Synthesis and discovery of novel piperidone-grafted mono- and bis-spirooxindole-hexahydropyrrolizines as potent cholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.01.066

Three-component reaction of a series of 1-acryloyl-3,5- bisbenzylidenepiperidin-4-ones with isatin and l-proline in 1:1:1 and 1:2:2 molar ratios in methanol afforded, respectively the piperidone-grafted novel mono- and bisspiro heterocyclic hybrids comprising functionalized piperidine, pyrrolizine and oxindole ring systems in good yields. The in vitro evaluation of cholinesterase enzymes inhibitory activity of these cycloadducts disclosed that monospiripyrrolizines (8a-k), are more active with IC₅₀ ranging from 3.36 to 20.07 μM than either the dipolarophiles (5a-k) or bisspiropyrrolizines (9a-k). The compounds, 8i and 8e with IC₅₀ values of 3.36 and 3.50 μM, respectively showed the maximum inhibition of acethylcholinesterase (AChE) and butrylylcholinestrase (BuChE). Molecular modeling simulation, disclosed the binding interactions of the most active compounds to the active site residues of their respective enzymes. The docking results were in accordance with the IC₅₀ values obtained from in vitro cholinesterase assay.

Full text of DOI:10.1016/j.bmc.2013.01.066

Bioorganic & Medicinal Chemistry 21 (2013) 1696–1707
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and discovery of novel piperidone-grafted
mono- and bis-spirooxindole-hexahydropyrrolizines
as potent cholinesterase inhibitors
b,
c
Yalda Kia a, Hasnah Osman a, , Raju Suresh Kumar , Vikneswaran Murugaiyah ,
⇑
⇑
Alireza Basiri c, Subbu Perumal d, Habibah A. Wahab e, Choi Sy Bing e
a School of Chemical Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia
b Department of Chemistry, College of Science, King Saud University, PO Box 2455, Riyadh, Saudi Arabia
c School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia
d Department of Organic Chemistry, School of Chemistry, Madurai Kamaraj University, Madurai, Tamil Nadu, India
e Laboratory of Pharmaceutical Design and Simulations, School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
Three-component reaction of a series of 1-acryloyl-3,5-bisbenzylidenepiperidin-4-ones with isatin and
Received 29 November 2012
Revised 23 January 2013
Accepted 24 January 2013
Available online 8 February 2013
L
-proline in 1:1:1 and 1:2:2 molar ratios in methanol afforded, respectively the piperidone-grafted novel
mono- and bisspiro heterocyclic hybrids comprising functionalized piperidine, pyrrolizine and oxindole
ring systems in good yields. The in vitro evaluation of cholinesterase enzymes inhibitory activity of these
cycloadducts disclosed that monospiripyrrolizines (8a–k), are more active with IC50 ranging from 3.36 to
20.07
l
M than either the dipolarophiles (5a–k) or bisspiropyrrolizines (9a–k). The compounds, 8i and 8e
Keywords:
AChE
BuChE
1,3-Dipolar cycloaddition
Selectivity and spiropyrrolizines
with IC50 values of 3.36 and 3.50
l
M, respectively showed the maximum inhibition of acethylcholinest-
erase (AChE) and butrylylcholinestrase (BuChE). Molecular modeling simulation, disclosed the binding
interactions of the most active compounds to the active site residues of their respective enzymes. The
docking results were in accordance with the IC50 values obtained from in vitro cholinesterase assay.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
short term memory seen in AD.7,8 Consequently, one of the strate-
gies for treating AD has been to enhance cholinergic function to
Alzheimer’s disease (AD) is the leading cause of dementia, in
particular among old people. Based on world Alzheimer’s reports,
there were 36 million people living with dementia worldwide in
2010, predicted to increase to 66 million by 2030 and 115 million
by 2050.1 Its prevalence rises sharply with the age, the incidence of
AD being substantial and is approximately 14 times higher among
persons older than 85 years than those between 65 and 69 years of
age.2 Age related dementia is considered to arise from the steady
loss of neurons that normally affects memory and thinking skills
and ultimately the ability to perform even simple tasks in life.3,4
Neuropathology of AD is generally characterized by the pres-
ence of two microscopic features: (i) extracellular amyloid plaques,
known as Ab plaques and (ii) neurofibrillary tangles (NFT) compris-
ing filaments of a phosphorylated form of microtubule associated
protein, tau.3,5,6 The secondary impact of these features are neuro-
degeneration and loss of cholinergic neurons in basal forebrain,
which are believed to underlie the cognitive deficit and loss of
sustain or prolong the action of remaining acetylcholine9 using
cholinesterase inhibitors.
The human brain contains two cholinesterase enzymes, (i) ace-
tylcholinesterase (AChE) encoded by a gene on chromosome 7 and
(ii) butyrylcholinesterase (BuChE) encoded by a gene on chromo-
some 3. Among these two, acetylcholinesterase has been studied
more and it is the only one consistently associated with cholinergic
transmission and cholinoceptive neurons.10 The therapeutic role of
cholinesterase inhibitors is mainly due to enhancement of cholin-
ergic transmission at cholinergic autonomic synapses.11 Acetylcho-
linesterase inhibitors such as galanthamine, donepezil,
rivastigmine are used as drugs for correcting neurotransmitter dis-
turbances or symptomatic treatment and many more drug candi-
dates are under development at various stages.12 All the known
AChE inhibitor drugs suffer from shortcomings such as low bio-
availability, short duration of biological action, narrow therapeutic
effects and high toxicity. Hence discovery and development of new
drug candidates as AChE inhibitors with enhanced potency and
diminished toxicity is imperative.
⇑
Corresponding authors. Tel.: +60 4 6533558; fax: +60 4 6574854.
E-mail addresses: osman.hasnah2012@yahoo.com (H. Osman), sraju@ksu.edu.sa
Multi-component reactions with pot, atom and step economies
offer a convenient tool for the assembly of highly functionalized
(R.S. Kumar).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.066

Y. Kia et al. / Bioorg. Med. Chem. 21 (2013) 1696–1707
1697
organic molecules of structural complexity and diversity in a single
procedural step resulting in convergence, atom economy, minimi-
zation of waste, flexibility and facile automation.13 Multi-compo-
nent 1,3-dipolar cycloaddition of azomethine ylides offers such a
versatile protocol to build highly functionalized complex N-
heterocycles.14
was derived by 1H and 13C NMR spectroscopic data and further
confirmed by single crystal X-ray crystallographic study
(Fig. 1).32 Dipolarophiles 5 are versatile synthons for the assembly
of spiro heterocycles, as they possess multi dipolarophilic func-
tions, viz. three C@C and C@O groups. Hence the study of reactivity
and product-selectivity of the cycloadditions of 5 are of consider-
able interest.
Pyrrolizines constitute an important class of heterocycles with
highly pronounced biological activities.15–17 Functionalized pyrr-
olizines with spirooxindole rings constitute prime sub-structures
of numerous alkaloids and pharmacologically important com-
pounds.18 Isatin and its derivatives possess antibacterial,19 antipro-
tozoal,20 antifungal,21 antiviral22 and anti-HIV activities23 and are
widely used as precursors for the synthesis of natural products.24
AChE and BuChE have similar structures and adopt a very sim-
ilar secondary structure topology with about 83.9% sequence sim-
ilarity.25 It is found that majority of the important functionality
binding sites as well as the structurally important residues are con-
served among AChE and BuChE. AChE and BuChE exhibit five
important regions for binding, namely active gorge,26 oxyanion
hole,27 anionic substrate site,28 an acyl binding site29 and periphe-
ral anionic site30 but there are some divergences between them. In
AChE, more aromatic residues are found in the gorge, whilst in
BuChE the gorge is lined with additional hydrophobic residues. De-
spite the different properties in the gorge region, Leu286 and
Val288 are responsible for the acyl binding site in BuChE, instead
of two Phe residues in AChE.30 The active site gorge in both AChE
and BuChE is located deep in the centre of the molecule with a nar-
row gorge made up of the catalytic triad Ser200, His440 and
Glu327 involved in hydrolyzing acetylcholine. In the present study,
we have introduced the multi-component 1,3-dipolar cycloaddi-
tion reactions for the synthesis of mono- and bispyrrolizines incor-
porating indolizine, piperidone and oxindole rings and reported
their AChE and BuChE inhibitory activities. Molecular docking sim-
ulation was also employed to disclose the mechanism of inhibition
for the most active compounds synthesized in the present work on
AChE and BuChE receptors. The important structural aspects of
AChE and BuChE receptors active sites were briefly discussed to en-
able understanding of their binding interactions with the most ac-
tive compounds.
With a series of dipolarophiles (5a–k) in hand, we commenced
our investigation on the three-component [3+2]-cycloaddition
reaction of 5 with isatin (6) and
dition reaction of an equimolar mixture of a series of 1-acryloyl-
3,5-diarylidenepiperidin-4-ones (5), isatin (6) and -proline (7)
L-proline (7). Initially, the cycload-
L
was performed in methanol under heating at reflux for 5 h. till
the reaction went to completion (TLC). This reaction afforded the
monospiropyrrolizines (8) (Scheme 2) as the sole product in 73–
91% yields. When the reaction of 5, 6 and 7 in a molar ratio 1:2:2
was performed for a longer duration of 15 h. the structurally more
complex bisspiropyrrolizines (9) were obtained in moderate yields
(53–74%). In both these reactions, the spiropyrrolizines (8) and (9)
that precipitated out as colourless solid from the reaction mixture
was filtered off and washed with cold methanol. The resulting
samples 8 and 9 were found to be of high purity as disclosed by
TLC and 1H NMR spectroscopic data analysis. Further treatment
of 8 or 9 with an excess of isatin and L-proline failed to afford
the trispiroheterocycles (Scheme 2).
Structure of the mono and bispiropyrrolizines 8 and 9 is in ac-
cord with the combustion data as well as IR, 1D and 2D NMR spec-
troscopic data (vide infra). The elemental analysis results are
within 0.4% of the theoretical values. The 1H NMR spectrum of
8a shows a doublet of doublets at 3.96 ppm with J = 11.66 and
7.09 Hz for H-3 of the pyrrolizine ring due to coupling with CH2-
4. The C,H-COSY correlation of H-3 assigns the carbon signal at
51.2 ppm to C-3. Further, H-3 shows HMBCs (Fig. 2) with (i) C@O
carbon linked to the N(100) at 164.1 ppm, (ii) the oxindole C@O at
180.8 ppm, (iii) the spiro carbon C-2 at 77.4 ppm, besides showing
correlation with the adjacent carbon C-4 at 35.3 ppm. The C,H-
COSY correlation of C-4 assigns the multiplets at 2.20–2.26 and
2.43–2.56 ppm to 4-CH2. The H,H-COSY correlation of 4-CH2 as-
signs the multiplet at 4.01–4.12 ppm to H-4a. By similar consider-
ations, the multiplets around 1.56–2.67 ppm is assigned to 5-, 6-,
and 7-CH2 hydrogens of the pyrrolizine ring. The multiplets around
4.24–4.66 ppm is due to 200-CH2 and 600-CH2 of the piperidone ring,
which show HMBCs with the carbon signal at 184.2 ppm due to C-
400 besides showing correlation with the carbonyl at 164.1 ppm.
From C,H-COSY correlation, the carbon signals at 42.3 and
46.5 ppm are assigned to C-600 and C-200, respectively. The singlets
at 7.53, 7.56 and 8.70 ppm are due to the arylmethylidene hydro-
gens and NH hydrogen of the oxindole. The aromatic hydrogens
appear as doublets and multiplets in region between 7.03 and
7.40 ppm. The 1H and 13C NMR chemical shifts of the bisspiropyrr-
olizines (9) were also assigned by similar straightforward consider-
ations. Further the structure and stereochemistry of mono and
bisspiropyrrolizines were confirmed by the single crystal X-ray
crystallographic analysis of 8a36 (Fig. 3) and 9k33 (Fig. 4), respec-
2. Results and discussion
2.1. Chemistry
The highly functionalized dipolarophiles viz. 1-acryloyl-3,5-
diarylidenepiperidin-4-ones (5) required for the synthesis of spiro-
heterocycles were prepared by the Claisen–Schmidt condensation
of 4-piperidone hydrochloride (1) with a series of aromatic alde-
hydes (Scheme 1) in the presence of HCl in acetic acid following
the literature procedure.31 Acylation of the N-unsubstituted 3,5-
bis[(E)-arylmethylidene]tetrahydro-4(1H)-pyridinones (3) with
acryloyl chloride furnished 5 in good yields. Among the synthe-
sized dipolarophiles, 5b, 5c, 5e and 5f are new and their structure
O
Ar
O
N
Ar
O
Cl
HCl, CH3COOH
NH
Ar
4
2 ArCHO
+
K2CO3
Acetone
K2CO3
Acetone
N
O
O
H.HCl
2
Ar
1
3
5
Scheme 1. Synthesis of highly functionalized dipolarophiles 5(a–k).

1698
Y. Kia et al. / Bioorg. Med. Chem. 21 (2013) 1696–1707
Figure 1. ORTEP diagram of 5g.
Ar
O
N
H
N
O
H
O
Ar
1 eq.
O
N
O
N
H
HN
6
O
5
O
Ar
Ar
1 eq.
COOH
N
H
( )- 8
6 (2 eq)
7 (2 eq)
MeOH, Reflux, 5 h
MeOH,
Reflux
15h
H
O
NH
N
O
O
H
N
H
H
N
O
N
NH
N
O
H
Ar
H
H
Ar
6 (excess)
7 (excess)
HN
Ar
N
O
O
O
H
HN
N
HN
O
Ar
10
(Not formed)
( )- 9
Scheme 2. Synthesis of mono and bisspiropyrrolizines 8(a–k) and 9(a–k).
m, 2.20 - 2.26
m, 2.43 - 2.56
dd, 3.96,
35.3
J =11.66, 7.09 Hz
51.2
m, 4.01 - 4.12
64.3
m, 4.43-4.57
m, 4.57-4.66
46.5
m, 1.56 - 1.69
164.1
O
m, 2.00 - 2.10
31.7
H
5
H
4
H
H
4a
6
O
Ar
Ar
4"
7
m, 1.76 - 1.94
27.8
s, 7.56
H
3
N
1
N
H
H
H
2
77.4
O
H
3"
N
O
N
1"
2'
2"
5"
m, 2.43 - 2.56
m, 2.56 - 2.67
47.7
6"
H
184.2
O
HN
1'
HN
s, 8.70
O
m, 4.24-4.37
m, 4.57-4.66
42.3
Ar
H
s, 7.53
H
Ar
Ar = Phenyl
Figure 2. Selected HMBCs and 1H and 13C chemical shifts of 8a.

Y. Kia et al. / Bioorg. Med. Chem. 21 (2013) 1696–1707
1699
experiment, also furnished 9. Further reaction of 9 with isatin
and -proline failed to afford the trispiro cycloadduct (10), ascrib-
L
able to the steric hindrance arising from the cycloadduct (9) for
further cycloaddition. The structure of 8 and 9 shows that the
cycloaddition reactions proceed regioselectively with the nucleo-
philic carbon of azomethine ylide preferentially adding to the
b-carbon of enone functions and stereoselectively affording
only one stereoisomer of both the mono- and bis-cycloadducts 8
and 9.
2.2. In vitro cholinesterase enzymes inhibitory activity
All the newly synthesized compounds were evaluated for their
AChE and BuChE inhibitory activities using Ellman’s method and
the results are summarized in Table 1. The data in this table show
that in the first series, 5, only 5c and 5f bearing ortho-methoxy and
meta-nitro moieties on aromatic ring, displayed good inhibitory
activity against AChE enzyme with IC50 = 8.72 and 9.76 lM, respec-
tively. The rest of the compounds in this series showed moderate
inhibitory activity against AChE with IC50 ranging from 11.81 to
22.04
itory activity against this enzyme, with activities ranging from
22.06 to 54.04 M. Compound 5f, also showed the highest selectiv-
lM. Regarding BuChE, all the compounds, showed low inhib-
Figure 3. ORTEP diagram of 8a.
l
ity for AChE enzyme in this group with 5.24 times more selectivity
toward AChE as compared to BuChE.
As for mono-spiropyrrolizine in series 8, compounds 8i carrying
para-chloro, 8g carrying ortho and para di-chloro, 8f carrying meta-
nitro and 8k carrying naphthyl moiety on aromatic ring, showed
noticeable inhibitory activity against AChE enzyme with
IC50 = 3.36, 4.11, 5.99 and 6.76 lM, respectively. Compound 8i also
displayed highest inhibitory activity and selectivity for AChE
among the compounds within this group and all the synthesized
compounds. It is observable that presence of chloro substituent
on aromatic ring has a significant influence on possessing AChE
inhibitory activity within these mono-cycloadducts as observed
for 8i and 8g. The rest of compounds in this series displayed mod-
erate AChE inhibitory with activities ranging from 11.92 to
34.37 lM.
As for BuChE, compound 8e, bearing fluoro substituent on ortho
position of aromatic ring showed highest inhibitory activity and
highest selectivity for this enzyme within mono-spiropyrrolizine
and all synthesized compounds (IC50 = 3.51 lM). Compounds 8j
bearing fluoro substituent on para position of aromatic ring, also
showed considerable BuChE inhibitory activity within this series
with IC50 = 3.99 lM. It can be postulated that presence of fluoro
Figure 4. ORTEP diagram of 9k.
substituent on aromatic ring of mono-spiropyrrolizine compounds,
remarkably enhances the BuChE inhibitory activity presumably
due to more appropriate interactions with active site residues of
this enzyme. The remaining compounds in this series, showed
moderate BuChE inhibitory activity ranging from 11.79 to
tively. As the cycloadduct 9 has two oxindolo-pyrrolizine rings, to
distinguish the 1H and 13C chemical shifts of the two rings, the ring
attached to 100-N–C@O is considered as ring A and the other ring at-
tached to C-300 of 4-piperidone moiety is considered as ring B.
The mechanism of the reaction involves the initial formation of
32.27 lM.
Considering the bis-spiropyrrolizines 9, interestingly deriva-
tives 9g and 9i with chlorine in the aromatic rings, again displayed
azomethine ylide 11 from isatin and L-proline, which adds che-
the highest inhibitory activities with IC50 = 6.96 and 7.92
lM,
moselectively to C@C bond of acryloyl moiety of 5 to form the
monospiropyrrolizine 8 (Scheme 3). This chemoselectivity is expli-
cable by the relatively less hindrance encountered for the cycload-
dition at the C@C bond of acryloyl moiety compared to the two
arylidiene C@C functions. In case of reaction of 5 with an excess
of 6 and 7 for a longer reaction time, the azomethine ylide further
adds to one of the C@C bonds of initially generated 8 affording the
dispiro cycloadduct (9). This is evident from the fact that (i) the
hexahydrospiroindoline-3,3’-pyrrolizine ring formed by the reac-
tion of the azomethine ylide to N-acryolyl group of 8 and 9 has
the same relative configuration and (ii) the reaction of 8 with
respectively. However the remaining compounds of series
showed moderate inhibitory activity against AChE. Regarding
BuChE, compounds 9k, 9j, 9e and 9g displayed considerable activ-
9
ities against BuChE with IC50 = 6.42, 8.29, 9.09 and 9.45
respectively.
lM,
In conclusion, it can be justified that mono-spiropyrrolizines
showed the best inhibitory activity in both AChE and BuChE com-
pared to the starting precursors (5) and bis-spiropyrrolizines (9),
probably owing to introduction of more functional groups into
the molecule which seems necessary to bind the inhibitors to the
active site residues. Moving from mono- to bis-spiropyrrolizines,
due to increase in the size of molecule, AChE inhibitory activity
one mole equivalent of each isatin and L-proline, in a separate

1700
Y. Kia et al. / Bioorg. Med. Chem. 21 (2013) 1696–1707
N
H
H
N
O
NH
N
O
H
O
H
Ar
COOH
O
N
N
N
H
N
N
H
H
7
Ar
5
O
11
O
+
O
H
HN
HN
O
O
O
N
Ar
N
Ar
H
6
( )- 8
( )- 9
O
N
H
11
Scheme 3. Mechanism of formation of spiropyrrolizines 8 and 9.
Table 1
Physical data, AChE and BuChE activities of 5(a–k), 8(a–k) and 9(a–k)
Entry
Compound
Ar
Yield (%)
AChE inhibition IC50
BuChE inhibition IC50
g/mL lM
Selectivity
AChEa BuChEb
l
g/mL
lM
l
1
2
3
4
5
6
7
8
9
5a
5b
5c
5d
5e
5f
5g
5h
5i
C6H5
87
81
83
74
76
84
83
85
82
82
91
5.83 0.29
17.68
17.39
8.72
16.32
11.81
9.76
15.75
18.06
22.04
15.12
13.84
7.26 0.15
10.72 0.27
11.24 0.32
14.54 0.22
19.73 0.29
21.44 0.27
13.62 0.21
12.52 0.17
15.44 0.31
11.16 0.19
9.92 0.24
22.06
30.02
28.92
36.62
54.04
51.16
29.37
35.07
38.19
30.58
23.12
1.25
1.73
3.32
2.24
4.58
5.24
1.86
1.94
1.73
2.02
1.67
0.80
0.58
0.30
0.45
0.22
0.19
0.54
0.51
0.58
0.49
0.60
2-CH3C6H4
2-(OCH3)C6H4
2-ClC6H4
6.21 0.22
3.44 0.08
6.49 0.12
4.31 0.15
4.11 0.24
7.31 0.19
6.81 0.29
8.72 0.38
5.54 0.17
5.94 0.11
2-FC6H4
3-(O2N)C6H4
2,4-Cl2C6H3
4-CH3C6H4
4-ClC6H4
4-FC6H4
1-Napthyl
10
11
5j
5k
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
9a
9b
9c
9d
9e
9f
C6H5
83
74
77
78
73
81
79
76
80
78
84
72
61
65
53
53
59
68
67
64
62
74
—
9.22 0.14
7.22 0.04
9.42 0.28
8.12 0.06
19.42 0.24
3.99 0.12
2.94 0.21
12.15 0.32
2.28 0.07
13.52 0.27
4.58 0.18
15.23 0.12
21.15 0.27
9.17 0.27
11.22 0.12
19.42 0.31
22.43 0.28
6.12 0.16
15.07 0.15
6.43 0.10
18.73 0.32
17.64 0.34
0.60 0.01
17.49
12.47
15.86
11.92
34.37
5.99
4.11
20.07
3.36
8.62 0.19
9.93 0.23
5.49 0.19
11.21 0.22
1.25 0.02
10.87 0.32
12.93 0.27
14.04 0.31
19.26 0.26
1.38 0.04
7.42 0.17
14.21 0.23
10.15 0.10
15.43 0.14
9.67 0.25
7.52 0.17
20.53 0.28
8.42 0.19
13.75 0.26
8.94 0.17
6.59 0.16
5.49 0.15
5.55 0.01
16.29
17.82
9.41
19.73
3.50
18.14
21.16
25.2
32.27
3.99
11.79
18.21
12.96
19.21
11.74
9.09
24.29
9.45
17.88
10.89
8.29
0.93
1.43
0.59
1.66
0.10
3.03
5.15
1.26
9.60
0.20
1.74
0.93
0.47
1.68
0.86
0.37
0.91
1.36
0.91
1.38
0.35
0.31
3.47
1.07
0.70
1.69
0.60
9.82
0.33
0.19
0.80
0.10
4.97
0.57
1.08
2.12
0.59
1.16
2.70
1.10
0.74
1.10
0.73
2.86
3.21
0.28
2-CH3C6H4
2-(OCH3)C6H4
2-ClC6H4
2-FC6H4
3-(O2N)C6H4
2,4-Cl2C6H3
4-CH3C6H4
4-ClC6H4
4-FC6H4
1-Naphthyl
C6H5
2-CH3C6H4
2-(OCH3)C6H4
2-ClC6H4
19.84
6.76
19.65
27.45
11.41
13.64
24.58
26.6
6.96
19.59
7.92
2-FC6H4
3-(O2N)C6H4
2,4-Cl2C6H3
4-CH3C6H4
4-ClC6H4
4-FC6H4
1-Naphthyl
Galanthamine
9g
9h
9i
9j
23.67
20.63
2.09
9k
Standard
6.42
19.34
a
Selectivity for AChE is defined as IC50(BuChE)/IC50(AChE).
Selectivity for BuChE is defined as IC50(AChE)/IC50(BuChE).
b
drops considerably, however BuChE inhibitory activity remains al-
most unchanged. This can be relevant to bigger active site gorge in
the BuChE enzyme, in comparison to AChE that accommodates
more bulky substrates/inhibitors. As mentioned earlier, presence
of chloro substituent on aromatic ring in mono and bis-cycload-
ducts, significantly improves both inhibitory activities and selec-
tivity for AChE enzyme. This effect is also noticeable for the
mono and bis-spiropyrrolizines which carry fluoro substituent on
aromatic ring, as their selectivity and inhibitory activity for BuChE
are superior as compared to other derivatives.
2.3. Docking simulation
Docking simulation is a popular approach for the preliminary
screening in structure based drug design. By performing docking
simulation, information on feasible conformations of the ligand
within the protein binding site can be obtained. This information
can also reflect the nature and quality of the interaction. In our
study, the grid box for docking simulation was built with enough
size to enable probing into the binding with peripheral and gorge
active sites. Docking simulations were carried out for the most ac-

Y. Kia et al. / Bioorg. Med. Chem. 21 (2013) 1696–1707
1701
Table 2
Binding interaction data for 8i and 8e to AChE and BuChE receptors
Entry
Ligand
Enzyme
Interacting site
Residue name
Bond type
Residue interacting moiety
Ligand interacting moiety
1
2
3
4
5
6
7
8
9
8i
TcAChE
PAS
PAS
PAS
PAS
Tyr70
Hydrophobic
Hydrophobic
Hydrophobic
H-bonding
4-Methoxyphenyl
Methoxy
Indole
Pyrrolizine ring
Ring 1
Pyrrolizine ring, ring 2 and 3
NH of pyrrolizine ring
Ring 1
Ring 1
Ring 2
Ring3
C@O of ring 2
Tyr121
Trp 279
Tyr334
Tyr334
Phe331
Phe330
Phe290
Arg289
C@O
PAS
p
,
p
p
-Stacking
-Stacking
4-Methoxyphenyl
Phenyl ring
Phenyl ring
Phenyl ring
N–H
Choline binding
Choline binding
Acyl binding
Side chain
p,
Hydrophobic
Hydrophobic
H-bonding
10
11
12
13
14
15
16
17
8e
BuChE
PAS
PAS
Trp 231
Phe398
Phe329
Gly116
Ala199
Trp82
p
,
p
-Stacking
Indole
Ring 1
Ring 1
Pyrrolizine ring
Ring 1
Ring 2
Oxo-indole and pyrrolizine ring
Ring 1
NH of pyrrolizine ring
Hydrophobic
Hydrophobic
Mild polar
Mild polar
Hydrophobic
Mild polar
Phenyl ring
Phenyl ring
N–H
Acyl binding
Oxyanion hole
Oxyanion hole
Choline binding
Catalytic triad
Side chain
N–H
Indole ring
Imidazole ring
C@O
His 438
Asp79
H-bonding
H
H
1
O
N
H
H
H
H
N
O
2
HN
O
H
H
3
tive inhibitors 8i and 8e, which displayed comparable potency to
AChE and five times more potency to BuChE enzymes as compared
to standard drug, galantamine. This strategy expected to disclose
the binding affinity of these inhibitors to their respective receptors
and their relative orientation inside the receptor and in contact
with active site residues.
The docking results on AChE and BuChE receptors were col-
lected using Schrodinger, Maestro 9.2 software for the most potent
inhibitors, 8i and 8e, to their respective receptors. As summarized
in Table 2, The most of the binding interactions within 8i and the
amino acid residues composing active site gorge in AChE receptor,
at catalytic triad are the other main residues which interact with
8e within active site gorge. As depicted in Figure 6 it seems that
this compound is completely accommodated inside the gorge ow-
ing to appropriate interactions with the key residues composing
active site in BuChE receptor. This suitable embedding probably in-
cur to prosperous prevention of substrates to insert into the gorge
rising to its significant inhibitory property.
In conclusion, information gathered from docking simulation
analysis for 8i and 8e, were in accordance with the IC50 values ob-
tained from the cholinesterase inhibition assay. In general, better
accommodation of inhibitor inside the active site gorge due to
more appropriate interactions with its amino acid residues, ensue
better inhibitory activities and lower IC50 values during in vitro
assay.
possess hydrogen bonding,
p,p-stacking and hydrophobic nature.
Concisely, this compound displayed strong
p,p-stacking interac-
tion with Tyr334 at peripheral anionic site and Phe331 at choline
binding site. Two strong hydrogen binding interaction with
Tyr334 (2.18 Å) and Arg289 (2.05 Å) residues which are located
at peripheral anionic site and side chain backbone, has been
docked this compound to the active site gorge. Hydrophobic inter-
actions with Tyr70, Tyr121 and Trp279 in peripheral anionic site
together with Phe290 at acyl binding pocket and Phe330 at choline
binding site are the other noticeable interactions which support
the accommodation of 8i inside the gorge. This compound via
interaction with residues composing peripheral anionic site
(PAS), presumably blocks the entrance of the gorge and prevents
the insertion/accommodation of substrate inside the active site of
the receptor (Fig. 5).
3. Conclusion
A series of piperidone-grafted novel mono- and bisspiropyrroli-
zines has been synthesized by the three-component [3+2]-cycload-
dition reactions of azomethine ylides to highly functionalized
dipolarophiles. This method employs simple, readily available
starting materials and facile work up procedure without requiring
column chromatographic purification. Mono-spiripyrrolizines 8i
and 8e, synthesized in the present study are found to be signifi-
cantly active in cholinesterase inhibitory assay with IC50 = 3.36
and 3.50 lM displayed respectively comparable potency to AChE
For 8e which is docked into BuChE receptor, interactions have
and five times more potent than BuChE enzymes as compared to
standard drug. The molecular modeling simulation for the most ac-
tive compounds completely coincided with IC50 data obtained from
in vitro assay. The synthesis and screening for biological activity of
further series of spirocycloadducts derived from different cyclic
and acyclic amino acids are currently under investigation in our re-
search group.
H-bonding,
ble 2). This compound showed strong
p
,
p
-stacking, hydrophobic and mild polar nature (Ta-
-stacking interaction
p,p
with Trp231 at peripheral anionic site and strong hydrogen bond-
ing with Asp70 (2.03 Å) located at side chain. Val 288, Leu 286 and
Phe 398 at peripheral anionic, Gly116, Gly117 and Ala199 at oxy-
anion hole, Trp82 and Phe 329 at choline binding site and His438

1702
Y. Kia et al. / Bioorg. Med. Chem. 21 (2013) 1696–1707
Figure 5. Orientations and interaction of 8i with main residues in AChE receptor.
matic), 7.74 (s, 1H, H-arylmethylidene), 7.85 (s, 1H, H-arylmethyl-
4. Experimental
idene). 13C NMR (125 MHz, CDCl3): 21.83, 21.89, 48.57, 127.17,
129.14, 129.72, 130.00, 131.25, 134.69, 137.61, 140.45, 165.94,
187.05.
4.1. General methods
The melting points were measured in open capillary tubes and
are uncorrected. FT-IR spectra were recorded on a Perkin Elmer
2000 instrument. The 1H, 13C and the 2D NMR spectra were re-
corded on a Bruker (Avance) 500 MHz NMR instrument using
TMS as internal standard, DMSO-d6 and CDCl3 as solvents. Stan-
dard Bruker software was used throughout. Chemical shifts are gi-
ven in parts per million (d-scale) and the coupling constants are
given in Hertz. Silica gel-G plates (Merck) were used for TLC anal-
ysis with a mixture of petroleum ether (60–80 °C) and ethyl ace-
tate as eluent. Elemental analyses were performed on a Perkin
Elmer 2400 Series II Elemental CHNS analyzer.
4.2.2. (3E,5E)-1-Acryloyl-3,5-bis(2-methoxyphenylmethylidene)
piperidin-4-one (5c)
Yellow solid; (1.79 g, 91%); mp 166–168 °C; IR (KBr) mmax: 3447,
1652, 1471 cmꢀ1; Anal. Calcd for C24H23NO4: C, 74.02; H, 5.95; N,
3.60. Found: C, 74.11; H, 6.05; N, 3.69. NMR (500 MHz, CDCl3): d
3.8 9 (s, 6H, OCH3), 4.90–5.08 (m, 4H, CH2), 5.61–5.69 (m, 1H,
CH), 6.09–6.18 (m, 1H, CH), 6.49–6.54 (m, 1H, CH), 7.05–7.54 (m,
8H, H-aromatic), 7.78 (s, 1H, H-arylmethylidene), 8.06 (s, 1H, H-
arylmethylidene). 13C NMR (125 MHz, CDCl3): 47.06, 47.47,
55.45, 127.63, 128.22, 128.54, 130.58, 132.88, 133.01, 135.56,
161.03, 186.47.
4.2. General procedure for the synthesis of 5
4.2.3. (3E,5E)-1-Acryloyl-3,5-bis(2-fluorophenylmethylidene)
piperidin-4-one (5e)
To a mixture of 3,5-diarylidenepiperidin-4-ones (4.82 mmol)
and K2CO3 (4.82 mmol) in acetone (10 ml) in an ice bath, acryloyl
chloride (7.24 mmol) was added dropwise under stirring. The reac-
tion was continued for 24 h. at ambient temperature. After com-
pletion of the reaction, by monitoring with TLC, the reaction
mixture was poured into ice, the resulting precipitate was filtered
off and washed with water to obtain pure 5 as yellow solid.
Yellow solid; (1.59 g, 81%); mp 120–122 °C; IR (KBr) mmax: 3444,
1651, 1463 cmꢀ1; Anal. Calcd for C22H17F2NO2: C, 72.32; H, 4.69; N,
3.83. Found: C, 72.31; H, 4.67; N, 3.81. NMR (500 MHz, CDCl3): d
4.67ꢀ4.91 (m, 4H, CH2), 5.52–5.61 (m, 1H, CH), 6.12–6.31 (m, 2H,
CH2), 7.09–7.48 (m, 8H, H-aromatic), 7.87 (s, 1H, H-arylmethyli-
dene), 7.91 (s, 1H, H-arylmethylidene). 13C NMR (125 MHz, CDCl3):
48.39, 124.34, 124.39, 124.71, 124.76, 126.94, 129.24, 131.13,
131.16, 131.28, 131.32, 131.45, 131.89, 165.93, 186.30.
4.2.1. (3E,5E)-1-Acryloyl-3,5-bis(2-methylphenylmethylidene)
piperidin-4-one (5b)
4.2.4. (3E,5E)-1-Acryloyl-3,5-bis(3-nitrophenylmethylidene)
piperidin-4-one (5f)
Yellow solid; (1.52 g, 89%); mp 100–102 °C; IR (KBr) mmax: 3451,
1650, 1463 cmꢀ1; Anal. Calcd for C24H23NO2: C, 80.64; H, 6.49; N,
3.92. Found: C, 80.59; H, 6.43; N, 3.87. NMR (500 MHz, CDCl3): d
2.34 (s, 3H, CH3), 2.36 (s, 3H, CH3), 4.70–4.99 (m, 4H, CH2), 5.50–
5.61 (m, 1H, CH), 6.12–6.30 (m, 2H, CH2), 7.16–7.32 (m, 8H, H-aro-
Yellow solid; (1.42 g, 79%); mp 147–149 °C; IR (KBr) mmax: 3449,
1654, 1470 cmꢀ1; Anal. Calcd for C22H17N3O6: C, 63.01; H, 4.09; N,
10.02. Found: C, 63.09; H, 4.14; N, 10.07. 1H NMR (500 MHz, CDCl3):

Y. Kia et al. / Bioorg. Med. Chem. 21 (2013) 1696–1707
1703
Figure 6. Orientations and interaction of 8e with main residues in BuChE receptor.
d 4.98–5.25 (m, 4H, CH2), 5.57–5.64 (m, 1H, CH), 6.04–6.12 (m, 1H,
CH), 6.41–6.53 (m, 1H, CH), 7.76–8.34 (m, 10H, H-aromatic, H-aryl-
methylidene). 13C NMR (125 MHz, CDCl3): 39.32, 124.25, 124.95,
127.31, 128.88, 130.55, 134.62, 136.53, 143.93, 166.52, 186.00.
4.57–4.66 (m, 2H, H-200, H-600), 7.03–7.40 (m, 14H, H-aromatic),
7.53 (s, 1H, H-phenylmethylidene), 7.56 (s, 1H, H-phenylmethylid-
ene), 8.70 (s, 1H, N–H). 13C NMR (125 MHz, CDCl3): 27.75, 31.73,
35.26, 42.30, 46.54, 47.74, 51.20, 64.28, 77.38, 110.29, 115.95,
116.24, 122.15, 125.49, 127.64, 128.96, 129.21, 131.35, 131.88,
132.65, 132.77, 136.68, 140.74, 164.10, 180.83, 184.20.
4.3. General procedure for the synthesis of (8) and (9)
A
mixture of 1-acryloyl-3,5-diarylidenepiperidin-4-ones (5,
4.3.2. 100-Carbonyl (spiro[2.3’]oxindole-hexahydro-1H-
pyrrolizine)-300,500-bis[(E)-2-methylpheylmethylidene]
tetrahydro-400(1H)-pyridinones (8b)
0.364 mmol), isatin (6, 0.364 mmol) and -proline (7, 0.364 mmol)
L
was dissolved in methanol (5 mL) and heated under reflux for 5 h.
The reaction progress was monitored by TLC analysis. After com-
pletion of the reaction, the precipitate obtained was filtered and
washed with cold methanol (25 ml) to afford the monospiropyrr-
olizines (8) as white solid. The same reaction employing (5,
0.364 mmol), (6, 0.728 mmol) and (7, 0.728 mmol) in refluxing
methanol (5 ml) for 15 h afforded the bisspiropyrrolizines (9) as
white solid. The purity of both (8) and (9) were checked using
TLC and 1H NMR techniques.
White solid; (0.125 g, 74%); mp 160–162 °C; IR (KBr) mmax: 3439,
1729, 1615 cmꢀ1; Anal. Calcd for C36H35N3O3: C, 77.53; H, 6.33; N,
7.53. Found: C, 77.41; H, 6.39; N, 7.56. 1H NMR (500 MHz, CDCl3): d
1.59–1.68 (m, 1H, H-5), 1.79–1.98 (m, 2H, H-6), 1.83–2.10 (m, 1H,
H-5), 2.12–2.26 (m, 1H, H-4), 2.28 (s, 3H, CH3), 2.36 (s, 3H, CH3),
2.42–2.56 (m, 2H, H- 4, H-7), 2.59–2.73 (m, 1H, H-7), 3.94–3.98
(m, 1H, H-3), 3.99–4.10 (m, 1H, H-4a), 4.34–4.56 (m, 3H, H-200, H-
600), 4.71 (d, J = 16.23 Hz, 1H, H-600), 6.79–7.38 (m, 12H, H-aro-
matic), 7.51–7.65 (m, 2H, H-arylmethylidene), 8.71 (br s, 1H, N–
H). 13C NMR (125 MHz, CDCl3): d 21.55, 27.51, 31.47, 35.41,
42.78, 46.62, 47.71, 51.07, 64.16, 73.31, 110.29, 122.03, 125.67,
127.59, 129.47, 130.41, 130.82, 131.79, 137.48, 137.69, 140.21,
141.11, 167.02, 181.05, 186.62.
4.3.1. 100-Carbonyl (spiro[2.30]oxindole-hexahydro-1H-
pyrrolizine)-300,500-bis[(E)-phenylmethylidene]
tetrahydro-400(1H)-pyridinones (8a)
White solid; (0.134 g, 83%); mp 228–230 °C; IR (KBr) mmax: 3432,
1719, 1611 cmꢀ1; Anal. Calcd for C34H31N3O3: C, 77.10; H, 5.90; N,
7.93. Found: C, 77.02; H, 5.95; N, 7.97. 1H NMR (500 MHz, CDCl3): d
1.56–1.69 (m, 1H, H-5), 1.76–1.94 (m, 2H, H-6), 2.00–2.10 (m, 1H,
H-5), 2.20–2.26 (m, 1H, H-4), 2.43–2.56 (m, 2H, H-4, H-7), 2.56–
2.67 (m, 1H, H-7), 3.96 (td, J = 11.66, 7.09 Hz, 1H, H-3), 4.01–4.12
(m, 1H, H-4a), 4.24–4.37 (m, 1H, H-600), 4.43–4.57 (m, 1H, H-200),
4.3.3. 100-Carbonyl (spiro[2.3’]oxindole-hexahydro-1H-
pyrrolizine)-300,500-bis[(E)-2-methoxy phenylmethylidene]
tetrahydro-400(1H)-pyridinones (8c)
White solid; (0.127 g, 77%); mp 252–254 °C; IR (KBr)
mmax: 3442,
1719, 1614 cmꢀ1; Anal. Calcd for C36H35N3O5: C, 73.33; H, 5.98; N,

1704
Y. Kia et al. / Bioorg. Med. Chem. 21 (2013) 1696–1707
7.13. Found: C, 73.31; H, 5.62; N, 7.24. 1H NMR (500 MHz, CDCl3): d
1.59–1.68 (m, 1H, H-5), 1.76–1.84 (m, 1H, H-6), 1.84–1.92 (m, 1H,
H-6), 2.01–2.09 (m, 1H, H-5), 2.23–2.35 (m, 1H, H-4), 2.43–2.54 (m,
2H, H-7, H-4), 2.63–2.71 (m, 1H, H-7), 3.71 (s, 3H, OCH3), 3.81 (s,
3H, OCH3), 3.96 (dd, J = 10.48, 7.33 Hz, 1H, H-3), 4.03–4.10 (m,
1H, H-4a), 4.32 (d, J = 16.55 Hz, 1H, H-600), 4.43 (d, J = 15.76 Hz,
1H, H-200), 4.53 (d, J = 15.76 Hz, 1H, H-200), 4.65 (d, J = 16.39 Hz,
1H, H-600), 6.80–7.35 (m, 12H, H-aromatic), 7.53 (s, 1H, H-aryl-
methylidene), 7.57 (s, 1H, H-arylmethylidene), 8.71 (br s, 1H, N–
H). 13C NMR (125 MHz, CDCl3): d 27.36, 31.37, 35.37, 42.66,
46.62, 47.81, 50.94, 55.33, 64.13, 73.29, 110.27, 114.21, 114.48,
122.01, 125.68, 127.08, 127.28, 127.58, 128.86, 129.34, 129.40,
132.27, 132.75, 136.98, 137.29, 141.04, 160.52, 160.73, 169.07,
180.77, 186.44.
110.14, 122.31, 125.59, 127.77, 129.02, 129.30, 129.53, 131.38,
131.47, 131.54, 131.69, 132.11, 132.86, 1323.11, 135.89, 136.25,
136.42, 137.09, 140.51, 168.92, 180.49, 186.71.
4.3.7. 100-Carbonyl (spiro[2.3’]oxindole-hexahydro-1H-
pyrrolizine)-300,500-bis[(E)-2,4-dichloro phenylmethylidene]
tetrahydro-400(1H)-pyridinones (8g)
White solid; (0.114 g, 79%); mp 210–212 °C; IR (KBr) mmax: 3434,
1701, 1642 cmꢀ1; Anal. Calcd for C34H27Cl4N3O3: C, 61.19; H, 4.08;
N, 6.30. Found: C, 61.11; H, 4.02; N, 6.37. 1H NMR (500 MHz, CDCl3)
d 1.53–1.64 (m, 1H, H-5), 1.80–1.95 (m, 2H, H-6), 2.00–2.11 (m, 1H,
H-5), 2.17–2.26 (m, 1H, H-4), 2.41–2.49 (m, 1H, H-7), 2.49–2.57 (m,
2H, H-4, H-7), 3.76 (d, J = 15.61 Hz, 1H, H-3), 3.93 (d, J = 15.61 Hz,
1H, H-600), 3.98–4.07 (m, 1H, H-4a), 4.34 (d, J = 17.18 Hz, 1H, H-
200), 4.75 (d, J = 17.18 Hz, 1H, H-200), 4.86 (d, J = 15.61 Hz, 1H, H-
600), 6.75–7.51 (m, 10H, H-aromatic), 7.66 (s, 1H, H-arylmethyli-
dene), 7.74 (br s, 2H, H-arylmethylidene and N–H). 13C NMR
(125 MHz, CDCl3): d 28.18, 32.08, 35.15, 42.04, 46.44, 47.51,
51.84, 64.45, 73.40, 110.24, 122.19, 125.28, 127.23, 127.62,
127.74, 129.64, 129.69, 129.97, 130.99, 131.15, 131.17, 132.37,
133.28, 133.57, 134.26, 135.59, 135.68, 135.74, 136.13, 140.09,
168.16, 180.44, 186.28.
4.3.4. 100-Carbonyl (spiro[2.30]oxindole-hexahydro-1H-
pyrrolizine)-300,500-bis[(E)-2-chlorophenylmethylidene]
tetrahydro-400(1H)-pyridinones (8d)
White solid; (0.132 g, 78%); mp 248–250 °C; IR (KBr) mmax: 3442,
1716, 1621 cmꢀ1; Anal. Calcd for C34H29Cl2N3O3: C, 68.23; H, 4.88;
N, 7.02. Found: C, 68.29; H, 4.83; N, 7.06. 1H NMR (500 MHz,
DMSO-d6): d 1.48–1.67 (m, 2H, H-5, H-6), 1.71–1.82 (m, 1H, H-6),
1.87–1.96 (m, 1H, H-5), 2.00–2.10 (m, 1H, H-4), 2.17–2.26 (m,
2H, H-4, H-7), 2.52–2.57 (m, 1H, H-7), 3.60 (t, J = 8.67 Hz, 1H, H-
3), 3.73–3.83 (m, 1H, H-4a), 4.09 (d, J = 16.87 Hz, 1H, H-600), 4.27
(d, J = 15.61 Hz, 1H, H-200), 4.49 (d, J = 15.61 Hz, 1H, H-200), 4.59 (d,
J = 16.87 Hz, 1H, H-600), 6.74–7.70 (m, 12H, H-aromatic), 7.70–
7.75 (m, 2H, H-arylmethylidene), 10.17 (s, 1H, N–H). 13C NMR
4.3.8. 100-Carbonyl (spiro[2.3’]oxindole-hexahydro-1H-
pyrrolizine)-300,500-bis[(E)-4-methylpheylmethylidene]
tetrahydro-400(1H)-pyridinones (8h)
White solid; (0.112 g, 76%); mp 233–235 °C; IR (KBr) mmax: 3447,
1714, 1614 cmꢀ1; Anal. Calcd for C36H35N3O3: C, 77.53; H, 6.33; N,
7.53. Found: C, 77.38; H, 6.41; N, 7.59. 1H NMR (500 MHz, CDCl3) d
1.56–1.65 (m, 1H, H-5), 1.74–1.92 (m, 2H, H-6), 1.80–2.08 (m, 1H,
H-5), 2.10–2.24 (m, 1H, H-4), 2.27 (s, 3H, CH3), 2.34 (s, 3H, CH3),
2.39–2.54 (m, 2H, H-4, H-7), 2.56–2.69 (m, 1H, H-7), 3.89–3.94
(m, 1H, H-3), 3.97–4.07 (m, 1H, H-4a), 4.33–4.55 (m, 3H, H-200, H-
600), 4.69 (d, J = 16.08 Hz, 1H, H-600), 6.75–7.34 (m, 12H, H-aro-
matic), 7.53–7.63 (m, 2H, H-arylmethylidene), 8.72 (br s, 1H, N–
H). 13C NMR (125 MHz, CDCl3): d 21.45, 27.45, 31.43, 35.37,
42.74, 46.62, 47.73, 51.04, 64.14, 73.31, 110.37, 122.04, 125.66,
127.56, 129.41, 130.39, 130.79, 131.76, 137.44, 137.63, 140.22,
140.97, 166.92, 180.84, 186.55.
(125 MHz, DMSO-d6)
d 26.50, 30.351, 48.56, 71.99, 109.69,
126.36, 126.92, 127.03, 127.32, 127.56, 128.79, 129.73, 129.92,
130.44, 130.86, 130.96, 131.73, 131.81, 132.28, 133.20, 133.85,
134.18, 142.11, 170.12, 181.15, 188.60.
4.3.5. 100-Carbonyl (spiro[2.3’]oxindole-hexahydro-1H-
pyrrolizine)-300,500-bis [(E)-2-fluoro phenylmethylidene]
tetrahydro-400(1H)-pyridinones (8e)
White solid; (0.115 g, 73%); mp 232–234 °C; IR (KBr) mmax:
3431, 1717, 1614 cmꢀ1
; Anal. Calcd for C34H29F2N3O3: C,
72.20; H, 5.17; N, 7.43. Found: C, 72.27; H, 5.11; N, 7.40. 1H
NMR (500 MHz, CDCl3) d 1.56–1.67 (m, 1H, H-5), 1.83–1.93
(m, 2H, H-6), 2.00–2.08 (m, 1H, H-4), 2.39–2.53 (m, 2H, H-4,
H-7), 2.65–2.79 (m, 1H, H-7), 3.84–3.95 (m, 1H, H-3),
3.98–4.10 (m, 2H, H-4a, H-600), 4.42 (d, J = 15.25 Hz, 1H, H-
200), 4.52 (d, J = 15.25 Hz, 1H, H-200), 4.82 (d, J = 15.61 Hz, 1H,
H-600), 6.73–7.45 (m, 12H, H-aromatic), 7.56 (s, 1H, N–H),
7.69 (s, 1H, H-arylmethylidene), 7.73 (s, 1H, H-arylmethyli-
4.3.9. 100-Carbonyl (spiro[2.3’]oxindole-hexahydro-1H-
pyrrolizine)-300,500-bis[(E)-4-chloro phenylmethylidene]
tetrahydro-400(1H)-pyridinones (8i)
White solid; (0.117 g, 80%); mp 166–168 °C; IR (KBr) mmax: 3442,
1729, 1616 cmꢀ1; Anal. Calcd for C34H29Cl2N3O3: C, 68.23; H, 4.88;
N, 7.02. Found: C, 68.28; H, 4.85; N, 7.07. 1H NMR (500 MHz, CDCl3)
d 1.55–1.68 (m, 1H, H-5), 1.76–1.93 (m, 2H, H-6), 2.00–2.10 (m, 1H,
H-5), 2.20–2.28 (m, 1H. H-4), 2.41–2.55 (m, 2H, H-4, H-7), 2.55–
2.62 (m, 1H, H-7), 3.97 (dd, J = 10.80, 7.01 Hz, 1H, H-3), 4.00–4.10
(m, 1H, H-4a), 4.25 (d, J = 15.76 Hz, 1H, H-600), 4.51–4.67 (m, 3H,
H-200, H-600), 6.76–7.39 (m, 12H, H-aromatic), 7.53 (s, 2H, H-aryl-
methylidene), 8.62 (br s, 1H, N–H). 13C NMR (125 MHz, CDCl3):
27.80, 31.77,35.26, 42.35, 46.69, 47.64, 51.38, 64.28, 73.30,
110.11, 122.25, 125.39, 127.73, 128.99, 129.30, 129.46, 131.31,
131.41, 131.55, 131.72, 131.83, 132.72, 132.78, 135.46, 136.01,
136.10, 136.72, 140.32, 168.59, 180.41, 186.62.
dene). 13C NMR (125 MHz, CDCl3):
d 27.26, 31.25, 35.30,
42.77, 46.62, 47.83, 50.98, 64.06, 73.07, 110.17, 115.79,
115.96, 116.14, 122.17, 122.40, 122.46, 124.20, 124.58,
125.27, 127.56, 127.69, 129.56, 129.66, 130.54, 130.70,
131.25, 131.32, 131.78, 131.82, 132.55, 132.94, 140.41,
159.93, 161.92, 168.92, 180.06, 185.86.
4.3.6. 100-Carbonyl (spiro[2.3’]oxindole-hexahydro-1H-
pyrrolizine)-300,500-bis [(E)-3-nitro phenylmethylidene]
tetrahydro-400(1H)-pyridinones (8f)
White solid; (0.125 g, 81%); mp 200–202 °C; IR (KBr) mmax: 3442,
1721, 1615 cmꢀ1; Anal. Calcd for C34H29N5O7: C, 65.91; H, 4.72; N,
11.30. Found: C, 65.85; H, 4.77; N, 11.38. 1H NMR (500 MHz, CDCl3)
d 1.59–1.69 (m, 1H, H-5), 1.81–1.97 (m, 2H, H-6), 2.05–2.14 (m, 1H,
H-5), 2.24–2.31 (m, 1H. H-4), 2.42–2.57 (m, 2H, H-4, H-7), 2.58–
2.64 (m, 1H, H-7), 4.02 (dd, J = 10.92, 7.12 Hz, 1H, H-3), 4.05–4.16
(m, 1H, H-4a), 4.29 (d, J = 15.76 Hz, 1H, H-600), 4.52–4.69 (m, 3H,
H-200, H-600), 6.78–7.28 (m, 12H, H-aromatic), 7.57 (s, 2H, H-aryl-
methylidene), 8.71 (br s, 1H, N–H). 13C NMR (125 MHz, CDCl3):
27.82, 31.82, 35.28, 42.33, 46.72, 47.68, 51.39, 64.32, 73.31,
4.3.10. 100-Carbonyl (spiro[2.3’]oxindole-hexahydro-1H-
pyrrolizine)-300,500-bis [(E)-4-fluoro phenylmethylidene]
tetrahydro-400(1H)-pyridinones (8j)
White solid; (0.129 g, 78%); mp 230–232 °C; IR (KBr) mmax: 3442,
1729, 1610 cmꢀ1; Anal. Calcd for C34H29F2N3O3: C, 72.20; H, 5.17;
N, 7.43. Found: C, 72.29; H, 5.14; N, 7.38. 1H NMR (500 MHz, CDCl3)
d 1.56–1.68 (m, 1H, H-5), 1.72–1.80 (m, 1H, H-6), 1.80–1.95 (m, 2H,
H-5, H-6), 1.95–2.05 (m, 1H, H-4), 2.13–2.20 (m, 1H, H-7), 2.33–
2.40 (m, 1H, H-4), 2.42–2.56 (m, 1H, H-7), 3.95 (dd, J = 10.96,

Y. Kia et al. / Bioorg. Med. Chem. 21 (2013) 1696–1707
1705
7.01 Hz, 1H, H-4a), 4.00–4.10 (m, 1H, H-600), 4.53 (d, J = 17.25 Hz,
1H, H-200), 4.67 (d, J = 17.25 Hz, 1H, H-200), 5.05 (d, J = 15.76 Hz,
1H, H-600), 6.71–7.45 (m, 12H, H-aromatic), 7.56 (s, 1H, H-aryl-
methylidene), 7.63 (s, 1H, H-arylmethylidene), 8.05–8.12 (br s,
1H, N–H). 13C NMR (125 MHz, CDCl3): d 27.69, 31.67, 35.29,
45.59, 47.68, 51.26, 64.23, 73.34, 110.15, 115.78, 115.96, 116.28,
122.16, 125.48, 127.66, 129.43, 130.54, 130.78, 130.96, 132.11,
132.18, 132.65, 132.71, 136.24, 136.78, 140.54, 162.14, 162.29,
164.13, 164.30, 168.74, 180.59, 186.72.
64.33, 66.24, 68.58, 113.56, 114.23, 126.12, 126.55, 132.71,
132.87, 133.84, 133.97, 134.15, 134.23, 135.49, 135.76, 135.91,
135.99, 141.73, 142.25, 147.67, 147.84, 177.78, 184.06, 184.22,
204.19.
4.3.14. Spiro-[2.3’]-oxindole-spiro[3.300]-100-carbonyl(spiro[2.3’]
oxindole-hexahydro-1H-pyrrolizine)-500-(2-methoxyphenylme-
thylidene) tetrahydro-400-(1H)-pyridinone-4-(2-meth-
oxyphenylmethylidene) hexahydro-1H-pyrrolizine (9c)
White solid; (0.131 g, 65%); mp 216–218 °C; IR (KBr) mmax: 3430,
4.3.11. 100-Carbonyl (spiro[2.3’]oxindole-hexahydro-1H-
pyrrolizine)-300,500-bis[(E)-naphthylmethylidene]
tetrahydro-400(1H)-pyridinones (8k)
1717, 1612 cmꢀ1; Anal. Calcd for C48H47N5O6: C, 72.98; H, 6.00; N,
8.87. Found: C, 72.91; H, 5.92; N, 8.80. 1H NMR (500 MHz, CDCl3) d
0.75–0.88 (m, 1H, H-200), 1.10–1.90 (m, 4H, H-6 of ring A, H-6 of
ring B, H-5 of ring B), 1.87–2.66 (m, 7H, H-5 of ring A, H-6 of ring
B, H-5 of ring B, H-4 of ring A, H-600), 2.78–3.74 (m, 8H, H-7 of ring
A, H-7 of ring B, H-200, H-600, H-3 of ring A, H-4a of ring A), 3.84 (s,
3H, CH3), 3.86 (s, 3H, CH3), 4.96–4.99 (m, 1H, H-4a of ring B), 6.50–
7.40 (m, 18H, H-aromatic, N–H), 7.58 (s, 1H, H-arylmethylidene).
13C NMR (125 MHz, CDCl3): d 27.41, 27.83, 31.37, 31.92 35.27,
42.61, 43.26, 46.52, 46.81, 47.73, 50.94, 55.33, 64.15, 66.29,
72.12, 73.29, 110.27, 110.52, 114.23, 114.57, 122.09, 125.74,
127.12, 127.31, 127.58, 128.77, 129.37, 129.44, 132.27, 132.78,
137.01, 137.35, 141.08, 160.50, 160.81, 169.07, 179.21, 180.77,
186.44.
White solid; (0.154 g, 84%); mp 210–212 °C; IR (KBr) mmax: 3431,
1716, 1617 cmꢀ1; Anal. Calcd for C42H35N3O3: C, 80.10; H, 5.60; N,
6.67. Found: C, 80.02; H, 5.53; N, 6.60. 1H NMR (500 MHz, CDCl3): d
1.51–1.61 (m, 1H, H-5), 1.73–1.81 (m, 1H, H-6), 1.81–1.91 (m, 1H,
H-6), 1.91–2.03 (m, 1H, H-5), 2.09 (m, 1H, H-4), 2.30–2.40 (m, 1H,
H-7), 2.43 (m, 1H, H-4), 2.61 (m, 1H, H-7), 3.66–3.75 (m, 1H, H-3),
3.89–4.02 (m, 1H, H-4a), 4.30 (d, J = 15.92 Hz, 1H, H-600), 4.36–4.45
(m, 2 H, H-200), 4.60 (d, J = 15.92 Hz, 1H, H-600), 6.60–8.00 (m, 21H,
H-aromatic and N–H), 8.31 (s, 1H, H-arylmethylidene), 8.34 (s, 1H,
H-arylmethylidene). 13C NMR (125 MHz, CDCl3): d 18.44, 27.18,
31.04, 35.12, 42.70, 46.31, 47.76, 51.13, 58.48, 63.96, 72.90,
109.93, 122.02, 124.32, 124.49, 125.31, 125.56, 125.64, 126.42,
126.60, 126.88, 127.32, 127.45, 127.56, 128.70, 129.00, 129.43,
129.82, 130.18, 131.19, 131.28, 131.77, 131.94, 132.64, 133.55,
133.62, 136.06, 136.12, 140.45, 159.38, 168.94, 179.84, 187.05.
4.3.15. Spiro-[2.3’]-oxindole-spiro[3.300]-100-carbonyl(spiro[2.3’]
oxindole-hexahydro-1H-pyrrolizine)-500-(2-chlorophenylme-
thylidene)tetrahydro-400-(1H)-pyridinone-4-(2-chloro-
phenylmethylidene) hexahydro-1H-pyrrolizine (9d)
4.3.12. Spiro-[2.3’]-oxindole-spiro[3.300]-100-carbonyl(spiro[2.3’]
oxindole-hexahydro-1H-pyrrolizine)-500-(phenylmethylidene)
tetrahydro-400-(1H)-pyridinone-4-(phenylmethylidene)
hexahydro-1H-pyrrolizine (9a)
White solid; (0.117 g, 53%); mp 150–152 °C; IR (KBr) mmax: 3428,
1721, 1611 cmꢀ1, Anal. Calcd for C46H41Cl2N5O4: C, 69.17; H, 5.17;
N, 8.77. Found: C, 69.10; H, 5.18; N, 8.75. 1H NMR (500 MHz, CDCl3)
d 1.35 (d, J = 13.60 Hz, 1H, H-200), 1.60–1.90 (m, 4H, H-6 of ring A,
H-6 of ring B, H-5 of ring B), 1.95–2.05 (m, 1H, H-5 of ring A),
2.10–2.40 (m, 5H, H-6 of ring B, H-5 of ring B, H-4 of ring A, H-5
of ring A), 2.42–2.53 (m, 1H, H-600), 2.60–2.80 (m, 2H, H-7 of ring
A, H-7 of ring B), 3.35 (d, J = 14 Hz, H-600), 3.51–3.64 (m, 2H, H-200,
H-7 of ring B), 3.80–3.90 (m, 1H, H-3 of ring A), 4.01–4.09 (m,
1H, H-4a of ring A), 4.90–5.00 (m, 1H, H-4a of ring B), 5.82–7.50
(m, 12H, H-aromatic), 7.65 (s, 1H, H-arylmethylidene), 8.07 (br s,
2H, N–H). 13C NMR (125 MHz, CDCl3): d 25.58, 25.76, 29.77,
29.91, 34.22, 43.80, 45.34, 47.50, 48.49, 50.01, 52.66, 64.11,
66.55, 67.58, 72.13, 109.44, 110.91, 120.71, 121.76, 126.38,
126.81, 127.98, 128.20, 129.50, 129.63, 129.72, 130.04, 130.58,
131.10, 131.46, 132.90, 133.29, 136.36, 136.44, 141.39, 143.00,
172.01, 178.50, 179.65, 201.95.
White solid; (0.110 g, 72%); mp 160–162 °C; IR (KBr) mmax: 3442,
1712, 1616 cmꢀ1; Anal. Calcd for C46H43N5O4: C, 75.70; H, 5.94; N,
9.60. Found: C, 75.65; H, 5.98; N, 9.64.1H NMR (500 MHz, CDCl3) d
1.15 (d, J = 14.15 Hz, 1H, H-200), 1.68–1.95 (m, 4H, H-6 of ring A, H-6
of ring B, H-5 of ring B), 2.00–2.60 (m, 6H, H-5 of ring A, H-6 of ring
B, H-5 of ring B, H-4 of ring A), 2.84 (d, J = 16.71 Hz, 1H, H-600),
2.90–3.00 (m, 1H, H-7 of ring A), 3.38–3.42 (m, 1H, H-7 of ring
B), 3.72 (d, J = 16.87 Hz, 1H, H-600), 3.95 (d, J = 14.19 Hz, 1H, H-200),
4.00–4.10 (m, 1H, H-3 of ring A), 4.22–4.32 (m, 1H, H-7 of ring
B), 4.40–4.45 (m, 1H, H-3 of ring A), 4.60–4.70 (m, 2H, H-4, H-4a
of ring B), 5.93–7.45(m, 18H, H-aromatic), 7.58 (s, 1H, H-phen-
ylmethylidene), 8.60 (s, 1H, N–H). 13C NMR (125 MHz, CDCl3): d
25.25, 25.91, 28.30, 29.38, 34.38, 43.95, 45.23, 47.94, 48.53,
51.64, 53.04, 64.31, 65.95, 68.05, 71.86, 72.67, 109.38, 110.71,
121.34, 122.27, 125.22, 126.14, 126.75, 126.92, 128.53, 128.84,
129.21, 129.53, 129.82, 130.21, 130.49, 130.68, 134.43, 137.96,
140.29, 141.23, 142.41, 173.09, 177.91, 179.97, 198.71.
4.3.16. Spiro-[2.3’]-oxindole-spiro[3.300]-100-carbonyl(spiro[2.3’]
oxindole-hexahydro-1H-pyrrolizine)-500-(2-fluorophenylme-
thylidene)tetrahydro-400-(1H)-pyridinone-4-(2-fluorob-
enzylidene) hexahydro-1H-pyrrolizine (9e)
4.3.13. Spiro-[2.3’]-oxindole-spiro[3.300]-100-carbonyl(spiro[2.3’]
oxindole-hexahydro-1H-pyrrolizine)-500-(2-methylphenylme-
thylidene)tetrahydro-400-(1H)-pyridinone-4-(2-meth-
ylphenylmethylidene) hexahydro-1H-pyrrolizine (9b)
White solid; (0.110 g, 53%); mp 158–160 °C; IR (KBr) mmax:
3431, 1715, 1612 cmꢀ1
; Anal. Calcd for C46H41F2N5O4: C,
72.14; H, 5.40; N, 9.14. Found: C, 72.11; H, 5.45; N, 9.17. 1H
NMR (500 MHz, CDCl3) d 1.33–1.42 (m, 1H, H-200), 1.67–2.24
(m, 5H, H-6 of ring A, H-6 of ring B, H-5 of ring A, H-5 of
ring B), 2.41–2.96 (m, 9H, H-600, H-7 of ring B, H-6 of ring
B, H-5 of ring B, H-4 of ring A, H-5 of ring A, H-7 of ring
A), 2.98–3.32 (m, 3H, H-200, H-600, H-7 of ring B), 3.82–3.94
(m, 1H, H-3 of ring A), 4.22–4.33 (m, 1H, H-4a of ring A),
4.51–4.74 (m, 2H, H-4a of ring B, H-4 of ring B), 5.05–7.15
(m, 16H, H-aromatic), 7.39 (s, 1H, H-arylmethylidene),
White solid; (0.109 g, 61%); mp 158–160 °C; IR (KBr) mmax: 3432,
1714, 1619 cmꢀ1; Anal. Calcd for C48H47N5O4: C, 76.07; H, 6.25; N,
9.24. Found: C, 76.17; H, 6.06; N, 9.14. 1H NMR (500 MHz, CDCl3) d
1.02–1.12 (m, 1H, H-200), 1.35–1.70 (m, 4H, H-6 of ring A, H-6 of
ring B, H-5 of ring B), 1.80–2.7 (m, 16H, H-600, H-7 of ring B, H-6
of ring B, H-5 of ring B, H-4a of ring A, H-5 of ring A, H-7 of ring
A, 2 ꢁ CH3), 3.40–3.55 (m, 2H, H-200, H-600), 3.60–4.25 (m, 3H, H-7
of ring B, H-3 of ring A, H-4a of ring A), 3.66–4.99 (m, 2H, H-4a
of ring B, H-4 of ring B), 5.76–7.96 (m, 15H, H-aromatic, H-aryl-
methylidene, N–H). 13C NMR (125 MHz, CDCl3): d 25.98, 26.05,
30.74, 31.99, 44.67, 45.07, 45.23, 45.50, 45.89, 53.75, 54.15,
7.31–7.47 (br s, 2H, N–H). 13C NMR (125 MHz, CDCl3):
d
25.31, 25.92, 27.56, 28.09, 35.89, 46.51, 47.23, 47.69, 57.35,
58.04, 59.76, 61.29, 63.84, 71.44, 73.67, 73.34, 75.05, 110.10,

1706
Y. Kia et al. / Bioorg. Med. Chem. 21 (2013) 1696–1707
110.58, 115.27, 115.39, 115.59, 115.67, 122.39, 123.19, 125.64,
128.19, 129.28, 129.82, 132.58, 132.74, 132.88, 132.92, 142.28,
172.96, 180.17, 180.59, 211.21.
135.67, 135.91, 135.99, 141.88, 142.05, 147.52, 147.84, 177.78,
185.17, 185.55, 205.15.
4.3.20. Spiro-[2.3’]-oxindole-spiro[3.300]-100-carbonyl(spiro[2.3’]
oxindole-hexahydro-1H-pyrrolizine)-500-(4-chlorophenylme-
thylidene)tetrahydro-400-(1H)-pyridinone-4-(4-chloro-
phenylmethylidene) hexahydro-1H-pyrrolizine (9i)
4.3.17. Spiro-[2.3’]-oxindole-spiro[3.300]-100-carbonyl(spiro[2.3’]
oxindole-hexahydro-1H-pyrrolizine)-500-(3-nitrophenylme-
thylidene)tetrahydro-400-(1H)-pyridinone-4-(3-nitrophenylme-
thylidene) hexahydro-1H-pyrrolizine (9f)
White solid; (0.124 g, 64%); mp 170–172 °C; IR (KBr) mmax: 3434,
White solid; (0.108 g, 59%); mp 160–162 °C; IR (KBr)
m
max: 3434,
1721, 1611 cmꢀ1; Anal. Calcd for C46H41Cl2N5O4: C, 69.17; H, 5.17;
N, 8.77. Found: C, 69.13; H, 5.17; N, 8.79. 1H NMR (500 MHz, CDCl3)
d 1.30–1.40 (m, 1H, H-200), 1.70–2.30 (m, 5H, H-6 of ring A, H-6 of
ring B, H-5 of ring B, H-5 of ring A), 2.35–3.00 (m, 9H, H-600, H-7 of
ring B, H-6 of ring B, H-5 of ring B, H-4 of ring A, H-5 of ring A, H-7
of ring A), 3.00–3.5 (m, 4H, H-200, H-600, H-7 of ring B, H-3 of ring A),
3.90–4.00 (m, 1H, H-4a of ring A), 4.55–4.65 (m, 1H, H-4 of ring B),
4.73–4.80 (m, 1H, H-4a of ring B), 5.10–7.52 (m, 14H, H-aromatic,
H-arylmethylidene, N–H). 13C NMR (125 MHz, CDCl3): d 25.69,
25.81, 27.55, 27.75, 30.00, 35.73, 46.30, 47.32, 47.68, 57.33,
58.11, 61.36, 61.87, 64.30, 71.42, 73.29, 74.68, 75.35, 110.01,
110.35, 121.54, 122.32, 125.60, 126.27, 126.91, 127.23, 128.17,
128.70, 129.45, 129.74, 129.85, 129.91, 130.41, 131.43, 132.53,
134.04, 142.18, 142.37, 172.82, 180.01, 180.87, 204.32.
1715, 1612 cmꢀ1; Anal. Calcd for C46H41N7O8: C, 67.39; H, 5.04; N,
11.96. Found: C, 67.45; H, 5.09; N, 12.02. 1H NMR (500 MHz, CDCl3)
d 0.67–0.73 (m, 1H, H-200), 1.45–2.25 (m, 4H, H-6 of ring B, H-5 of
ring B, H-6 of ring A), 2.26–2.77 (m, 10H, H-6 of ring B, H-5 of ring
B, H-7 of ring B, H-600, H-4 of ring A, H-5 of ring A, H-7 of ring A),
3.97–4.15 (m, 5H, H-200, H-600, H-7 of ring B, H-3 of ring A, H-4a
of ring A), 4.50–5.17 (m, 5H, H-4a of ring B, H-4 of ring B), 6.02–
7.73 (m, 16H, H-aromatic), 7.87 (s, 1H, H-arylmethylidene), 8.11
(s, 1H, N–H), 8.19 (s, 1H, N–H). 13C NMR (125 MHz, CDCl3): d
29.10, 29.25, 30.23, 30.64, 31.52, 39.45, 61.42, 63.36, 64.88,
67.20, 74.86, 76.63, 76.75, 113.16, 113.95, 125.96, 126.30, 126.48,
129.08, 129.49, 129.64, 129.90, 131.56, 133.14, 133.54, 139.35,
139.51, 140.15, 140.37, 145.37, 145.64, 145.73, 151.43, 151.51,
176.36, 183.11, 184.01, 209.05.
4.3.21. Spiro-[2.3’]-oxindole-spiro[3.300]-100-carbonyl(spiro[2.3’]
oxindole-hexahydro-1H-pyrrolizine)-500-(4-fluorophenylme-
thylidene)tetrahydro-400-(1H)-pyridinone-4-(4-fluor-
ophenylmethylidene) hexahydro-1H-pyrrolizine (9j)
4.3.18. Spiro-[2.3’]-oxindole-spiro[3.300]-100-carbonyl(spiro[2.3’]
oxindole-hexahydro-1H-pyrrolizine)-500-(2,4-dic-
hlorophenylmethylidene) tetrahydro-400-(1H)-
pyridinone-4-(2,4-dichloro phenylmethylidene)
hexahydro-1H-pyrrolizine (9g)
White solid; (0.118 g, 62%); mp 170–172 °C; IR (KBr) mmax: 3430,
1716, 1616 cmꢀ1; Anal. Calcd for C46H41F2N5O4: C, 72.14; H, 5.40;
N, 9.14. Found: C, 72.10; H, 5.43; N, 9.11. 1H NMR (500 MHz, CDCl3)
d 1.30–1.40 (m, 1H, H-200), 1.65–2.20 (m, 5H, H-6 of ring A, H-6 of
ring B, H-5 of ring B, H-5 of ring A), 2.35–2.91 (m, 9H, H-600, H-7 of
ring B, H-6 of ring B, H-5 of ring B, H-4 of ring A, H-5 of ring A, H-7
of ring A), 3.00–3.40 (m, 3H, H-200, H-600, H-7 of ring B), 3.80–3.90
(m, 1H, H-3 of ring A), 4.20–4.30 (m, 1H, H-4a of ring A), 4.52–
4.72 (m, 2H, H-4a of ring B, H-4 of ring B), 5.05–7.12 (m, 16H, H-
aromatic), 7.36 (s, 1H, H-arylmethylidene), 7.35–7.45 (br s, 2H,
N–H). 13C NMR (125 MHz, CDCl3): d 25.61, 25.89, 27.52, 27.71,
35.78, 46.30, 47.32, 47.61, 57.31, 57.97, 59.70, 61.24, 63.79,
71.35, 73.09, 73.34, 74.84, 110.09, 110.56, 115.27, 115.35, 115.55,
115.63, 122.32, 122.94, 125.63, 128.19, 129.21, 129.75, 132.58,
132.69, 132.82, 132.92, 142.20, 172.93, 180.11, 180.50, 212.38.
White solid; (0.132 g, 68%); mp 140–142 °C; IR (KBr) mmax: 3429,
1717, 1611 cmꢀ1, Anal. Calcd for C46H39Cl4N5O4: C, 63.68; H, 4.53;
N, 8.07. Found: C, 63.61; H, 4.54; N, 8.11. 1H NMR (500 MHz, CDCl3)
d 1.03–1.12 (m, 1H, H-200), 1.26–2.12 (m, 5H, H-6 of ring A, H-6 of
ring B, H-5 of ring B, H-5 of ring A), 2.22–3.23 (m, 9H, H-600, H-7 of
ring B, H-6 of ring B, H-5 of ring B, H-4 of ring A, H-5 of ring A, H-7
of ring A), 3.27–3.86 (m, 4H, H-600, H-200, H-7 of ring B, H-3 of ring
A), 3.93–4.08 (m, 1H, H-4a of ring A), 4.10–4.81 (m, 2H, H-4 of ring
B, H-4a of ring B), 5.47–7.67 (m, 15H, H-aromatic, H-arylmethyli-
dene), 7.76 (s, 1H, N–H), 8.03 (s, 1H, N–H). 13C NMR (125 MHz,
CDCl3): d 28.32, 28.53, 32.41, 32.77, 35.15, 42.04, 43.09, 46.51,
46.84, 47.25, 49.11, 64.45, 66.24, 66.91, 71.15, 73.40, 110.11,
110.93, 122.24, 125.33, 127.42, 127.75, 127.89, 129.72, 129.83,
130.12, 131.25, 131.42, 131.48, 132.56, 133.43, 133.67, 134.35,
135.71, 135.92, 136.08, 136.31, 140.19, 168.15, 179.11, 180.51,
187.03.
4.3.22. Spiro-[2.3’]-oxindole-spiro[3.300]-100-carbonyl(spiro[2.3’]
oxindole-hexahydro-1H-pyrrolizine)-500-(naphthalen-1-
ylphenylmethylidene)tetrahydro-400-(1H)-pyridinone-4-
(naphthalen-1-ylphenylmethyliden) hexahydro-1H-pyrrolizine
(9k)
4.3.19. Spiro-[2.3’]-oxindole-spiro[3.300]-100-carbonyl(spiro[2.3’]
oxindole-hexahydro-1H-pyrrolizine)-500-(4-methylphenylme-
thylidene)tetrahydro-400-(1H)-pyridinone-4-(4-meth-
ylphenylmethylidene) hexahydro-1H-pyrrolizine (9h)
White solid; (0.142 g, 74%); mp 220–222 °C; IR (KBr) mmax: 3428,
1718, 1619 cmꢀ1; Anal. Calcd for C54H47N5O4: C, 78.14; H, 5.71; N,
8.44. Found: C, 78.10; H, 5.65; N, 8.48. 1H NMR (500 MHz, CDCl3) d
0.80 (d, J = 13.87 Hz, 1H, H-200), 1.54–1.65 (m, 1H, H-6 of ring A),
1.71 - 1.82 (m, 2H, H-5 of ring B, H-6 of ring A), 1.84–1.92 (m,
1H, H-6 of ring B), 2.00–2.09 (m, 1H, H-5 of ring A), 2.10–2.20
(m, 3H, H-5 of ring A, H-6 of ring B, H-5 of ring B), 2.25–2.40 (m,
2 H, H-4 of ring A), 2.48 (d, J = 16.71 Hz, 1H, H-600), 2.67–2.75 (m,
2H, H-7 of ring A, H-7 of ring B), 3.41 (d, J = 16.87 Hz, 1H, H-600),
3.63 (d, J = 13.87 Hz, 1H, H-200), 3.70–3.78 (m, 1H, H-7 of ring B),
3.82 (t, J = 8.20 Hz, 1H, H-3 of ring A), 4.12–4.20 (m, 1H, H-4a of
ring A), 5.05 (d, J = 9.62 Hz, 1H, H-4 of ring B), 5.24–5.32 (m, 1H,
H-4a of ring B), 5.58–8.08 (m, 24H, H-aromatic, N–H), 8.11 (s,
White solid; (0.124 g, 67%); mp 184–186 °C; IR (KBr) mmax: 3434,
1712, 1613 cmꢀ1; Anal. Calcd for C48H47N5O4: C, 76.07; H, 6.25; N,
9.24. Found:, C, 76.16; H, 6.07; N, 9.11. 1H NMR (500 MHz, CDCl3) d
1.15 (d, J = 14.17 Hz, 1H, H-200), 1.10–1.80 (m, 4H, H-6 of ring A, H-6
of ring B, H-5 of ring B), 1.83 (s, 3H, CH3), 2.04 (s, 3H, CH3), 2.10-
2.70 (m, 7H, H-5 of ring A, H-6 of ring B, H-5 of ring B, H-4 of ring
A, H-600), 2.90–3.00 (m, 1H, H-7 of ring A), 3.30–3.45 (m, 1H, H-7 of
ring B), 3.72 (d, J = 16.87 Hz, 1H, H-600), 3.95 (d, J = 14.17 Hz, 1H, H-
200), 4.05–4.15 (m, 1H, H-3 of ring A), 4.20–4.30 (m, 1H, H-7 of ring
B), 4.40–4.45 (m, 1H, H-4a of ring B), 4.50–4.60 (m, 1H, H-4 of ring
B), 6.30–7.30 (m, 12H, H-aromatic), 9.32 (s, 1H, H-arylmethyli-
dene), 9.56 (s, 1H, N–H), 9.66 (s, 1H, N–H).13C NMR (125 MHz,
CDCl3): d 26.02, 26.13, 30.63, 32.25, 44.65, 44.93, 45.21, 45.48,
45.76, 53.64, 54.05, 64.54, 66.18, 68.49, 114.86, 115.23, 126.36,
126.50, 132.79, 132.84, 133.76, 133.93, 134.07, 134.17, 135.49,
1H, H-naphthylmethylidene). 13C NMR (125 MHz, CDCl3):
d
24.03, 24.24, 28.40, 28.66, 33.10, 43.47, 44.42, 47.45, 47.80,
48.81, 49.80, 63.34, 66.30, 67.05, 71.59, 73.52, 108.28, 110.33,
120.10, 121.63, 123.38, 123.86, 124.06, 124.09, 124.57, 125.01,
125.47, 126.06, 126.13, 126.24, 126.53, 127.05, 127.33, 127.69,

Y. Kia et al. / Bioorg. Med. Chem. 21 (2013) 1696–1707
1707
128.39, 128.63, 129.63, 129.84, 130.29, 131.19, 132.56, 132.90,
Acknowledgements
133.33, 133.49, 133.74, 138.24, 139.91, 142.26, 171.95, 177.13,
178.66, 201.64.
The authors thank the Malaysian government and Universiti
Sains Malaysia for RUT grant [1001/PKIMIA/855006] and RU grant
[1001/PKIMIA/811221], to conduct this research. Y.K. thanks Uni-
versity Sains Malaysia for financial support by way of a fellowship.
4.4. In vitro cholinesterase enzymes inhibitory activity
The test samples for cholinesterase enzymes inhibitory poten-
tial was evaluated using modified Ellman’s method as described
by Ahmed and Gilani.34 Galantamine was used as positive control.
Solutions of test samples and galantamine were prepared in DMSO
at an initial concentration of 1 mg/mL (1000 ppm). The concentra-
tion of DMSO in final reaction mixture was 1%. At this concentra-
tion, DMSO has no inhibitory effect on both acetylcholi-nesterase
and butyrylcholinesterase enzymes.35
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.066.
References and notes
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l
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line esterase from equine serum and S-butyrylthiocholine chloride
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Union Road, Cambridge CB2 1EZ, UK [fax: +44 (0)1223 336033 or e-mail:
deposit@ccdc.cam.ac.uk].
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