Design, synthesis and cholinesterase inhibitory evaluation study of fluorescent N-benzylpiperidine-4-one derivatives

Article abstract of DOI:10.1007/s00044-016-1619-6

Some N-benzylpiperidine-4-one derivatives were synthesized via one-pot three-component protocols in excellent yields, and were evaluated for their potential cholinesterase inhibition. The mono-substituted derivatives except 4b showed butyrylcholinesterase

Full text of DOI:10.1007/s00044-016-1619-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1619-6
ORIGINAL RESEARCH
Design, synthesis and cholinesterase inhibitory evaluation study of
fluorescent N-benzylpiperidine-4-one derivatives
Dileep Kumar Sukumarapillai1 Khaw Kooi-Yeong2 Yalda Kia2
●
●
●
Vikneswaran Murugaiyah2 Sathiyanarayanan Kulathu Iyer1
●
Received: 7 December 2015 / Accepted: 15 June 2016
© Springer Science+Business Media New York 2016
Abstract Some N-benzylpiperidine-4-one derivatives were
synthesized via one-pot three-component protocols in
excellent yields, and were evaluated for their potential
cholinesterase inhibition. The mono-substituted derivatives
except 4b showed butyrylcholinesterase selective, while
unsubstituted and di-substituted derivatives showed acet-
ylcholinesterase selective inhibitions. It is interesting to note
that all the mono-substituted derivatives showing butyr-
ylcholinesterase inhibitory activity were more potent than
galanthamine. Compound 4c bearing ortho-methoxy group
showed the most potent, mixed-mode inhibitory activity on
both acetylcholinesterase and butyrylcholinesterase with
IC50 5.67 and 0.87 μM, respectively. Molecular docking
revealed that compound 4c had hydrophobic interactions
and hydrogen bonding at the catalytic triad and choline
binding sites of the enzymes. In conclusion, this simple and
cheaper synthesis method yielded derivatives with good
cholinesterase inhibition and favorable photophysical
properties.
Introduction
Alzheimer’s disease (AD) is an irreversible, progressive
brain disease that slowly destroys memory and thinking
skills, and eventually even the ability to carry out the sim-
plest tasks (Federica et al., 2011; Hardy, 2006). World
Alzheimer’s report 2014 reveals that control of diabetes and
high blood pressure, as well as measures to encourage
smoking cessation and to reduce cardiovascular risk, have
the potential to reduce the risk of dementia even in late life
(Martin et al., 2014). AD is characterized by the develop-
ment of amyloid plaques and neurofibrillary tangles, the
loss of connections between nerve cells or neurons in the
brain and the death of these nerve cells, in particular the
cholinergic neurons in basal forebrain leading to impair-
ment of memory and other cognitive dysfunctions
(Querfurth and LaFerla, 2010; Terry and Buccafusco,
2003). Postmortem studies supported the reduced choline
uptake, acetylcholine (ACh) synthesis and release, and
progressive loss of cholinergic neurons are responsible for
AD. These findings formed the basis of “cholinergic defi-
cient hypothesis” that claims the symptoms of AD are
caused by decreased ACh in the pre-synaptic region; thus,
conservation of the remaining ACh should improve the
memory and cognition impairment in AD (Giacobini,
2004). The best pharmacological approach to restore the
cholinergic function is by preventing the breakdown of ACh
in the cholinergic neurons by cholinesterase (ChE) (Tabet,
2006).
●
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Keywords N-benzylpiperidin-4-one Alzheimer’s disease
●
●
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Cholinesterase Enzyme kinetics Molecular docking
Fluorescence
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1619-6) contains supplementary material,
which is available to authorized users.
* Sathiyanarayanan Kulathu Iyer
Two types of ChE enzymes have been found in the
central nervous system, namely, acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE) (Tumiatti et al.,
2010). Current treatment of AD mainly focuses on the
inhibition of AChE activity in order to rectify the deficiency
of cerebral ACh (Decker et al., 2008; Kamal et al., 2008).
sathiya_kuna@hotmail.com
1
School of Advanced Sciences, Vellore Institute of Technology
University, Vellore 632014, India
2
Discipline of Pharmacology, School of Pharmaceutical Sciences,
Universiti Sains Malaysia, Penang 11800, Malaysia

Med Chem Res
The overall structure of human BChE is very similar to that
of AChE. Most differences between BChE and TcAChE are
confined to the residues composing the gorge, whereas in
BChE it is replaced with hydrophobic Leu286 and Val288.
These changes make it possible for the binding of bulkier
butyrate substrate moiety and inhibitors in BChE (Nicolet
et al., 2003).
N-benzylpiperidin-4-one derivatives are a versatile group
of organic compounds that were extensively used as
excellent ligands in pharmaceutical and synthetic applica-
tions as an important precursor in new drug discovery
(Shanmugam et al., 2013; Catto et al., 2013; Lee et al.,
2011; Huang et al., 2011; Masoumeh et al., 2012). Syn-
thetic, biologically active compounds having piperidin-4-
one moiety find good applications in pharmaceutical areas
such as CCR5 antagonist for the treatment of HIV, anti-
inflammatory agents, antitumor agents, brain-penetrant
inhibitors of LRRK2, and AChE inhibitors (Amr et al.,
2006; Joshi et al., 2010; Rajendra Prasad et al., 2005). Some
coumarin-3-carboxamides of N-benzylpiperidine also
act as AChE inhibitor (Ali et al., 2013). Due to
the significance of N-benzylpiperidine-4-one derivatives,
in the current study, we synthesized a series of N-benzyl-
piperidine-4-ones possessing 6-amino-2-benzyl-8-phenyl-
Fig. 1 Structure of available cholinesterase inhibitors and designed
compounds
However, the role of BChE in the progression of AD has
been recently found, and BChE inhibitors could recover
cholinergic activity through restoring the AChE/BChE
activity ratios in healthy brain (Basiri et al., 2013). The
FDA has approved four drugs, namely, galanthamine,
donepezil, rivastigmine, and tacrine, for symptomatic
treatment of AD (Fig. 1) (Kavanagh et al., 2011; Pepeu and
Giovannini, 2009; Birks et al., 2000; Racchi et al., 2004;
Leon et al., 2013). Among these four drugs, donepezil and
galanthamine are more effective for AChE while rivas-
tigmine is effective against both AChE and BChE to the
same extent. Tacrine was later abandoned because of its
hepatotoxicity, gastrointestinal adverse reactions, poor
oral bioavailability, and frequent dosing requirements
(Giacobini, 2001). Like AChE inhibitors, BChE inhibitors
were also found to reduce the amyloid precursor protein
levels in animals with a cholinergic lesion in the forebrain,
i.e., a selective BChE inhibitor significantly increases the
ACh level in the brain (Haroutunian et al., 1997).
In modern drug designing, molecular docking is routi-
nely used for understanding drug-receptor interaction
(Kitchen et al., 2004). Molecular docking provides useful
information about drug-receptor interactions, and is fre-
quently used for predicting the binding orientation of small
molecule drug candidates to their protein targets (Koellner
et al., 2002). AChE’s active site is located at the bottom of a
long and narrow gorge. ACh and substrate guidance down
the gorge are facilitated by cation–π interactions with aro-
matic sidechains residues such as phenylalanine, trypto-
phan, and tyrosine lining gorge wall (Koellner et al., 2002).
1,2,3,4-tetrahydroisoquinoline-5,7-dicarbonitriles,
which
are less explored (Alvarez and Marco, 2011). In addition,
cholinesterase inhibitory activities and photophysical
properties for all the derivatives were studied. Enzyme
kinetic and molecular docking analyses were also per-
formed for the most active derivative to find out the mode of
inhibition and ligand–protein interactions.
Results and discussion
Synthesis
For the initial optimization of the reaction conditions,
N-benzylpiperidine-4-one, benzaldehyde, malononitrile,
and 5 mol % of catalyst were selected as prototype. Among the
chosen solvents, ethanol (EtOH) was the best for the synthesis
of N-benzylpiperidine-4-one derivatives, as shown in Table 1.
Although acetonitrile, chloroform, dichloromethane, methanol,
and isopropanol allowed the reaction to progress in a good
manner, the yield was lesser than that obtained using EtOH as
solvent. Tetrahydrofuran (THF) and ethyl acetate gave low
yields and no desired product was obtained with water. By
screening a wide range of catalysts, we found out that the
product 4a could be obtained in good yields ranging from 54
to 96 % in different mole ratios of sodium hydroxide (NaOH)
(1, 2, 5, and 10 mol %) and the results are summarized in
Table 2. Better yields were obtained (up to 96 %) by carrying
out the reaction using 5 mol % of NaOH in EtOH as solvent.

Med Chem Res
Table 1 Initial solvent effect studies for synthesis of N-
Table 2 Screening of catalysts
benzylpiperidin-4-one derivatives with 5 mol % catalyst
Entry
Catalyst (mol %)
Time (h)
Yield (%)
Entry
Solvent
Catalyst
Time (h)
Yield (%)
1
2
3
4
5
6
7
8
9
KOH
10
24
24
24
24
7
70
19
21
27
25
54
73
96
96
1
2
3
4
5
6
7
8
9
CH3CN
CH2Cl2
CHCl3
EtOAc
MeOH
EtOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
5.5
5
72
Triethylamine
Pyridine
78
6
76
Piperidine
12
6
42
Morpholine
81
NaOH (1 mol %)
NaOH (2 mol %)
NaOH (5 mol %)
NaOH (10 mol %)
5
96
6
i-PrOH
THF
7.5
10
12
77
5
63
5
H2O
Traces
Conditions: N-benzylpiperidine-4-one (0.005 mol), malononitrile
(0.01 mol), benzaldehyde (0.005 mol), and 5 mol % catalyst in ethanol
(10 mL) solvent, refluxing on a water bath
Conditions: N-benzylpiperidine-4-one (0.005 mol), malononitrile
(0.01 mol), benzaldehyde (0.005 mol), and 5 mol % NaOH in 10 mL
solvent, refluxing on a water bath
Scheme 1 Synthesis of 6-
amino-2-benzyl-8-phenyl-
1,2,3,4-tetrahydroisoquinoline-
5,7-dicarbonitriles (4)
Table 3 Synthesis of N-benzylpiperidin-4-one derivatives (4)
The synthetic route from the starting materials to the
target compounds is shown in Scheme 1. A series of deri-
vatives have been synthesized by one-pot three-component
reactions of N-benzylpiperidine-4-one, malononitrile, and
aldehyde in the presence of catalytic amount of NaOH. The
mixture was refluxed on a water bath. The reaction was
completed in 5 h and afforded the product in good yield.
Structural elucidation of all compounds was done by FT-IR,
1H NMR, 13C nuclear magnetic resonance (NMR) spec-
troscopy techniques and elemental analysis. The synthetic
detail and yields for all the derivatives are summarized in
Table 3.
Product
R
% Yield
m.p. (°C)
4a
4b
4c
4d
4e
4f
H
96
93
90
93
91
94
89
88
89
92
90
88
186–188
183–185
181–183
185–187
190–192
186–188
191–193
182–184
186–190
188–190
184–186
189–191
2 Me
2 OMe
2 F
2 Naphthyl
4 Me
4g
4h
4i
4 OMe
4 OEt
4 Cl
4j
4 CN
Pharmacology
4k
4l
4 Br
3,4 di OMe
Cholinesterase inhibitory activity
All the reactions were carried out by employing 0.005 mol of
N-benzyl piperidine-4-one, 0.01 mol of malononitrile, and 0.005
mol of aldehyde in 10 mL of absolute ethanol and 5 mol % NaOH
The cholinesterase inhibitory activity of the synthesized
compounds is summarized in Table 4. In general, most of
the compounds exhibited good to moderate cholinester-
ase inhibitory activity. IC50 determination was carried out
only for those compounds having more than 50 % inhi-
bition at 10 μg/mL. The unsubstituted derivative (4a)
showed the least inhibitory activity, while the di-
substituted was less active than the mono-substituted
Primary screening studies of a variety of catalysts revealed that
potassium hydroxide (KOH) also act as a catalyst. KOH (5
mol %) was employed as catalyst for the three-component
reaction but moderate yield was obtained. Lower yields were
obtained with the use of triethylamine, pyridine, piperidine,
and morpholine.

Med Chem Res
Table 4 Cholinesterase
inhibitory activity of 4 (a–l)
Sample
% of inhibition at 10 μg/mL AChE inhibition,
IC50 (μM)
BChE inhibition,
IC50 (μM)
Selectivity for
AChE
BChE
AChEa BChEb
—
—
4a
30.27 0.003 11.24 0.007
33.22 0.001 4.72 0.013
91.57 0.005 96.43 0.12
—
—
—
4b
—
—
—
4c
5.61 0.22
0.87 0.03
0.94 0.03
15.26 0.61
2.15 0.08
2.67 0.10
15.96 0.63
—
0.16
0.04
—
6.44
22.72
—
4d
55.39 0.001 98.73 0.002 21.36 0.85
35.14 0.005 56.76 0.002
4e
—
4f
62.81 0.005 65.39 0.013 8.51 0.34
61.10 0.000 65.61 0.000 8.80 0.35
0.25
0.30
—
3.96
3.30
—
4g
4h
41.01 0.005 54.49 0.011
36.78 0.006 3.26 0.002
45.68 0.004 53.99 0.009
—
—
—
4i
—
—
4j
18.32 0.73
—
—
—
4k
70.92 0.002 31.75 0.005 10.81 0.43
61.54 0.011 30.41 0.007 22.67 0.90
2.09 0.04
—
—
4l
—
—
—
Galanthamine
19.34 0.10
9.25
0.11
Data presented as Mean SD (n = 3)
— not determined
a
Selectivity for AChE is defined as IC50(BChE)/IC50(AChE)
b
Selectivity for BChE is defined as IC50(AChE)/IC50(BChE)
Table 5 Kinetic parameters of cholinesterase inhibitions by 4c and 4d
Of all the synthesized derivatives, compound 4c with
ortho-methoxy substitution exhibited the most potent inhi-
bitor against both AChE and BChE. It was 2.7-fold less
potent against AChE, but 22-fold more potent on BChE
than galanthamine. In general, the monosubstituted deriva-
tives except 4b and 4i showed BChE selective inhibitions,
while unsubstituted and disubstituted derivatives showed
AChE selective inhibitions. It was interesting to note that all
the monosubstituted derivatives showing BChE inhibitory
activity were more potent than galanthamine, which is an
AChE selective inhibitor. BChE has gain growing impor-
tance in AD. As the disease progresses, AChE level pro-
gressively decline in certain brain regions, while BChE
remains unchanged or even increased. Thus, in the later
stage of the disease, BChE compensated for the role of
hydrolyzing Ach (Cokugras, 2003). Owing to the growing
relevance of BChE, an agent that selectively inhibits BChE
would be beneficial especially during the later stages of AD
(Liew et al., 2015).
Sample Concentration μM Vmax
Km
Ki
(OD412/min) (mM)
AChE 4c
BChE 4c
4d
0
0.01
0.01
0.01
0.01
0.02
0.02
0.01
0.01
0.02
0.02
0.02
0.01
0.20
0.23
0.21
0.23
0.30
0.37
0.62
0.64
0.43
0.76
0.77
0.83
5.03
1.28
2.56
5.12
0
0.88
0.88
1.28
2.56
5.12
0
1.32
2.63
5.26
derivatives. Mono substitution at ortho position with
methoxy group gave better inhibitory activity than methyl
group. In contrast, mono substitution at para position
with methyl gave slightly better inhibitory activity than
methoxy group. Among the halogen substituted deriva-
tives, fluoro substitution at ortho position showed
potent BChE inhibitory activity with IC50 at sub-
micromolar level. The results suggest no clear relation-
ship between the presence of electron withdrawing
groups (e.g., chloro and fluoro) or electron donating
groups (e.g., methoxy and methyl) with the inhibitory
activity of the derivatives.
Enzyme kinetics and mode of inhibition
Lineweaver–Burk and Dixon plots analyses were performed
to determine the mode of enzyme inhibition and kinetics of
the most active cholinesterase inhibitors, 4c and 4d. The
inhibition data and types of inhibitions are summarized as
kinetic data (Table 5) and Lineweaver–Burk plots (Fig. 2),
respectively. The results suggest that 4c is a mixed-mode
inhibitor of AChE, where it can bind to the enzyme’s active
or allosteric sites. Likewise, both 4c and 4d were mixed-

Med Chem Res
Fig. 2 Lineweaver–Burk plots
of cholinesterase inhibition by
4c and 4d. (A)
Acetylcholinesterase,
(B) butyrylcholinesterase, (1) 4c
(2) 4d

Med Chem Res
Fig. 3 Binding orientations and
interactions of 4c and 4d with
protein residues at the active site
of acetylcholinesterase and
butyrylcholinesterase. (A)
Acetylcholinesterase, (B)
butyrylcholinesterase, (1) 4c
(2) 4d
Table 6 Binding interactions
data for 4c and 4d docked into
active site gorge of TcAChE and
hBChE
Ligand Enzyme Binding
Interacting site Residue Type of
interaction
Distance (Å) Ligand
energy
(kcal)
interacting
moiety
4c
4c
AChE
BChE
−12.17
−11.54
Catalytic triad His 440 Hydrophobic
3.52
3.79
2.35
3.09
3.02
Aromatic ring
Piperidone ring
Aromatic ring
CN
Choline
Catalytic
triad
Trp 82 Hydrophobic
His 438 Hydrophobic
Ser 198 Hydrogen
4d
BChE
−13.51
Choline
Trp 82 Hydrophobic
Aromatic ring
mode inhibitors of BChE. The Ki values obtained were in
agreement with their respective IC50 values.
His438 of the catalytic triad of BChE. These interactions
between 4c and the active site of BChE were further
strengthened by a hydrogen bonding between Ser198 at
catalytic triad site and the cyano moiety of the compound.
In contrast, compound 4d, despite having almost equal
potent inhibition with 4c on BChE, only displayed a
hydrophobic interaction between the aromatic ring and
Trp82 at the choline binding site. Overall, the molecular
findings revealed that there were a few interactions at the
active site of the enzymes, in line with the mixed-mode
inhibition of both 4c and 4d. This suggests that the inter-
actions at allosteric sites of the enzyme could be playing an
important role in their potent inhibitory activity.
Enzyme inhibition kinetics was carried out in the absence
and presence of 4c (0, 1.28, 2.56, and 5.12 μM), and 4d (0,
1.32, 2.63, and 5.26 μM) at various concentrations of sub-
strates (3.5, 7, 14, and 28 mM). Kinetic parameters
including Michaelis–Menten (Km) constant and maximum
velocity (Vmax) were derived from the Lineweaver–Burk
plot, while inhibition constant (Ki) was derived from Dixon
plot.
Docking studies
Docking studies were performed in order to obtain further
insight into the binding mode of the compounds and also to
gain the structural modifications on the inhibitory activities
of the compounds against AChE and BChE. The binding
orientations and interactions data are summarized in Fig. 3
and Table 6. The active site in TcAChE and human BChE is
located deep in the center of the enzyme with a narrow
gorge. The region is made up of five important regions that
accommodate and hydrolyze ACh, namely, the catalytic
triad, oxyanion hole, choline binding site, acyl binding
pocket, and peripheral anionic site (Basiri et al., 2013).
In TcAChE, compound 4c displayed a single hydro-
phobic interaction between aromatic ring attached to the
nitrogen atom of the piperidone moiety and the His440
residue at the catalytic triad. Likewise, in BChE the same
aromatic ring also had hydrophobic interaction with the
Trp82 of choline binding site. In addition, there was another
hydrophobic interaction between the piperidone ring and
Photophysical studies of N-benzylpiperidin-4-one
derivatives
The optical properties of the compounds such as absorption
(λmax), emission (λem), Stokes shift, and quantum yields
were measured in dimethyl sulfoxide (DMSO) and the data
are summarized in Table 7. The absorption and fluorescence
spectra of all the products dissolved in DMSO are shown in
Figs. 4 and 5 respectively. All the compounds showed
absorption maximum with wavelength ranging from 290 to
365 nm. Most of the derivatives displayed two absorption
maxima except 4c, 4d, and 4i which showed single band.
Highest energy absorption band I in the region of 280–300
nm and lowest energy absorption band II in the region of
330–365 nm were observed. The lowest energy transition
band II could be attributed to the transition from singlet
ground (S0) to the first excited state (S1) S0→S1.

Med Chem Res
Table 7 Photophysical properties of N-benzylpiperidin-4-one
derivatives
It can be understood from Fig. 5 that the compounds are
fluorescent in solution and most of the compounds dis-
played almost similar emission spectra in the range of
417–507 nm with the exception of 4a and 4d, which
showed two emissions maxima. The Stokes shift value was
calculated to be in the range of 3757–9975 cm−1 for this
library. The higher Stokes shift value suggested significant
structural changes between the ground and excited states.
Further, the large emission shift from absorption maximum
may be due to the presence of intramolecular charge transfer
character, which would also be responsible for the observed
larger Stokes shift values. Quantum yields were determined
in a solution using quinine sulfate as reference φ = 0.55 in
H2SO4 1 N, λex = 366 nm for dyes, and it is clear that all the
derivatives exhibit low to moderate quantum yields.
Code
aλabs
aλfl
Δss (cm−1
)
bΦfl
4a
4b
4c
4d
4e
4f
4g
4h
4i
290, 360
310, 360
345
427, 507
417
4359, 8054
3797
0.12
0.10
0.05
0.04
0.27
0.11
0.14
0.17
0.32
0.12
0.09
0.02
417, 460
424
5005, 7246
3812
365
325, 360
300, 330
300, 330
290, 365
295
425
4248
424
6718
370, 433
423
3276, 7208
3757
418
9975
4j
290, 365
290, 365
295, 365
448
5076
4k
4l
429
4087
428
4033
a
Measured in the concentration of 2 × 10−5 mol in DMSO solvent
b Quantum yields were measured using quinine sulfate as reference φ
= 0.55 in H2SO4 1 N, λex = 366 nm
Experimental section
General
All the chemicals were purchased from commercial sources
and were used without further purification unless otherwise
1
specified. H and 13C NMR spectra were taken on Bruker
400 MHz using DMSO-d6 as the solvent with tetra-
methylsilane as an internal standard. The melting points (m.p.)
were measured on Guna capillary-based melting point appa-
ratus, and were not corrected. HRMS values were obtained on
Joel GCMate II GC-Mass Spectrometer. Fourier transform
infrared spectroscopy spectra of the synthesized organic
compounds were recorded using a JASCO-4100 spectrometer
instrument. UV-visible spectra were taken using Hitachi U-
2910 spectrophotometer. Fluorescence spectra in solution were
measured using Hitachi F-7000 fluorescence spectrometer.
Fig. 4 UV-vis spectra of compounds 4a–l in dimethyl sulfoxide
solution
General procedure for the synthesis of compounds 6-amino-
2-benzyl-8-phenyl-1,2,3,4-tetra hydroisoquinoline-5,7-
dicarbonitriles (4)
A mixture of malononitrile (0.01 mol), aldehyde (0.005 mol)
and N-benzylpiperidine-4-one (0.005 mol) were dissolved in
10 mL of absolute ethanol and catalytic amount of NaOH was
added and heated on the water bath at 70 °C for 5 h. The
completion of reaction was examined by thin layer chroma-
tography; the precipitate was filtered and dried in vacuo. The
product was then crystallized from ethanol:THF (1:1) solvent
to afford compounds in excellent yield (Table 3).
6-Amino-2-benzyl-8-phenyl-1,2,3,4-tetrahydroisoquinoline-
5,7-dicarbonitrile (4a)
Pale yellow solid; yield 96 %; m.p. 186–188 °C; IR (KBr
Fig. 5 Fluorescence spectra of compounds 4a–l in dimethyl sulfoxide
solution
cm−1): 3414, 3344, 3240, 3160, 2337, 2216, 1649, 1582,

Med Chem Res
1
1485, 1344, 1261, 1128, 1061, 983, 802, 777, 750, and 698;
1H NMR (DMSO-d6, 400 MHz, δ ppm): 7.46–7.42 (m, 3H,
ArH), 7.25–7.22 (m, 4H, ArH), 7.20–7.16 (m, 3H, ArH),
6.52 (s, 2H, NH2), 3.46 (s, 2H, H-1), 3.03 (s, 2H, benzylic-
CH2), 2.90–2.87 (t, J = 6 Hz, 2H, H-4), 2.64–2.61 (t, J = 6
Hz, 2H, H-3); 13C NMR (DMSO d6, 100 MHz, δ ppm):
151.0 (C6–NH2), 148.3, 144.8, 137.8, 128.7, 128.6, 128.5,
128.1, 128.0, 126.9, 122.2 (aromatic carbons), 115.5 (C7–
CN), 115.1 (C5–CN), 95.4 (C7), 95.3 (C5), 60.9 (benzylic-
CH2), 53.3 (C1), 48.1 (C3), 28.8 (C4); HRMS calculated: [M
+H]+ m/z 365.1688; found: 365.1690.
1446, 1348, 1261, 1201, 1139, 823, 742, 696. H NMR
(DMSO-d6, 400 MHz, δ ppm): 7.53–7.51 (t, J = 6 Hz, 1H,
ArH), 7.37–7.19 (m, 8H, ArH), 6.67 (s, 2H, NH2), 3.49 (s,
2H, benzylic-CH2), 3.10–2.92 (m, 4H, H-3 & H-1),
2.66–2.63 (t, J = 6 Hz, 2H, H-4); 13C NMR (DMSO-d6,
100 MHz, δ ppm): 156.2 (C6–NH2), 150.4, 147.2, 143.0,
136.8, 135.7, 134.0, 133.7, 132.1, 130.1, 128.5, 121.2,
120.4 (aromatic carbons), 101.5 (C7–CN), 101.0 (C5–CN),
66.1 (C7 & C5), 57.9 (benzylic-CH2), 53.4 (C1 & C3), 34.0
(C4); HRMS calculated [M+H]+ m/z 383.1594; found
383.1598.
6-Amino-2-benzyl-8-(o-tolyl)-1,2,3,4-
6-Amino-2-benzyl-8-(naphthalen-2-yl)-1,2,3,4-
tetrahydroisoquinoline-5,7-dicarbonitrile (4b)
tetrahydroisoquinoline-5,7-dicarbonitrile (4e)
Pale yellow solid; yield 93 %; m.p. 183–185 °C: IR (KBr
Brown solid; yield 91 %; m.p. 190–192 °C; IR (KBr cm−1):
3421, 3332, 2802, 2212, 1639, 1595, 1566, 1514, 1396,
1359, 1321, 1228, 1205, 1124, 804, 781, 736, 694. 1H
NMR (DMSO-d6, 400 MHz, δ ppm): 8.52–8.50 (t, J = 6 Hz,
1H, ArH), 7.97–7.94 (t, J = 6 Hz, 2H, ArH), 7.79–7.77
(d, J = 8 Hz, 1H, ArH), 7.56–7.47 (m, 5H, ArH), 7.25–7.14
(m, 5H, ArH & NH2), 3.47 (s, 2H, H-1), 3.00 (s, 2H,
benzylic-CH2), 2.90–2.88 (t, J = 6 Hz, 2H, H-4), 2.63–2.61
(t, J = 6 Hz, 2H, H-3) ; 13C NMR (DMSO-d6, 100 MHz, δ
ppm): 156.2 (C6–NH2), 144.6, 137.8, 133.5, 132.4, 130.2,
129.4, 128.6, 127.8, 126.5, 125.5, 123.8 (aromatic carbons),
116.1 (C7–CN), 112.8 (C5–CN), 111.9 (C7), 80.5 (C5), 60.8
(benzylic-CH2), 52.3 (H-1), 42.6 (H-3), 25.1 (H-4); HRMS
calculated [M+H]+ m/z 415.1844; found 415.1849.
cm−1): 3340, 3194, 3054, 2962, 2929, 2872, 2524, 2385,
1
2216, 1668, 1568, 1446, 1282, 1217, 1114, 927, 742. H
NMR (DMSO-d6, 400 MHz, δ ppm): 7.72 (s, 3H, ArH),
7.58–7.47 (t, J = 6 Hz, 4H, ArH), 7.23–7.12 (q, J = 8 Hz,
4H, ArH & NH2), 4.49–4.45 (t, J = 6 Hz, 2H, H-4),
4.01–3.95 (t, J = 6 Hz, 2H, H-3), 3.76 (s, 2H, benzylic-
CH2), 3.49 (s, 2H, H-1), 2.33 (s, 3H, CH3); 13C NMR
(DMSO-d6, 100 MHz, δ ppm): 158.4 (C6–NH2), 136.7,
135.5, 134.4, 131.3, 130.3, 129.4, 128.8, 126.1, 125.2
(aromatic carbons), 118.5 (C7–CN), 114.4 (C5–CN), 88.6
(C7), 76.0 (C5), 56.0 (benzylic-CH2), 54.4 (C1), 47.7 (C3),
29.9 (C4), 19.2 (CH3); HRMS calculated [M+H]+ m/z
379.1844; found 379.1848.
6-Amino-2-benzyl-8-(2-methoxyphenyl)-1,2,3,4-
6-Amino-2-benzyl-8-(p-tolyl)-1,2,3,4-
tetrahydroisoquinoline-5,7-dicarbonitrile (4c)
tetrahydroisoquinoline-5,7-dicarbonitrile (4f)
Reddish brown solid; yield 90 %; m.p. 181–183 °C; IR (KBr
Yellow solid; yield 94 %; m.p. 186–188 °C; IR (KBr cm−1):
3340, 3236, 3026, 2922, 2818, 2765, 2677, 2214, 1635,
1568, 1516, 1446, 1363, 1228, 1192, 964, 823, 742, 698.
1H NMR (DMSO-d6, 400 MHz, δ ppm): 7.28–7.20 (m, 7H,
ArH), 7.15–7.13 (d, J = 8 Hz, 2H, ArH), 6.54 (s, 2H, NH2),
3.48 (s, 2H, benzylic-CH2), 3.09–2.90 (m, 4H, H-3 & H-1),
2.66–2.64 (t, J = 6 Hz, 2H, H-4), 2.36 (s, 3H, CH3); 13C
NMR (DMSO-d6, 100 MHz, δ ppm): 151.0 (C6–NH2),
148.4, 144.6, 138.1, 137.8, 133.1, 129.1, 128.5, 127.9,
126.9, 122.4 (aromatic carbons), 115.6 (C7–CN), 115.1(C5–
CN), 95.6 (C7), 95.2 (C5), 60.8 (benzylic-CH2), 53.5 (C1),
48.0 (C3), 28.8 (C4), 20.8 (CH3); HRMS calculated [M+H]+
m/z 379.1844; found 379.1848.
cm−1): 3327, 3210, 3018, 2980, 2313, 1637, 1433, 1375,
1
1228, 1207, 1016, 758, 696.30. H NMR (DMSO-d6, 400
MHz, δ ppm): 7.51 (s, 2H, ArH), 7.41 (s, 2H, ArH), 7.41–7.35
(m, 2H, ArH), 7.29–7.19 (m, 3H, ArH), 6.99 (s, 2H, NH2),
3.76 (s, 3H, O–CH3), 3.47 (s, 2H, H-1), 3.05 (s, 2H, benzylic-
CH2 ), 2.92- 2.89 (t, J = 6 Hz, 2H, H-4), 2.66–2.63 (t, J = 6
Hz, 2H, H-3); 13C NMR (DMSO-d6, 100 MHz, δ ppm): 157.5
(C6–NH2), 145.0, 137.9, 134.4, 130.7, 128.5, 128.0, 127.5,
127.4, 126.8, 121.1, 120.2 (aromatic carbons), 117.8 (C7–CN),
115.7 (C3-phenyl carbon), 111.8 (C5–CN), 80.1 (C7), 60.8
(C5), 55.7 (benzylic-CH2), 54.4 (O–CH3), 52.2 (C1), 42.2 (C3),
34.3 (C4); HRMS calculated [M+H]+ m/z 395.1794; found
395.1797.
6-Amino-2-benzyl-8-(4-methoxyphenyl)-1,2,3,4-
6-Amino-2-benzyl-8-(2-fluorophenyl)-1,2,3,4-
tetrahydroisoquinoline-5,7-dicarbonitrile (4d)
tetrahydroisoquinoline-5,7-dicarbonitrile (4g)
Reddish brown solid; yield 89 %; m.p. 191–193 °C; IR
(KBr cm−1): 3334, 3238, 3016, 2949, 2393, 2187, 2160,
1606, 1556, 1537, 1510, 1444, 1375, 1244, 1174, 972, 833,
Brown solid; yield 93 %; m.p. 185–187 °C; IR (KBr cm−1):
3435, 3350, 3250, 2926, 2756, 2216, 1647, 1564, 1494,

Med Chem Res
1
744. H NMR (DMSO-d6, 400 MHz, δ ppm): 7.55–7.46 (t,
7.28–7.18 (m, 5H, ArH), 6.68 (s, 2H, NH2), 3.48 (s, 2H, H-
1), 3.02 (s, 2H, benzylic-CH2), 2.91–2.89 (t, J = 6 Hz, 2H,
H-4), 2.65–2.62 (t, J = 6 Hz, 2H, H-3); 13C NMR (DMSO-
d6, 100 MHz, δ ppm): 151.0 (C6–NH2), 146.4, 145.2, 140.9,
137.7, 132.5, 129.4, 128.5, 128.1, 126.9, 121.9 (aromatic
carbons), 118.3 (Phenyl C–CN), 115.3(C7–CN), 114.9
(C5–CN) 111.7 (Phenyl C–CN), 96.0 (C7), 94.8 (C5), 60.8
(benzylic-CH2), 53.1 (C1), 47.9 (C3), 28.8 (C4); HRMS
calculated [M+H]+ m/z 390.1640; found 390.1645.
J = 4 Hz, 6H, ArH), 6.96–6.82 (q, J = 8 Hz, 5H, ArH &
NH2), 3.77(s, 3H, OCH3), 3.52 (s, 2H, benzylic-CH2),
3.13–2.94 (m, 4H, H-3 & H-1) 2.69–2.67 (t, J = 6 Hz, 2H,
H-4); 13C NMR (DMSO-d6, 100 MHz, δ ppm): 161.7,
159.6 (C6–NH2), 139.0, 131.2, 129.7, 129.5, 128.9, 128.3,
128.0 (aromatic carbons), 115.8 (C7–CN), 114.6 (C5–CN),
114.4 (C3-phenyl carbon), 114.1 (C5-phenyl carbon), 81.4
(C7), 75.9 (C5), 55.7 (benzylic-CH2), 55.1 (C1), 54.4 (O–
CH3), 49.2 (C3) 28.4 (C4); HRMS calculated [M+H]+ m/z
395.1794; found 395.1797.
6-Amino-2-benzyl-8-(4-bromophenyl)-1,2,3,4-
tetrahydroisoquinoline-5,7-dicarbonitrile (4k)
6-Amino-2-benzyl-8-(4-ethoxyphenyl)-1,2,3,4-
tetrahydroisoquinoline-5,7-dicarbonitrile (4h)
Off-white solid; yield 90 %; m.p. 184–186 °C; IR (KBr cm−1):
3568, 3313, 3211, 3199, 2935, 2671, 2461, 2214, 1635, 1588,
1454, 1284, 1228, 1118, 1014, 918, 758, 702. H NMR
Off white solid; yield 88 %; m.p. 182–184 °C; IR (KBr cm−1):
3429, 3332, 3238, 2839, 2725, 2652, 2324, 2216, 2162, 1834,
1647, 1556, 1444, 1373, 1178, 1043, 914. 1H NMR (DMSO-
d6, 400 MHz, δ ppm): 7.56–7.45 (t, J = 8 Hz, 7H, ArH),
7.10–6.72 (m, 4H, ArH), 4.46 (s, 2H, H-1), 4.07–4.02 (m, 4H,
H-4 & OCH2–CH3), 3.48–3.36 (t, J = 6 Hz, 2H, H-3), 3.11 (s,
2H, benzylic-CH2), 1.35–1.31 ( t, J = 8 Hz, 3H, OCH2-CH3);
13C NMR (DMSO-d6, 100 MHz, δ ppm): 163.1, 161.7, 158.9,
157.9 (C6–NH2), 139.0, 131.2, 129.7, 129.5, 128.9, 128.3,
127.9 (aromatic carbons), 114.5 (C7–CN), 114.3 (C5–CN),
81.4 (C7 & C5), 63.1 (OCH2–CH3), 55.6 (benzylic-CH2), 54.3
(C1), 49.2 (C3), 28.4 (C4), 14.5 (OCH2–CH3); HRMS calcu-
lated [M+H]+ m/z 409.1950; found 409.1953.
1
(DMSO-d6, 400 MHz, δ ppm): 7.92–7.90 (d, J = 6 Hz, 2H,
ArH), 7.48–7.46( d, J = 8 Hz, 2H, ArH), 7.22–7.12 (m, 5H,
ArH), 6.62 (s, 2H, NH2), 3.42 (s, 2H, benzylic-CH2), 2.96 (s,
2H, H-1), 2.85–2.83 (t, J = 6 Hz, 2H, H-4), 2.59–2.56 (t, J = 6
Hz, 2H, H-3) ; 13C NMR (DMSO-d6, 100 MHz, δ ppm): 151.8
(C6–NH2), 147.6, 142.2, 134.0, 131.9, 131.1, 130.3, 128.6,
122.9 (aromatic carbons), 114.9 (C7–CN), 114.5 (C5–CN),
96.2 (C7), 95.3 (C5), 56.8 (benzylic-CH2), 48.8 (C1), 47.9 (C3),
24.7 (C4); HRMS calculated [M+H]+ m/z 443.0793; found
443.0797.
6-Amino-2-benzyl-8-(3,4-dimethoxyphenyl)-1,2,3,4-
tetrahydroisoquinoline-5,7-dicarbonitrile (4l)
6-Amino-2-benzyl-8-(4-chlorophenyl)-1,2,3,4-
tetrahydroisoquinoline-5,7-dicarbonitrile (4i)
Reddish brown solid; yield 88 %; m.p. 189–191 °C; IR
(KBr cm−1): 3329, 2999, 2937, 2837, 2478, 2414, 2385,
2324, 2187, 2162, 1599, 1554, 1514, 1442, 1377, 1296,
Pale yellow solid; yield 89 %; m.p. 186–190 °C; IR (KBr
cm−1): 3329, 3003, 2935, 2385, 2120, 1598, 1562, 1431, 1259,
1217, 1141, 1020, 748, 700. 1H NMR (DMSO-d6, 400 MHz,
δ ppm): 7.99–7.97 (d, J = 8 Hz, 2H, ArH), 7.55–7.53 (d, J = 8
Hz, 2H, ArH), 7.30–7.20 (m, 5H, ArH), 6.70 (s, 2H, NH2),
3.50 (s, 2H, H-1), 3.04 (s, 2H, benzylic-CH2), 2.92–2.90 (t, J
= 6 Hz, 2H, H-4), 2.66–2.64 (t, J = 6 Hz, 2H, H-3); 13C NMR
(DMSO-d6, 100 MHz, δ ppm): 158.4 (C6–NH2), 133.7, 132.9,
132.4, 131.3, 131.1, 129.5, 128.9, 128.8, 127.7 (aromatic
carbons), 114.3 (C7–CN & C5–CN), 88.6 (C7), 76.0 (C5), 56.3
(benzylic-CH2), 54.5 (C1), 53.7 (C3), 28.8 (C4); HRMS cal-
culated [M+H]+ m/z 399.1298; found 399.1301.
1
1217, 1141, 1020, 748. H NMR (DMSO-d6, 400 MHz, δ
ppm): 7.46–7.43 (m, 2H, ArH), 7.27–7.18 (m, 6H, ArH),
6.54 (s, 2H, NH2), 3.95 (s, 3H, O–CH3), 3.80 (s, 3H,
O–CH3), 3.47 (s, 2H, H-1), 3.05 (s, 2H, benzylic-CH2),
2.92–2.89 (t, J = 6 Hz, 2H, H-4), 2.66–2.63 (t, J = 6 Hz, 2H,
H-3); 13C NMR (DMSO-d6, 100 MHz, δ ppm): 158.0 (C6-
NH2), 149.4, 148.8, 139.3, 131.2, 129.8, 129.6, 128.9,
128.3, 120.5 (aromatic carbons), 115.9 (C7-CN), 114.6 (C5-
CN), 111.6 (C2-phenyl carbon), 109.5 (C5-phenyl carbon),
81.4 (C7), 75.8 (C5), 55.7 (benzylic-CH2), 55.5 (O–CH3,
54.3 (O–CH3), 49.2 (C1), 43.6 (C3), 28.6 (C4); HRMS
calculated [M+H]+ m/z 425.1899; found 425.1903.
6-Amino-2-benzyl-8-(4-cyanophenyl)-1,2,3,4-
tetrahydroisoquinoline-5,7-dicarbonitrile (4j)
Pharmacology
Reddish brown solid; yield 92 %; m.p. 188–190 °C; IR
(KBr cm−1): 3342, 3236, 3197, 3026, 2816, 2767, 2727,
2222, 1608, 1556, 1454, 1369, 1246, 1136, 1053, 941, 827,
744. 1H NMR (DMSO-d6, 400 MHz, δ ppm): 7.98–7.96 (d,
J = 8 Hz, 2H, ArH), 7.54–7.52 (d, J = 8 Hz, 2H, ArH),
Cholinesterases inhibitory activity
Enzymes and chemicals Acetylthiocholine iodide, AChE
from electric eel, 5,5-dithiobis[2-nitrobenzoic acid] (DTNB),

Med Chem Res
BChE from equine serum, S-butyrylthiocholinechloride, and
galanthamine were purchased from Sigma Chemicals (St.
Louis, MO, USA). All the other reagents used were of ana-
lytical grade.
Conclusion
A series of new N-benzylpiperidin-4-one derivatives were
synthesized via one-pot three-component protocols and
evaluated for AChE and BChE inhibitory activity. The
advantages of this method are cost effectiveness, simple
work up and production of higher yields in a short duration.
No column chromatography is required. Compound 4c
showed the most potent inhibitory activity on both AChE
and BChE with IC50 5.67 and 0.87 μM, respectively. It
showed a mixed-mode inhibition against both enzymes.
Molecular docking revealed that compound 4c had hydro-
phobic interactions and hydrogen bonding at the catalytic
triad and choline binding sites of the enzymes. The prepared
N-benzylpiperidin-4-one derivatives showed excellent
fluorescent properties, which can also be used in
fluorescence-based sensor applications.
Enzyme assay Cholinesterase inhibitory activity of the test
samples was determined by Ellman’s method (Khaw et al.,
2014). Galanthamine was used as the positive control.
Solutions of test samples and galanthamine were prepared
in DMSO. The final concentration of DMSO in reaction
mixture was 1 %. At this concentration, DMSO has no
inhibitory effect on both AChE and BChE. For AChE
assay, 140 μL of 0.1 M sodium phosphate buffer (pH 8) was
first added to each well of a 96-well microplate followed by
20 μL of the test sample and 20 μL of 0.09 unit/mL AChE.
After preincubation at room temperature, 10 μL of 10 mM
DTNB was added into each well followed by 10 μL of 14
mM acetylthiocholine iodide as substrate. Absorbance of
the colored end product was measured using BioTek Power
Wave X 340 Micro plate Spectrophotometer at 412 nm
for 30 min after the initiation of enzymatic reaction. For
BChE inhibitory assay, the same procedure as described
above was followed except for the use of enzyme and
substrate, which were BChE from equine serum and
S-butyrylthiocholine chloride, respectively. A set of five
concentrations was used to estimate the 50 % inhibitory
concentration (IC50). Each test was conducted in triplicate.
Acknowledgements Dileep K. S. expresses his thanks to VIT
management for providing financial support through research asso-
ciateship. The DST—FIST NMR facility at VIT University is duly
acknowledged.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Enzyme kinetics and mode of inhibition The enzyme
inhibition kinetics was carried out in the absence and pre-
sence of 4c (0, 1.28, 2.56 and 5.12 μM), and 4d (0, 1.32,
2.63 and 5.26 μM) at various concentrations of substrates
(3.5, 7, 14 and 28 mM). Kinetic parameters including Km
and Vmax were derived from the Lineweaver–Burk plot
while Ki was derived from Dixon plot.
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