Synthesis and screening for acetylcholinesterase inhibitor activity of some novel 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-ones: Derivatives of irbesartan key intermediate

Article abstract of DOI:10.1016/j.bmc.2007.07.014

The association of bioactive nucleus with other pharmacological agents is hoped to improve the efficacy of the treatment by combining the effects of different pharmacological mechanisms of action. Keeping this in view, a series of 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-one derivatives have been synthesized by interaction of 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-one with different bioactive aralkyl halides in presence of powdered potassium carbonate by two different methods viz., conventional and microwave irradiation. The yields under conventional and microwave irradiation methods were in the range of 60-65% and 80-90%, respectively. The structure elucidation of the new compounds has been carried out with the help of elemental analysis and spectral data. All the synthesized compounds have been screened for their efficacy as acetylcholinesterase (AChE) inhibitor. AChE inhibitory activity study was carried out by using Ellman colorimetric assay with neostigmine as a reference standard against targets from different species, such as pure electric eel AChE, human serum AChE, and rat brain AChE. Among the compounds synthesized, compounds 5a, 5b, 5j showed good inhibition against AChE.

Full text of DOI:10.1016/j.bmc.2007.07.014

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7391–7398
Synthesis and screening for acetylcholinesterase inhibitor activity
of some novel 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-ones:
Derivatives of irbesartan key intermediate
C. V. Kavitha, S. L. Gaonkar, J. N. Narendra Sharath Chandra,
C. T. Sadashiva and K. S. Rangappa*
Department of Studies in Chemistry, University of Mysore, Manasagangothri, Mysore 570006, India
Received 7 June 2007; revised 15 July 2007; accepted 18 July 2007
Available online 19 August 2007
Abstract—The association of bioactive nucleus with other pharmacological agents is hoped to improve the efficacy of the treatment
by combining the effects of different pharmacological mechanisms of action. Keeping this in view, a series of 2-butyl-1,3-diaza-
spiro[4,4]non-1-en-4-one derivatives have been synthesized by interaction of 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-one with
different bioactive aralkyl halides in presence of powdered potassium carbonate by two different methods viz., conventional and
microwave irradiation. The yields under conventional and microwave irradiation methods were in the range of 60–65% and
80–90%, respectively. The structure elucidation of the new compounds has been carried out with the help of elemental analysis
and spectral data. All the synthesized compounds have been screened for their efficacy as acetylcholinesterase (AChE) inhibitor.
AChE inhibitory activity study was carried out by using Ellman colorimetric assay with neostigmine as a reference standard against
targets from different species, such as pure electric eel AChE, human serum AChE, and rat brain AChE. Among the compounds
synthesized, compounds 5a, 5b, 5j showed good inhibition against AChE.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
O
N
N
N
NH
The rapid synthesis of a diverse range of libraries of
small organic molecules for biochemical uses and drug
discovery has become an active sphere of research.1,2
Due to its high biological profile 2-butyl-1,3-diaza-spiro-
[4,4]non-1-en-4-one 3 may be considered as an attrac-
tive template among small molecule libraries.
Irbesartan (SR47436, BMS-186295), chemically desig-
nated as 2-butyl-3-[[29-(1H-tetrazole-5-yl)[1,19-biphe-
nyl]-4-yl]methyl]-1,3-diaza-spiro[4,4]non-1-en-4-one 6,
is a potent, long-acting angiotensin II (AII)1 receptor
antagonist, with a high selectivity for the AT1 subtype.3
The chemical structure of irbesartan is shown in Fig. 1.
Irbesartan and other AII receptor antagonists have a
potentiality to be advantageous in terms of safety
and tolerability over earlier classes of drugs for the
N
N
H3C
(6)
Figure 1.
treatment of hypertension, diabetic nephropathy, and
heart failure.4–6
Alzheimer’s disease (AD) is a neurodegenerative disease
characterized by a low concentration of acetylcholine
(ACh) in the hippocampus and cortex.7 Inhibition of
AChE, an enzyme responsible for the metabolic break-
down of ACh, is considered appropriate for improving
AD.8,9 Currently available AChE inhibitors can treat
only a mild to moderate levels of AD disease.10 There
is still a need to develop more efficient drugs for AD.
Spiro compounds represent an important class of
Keywords: 2-Butyl-1,3-diaza-spiro[4,4]non-1-en-4-one; Aralkyl halides;
Irbesartan; Acetylcholinesterase inhibitors.
*
Corresponding author. Tel.: +91 821 2419661; fax: +91 821
2412191; e-mail: rangappaks@gmail.com
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.014

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C. V. Kavitha et al. / Bioorg. Med. Chem. 15 (2007) 7391–7398
4]non-1-en-4-one 3 in presence of powdered potassium
N
carbonate in DMF (Scheme 1). The compounds 5(a–j)
were synthesized in two different ways viz., conventional
and microwave irradiation. Compared to conventional
method, microwave heating offers more advantages such
as reduced reaction time (65–75 s), low cost, simplicity
in processing, reduced pollution and high yield. The
yields under conventional and microwave irradiation
methods were in the range of 60–65% and 80–90%,
respectively. The synthesized compounds were charac-
O
N
ZSET1446
(7)
1
Figure 2.
terized by the spectral (IR, H NMR) and elemental
analyzes.
naturally occurring substances characterized by their
highly pronounced biological properties.11–13 The asym-
metric characteristic of the molecule resulting from the
chiral spiro carbon is one of the important criteria for
the biological activities.14 Of late, the effects of azaindo-
lizinone derivative ZSET1446, spiro[imidazo[1,2-a]pyri-
dine-3,2-indon]-2(3H)-one 7, have been assessed in rats
with learning deficits induced by Ab1-40 or scopolamine
suggesting that ZSET1446 (Fig. 2) may be a potential
candidate for being developed as a therapeutic agent
to treat cognitive impairment associated with conditions
such as Alzheimer’s disease.15 Title compounds have
been taken from these observations to screen their
AChE inhibitory activity.
IR spectrum of the compound 2 showed bands at 3340
and 2224 cmꢀ1 due to –NH2 and CN groups, respec-
1
tively. H NMR of 2 showed doublet at d 2.4 for two
protons due to –NH2 group.
IR spectrum of compound 3 showed bands at 1710 cmꢀ1
due to carbonyl group. The band at 2224 cmꢀ1 disap-
peared and the new band appeared at 1480 cmꢀ1 due
1
to C@N stretching. H NMR of 3 showed singlet at d
7.8 for 1 proton due to –NH and showed peaks for new-
ly formed ring in the expected region.
IR spectrum of all the compounds 5(a–j) showed bands
at 1730–1740 cmꢀ1 due to carbonyl group. Compound
5a showed the band at 2224 cmꢀ1 due to CN group of
1
biphenyl ring. The band at 3320 cmꢀ1 disappeared. H
2. Results and discussion
2.1. Chemistry
NMR of all the compounds 5(a–j) showed absence of
peak at d 7.8 for 1 proton due to –NH group and a
new singlet was observed at d 4.3–4.4 for two protons
indicating the formation of the product. All other pro-
tons are observed in the expected region. The formation
of the product was further confirmed by correct elemen-
tal analyzes.
The bioactive intermediate 3 was synthesized in accor-
dance with the reported procedure.16 The synthesis of
the title compounds was done by condensing different
bioactive aralkyl halides with 2-butyl-1,3-diaza-spiro[4-
.HCl
O
O
Cl
O
NC
NH2
O
HCOONH4
KCN
R-X
4(a-j)
R
N
N
HN
.HCl
N
Anhydrous K2CO3
DMF
5(a-j)
1
2
3
Br
Br
Br
Br
O
O
O
Br
CH3
O
Cl
N
Where R-X=
Cl
CN
O
MeO
NO2
OMe
Cl
(4f)
(4e)
(4c)
(4d)
(4a)
(4b)
Br
Br
Br
Br
N
N
N
O
O
CH3
N
CH3
O
CH3
O
Br
CH3
(4j)
(4i)
(4h)
(4g)
Scheme 1.

C. V. Kavitha et al. / Bioorg. Med. Chem. 15 (2007) 7391–7398
7393
Table 1. Reaction condition and physical data of 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-one derivatives 5(a–j)
Compound
R
Reaction time
Conventional (h)
Yield (%)
Conventional
Mp (ꢁC)
MWirr. (S)
MWirr.
5a
6
5
6
75
65
60
65
90
126
134
106
CN
Br
5b
5c
65
75
87
84
MeO
OMe
NO2
O
N
O
5d
5e
6
6
65
65
62
65
83
85
127
128
CH3
O
O
O
5f
5
70
60
83
122
Cl
N
N
N
N
5g
5
70
63
90
109
5h
5
65
63
85
118
O
O
CH3
5i
6
65
65
88
136
CH3
CH3
CH3
O
O
(continued on next page)

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C. V. Kavitha et al. / Bioorg. Med. Chem. 15 (2007) 7391–7398
Table 1 (continued)
Compound
R
Reaction time
Conventional (h)
Yield (%)
Conventional
Mp (ꢁC)
MWirr. (S)
MWirr.
5j
5
70
65
84
121
Br
2.2. Pharmacology
Inhibition of human serum AChE by spiroimidazol-4-
one derivatives
Screening of cholinesterase inhibition was done by
determining the IC50 value, which is the diazaspiro
(ligand) concentration that causes 50 percent inhibition
of the cholinesterase activity measured with a given sub-
strate at a specified substrate concentration. The IC50
values were determined for inhibition of human serum,
for rat brain homogenate, for electric Eel AChE, mea-
sured with 1.0 mM acetylthiocholine (ATCh) as sub-
strate, at 37 ꢁC and pH 7.4.
100
90
5a
80
70
60
50
40
30
20
10
0
5b
5j
Neostigmine
The inhibitory studies of the newly synthesized com-
pounds against AChE using different sources such as
rat brain homogenate AChE, human serum AChE,
and electric eel AChE are shown in Figures 3–5, respec-
tively. Activities of the synthesized compounds were
compared with the inhibitory activity shown by the
known standard inhibitor neostigmine. The effects of
different or versatile aromatic substituents are explored
for their inhibitory activity against AChE.
0
50
100
150
200
Inhibitor concentration (nM)
Figure 4.
Inhibition of electric eel AChE by spiroimidazol-
4-one derivatives
Among the molecules screened for the AChE inhibitory
activity, the compound 5j (IC50 = 70, 70 and 67.5 nM)
with bromo group at the 4th position shows highest
AChE inhibitory activity. The compounds 5b and 5a
with bromo group at the 2nd position and cyano group
on the biphenyl ring, respectively, are also effective in
blocking the AChE enzyme activity (IC50 = 77.5, 82.5,
100
90
80
70
60
50
40
30
20
5a
5b
5j
Neostigmine
Inhibition of Rat brain homogenate AChE by
spiroimidazol-4-one derivatives
100
90
5a
5b
80
70
10
0
5j
60
Neostigmine
0
50
100
150
200
50
40
30
20
10
0
Inhibitor concentration (nM)
Figure 5.
80.0; 90.0, 82.5, 90.0 nM, respectively) (Table 2). But
the compounds 5(g–i) with biphenyl ring with bulky
substituent did not show any inhibitory activity. Smaller
the substituent, the more favorable activity at the biphe-
nyl methyl site of the molecule (5a). The compounds 5c
and 5f show moderate activity. This is probably due to
the presence of electron withdrawing nitro group (5c)
0
50
100
150
200
Inhibitor concentration (nM)
Figure 3.

C. V. Kavitha et al. / Bioorg. Med. Chem. 15 (2007) 7391–7398
7395
Table 2. Comparative inhibitory activities of spiroimidazol-4-one
derivatives 5(a–j) against AChE from different sources
thus concluded that under microwave heating, the prod-
ucts 5(a–j) were conveniently and efficiently prepared in
good yields, typically in the range of 80–90%. The AChE
inhibitory activity data reveal that, the compounds 5j, 5b,
and 5a are active site-directed irreversible inhibitors of
AChE activity. These results could be useful in designing
new AChE inhibitors, resulting in greater selectivity as
well as an increase in new inhibitor potency.
Compound
Rat brain
homogenate
IC50 (nM)
Human
serum
IC50 (nM)
Electric
eel
IC50 (nM)
5a
5b
90.0
77.5
5
3
6
82.5
82.5
5
5
6
90.0
80.0
6
5
7
5c
5d
133.6
135.4
149.2
233.8 15
216.2 12
280.4 19
356.5 24
243.2 17
270.6 18
230.8 16
213.5 13
286.4 19
383.5 25
240.6 17
268.8 17
250.1 15
212.4 13
279.6 19
373.2 24
235.7 18
276.8 17
5e
5f
5g
4. Experimental
5h
5i
The melting points were determined on SELACO-650
hot stage apparatus and remain uncorrected. IR (KBr)
spectra were recorded on a Jasco FT/IR-4100 Fourier
transform infrared spectrometer, 1H NMR were re-
corded on Shimadzu AMX 400 spectrometer by using
CDCl3 as solvent and TMS as an internal standard
(Chemical shift in ppm). Elemental analyzes were per-
formed on a vario-EL instrument. Thin layer chroma-
tography (TLC) was conducted on 0.25 mm silica gel
plates (60F254, Merck). Visualization was made with
ultraviolet light. All extracted solvents were dried over
anhydrous Na2SO4 and evaporated with a BUCHI ro-
tary evaporator. Reagents were obtained commercially
and used as they were received.
5j
Neostigmine
70.0
35.0
3
2
70.0
45.0
3
4
67.5
47.5
3
5
and chlorine atom at 4th position of the substituent and
also because of the ester spacer at the 1st position of the
substituent R. Compounds 5d and 5e having fused rings
did not show good inhibitory activity.
It may be observed from the SAR studies that, the spi-
roimidazol-4-one ring containing single or non-fused
aromatic rings (5j, 5b, and 5a) show a better activity
than the fused aromatic rings (5d and 5e). The order
of potency is 5j > 5b > 5a. It may be concluded that,
for an effective binding and blocking of the AChE activ-
ity, the molecule requires to bind with the peripheral site
and the active site of the enzyme and it may be possible
that the spiroimidazol-4-one binds at the active site
gorge and the different substituents bind to peripheral
site separated by the spacer such as methylene or acyl
or ester groups. Furthermore, the bulky n-butyl chain
present on second position of the spiroimidazol-4-one
enhances the hydrophobicity which may help in binding
to the active site of the enzyme. Therefore, it may be
summarized that, the substitution of other smaller rings
with smaller electron withdrawing groups on spiro-
imidazol-4-one basic nucleus needs to be studied for bet-
ter AChE inhibitory activity. The structures of the
potent AChE inhibitor are shown in Figure 6.
4.1. Synthesis of 1-amino-1-cyano-cyclopentane hydroch-
loride (2)
A mixture of cyclopentanone (20 g, 238 mmol) and
ammonium formate (15 g, 238 mmol) was stirred in
125 ml water to obtain a clear solution. Potassium cya-
nide (15.5 g, 238 mmol) was spooned into the solution
over 5 min with a little heat evolution; an oily phase be-
gan to separate. The reaction mixture was stirred at
ambient temperature for about 24 h. The upper layer
was separated and it was diluted to 75 ml with toluene
and then dried over anhydrous sodium sulfate. The tol-
uene mixture was saturated with dry hydrogen chloride
gas, giving separation of solid (a precipitate). The solid
was filtered, washed with toluene and hexane, and dried
at 50 ꢁC under vacuum. Yield 68%; mp: 156–158 ꢁC.
IR mmax (KBr): 3340, 2985, 2224, 1480 cmꢀ1
.
3. Conclusions
1H NMR (CDCl3, 400 MHz) d: 1.5–1.95 (m, 8H, cyclo-
pentyl), 2.4 (d, 2H, –NH2).
In conclusion it may be said that, we have succeeded in
synthesizing the novel 2-butyl-1,3-diaza-spiro[4,4]non-1-
en-4-one derivatives 5(a–j) under both conventional and
microwave irradiation technique (solution phase). It is
Anal. Calcd for C6H10N2: C, 65.42; H, 9.15; N, 25.43.
Found: C, 65.43; H, 9.18; N, 25.42%.
NC
Br
N
O
N
O
N
O
N
N
N
OMe
OMe
Br
5a
Figure 6. Structures of potent AChE inhibitor.
5j
5b

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C. V. Kavitha et al. / Bioorg. Med. Chem. 15 (2007) 7391–7398
4.2. Synthesis of 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-one
monohydrochloride (3)
3 (1 g, 5.15 mmol), different aralkyl halides 4(a–j)
(1.2 equiv), and DMF (10 ml) was irradiated in the
microwave oven at 20% power level (60 W) for 65–
75 s. After completion of the reaction (tlc), 10 equivalent
of water was added to the cooled (rt) contents of the
flask. Using the above workup procedure isolated the
pure products. The reaction condition and physical data
of the synthesized compounds are depicted in Table 1.
To the solution of 1-amino-1-cyanocyclopentane (15 g,
136 mmol) in 150 ml of toluene triethylamine (20.6 g,
204 mmol; 1.5 equiv) was added and valeroyl chloride
(19.7 g, 163 mmol; 1.2 equiv) was added to this solution
dropwise, under cooling, at 20 ꢁC. After 10–15 min the
temperature was raised to 80 ꢁC and the mixture was
stirred at that temperature for about 2 h. The reaction
mixture was then cooled to room temperature and was
extracted consecutively with 50 ml of water, 40 ml of
2% hydrochloric acid solution, and 40 ml of water.
The toluene solution was evaporated under vacuo. To
the residue a solution of potassium hydroxide (3.8 g,
68 mmol; 0.5 equiv) in 50 ml of methanol was added
at first and then under cooling a 30 ml of 30% hydrogen
peroxide solution. After the addition of hydrogen perox-
ide, the temperature was maintained at 50 ꢁC for about
30 min. The solution was cooled to room temperature
and added potassium hydroxide (15.3 g, 272 mmol;
2 equiv) solution. The mixture was heated under reflux
condition for about 2 h. The reaction was then frozen
by adding 30 g of ammonium chloride and methanol
was distilled out. The reaction mixture was then ex-
tracted with ethyl acetate (50· 3 ml). The combined or-
ganic phase was evaporated in vacuo, the residue was
dissolved in 5-fold amount of acetone, the resulting
solution was filtered, its pH was adjusted to 1–2 by the
addition of concd hydrochloric acid solution. The sus-
pension thus obtained was stirred for 1 h, cooled to
0 ꢁC. The crystals were filtered off, washed with cold ace-
tone, and dried to obtain the product 3. Yield 55%; mp:
68 ꢁC
4.3.1. Synthesis of 40-(2-butyl-4-oxo-1,3-diaza-spiro[4,4]-
non-1-en-3-ylmethyl)-biphe-nyl-2-carbonitrile (5a). The
product 5a was obtained from 2-butyl-1,3-diaza-spiro-
[4,4]non-1-en-4-one 3 (1 g, 5.15 mmol) and 40-bromo-
methyl-biphenyl-2-carbonitrile 4a (1.7 g, 6.18 mmol).
IR mmax (KBr): 3095, 2224, 1735, 1574, 1480 cmꢀ1
.
1H NMR (CDCl3 400 MHz) d: 0.88–0.92 (t, 3H, –CH3),
1.2–1.4 (m, 6H, –CH2–CH2–CH2–), 1.51–1. 81 (m, 8H,
–cyclopentane), 4.34 (s, 2H, –CH2–), 7.15–8.00 (m, 8H,
Ar-H).
Anal. Calcd for C25H27N3O: C, 77.89; H, 7.06; N, 10.90.
Found: C, 77.91; H, 7.04; N, 10.89%.
4.3.2. Synthesis of 3-(2-bromo-4,5-dimethoxybenzyl)-2-
butyl-1,3-diazaspiro[4,4]non-1-en-4-one (5b). The prod-
uct 5b was obtained from 2-butyl-1,3-diaza-spiro[4,4]-
non-1-en-4-one 3 (1 g, 5.15 mmol) and 1-bromo-2-
bromomethyl-4,5-dimethoxy-benzene 4b (1.9 g, 6.17
mmol).
IR mmax (KBr): 3105, 2940, 1739, 1572, 1482, 1455,
1367 cmꢀ1
.
IR mmax (KBr): 2995, 1710, 1480, 1455, 1365 cmꢀ1
.
1H NMR (CDCl3 400 MHz) d: 0.88–0.92 (t, 3H, –CH3),
1.2–1.4 (m, 6H, –CH2–CH2–CH2–), 1.51–1. 81 (m, 8H,
–cyclopentane), 3.81 (s, 6H–OCH3), 4.34 (s, 2H, –CH2–),
6.46 (s,1H, Ar-H), 6.75 (s, 1H, Ar-H).
1H NMR (CDCl3, 400 MHz) d: 0.88–0.92 (t, 3H, –CH3),
1.2–1.4 (m, 6H, –CH2–CH2–CH2), 1.5–1.95 (m, 8H,
cyclopentyl), 7.8–8.2 (s, 1H, –NH).
Anal. Calcd for C20H27BrN2O3: C, 56.74; H, 6.43; N,
6.62. Found: C, 56.73; H, 6.43; N, 6.64%.
Anal. Calcd for C11H18N2O: C, 68.01; H, 9.34; N, 14.42.
Found: C, 68.03; H, 9.35; N, 14.45%.
4.3.3. Synthesis of 2-butyl-3-(4-nitrobenzyl)-1,3-diazaspi-
ro[4,4]non-1-en-4-one (5c). The product 5c was obtained
from 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-one 3 (1 g,
5.15 mmol) and 1-bromomethyl-4-nitro-benzene 4c
(1.3 g, 6.17 mmol).
4.3. General procedures for the synthesis of 2-butyl-1,3-
diaza-spiro[4,4]non-1-en-4-one derivatives 5(a–j)
[a] Conventional method. A mixture of 2-butyl-1,3-dia-
za-spiro[4,4]non-1-en-4-one 3 (1 equiv), aralkyl halides
4(a–j) (1.2 equiv), and powdered potassium carbonate
(1.2 equiv) in N,N-dimethylformamide (10 ml) was stir-
red at room temperature for about 6–8 h. The reaction
was monitored by thin layer chromatography. After
the completion of the reaction, deionized water was
added to the reaction mixture and extracted with ethyl
acetate (3· 10 ml). The combined organic layer was
dried over anhydrous Na2SO4. The crude products
5(a–j) obtained on evaporation of the solvent under re-
duced pressure were purified by column chromatogra-
phy using hexane and ethyl acetate as eluents.
IR mmax (KBr): 3100, 2935, 1732, 1565, 1480, 1445,
1355 cmꢀ1
.
1H NMR (CDCl3 400 MHz) d: 0.88–0.92 (t, 3H, –CH3),
1.2–1.4 (m, 6H, –CH2–CH2–CH2–), 1.51–1.81(m, 8H,
–cyclopentane), 4.34 (s, 2H, –CH2–), 7.15–7.18 (d, 4H,
Ar-H).
Anal. Calcd for C18H25N3O3: C, 65.24; H, 7.60; N,
12.68. Found: C, 65.24; H, 7.61; N, 12.68%.
4.3.4. Synthesis of 2-[2-(2-butyl-4-oxo-1,3-diazaspi-
ro[4,4]non-1-en-3-yl)ethyl]isoin-dole-1,3-dione (5d). The
product 5d was obtained from 2-butyl-1,3-diaza-spir-
[b] Microwave irradiation method. A 25 ml conical flask
charged with 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-one

C. V. Kavitha et al. / Bioorg. Med. Chem. 15 (2007) 7391–7398
7397
o[4,4]non-1-en-4-one 3 (1 g, 5.15 mmol) and 2-(2-chloro-
ethyl)-isoindole-1,3-dione 4d (1.2 g, 6.18 mmol).
4.3.8. Synthesis of 40-(2-butyl-4-oxo-1,3-diazaspiro[4,4]
non-1-en-3-ylmethyl)-biphe-nyl-2-carboxylic acid methyl
ester (5h). The product 5h was obtained from 2-butyl-1,3-
diaza-spiro[4,4]non-1-en-4-one 3 (1 g, 5.15mmol) and 40-
bromomethyl-biphenyl-2-carboxylic acid methyl ester 4h
(1.9 g, 6.18 mmol).
IR mmax (KBr): 3099, 2932, 1734, 1578, 1495, 1465,
1365 cmꢀ1
.
1H NMR (CDCl3 400 MHz) d: 0.88–0.92 (t, 3H, –CH3),
1.2–1.4 (m, 6H, –CH2–CH2–CH2–), 1.51–1.81 (m, 8H,
–cyclopentane), 3.65–3.85 (t, 4H, –CH2–CH2–), 7.72–
8.35 (m, 4H, Ar-H).
IR mmax (KBr): 3105, 2937, 1740, 1576, 1467, 1465,
1354 cmꢀ1
.
1H NMR (CDCl3 400 MHz) d: 0.88–0.92 (t, 3H, –CH3),
1.2–1.4 (m, 6H, –CH2–CH2–CH2–), 1.51–1.81(m, 8H,
–cyclopentane), 3.92–3.93 (s, 3H, –OCH3), 4.34 (s, 2H,
–CH2-), 7.11–8.00 (m, 8H, Ar-H).
Anal. Calcd for C21H25N3O3: C, 68.64; H, 6.86; N,
11.44. Found: C, 68.63; H, 6.87; N, 11.45%.
4.3.5. Synthesis of 2-butyl-3-(6-methylbenzo[1,3]dioxole-5-
ylmethyl)-1,3-diazaspiro [4,4]non-1-en-4-one (5e). The
product 5e was obtained from 2-butyl-1,3-diaza-spiro[4,4]-
non-1-en-4-one 3 (1 g, 5.15 mmol) and 5-chloromethyl-6-
methyl-benzo[1,3]dioxole 4e (1.4 g, 6.18 mmol).
Anal. Calcd for C26H30N2O3: C, 74.61; H, 7.22; N, 6.69.
Found: C, 74.61; H, 7.23; N, 6.69%.
4.3.9. Synthesis of 40-(2-butyl-4-oxo-1,3-diazaspiro[4,4]-
non-1-en-3-ylmethyl)-biphenyl-2-carboxylic acid tert-
butyl ester (5i). The product 5i was obtained from 2-bu-
tyl-1,3-diaza-spiro[4,4]non-1-en-4-one 3 (1 g, 5.15 mmol)
and 40-bromomethyl-biphenyl-2-carboxylic acid tert-bu-
tyl ester 4i (2.1 g, 6.17 mmol).
IR mmax (KBr): 3095, 2930, 1735, 1574, 1480, 1465,
1369 cmꢀ1
.
1H NMR (CDCl3 400 MHz) d: 0.88–0.92 (t, 3H, –CH3),
1.2–1.4 (m, 6H, –CH2–CH2–CH2–), 1.51-1.81(m, 8H,
–cyclopentane), 4.34 (s, 2H, –CH2–), 5.8 (s, 2H, –CH2-),
6.46 (s, 3H, Ar-H), 5.8 (s, 3H, –CH3).
IR mmax (KBr): 3017, 2945, 1740, 1578, 1493 cmꢀ1
.
1H NMR (CDCl3 400 MHz) d: 0.88–0.92 (t, 3H, –CH3),
0.97–1.00 (s, 9H, –C(CH3)3), 1.2–1.4 (m, 6H, –CH2–
CH2–CH2–), 1.51–1.81(m, 8H, –cyclopentane), 4.34 (s,
2H, –CH2–), 7.12–7.89 (m, 8H, Ar-H).
Anal. Calcd for C20H26N2O3: C, 70.15; H, 7.65; N, 8.18.
Found: C, 70.13; H, 7.65; N, 8.19%.
4.3.6. Synthesis of 2-butyl-3-[2-(4-chlorophenyl)-2-oxoeth-
yl]-1,3-diazaspiro[4,4]non-1-en-4-one (5f). The product 5f
was obtained from 2-butyl-1,3-diaza-spiro[4,4]non-1-en-
4-one 3 (1 g, 5.15 mmol) and 2-Bromo-1-(4-chloro-
phenyl)ethanone 4f (1.4 g, 6.18 mmol).
Anal. Calcd for C29H36N2O3: C, 75.62; H, 7.88; N, 6.08.
Found: C, 75.61; H, 7.87; N, 6.09%.
4.3.10. Synthesis of 3-(4-bromobenzyl)-2-butyl-1,3-diaza-
spiro[4,4]non-1-en-4-one (5j). The product 5j was ob-
tained from 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-one
3 (1 g, 5.15 mmol) and 1-bromo-4-bromomethyl-ben-
zene 4j (1.5 g, 6.18 mmol).
IR mmax (KBr): 3115, 2940, 1743, 1572, 1488, 1455, 1345 cmꢀ1.
1H NMR (CDCl3 400 MHz) d: 0.88–0.92 (t, 3H, –CH3),
1.2–1.4 (m, 6H, –CH2–CH2–CH2–), 1.51–1.81 (m, 8H,
–cyclopentane), 4.34 (s, 2H, –CH2–), 7.65 (dd, 2H, Ar-
H), 7.88 (dd, 2H, Ar-H).
IR mmax (KBr): 3098, 2941, 1740, 1579, 1478, 1462,
1345 cmꢀ1
.
Anal. Calcd for C19H23ClN2O2: C, 65.79; H, 6.68; N,
8.08. Found: C, 65.78; H, 6.69; N, 8.10%.
1H NMR (CDCl3 400 MHz) d: 0.88–0.92 (t, 3H, –CH3),
1.2–1.4 (m, 6H, –CH2–CH2–CH2–), 1.51–1.81 (m, 8H,
–cyclopentane), 4.34 (s, 2H, –CH2–), 7.25 (dd, 2H, Ar-
H), 7.46 (dd, 2H, Ar-H).
4.3.7. Synthesis of 2-butyl-3-[20-(1-trityl-tetrazol-5-
yl)biphenyl-4-ylmethyl]-1,3-diaza spiro[4,4]non-1-en-4-
one (5g). The product 5g was obtained from 2-butyl-1,
3-diaza-spiro[4,4]non-1-en-4-one 3 (1 g, 5.15 mmol) and
5-(40-bromomethyl-biphenyl-2-yl)-1-trityl-1H-tetrazole 4g
(3.4 g, 6.18 mmol).
Anal. Calcd for C18H23BrN2O: C, 59.51; H, 6.38; N,
7.71. Found: C, 59.51; H, 6.39; N, 7.70%.
5. Biology: in vitro acetylcholinesterase assay
IR mmax (KBr): 3114, 2945, 1740, 1570, 1486, 1451, 1340 cmꢀ1.
The cholinesterase assay method of Ellman et al.17 was
used to determine the in vitro cholinesterase activity.
The activity was measured by increase in absorbance
at 412 nm due to the yellow color produced from the
reaction of acetylthiocholine iodide with the dith-
iobisnitrobenzoate (DTNB) ion. AChE was obtained
from the brain of Wistar rats by homogenizing under
a Teflon blender for 10 min in 0.1 M KH2PO4 buffer,
1H NMR (CDCl3 400 MHz) d: 0.88–0.92 (t, 3H, –CH3),
1.2–1.4 (m, 6H, –CH2–CH2–CH2–), 1.51–1.81(m, 8H,
–cyclopentane), 4.34 (s, 2H, –CH2–), 6.79–8.05 (m,
24H, Ar-H).
Anal. Calcd for C44H42N6O: C, 78.78; H, 6.31; N, 12.53.
Found: C, 78.79; H, 6.32, N, 12.52%.

7398
C. V. Kavitha et al. / Bioorg. Med. Chem. 15 (2007) 7391–7398
pH 8. A stock solution of the enzyme in 0.1 M KH2PO4
buffer (pH 8) was kept frozen. For each assay 300 lg of
enzyme was used. Acetylthiocholine iodide was pre-
pared daily using 0.1 M KH2PO4 buffer (pH 7). A
0.01 M solution of DTNB was prepared in 0.1 M
KH2PO4 buffer (pH 7). Crude human AChE was pre-
pared by mixing 9 ml of fresh blood (collected from
healthy volunteers by vein puncture) with 1 ml of 3.8
% (w/v) trisodium citrate and centrifuging at 3000 rpm
at 0 ꢁC for 20 min. The supernatant was used as a source
of AChE. Electric eel AChE was obtained from Sigma
Laboratory and similar procedure was employed for
the assay as that of rat brain AChE.
tained from the instrument granted by DST-FIST and
IR data by the Instrument FT-IR by UGC-SAP greatly
acknowledged. We thank NMR Research Centre, IISC,
Bangalore, for the NMR spectral analysis.
References and notes
1. Thompson, L. A.; Ellman, J. A. Chem. Rev. 1996, 96, 555.
2. Nefzi, A.; Ostresh, J. M.; Houghten, R. A. Chem. Rev.
1997, 97, 449.
3. Cazaubon, C.; Gougat, J.; Bousquet, F.; Guiraudou, P.;
Gayraud, R.; Lacour, C.; Roccon, A.; Galindo, G.;
Barthelemy, G.; Gautret, B.; Bernhart, C.; Perreaut, P.;
Breliere, J-C.; Le Fur, G.; Nisato, D. J. Pharmacol. Exp.
Ther. 1993, 265, 826.
5.1. Experimental conditions and kinetics
4. Eberhardt, R. T.; Kevak, R. M.; Kang, P. M.; Frishman,
W. H. J. Clin. Pharmacol. 1993, 33, 1023.
5. Gottlieb, S. S.; Dickstein, K.; Fleck, E.; Kostis, J.; Levine,
T. B.; LeJemtel, T.; DeKock, M. Circulation 1993, 88,
1602.
6. Lacourcie‘re, Y.; Brunner, H.; Irwin, R.; Karlberg, B. E.;
Ramsay, L. E.; Snavely, D. B.; Dobbins, T. W.; Faison, E.
P.; Nelson, E. B. J. Hypertens. 1994, 12, 1387.
7. Terry, A. V.; Buccafusco, J. J. J. Pharmacol. Exp. Ther.
2003, 306, 821.
8. Giacobini, E. Pharmacol. Res. 2004, 50, 433.
9. Johnson, C. N.; Ronald, A.; Upton, N. Drug Discovery
Today Therap. Strategies 2004, 1, 13.
10. Grossberg, G. T. Curr. Ther. Res. 2004, 64, 216.
11. Longeon, A.; Guyot, M.; Vacelet, J. Experentia 1990, 46,
548.
12. Kobayashi, J.; Tsuda, M.; Agemi, K.; Shigemiri, H.;
Ishibashi, M.; Sasaki, T.; Mikami, Y. Tetrahedron 1991,
47, 6617.
Enzyme activity was measured using a Shimadzu Spec-
trophotometer. The assay medium contained phosphate
buffer, pH 8.0 (2.6 ml), DTNB (0.1 ml), 5 ll of enzyme,
20 ll of 0.075 M substrate. The activity was determined
by measuring the increase in absorbance at 412 nm at
1 min intervals for 10 min at 37 ꢁC. In dose-dependent
inhibition studies, the substrate was added to the assay
medium containing enzyme, buffer, and DTNB with
inhibitor after 10 min of incubation time. Calculations
were performed according to the method of the equation
in Ellman et al.17 All experiments were carried out in
duplicate and the mean values are reported here. The
relative activity is expressed as percentage ratio of en-
zyme activity in the absence of inhibitor.
5.1.1. Protein estimation. Protein content was deter-
mined by the Lowry method18 using bovine serum albu-
min as standard.
13. James, D. M.; Kunze, H. B.; Faulkner, D. J. J. Nat. Prod.
1991, 54, 1137.
14. Takahashi, K.; Witkop, B.; Brossi, A.; Maleque, M. A.;
Albuquerque, E. X. Helv. Chim. Acta 1982, 65, 252.
15. Yamaguchi, Y.; Miyashita, H.; Tsunekawa, H.; Mouri,
A.; Kim, H. C.; Saito, K.; Matsuno, T.; Kawashima, S.;
Nabeshima, T. J. Pharmacol. Exp. Ther. 2006, JPET
#98640 1-45.
16. Attila, K. T; Csaba, H; Bertrand, C; Attila, N; Peter, A;
Karoly, G; Istvan, M; Dervalicsne, Z. I; Katalin, D;
Lajosne, P; Antal, G; Attila, S; Zsuzsanna, N; Zoltan, M;
Endre, K. United States Patent 6,239,286, 2001, Sanofi-
Synthelabo (Paris, FR).
5.1.2. IC50 determination. The inhibition by spiro-
imidazol-4-one derivatives was studied in the presence
of different concentrations of compounds and the per-
centage inhibition of enzyme activity was calculated.
The results obtained were analyzed with the values ob-
tained in comparison to that of neostigmine.
Acknowledgments
17. Ellman, G. L.; Courtney, K. D.; Andress, V.; Earther-
stone, F., Jr. Mol. Biochem. Pharmacol. 1961, 7, 88.
18. Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L.; Randall,
R. J. J. Biol. Chem. 1951, 193, 265.
The authors are grateful to Department of Science and
Technology, New Delhi, for financial support under
the project SR/SO/HS-58/2003. The CHNS data ob-