 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 Labo-
ratory 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 anticonvul-
sant activity and neurotoxicity were described elsewhere
[28,29].
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