An expedient synthesis, acetylcholinesterase inhibitory activity, and molecular modeling study of highly functionalized hexahydro-1,6-naphthyridines

Article abstract of DOI:10.1155/2015/965987

A series of hexahydro-1,6-naphthyridines were synthesized in good yields by the reaction of 3,5-bis[(E)-arylmethylidene] tetrahydro-4(1H)-pyridinones with cyanoacetamide in the presence of sodium ethoxide under simple mixing at ambient temperature for 6-10 minutes and were assayed for their acetylcholinesterase (AChE) inhibitory activity using colorimetric Ellman's method. Compound 4e with methoxy substituent at ortho-position of the phenyl rings displayed the maximum inhibitory activity with IC₅₀ value of 2.12 μM. Molecular modeling simulation of 4e was performed using three-dimensional structure of Torpedo californica AChE (TcAChE) enzyme to disclose binding interaction and orientation of this molecule into the active site gorge of the receptor.

Full text of DOI:10.1155/2015/965987

Hindawi Publishing Corporation
BioMed Research International
Volume 2015, Article ID 965987, 9 pages
http://dx.doi.org/10.1155/2015/965987
Research Article
An Expedient Synthesis, Acetylcholinesterase
Inhibitory Activity, and Molecular Modeling Study of Highly
Functionalized Hexahydro-1,6-naphthyridines
Abdulrahman I. Almansour,1 Raju Suresh Kumar,1 Natarajan Arumugam,1
Alireza Basiri,2 Yalda Kia,3 and Mohamed Ashraf Ali4
1 Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
2School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia
3School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia
4Pharmacogenetic and Novel erapeutic Institute for Research in Molecular Medicine, Universiti Sains Malaysia,
11800 Penang, Malaysia
Correspondence should be addressed to Raju Suresh Kumar; rajusures@gmail.com
Received 17 November 2014; Revised 9 January 2015; Accepted 9 January 2015
Academic Editor: Miroslav Pohanka
Copyright © 2015 Abdulrahman I. Almansour et al. is is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
A series of hexahydro-1,6-naphthyridines were synthesized in good yields by the reaction of 3,5-bis[(E)-arylmethylidene]
tetrahydro-4(1H)-pyridinones with cyanoacetamide in the presence of sodium ethoxide under simple mixing at ambient
temperature for 6–10 minutes and were assayed for their acetylcholinesterase (AChE) inhibitory activity using colorimetric Ellman’s
method. Compound 4e with methoxy substituent at ortho-position of the phenyl rings displayed the maximum inhibitory activity
with IC value of 2.12 ꢀM. Molecular modeling simulation of 4e was performed using three-dimensional structure of Torpedo
50
californica AChE (TcAChE) enzyme to disclose binding interaction and orientation of this molecule into the active site gorge of the
receptor.
1. Introduction
efficacy of treatment and broaden the indications [6]. Effects
of cholinesterase inhibitors are mainly due to enhancement of
cholinergic transmission at cholinergic autonomic synapses
and at the neuromuscular junction [7].
Alzheimer’s disease (AD) is associated with loss of cholinergic
neurons in basal forebrain, which results in loss or failure of
memory which slowly worsens and eventually incapacitates
the patients [1, 2]. According to the World Alzheimer report,
AD is one among the most significant social, health, and
economic crises of the 21st century [3]. Although the exact
factors initiating AD are unclear, genetic and environmental
factors have been implicated [4]. In general, pharmacological
therapies have twin objectives: (i) to prevent the loss of the
neurons and (ii) to restore the cholinergic functions of AD
patients. Cholinesterase inhibitors have been used clinically
for symptomatic treatment of AD [5]. Acetylcholinesterase
(AChE) enzyme is involved in the breakdown of acetylcholine
in the brain and inhibition of this enzyme may increase the
AChE inhibitors are one of the most actively investi-
gated classes of compounds in the search for an effective
treatment of AD. Although there are many ongoing research
activities in the search of drugs for treating AD, only few
drugs like galantamine, donepezil, and rivastigmine are now
available [8], and these drugs do not show potential cure
rates; additional treatments are still being developed. e
treatment of AD still remains an area of significant unmet
need, with drugs that only target the symptoms of the disease.
erefore, there is considerable need for disease-modifying
therapies. To meet the need of disease-modifying drugs
for AD, in recent years, new approaches have emerged in

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medicinal chemistry. In particular the concept has recently
been proposed that due to the multifactorial and complex
etiology of AD, the modulation of a single factor might not
be sufficient to produce the desired efficacy. Researchers are
now paying attention to the design of structures that could be
able to simultaneously interact with different targets involved
in the pathogenic process [9, 10].
Naphthyridine structural motif has been extensively syn-
thesized and incorporated into biologically active molecules
[11]. In particular, 1,6-naphthyridines exhibit a broad spec-
trum of biological activities such as being inhibitor of HIV-
1 integrase [12, 13], HCMV [14, 15], FGF receptor-1 tyrosine
kinase [16], and the enzyme acetylcholinesterase [17]. Many
routes for the syntheses of 1,6-naphthyridines derivatives
have previously been reported [18–21]. In continuation of our
previous work towards the synthesis of novel hybrid hetero-
cycles employing new synthetic methodologies and/or their
potential as inhibitors of AChE [22–26], herein we report
the synthesis and AChE inhibitory activities of nitrogen
heterocyclic hybrids comprising naphthyridine structural
motif.
146.85, 159.18, 162.83. EIMS: m/z 341 [M+1]. Anal. calcd for
C H N O: C, 77.86; H, 5.05; N, 12.38; found: C, 77.69; H,
22 17
3
5.22; N, 12.25%.
(E)-8-(2-Methylbenzylidene)-2-oxo-4-(o-tolyl)-1,2,5,6,7,8-hex-
ahydro-1,6-naphthyridine-3-carbonitrile (4b). Yellow solid; IR
(KBr) ]max 3380, 2948, 2210, 1640, 1627 cm−1; 1H NMR
(300 MHz, DMSO): ꢁ 2.13 (s, 3H, CH ), 2.30 (s, 3H, CH ),
H
3
3
3.14 (d, J = 15.9 Hz, 1H, 5-CH ), 3.33 (d, J = 15.9 Hz, 1H,
2
5-CH ), 3.62 (d, J = 15.6 Hz, 1H, 7-CH ), 3.68 (d, J =
2
2
15.6 Hz, 1H, 7-CH ), 7.04–7.35 (m, 8H, Ar-H), 7.80 (s, 1H,
2
Arylmethylidene-H), 8.14 (s, 1H, NH). 13C NMR (75 MHz,
DMSO): ꢁ 19.75, 20.56, 45.43, 45.82, 79.76, 101.32, 115.23,
C
116.91, 126.21, 127.03, 127.79, 128.89, 129.33, 129.53, 129.92,
130.78, 131.19, 134.76, 135.42, 135.55, 137.91, 146.96, 159.08,
162.92. EIMS: m/z 369 [M+1]. Anal. calcd for C H N O: C,
24 21
3
78.45; H, 5.76; N, 11.44; found: C, 78.32; H, 5.98; N, 11.29%.
(E)-8-(2-Chlorobenzylidene)-4-(2-chlorophenyl)-2-oxo-1,2,5,6,
7,8-hexahydro-1,6-naphthyridine-3-carbonitrile (4c). Pale yel-
low solid; IR (KBr) ]max 3384, 2950, 2214, 1642, 1625 cm−1;
1H NMR (300 MHz, DMSO): ꢁ 3.16 (d, J = 15.6 Hz, 1H, 5-
H
CH ), 3.34 (d, J = 15.6 Hz, 1H, 5-CH ), 3.61 (d, J = 15.6 Hz,
2
2
2. Materials and Methods
1H, 7-CH ), 3.68 (d, J = 15.6 Hz, 1H, 7-CH ), 7.10–7.36 (m, 8H,
2
2
Ar-H), 7.81 (s, 1H, Arylmethylidene-H), 8.12 (s, 1H, NH). 13C
NMR (75 MHz, DMSO): ꢁ 45.38, 45.95, 79.70, 101.64, 115.21,
116.90, 126.28, 127.01, 127.C85, 128.90, 129.31, 129.54, 129.93,
130.69, 131.26, 134.73, 135.40, 135.52, 137.90, 146.91, 159.13,
162.90. EIMS: m/z 408 [M+1]. Anal. calcd for C H Cl N O:
2.1. Chemistry
General Methods. Melting points were measured in KRUSS
melting point meter using open capillary tubes and are
1
uncorrected. H and 13C NMR spectra were recorded on a
22 15
2
3
Bruker 300 MHz instrument in DMSO using TMS as internal
standard. Standard Bruker sofware was used throughout.
Chemical shifs are given in parts per million (ꢁ-scale) and
the coupling constants are given in Hertz. IR spectra were
recorded in a Perkin Elmer system 2000 FT IR instrument
(KBr). Elemental analyses were performed on a Perkin Elmer
2400 Series II Elemental CHNS analyser. Mass spectra were
recorded in Agilent technologies 7820A GC-MS system.
C, 64.72; H, 3.70; N, 10.29; found: C, 64.80; H, 3.87; N, 10.48%.
(E)-8-(2-Bromobenzylidene)-4-(2-bromophenyl)-2-oxo-1,2,5,6,
7,8-hexahydro-1,6-naphthyridine-3-carbonitrile (4d). Pale yel-
low solid; IR (KBr) ]max 3381, 2950, 2210, 1644, 1626 cm−1;
1H NMR (300 MHz, DMSO): ꢁ 3.18 (d, J = 15.9 Hz, 1H, 5-
H
CH ), 3.34 (d, J = 15.9 Hz, 1H, 5-CH ), 3.62 (d, J = 15.9 Hz,
2
2
1H, 7-CH ), 3.69 (d, J = 15.6 Hz, 1H, 7-CH ), 7.16–7.39 (m, 8H,
2
2
Ar-H), 7.82 (s, 1H, Arylmethylidene-H), 8.09 (s, 1H, NH). 13
C
2.1.1. General Procedure for the Synthesis of Naphthyridines
(4a–k). A mixture of 3,5-bis[(E)-arylmethylidene]tetrahy-
dro-4(1H)-pyridinones (1 mmol) and 2-cyanoacetamide
(1 mmol) in ethanol (200 ꢀL) in the presence of catalytic
amount of sodium ethoxide (5 mg) were ground well in a
semimicro boiling tube at ambient temperature for about
6–10 min. Afer completion of the reaction as evident from
TLC, water (50 mL) was added to the reaction mixture and
the product was filtered, washed with water, and dried in
vacuo.
NMR (75 MHz, DMSO): ꢁ 44.93, 45.80, 79.78, 101.61, 115.20,
116.96, 126.25, 127.05, 127.C81, 128.98, 129.35, 129.62, 129.92,
130.73, 131.24, 134.71, 135.43, 135.51, 137.93, 146.90, 159.17,
162.92. EIMS: m/z 498 [M+1]. Anal. calcd for C H Br N O:
22 15
2
3
C, 53.15; H, 3.04; N, 8.45; found: C, 53.38; H, 3.16; N, 8.37%.
(E)-8-(2-Methoxybenzylidene)-4-(2-methoxyphenyl)-2-oxo-1,2,
5,6,7,8-hexahydro-1,6-naphthyridine-3-carbonitrile (4e). Yel-
low solid; IR (KBr) ]max 3385, 2944, 2210, 1641, 1627 cm−1; 1H
NMR (300 MHz, DMSO): ꢁ 3.14 (d, J = 15.6 Hz, 1H, 5-CH ),
H
2
3.31 (d, J = 15.6 Hz, 1H, 5-CH ), 3.60 (d, J = 15.6 Hz, 1H, 7-
(E)-8-Benzylidene-2-oxo-4-phenyl-1,2,5,6,7,8-hexahydro-1,6-
naphthyridine-3-carbonitrile (4a). Pale yellow solid; IR (KBr)
2
CH ), 3.68 (d, J = 15.6 Hz, 1H, 7-CH ), 3.74 (s, 3H, OCH ),
2
2
3
3372, 2945, 2213, 1642, 1625 cm−1; 1H NMR (300 MHz,
3
3.82 (s, 3H, OCH ), 7.11–7.38 (m, 8H, Ar-H), 7.83 (s, 1H,
]
max
Arylmethylidene-H), 8.10 (s, 1H, NH). 13C NMR (75 MHz,
DMSO): ꢁ 3.15 (d, J = 15.9 Hz, 1H, 5-CH ), 3.35 (d, J =
15.9 Hz, 1HH, 5-CH ), 3.60 (d, J = 15.6 Hz, 1H, 7-CH ), 3.69 (d,
2
DMSO): ꢁ 45.39, 45.93, 55.51, 55.70, 79.70, 101.67, 114.20,
C
2
2
114.81, 115.20, 116.94, 126.31, 127.81, 128.93, 129.56, 130.66,
131.29, 134.70, 135.42, 137.90, 146.95, 159.07, 159.14, 160.04,
162.95. EIMS: m/z 401 [M+1]. Anal. calcd for C H N O :
J = 15.6 Hz, 1H, 7-CH ), 7.07–7.38 (m, 10H, Ar-H), 7.81 (s, 1H,
2
Arylmethylidene-H), 8.15 (s, 1H, NH). 13C NMR (75 MHz,
DMSO): ꢁ 45.48, 45.76, 79.31, 101.70, 115.22, 116.94, 126.21,
C
24 21
3
3
127.81, 128.84, 129.50, 130.78, 131.10, 134.71, 135.54, 137.90,
C, 72.16; H, 5.30; N, 10.52; found: C, 72.30; H, 5.53; N, 10.39%.

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3
(E)-8-(2,4-Dichlorobenzylidene)-4-(2,4-dichlorophenyl)-2-oxo-
1H, 7-CH ), 3.70 (d, J = 15.9 Hz, 1H, 7-CH ), 7.13–7.45 (m, 8H,
2
2
1,2,5,6,7,8-hexahydro-1,6-naphthyridine-3-carbonitrile (4f ).
Ar-H), 7.84 (s, 1H, Arylmethylidene-H), 8.11 (s, 1H, NH). 13C
NMR (75 MHz, DMSO): ꢁ 45.37, 45.93, 79.72, 101.64, 114.36,
114.58, 115.23, 116.85, 126.C37, 127.84, 128.92, 129.45, 130.72,
131.35, 134.78, 135.43, 137.94, 146.96, 159.25, 160.23, 161.18,
162.95. EIMS: m/z 377 [M+1]. Anal. calcd for C H F N O:
Pale yellow solid; IR (KBr) ]max 3380, 2951, 2219, 1646,
1
1628 cm−1; H NMR (300 MHz, DMSO): ꢁ 3.18 (d, J =
H
15.9 Hz, 1H, 5-CH ), 3.37 (d, J = 15.9 Hz, 1H, 5-CH ), 3.60
2
2
(d, J = 15.6 Hz, 1H, 7-CH ), 3.68 (d, J = 15.6 Hz, 1H, 7-CH ),
22 15
2
3
2
2
C, 70.39; H, 4.03; N, 11.19; found: C, 70.65; H, 4.27; N, 11.10%.
7.14–7.42 (m, 6H, Ar-H), 7.82 (s, 1H, Arylmethylidene-H),
8.09 (s, 1H, NH). 13C NMR (75 MHz, DMSO): ꢁ 45.35,
C
(E)-4-(Naphthalen-1-yl)-8-(naphthalen-1-ylmethylene)-2-oxo-
1,2,5,6,7,8-hexahydro-1,6-naphthyridine-3-carbonitrile (4k).
45.97, 79.65, 101.62, 115.26, 116.81, 126.30, 127.12, 127.87, 128.94,
129.43, 129.60, 129.94, 130.74, 131.27, 134.72, 135.47, 135.53,
137.94, 146.97, 159.16, 162.95. EIMS: m/z 478 [M+1]. Anal.
calcd for C H Cl N O: C, 55.38; H, 2.75; N, 8.81; found: C,
Pale yellow solid; IR (KBr) ]max 3384, 2946, 2214, 1645,
1629 cm−1; H NMR (300 MHz, DMSO): ꢁ 3.17 (d, J =
1
H
22 13
4
3
15.9 Hz, 1H, 5-CH ), 3.41 (d, J = 15.9 Hz, 1H, 5-CH ), 3.61
55.29; H, 2.92; N, 8.70%.
2
2
(d, J = 15.6 Hz, 1H, 7-CH ), 3.70 (d, J = 15.6 Hz, 1H, 7-CH ),
2
2
7.02–7.65 (m, 14H, Ar-H), 7.91 (s, 1H, Arylmethylidene-H),
(E)-8-(3-Nitrobenzylidene)-4-(3-nitrophenyl)-2-oxo-1,2,5,6,7,
8.17 (s, 1H, NH). 13C NMR (75 MHz, DMSO): ꢁ 45.31, 45.84,
8-hexahydro-1,6-naphthyridine-3-carbonitrile (4g). Yellow
C
solid; IR (KBr) ]max 3389, 2942, 2210, 1645, 1626 cm−1; H
1
79.68, 101.65, 115.28, 116.80, 123.46, 124.03, 125.12, 125.57,
126.30, 126.42, 126.80, 127.20, 127.85, 128.22, 129.12, 129.48,
129.94, 130.79, 131.43, 132.10, 132.67, 134.73, 135.54, 135.68,
137.92, 146.90, 159.28, 162.94. EIMS: m/z 441 [M+1]. Anal.
calcd for C H N O: C, 81.98; H, 4.82; N, 9.56; found: C,
NMR (300 MHz, DMSO): ꢁ 3.21 (d, J = 15.9 Hz, 1H, 5-CH ),
H
2
3.39 (d, J = 15.9 Hz, 1H, 5-CH ), 3.61 (d, J = 15.6 Hz, 1H, 7-
2
CH ), 3.70 (d, J = 15.6 Hz, 1H, 7-CH ), 7.12–7.38 (m, 8H, Ar-
2
2
H), 7.80 (s, 1H, Arylmethylidene-H), 8.11 (s, 1H, NH). 13C
30 21
3
81.80; H, 4.95; N, 9.48%.
NMR (75 MHz, DMSO): ꢁ 45.32, 45.95, 79.66, 101.60, 115.24,
C
116.79, 126.31, 127.15, 127.86, 128.93, 129.41, 129.64, 129.96,
130.75, 131.30, 134.71, 135.49, 135.61, 137.92, 146.95, 159.14,
162.93. EIMS: m/z 431 [M+1]. Anal. calcd for C H N O :
2.2. In Vitro Cholinesterase Enzymes Inhibitory Assay. Cho-
linesterase inhibitory activity of the synthesized compounds
was evaluated using the Ellman’s microplate assay [27]. For
acetylcholinesterase (AChE) inhibitory assay, 140 ꢀL of 0.1 M
sodium phosphate buffer (pH 8) was first added to a 96-
well microplate followed by 20 ꢀL of test samples and 20 ꢀL
of 0.09 units/mL acetylcholinesterase enzyme from Elec-
trophoruselectricus (Sigma). Afer 15 minutes of incubation
at 25∘C, 10 ꢀL of 10 mM 5,5ꢀ-dithiobis-2-nitrobenzoic acid
(DTNB) was added into each well followed by 10 ꢀL of
acetylthiocholine iodide (14 mM). At 30 minutes afer the ini-
tiation of enzymatic reaction, absorbance of the colored end-
product was measured using BioTek Power Wave X 340
Microplate Spectrophotometer at 412 nm.
22 15
5
5
C, 61.54; H, 3.52; N, 16.31; found: C, 61.27; H, 3.75; N, 16.18%.
(E)-8-(4-Methylbenzylidene)-2-oxo-4-(p-tolyl)-1,2,5,6,7,8-
hexahydro-1,6-naphthyridine-3-carbonitrile (4h). Pale yellow
solid; IR (KBr) ]max 3387, 2940, 2212, 1647, 1625 cm−1; H
1
NMR (300 MHz, DMSO): ꢁ 2.25 (s, 3H, CH ), 2.29 (s, 3H,
H
3
CH ), 3.20 (d, J = 15.9 Hz, 1H, 5-CH ), 3.41 (d, J = 15.9 Hz,
3
2
1H, 5-CH ), 3.63 (d, J = 15.6 Hz, 1H, 7-CH ), 3.72 (d, J =
2
2
15.6 Hz, 1H, 7-CH ), 7.12–7.40 (m, 8H, Ar-H), 7.83 (s, 1H,
2
Arylmethylidene-H), 8.12 (s, 1H, NH). 13C NMR (75 MHz,
DMSO): ꢁ 21.4, 21.8, 45.35, 45.97, 79.68, 101.64, 115.25,
C
116.80, 126.32, 127.18, 127.82, 128.91, 129.45, 129.96, 130.69,
131.35, 134.73, 135.47, 135.64, 137.90, 139.21, 146.94, 159.17,
162.90. EIMS: m/z 369 [M+1]. Anal. calcd for C H N O: C,
Galantamine was used as positive control. Test samples
and galantamine were prepared in DMSO at an initial
concentration of 1 mg/mL (1000 ppm). e concentration of
DMSO in final reaction mixture was 1%. At this concentra-
tion, DMSO has no inhibitory effect on acetylcholinesterase
enzyme.
e initial screening was carried out at 10 ꢀg/mL of test
samples in 1% DMSO and each test was conducted in
triplicate. Absorbencies of the test samples were corrected
by subtracting the absorbance of their respective blank. Per-
centage enzyme inhibition is calculated using the following
formula:
24 21
3
78.45; H, 5.76; N, 11.44; found: C, 78.66; H, 5.87; N, 11.35%.
(E)-8-(4-Chlorobenzylidene)-4-(4-chlorophenyl)-2-oxo-1,2,5,6,
7,8-hexahydro-1,6-naphthyridine-3-carbonitrile (4i). Yellow
solid; IR (KBr) ]max 3385, 2942, 2210, 1645, 1627 cm−1; H
1
NMR (300 MHz, DMSO): ꢁ 3.17 (d, J = 15.9 Hz, 1H, 5-CH ),
H
2
3.40 (d, J = 15.9 Hz, 1H, 5-CH ), 3.62 (d, J = 15.6 Hz, 1H, 7-
2
CH ), 3.71 (d, J = 15.6 Hz, 1H, 7-CH ), 7.16–7.48 (m, 8H, Ar-
2
2
H), 7.87 (s, 1H, Arylmethylidene-H), 8.15 (s, 1H, NH). 13C
NMR (75 MHz, DMSO): ꢁ 45.39, 45.94, 79.70, 101.65, 115.26,
C
116.81, 126.34, 127.21, 127.85, 128.94, 129.48, 129.91, 130.76,
131.39, 134.71, 135.51, 135.60, 137.90, 146.92, 159.25, 162.97.
EIMS: m/z 409 [M+1]. Anal. calcd for C H Cl N O: C,
Percentage of inhibition
22 15
2
3
64.72; H, 3.70; N, 10.29; found: C, 64.95; H, 3.84; N, 10.21%.
(Absorbance of sample − Absorbance of control)
Absorbance of control × 100
=
.
(E)-8-(4-Fluorobenzylidene)-4-(4-fluorophenyl)-2-oxo-1,2,5,6,
7,8-hexahydro-1,6-naphthyridine-3-carbonitrile (4j). Pale yel-
low solid; IR (KBr) ]max 3387, 2940, 2213, 1645, 1624 cm−1;
(1)
1H NMR (300 MHz, DMSO): ꢁ 3.15 (d, J = 15.9 Hz, 1H, 5-
Subsequently, the determination of IC was carried out using
50
H
CH ), 3.37 (d, J = 15.9 Hz, 1H, 5-CH ), 3.63 (d, J = 15.6 Hz,
a set of five concentrations.
2
2

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BioMed Research International
Ar
7
Ar
O
9
CN
H
N
1
8
H2N
O
O
8a
4a
4
2
O
2 ArCHO
3
3
HCl,
CH3COOH
HN
HN
6
HCl·HN
NaOEt
4
Ar
6–10min
5
N
Ar
1
2
Scheme 1: Synthesis of naphthyridines (4a–k).
2.3. Molecular Modeling. Using Glide (version 5.7, Schro¨-
dinger, LLC, New York, NY, 2011), most active compound was
docked onto the active site of TcAChE derived from three-
dimensional structure of the enzyme complex with anti-
Alzheimer’s drug, galantamine (PDB ID: 4EVE).
the multiplets around 7.04–7.35 ppm are due to aromatic
protons. In the 13C NMR spectrum, the chemical shifs at
19.75 and 20.56 ppm were due to the two –CH carbons
3
whilst the two methylene carbons of the piperidine ring
resonated at 45.43 and 45.82 ppm. e aromatic carbons
resonated at 101.32–162.92 ppm. e molecular ion peak at
369 [M+1] confirms the presence of compound 4b. e
elemental analysis result of 4b was within 0.4% of the
theoretical values. e structure of other 2-pyridones was
also assigned by similar considerations. Presumably, the 1,6-
naphthyridine (4) is formed via a cascade heterocyclization
mechanism involving an initial Michael addition followed by
cyclization and air oxidation as reported by Jain et al. [29].
Water molecules and hetero groups were deleted from
˚
enzyme beyond the radius of 5 A of reference ligand (galan-
tamine), resulting protein structure refined and minimized
by Protein Preparation Wizard using OPLS-2005 force field.
Receptor Grid Generation program was used to prepare
TcAChE grid and the ligand was optimized by LigPrep pro-
gram by using OPLS-2005 force field to generate lowest
energy state. Docking stimulations were carried out on
bioactive compound, handed in 5 poses per ligand, in which
the best pose with highest score was displayed for each ligand.
3.2. In Vitro Evaluation. All newly synthesized 1,6-naphthy-
ridines were evaluated in vitro for their inhibitory potential
against AChE enzyme from electric eel using colorimetric
Ellman’s method (Table 1). All the 1,6-naphthyridines (4a–k)
3. Results and Discussion
3.1. Chemistry. In the present investigation, the reaction of
a series of bisarylmethylidene piperidones with 2-cyano-
acetamide in the presence of sodium ethoxide with few drops
of ethanol under simple mixing at ambient temperature
for 6–10 min afforded functionalized 1,6-naphthyridines in
good yields (65–78%; Scheme 1). e prerequisite bisaryl-
methylidene piperidones were synthesized following the
literature reported method [28]. In a typical reaction, an
equimolar mixture of 3,5-bis[(E)-2-methylphenylmethylid-
ene]tetrahydro-4(1H)-pyridinones (2b) and 2-cyanoacetam-
ide (3) in catalytic amount of sodium ethoxide were ground
well in a semimicro boiling tube with few drops of ethanol at
ambient temperature for about 7 min and afer completion of
the reaction water was added to the mixture and the product
was filtered and dried in vacuo. In this case, the 2-pyridone
was obtained as a sole reaction product and does not require
column chromatography for purification. Easy availability
of the reagents, short reaction time, and simple reaction
condition rendered this method more attractive from the
viewpoint of green chemistry.
displayed good to moderate inhibitory activities with IC
50
values ranging from 2.12 to 24.72 ꢀM, irrespective of the
position of the substituent on the aryl ring. Among the
1,6-naphthyridines, compounds 4i with p-chloro, 4k with 1-
naphthyl, 4h with p-methyl, and 4b with o-methyl substituent
in the aromatic ring displayed good activities (<10 ꢀM) with
IC 3.86 ꢀM, 6.86 ꢀM, 7.16 ꢀM, and 7.20 ꢀM, respectively.
50
Compounds 4a, 4c, 4d, 4g, and 4j displayed moderate activ-
ities (IC = 11.18–18.16 ꢀM) while compound 4f displayed
50
the lowest activity (IC = 24.72 ꢀM). Compound 4e with
50
o-methoxy phenyl rings displayed the highest activity with
IC value of 2.12 ꢀM, comparable to the standard drug
50
galantamine (IC = 2.09 ꢀM). It is also observed that the
50
AChE inhibitory activities were directly correlated to the size
of substituents in the phenyl ring. For instance, derivative
bearing bulky moieties, such as m-nitro, o,p-dichloro, and
p-bromo, displayed lower inhibition than the derivatives
carrying smaller functions irrespective of their position in the
phenyl ring.
˚
e active site of AChE enzyme is located inside a 20 A
e structure of 1,6-naphthyridines was elucidated using
long, narrow gorge which is dominantly composed of amino
acids possessing aromatic side chains such as tryptophan and
tyrosine. erefore, the derivatives bearing a relatively small
and/or electron donating moieties in phenyl rings, such as
methyl and methoxy, displayed better inhibitory activities
than derivatives carrying bulky and/or electron withdrawing
groups, plausibly due to the better insertion into the active
site channel and also more efficient binding interaction with
aforementioned aromatic residues. However with limited
1
IR, NMR, and CHN analysis. In the H NMR spectrum of
4b, the two doublets at 3.14 and 3.33 ppm with J = 15.9 Hz
are due to 5-CH protons while the other doublets at 3.62
2
and 3.68 ppm with J = 15.6 Hz are due to 7-CH protons
2
of the piperidine ring. e singlets at 8.14 and 7.80 ppm
can be attributed to the NH of 2-pyridone ring and aryl-
methylidene proton, respectively. e two –CH protons of
3
aromatic ring appear as singlets at 2.13 and 2.30 ppm while

BioMed Research International
Table 1: Physical data and AChE inhibitory activity of naphthyridines (4a–k).
5
Yield
(%)
AChE inhibition
(IC50 SD) mol/L
Entry
Product
Reaction time (min)
mp ∘C
H
N
O
1
8
75
72
76
211-212
11.18 0.02
7.20 0.02
15.21 0.1
HN
4a
N
H
N
O
2
3
4
7
7
6
229-230
220-221
208-209
HN
4b
N
Cl
H
N
O
HN
N
N
N
Cl
4c
Br
H
N
O
70
18.16 0.02
HN
Br
4d
O
H
N
O
5
9
67
215-216
2.12 0.02
HN
O
4e

6
BioMed Research International
Table 1: Continued.
Yield
(%)
AChE inhibition
mp ∘C
Entry
Product
Reaction time (min)
(IC50 SD) mol/L
Cl
Cl
H
N
O
HN
6
6
72
225-226
24.72 0.01
N
Cl
4f
Cl
NO2
H
N
O
7
6
70
206-207
16.86 0.02
HN
N
4g
O2N
H
O
N
HN
8
7
73
235-236
7.16 0.02
N
4h
Cl
H
N
O
HN
9
6
78
221-222
3.86 0.02
N
4i
Cl

BioMed Research International
7
Table 1: Continued.
Yield
(%)
AChE inhibition
(IC50 SD) mol/L
Entry
Product
Reaction time (min)
mp ∘C
F
H
N
O
HN
10
7
74
218-219
14.16 0.02
N
4j
F
H
N
O
11
10
65
—
204-205
6.86 0.02
2.09 0.02
HN
N
4k
12
Galantamine.HBr
—
—
Tyr334
substituent on the aryl ring, it is difficult to ascertain the
exact structure activity relationship based on their activities
observed.
Phe330
3.3. Docking Studies. e most active AChE inhibitor, 4e, was
docked into the active site of AChE enzyme derived from
crystal structure of Torpedo californica AChE (TcAChE). e
docking analysis revealed that this compound is properly
inserted into the active site of AChE enzyme with free
binding energy of 8.71 kcal/mol and strongly bound to the
residues comprising aromatic side chains such as Tyr70 (H-
Tyr70
Tyr121
His440
˚
bonding 1.16 A), Tyr121 (hydrophobic), Tyr334 (hydrophobic)
Gly118
at peripheral anionic site as well as Phe330 (hydrophobic),
and Trp84 (ꢂ,ꢂ-stacking) at choline binding site of the
enzyme (Figure 1). 4e also exhibited mild polar interaction
with Gly116 and Gly117 at oxyanion hole of the AChE enzyme.
e crystal structure of the TcAChE in complex with available
AD drugs such as galantamine and huperzine A showed sim-
ilar interactions with residues composing peripheral anionic
site along with stacking against Trp84 at bottom of the gorge.
It seems that the presence of methoxy group in 4e has notable
influence on proper positioning of this compound in AChE
active site. is orientation effectively avoids insertion and
hydrolysis of substrate inside the AChE active site channel
and completely coincides with the activity observed for this
compound.
Trp84
Gly117
Figure 1: Binding interaction of 4e with active site of AChE
receptor. (Hydrogen atoms are not shown for clarity.)
4. Conclusion
A series of novel 1,6-naphthyridines were synthesized in
good yields and evaluated for their inhibitory potentials
against AChE enzyme, using colorimetric Ellman’s method.

8
BioMed Research International
Among them, compound 4e displayed the highest AChE
[12] J. Y. Melamed, M. S. Egbertson, S. Varga et al., “Synthesis of
5-(1-H or 1-alkyl-5-oxopyrrolidin-3-yl)-8-hydroxy-[1,6]-naph-
thyridine-7-carboxamide inhibitors of HIV-1 integrase,” Bioor-
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5310, 2008.
[13] M. S. Egbertson, H. M. Moritz, J. Y. Melamed et al., “A potent
and orally active HIV-1 integrase inhibitor,” Bioorganic and
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inhibition with remarkable IC value of 2.12 ꢀM, comparable
50
to standard drug, galantamine. Molecular modeling analysis
for this compound manifested its orientation inside the active
site cavity and its effective binding interactions to the residues
lining the active site channel, which coincided with its in vitro
activity.
[14] G. Falardeau, H. Lachance, A. S. Pierre et al., “Design and syn-
thesis of a potent macrocyclic 1,6-napthyridine anti-human
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[15] L. Chan, H. Jin, T. Stefanac et al., “Discovery of 1,6-naphthy-
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Conflict of Interests
e authors confirm that this paper content has no conflict of
interests.
Acknowledgment
[16] A. M. ompson, C. J. C. Connolly, J. M. Hamby et al., “3-
(3,5-Dime-thoxyphenyl)-1,6-naphthyridine-2,7-diamines and
related 2-urea derivatives are potent and selective inhibitors
of the FGF receptor-1 tyrosine kinase,” Journal of Medicinal
Chemistry, vol. 43, no. 22, pp. 4200–4211, 2000.
[17] S. Vanlaer, A. Voet, C. Gielens, M. de Maeyer, and F. Comper-
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of huperzine A: synthesis, modelling studies and evaluation as
inhibitors of acetylcholinesterase,” European Journal of Organic
Chemistry, no. 5, pp. 643–654, 2009.
e authors acknowledge the Deanship of Scientific Research
at King Saud University for the Research Grant RGP-VPP-
026.
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