Synthesis and pharmacological evaluation of donepezil-based agents as new cholinesterase/monoamine oxidase inhibitors for the potential application against Alzheimer’s disease

Article abstract of DOI:10.1080/14756366.2016.1201814

In a continuing effort to develop multitargeted compounds as potential treatment agents against Alzheimer's disease (AD), a series of donepezil-like compounds were designed, synthesized and evaluated. In vitro studies showed that most of the designed compounds displayed potent inhibitory activities toward AChE, BuChE, MAO-B and MAO-A. Among them, w18 was a promising agent with balanced activities, which exhibited a moderate cholinesterase inhibition (IC₅₀, 0.220 μM for eeAChE; 1.23 μM for eqBuChE; 0.454 μM for hAChE) and an acceptable inhibitory activity against monoamine oxidases (IC₅₀, 3.14 μM for MAO-B; 13.4 μM for MAO-A). Moreover, w18 could also be a metal-chelator, and able to cross the blood–brain barrier with low cell toxicity on PC12 cells. Taken together, these results suggested that w18 might be a promising multitargeted compound for AD treatment.

Full text of DOI:10.1080/14756366.2016.1201814

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and pharmacological evaluation of
donepezil-based agents as new cholinesterase/
monoamine oxidase inhibitors for the potential
application against Alzheimer’s disease
Fan Li, Zhi-Min Wang, Jia-Jia Wu, Jin Wang, Sai-Sai Xie, Jin-Shuai Lan, Wei Xu,
Ling-Yi Kong & Xiao-Bing Wang
To cite this article: Fan Li, Zhi-Min Wang, Jia-Jia Wu, Jin Wang, Sai-Sai Xie, Jin-Shuai Lan, Wei
Xu, Ling-Yi Kong & Xiao-Bing Wang (2016): Synthesis and pharmacological evaluation of
donepezil-based agents as new cholinesterase/monoamine oxidase inhibitors for the potential
application against Alzheimer’s disease, Journal of Enzyme Inhibition and Medicinal Chemistry,
DOI: 10.1080/14756366.2016.1201814
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1201814
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J Enzyme Inhib Med Chem, Early Online: 1–13
2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/14756366.2016.1201814
!
RESEARCH ARTICLE
Synthesis and pharmacological evaluation of donepezil-based agents as
new cholinesterase/monoamine oxidase inhibitors for the potential
application against Alzheimer’s disease
Fan Li, Zhi-Min Wang, Jia-Jia Wu, Jin Wang, Sai-Sai Xie, Jin-Shuai Lan, Wei Xu, Ling-Yi Kong, and
Xiao-Bing Wang
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, People’s Republic of
China
Abstract
Keywords
In a continuing effort to develop multitargeted compounds as potential treatment agents
against Alzheimer’s disease (AD), a series of donepezil-like compounds were designed,
synthesized and evaluated. In vitro studies showed that most of the designed compounds
displayed potent inhibitory activities toward AChE, BuChE, MAO-B and MAO-A. Among them,
w18 was a promising agent with balanced activities, which exhibited a moderate cholinester-
ase inhibition (IC50, 0.220 mM for eeAChE; 1.23 mM for eqBuChE; 0.454 mM for hAChE) and an
acceptable inhibitory activity against monoamine oxidases (IC50, 3.14 mM for MAO-B; 13.4 mM for
MAO-A). Moreover, w18 could also be a metal-chelator, and able to cross the blood–brain
barrier with low cell toxicity on PC12 cells. Taken together, these results suggested that w18
might be a promising multitargeted compound for AD treatment.
Alzheimer’s disease, donepezil,
cholinesterase, monoamine oxidase,
molecular modeling
History
Received 25 April 2016
Revised 2 June 2016
Accepted 4 June 2016
Published online 6 July 2016
Introduction
and BuChE are important target, and inhibition of both of them
will be more beneficial to the treatment of AD.
Alzheimer’s disease (AD), the most common cause of dementia in
elderly people, is a complex and progressive neurodegenerative
disorder characterized by memory loss, decline in language ability
and other cognitive imparment1,2. Over 100 years, the exact
etiology of AD still remain elusive, multiple factors such as
b-amyloid deposite, dyshomeostasis of biometal, oxidative and
low levels of acetylcholine (ACh) are considered to play
significant roles in the pathophysiology of AD3.
Among the multiple factors that induce AD, the cholinergic
hypothesis has been proposed to explain the mechanism of AD
development4. This hypothesis asserts that dysfunction of cho-
linergic system, mainly decline of acetylcholine (ACh) level,
leads to the memory and cognitive deficits associated with AD,
and inhibiting the cholinesterase (ChE) responsible for the
hydrolysis of ACh is therefore supposed to be clinically beneficial
to patients5,6. Two types of ChEs, acetycholinesterase (AChE) and
butyrylcholinesterase (BuChE), existed in the central nervous
system of human. Compared to BuChE, AChE is more active and
can hydrolyze the major ACh in healthy brains7. However, in the
case of AD, BuChE is a major modulator in regulating the ACh
level8,9. As AD progresses, the activity of AChE is found to be
decreased, and that of BuChE is significantly increased in the
hippocampus and temporal cortex10,11. Consequently, both AChE
In addition, many studies have found that monoamine oxidase
(MAO) also play a very important role in the pathogenesis of AD,
as the increase of MAO in brain may result in a cascade of
biochemical events leading to neuronal dysfunction12,13. MAOs
are important FAD-dependent enzymes (flavoenzymes), which
have two functional isozymic forms, namely MAO-A and MAO-
B, identified by their different substrate and inhibitor specifi-
city14,15
. Catecholaminergic neurons predominantly contain
MAO-A, while MAO-B is located in serotonergic glia and
neurons16,17. The MAOs have been used as drug targets and
inhibitors of these enzymes are used to treat neuropsychiatric
syndromes18,19. Selective MAO-B inhibitors have been applied to
treat the neurodegenerative disorders such as Alzheimer’s and
Parkinson’s diseases18, while selective MAO-A inhibitors have
been revealed to treat depression and anxiety19. Several lines of
evidence indicate that, AD patients also commonly present
depressive symptom20. Based on these aspects, simultaneous
inhibition of both MAO-A and MAO-B, could provide additional
benefits in AD therapy.
At present, there are three FDA-approved drugs for AD
treatment, these anti-AChE agents include galanthamine, done-
pezil, and rivastigmine, which can only provide a temporary
symptom alleviation instead of preventing or slowing the
progressive neurodegeneration21–23. However, the multiple etiol-
ogies of AD make single-target strategy difficult to shed good
therapeutic effect. Thus, multi-target-directed ligand (MTDL)
Address for correspondence: Ling-Yi Kong and Xiao-Bing Wang, State
Key Laboratory of Natural Medicines, Department of Natural Medicinal
Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing
210009, People’s Republic of China. Tel/Fax: +86-25-83271405. E-mail:
cpu_lykong@126.com; xbwang@cpu.edu.cn
raises as an effective strategy for the treatment of AD24,25
Attempts to combine anti-AChE and anti-MAO activities in one
.

2
F. Li et al.
J Enzyme Inhib Med Chem, Early Online: 1–13
molecular entity have previously been reported26. For example, N- chelation and penetration of the blood–brain barrier (BBB). The
pyrimidine-4-acetylaniline derivatives possessing AChE and structure–activity relationships were discussed based on
reversible MAO-A inhibitory activity in vitro have been reported, the pharmacological activities. Moreover, to further investigate
and our group has also reported the synthesis of tacrine–coumarin the interaction mechanism with ChEs and MAOs, kinetic analysis
hybrids as multitargeted agents against AD27–29
.
and molecular modeling studies were also performed.
Recently, donepezil, an effective anti-AChE drug for AD
treatment, has attracted considerable attention30,31. Replacing the
indanone fragment of donepezil with additional bioactive mol-
ecules to produce multitargeted inhibitors is a good strategy. For
example, donepezil and N-[(5-(benzyloxy)-1-methyl-1H-indol-2-
yl)methyl]-N-methylprop-2-yn-1-amine hybrids have been
designed as multitargeted agents capable of inhibiting ChEs and
MAOs32. On the other hand, lazabemide and its analog Ro 16–
6491 are reversible MAO inhibitors with remarkably high potency
and selectivity for MAO-B33,34. They could serve as adjuvants in
the therapy of AD and other degenerative brain disorders35.
Moreover, moclobemide, a another reversible and short-acting
preferential MAO-A inhibitor, and it has been shown to have
antidepressant effects on human36,37. The neurochemical and
pharmacological characteristics of these carboxamide derivatives
lazabemide, Ro16–6491 and moclobemide have been studied as to
Materials and methods
Materials
All common reagents and solvents were obtained from commercial
suppliers and used without purification. Reaction progress was
monitored using analytical thin layer chromatography (TLC) on
precoated silica gel GF254 plates (Qingdao Haiyang Chemical
Plant, Qing-Dao, China), and the spots were detected under UV
light (254 nm). Melting points were determined on an XT-4
micromelting point instrument and uncorrected. IR (KBr-disc)
spectra were recorded by Bruker Tensor 27 spectrometer (Bruker,
Karlsruhe, Germany). Column chromatography was performed on
1
silica gel (90–150 mm; Qingdao Marine Chemical Inc.). H NMR
and 13C NMR spectra were measured on a Bruker ACF-500
spectrometer (Bruker, Karlsruhe, Germany) at 25 ꢀC and refer-
enced to TMS. Chemical shifts are reported in ppm (ꢀ) using the
residual solvent line as internal standard. Mass spectra were
obtained on a MS Agilent 1100 (Agilent Technologies, Santa
Clara, CA) Series LC/MSD Trap mass spectrometer (ESI-MS) and
a Mariner ESI-TOF spectrometer (HRESIMS), respectively.
effect monoamine levels in human brain33–37
.
Given the activities of them, and in an attempt to obtain new
multi-targeted molecules with both ChEs and MAOs inhibitory
activity for the treatment of AD38,39, a series of novel compounds
have been designed and synthesized. The strategy is to retain the
1-benzylpiperidine fragment from donepezil with ChEs inhibition
and introduce the benzamide or 2-picolinamide moiety from
lazabemide, Ro16–6491 and moclobemide with MAOs inhibitory
activity (Figure 1). Besides, we also introduces a 2-thiophene-
carboxamide moiety for its structural similarity to benzamide.
Since the length of the linker could affect the accommodation of
the hybrid in AChE40, we changed the length of carbon spacer to
obtain optional conformation that could make the activity of
designed compounds better. Although a part of donepezil-like
compounds have been known, their biological activities such as
General procedures for the preparation of compounds w1–23
A solution of 1a–u (1.0 mmol) and 1, 0-carbonyldiimidazole
(1.2 mmol) in 10 mL of anhydrous CH2Cl2 was stirred at room
temperature for 1 h. The 1-benzylpiperidin-4-amine or 2-(1-
benzylpiperidin-4-yl) ethanamine (1.0 mmol) was added to the
solution, and stirring was continued overnight. The reaction
mixture was diluted with H2O and extracted with CH2Cl2. The
organic extracts were combined, washed with brine, and dried
with anhydrous Na2SO4, and the solvent was evaporated in vacuo
to give the crude product, which was purified by silica gel
chromatography with CH2Cl2:MeOH ¼ 30:1 as an eluent to afford
corresponding target compounds.
the inhibition of ChEs and MAOs have not been determined41–43
.
Meanwhile, the amide of 2-picolinamide moiety also has the
ability to chelate metal ions44,45. In this study, we described the
design, synthesis, and evaluation of series of donepezil-like
compounds which were found to show potential abilities,
including the inhibition of ChEs, inhibition of MAOs, metal
N-(1-benzylpiperidin-4-yl)benzamide (w1). Yield 68%, white
solid, m.p. 171–173 ꢀC; IR (KBr) ꢁ 3303, 2916, 2792, 1632,
Figure 1. Design strategy for donepezil-like
compounds.

DOI: 10.1080/14756366.2016.1201814
Donepezil-based agents
3
1548, 1381, 1341, 743, 701 cmꢁ1; 1H NMR (500 MHz, DMSO) ꢀ 8.42 (t, J ¼ 5.3 Hz, 1H), 7.85 (d, J ¼ 7.3 Hz, 2H), 7.52 (t,
8.24 (d, J ¼ 7.7 Hz, 1H), 7.85 (d, J ¼ 7.2 Hz, 2H), 7.53 (t, J ¼ 7.3 Hz, 1H), 7.47 (t, J ¼ 7.3 Hz, 2H), 7.36–7.27 (m, 4H),
J ¼ 7.2 Hz, 1H), 7.46 (t, J ¼ 7.4 Hz, 2H), 7.37–7.30 (m, 4H), 7.29– 7.25 (t, J ¼ 6.9 Hz, 1H), 3.44 (s, 2H), 3.30 (dd, J ¼ 13.3, 6.9 Hz,
7.25 (m, 1H), 3.85–3.71 (m, 1H), 3.48 (s, 2H), 2.84 (d, 2H), 2.79 (d, J ¼ 11.3 Hz, 2H), 1.90 (t, J ¼ 11.0 Hz, 2H), 1.68 (d,
J ¼ 11.0 Hz, 2H), 2.04 (t, J ¼ 11.0 Hz, 2H), 1.79 (d, J ¼ 10.6 Hz, J ¼ 12.0 Hz, 2H), 1.48 (d, J ¼ 7.2 Hz, 2H), 1.33–1.28 (m, 1H),
2H), 1.60 (qd, J ¼ 12.1, 3.5 Hz, 2H). 13C NMR (125 MHz, 1.17 (qd, J ¼ 12.3, 3.4 Hz, 2H). 13C NMR (125 MHz, DMSO) ꢀ
DMSO) ꢀ 166.21, 138.96, 135.22, 131.48, 129.26, 128.63, 166.56, 139.17, 135.26, 131.40, 129.21, 128.67, 128.55, 127.58,
127.72, 127.36, 62.58, 52.70, 47.39, 31.89. ESI-MS m/z: 295.12 127.22, 63.02, 53.75, 37.40, 36.39, 33.52, 32.38. ESI-MS m/z:
[M + H]+; HRMS: calcd for C19H22N2O [M + H]+ 295.1805, 323.19
found 295.1803.
[M + H]+;
HRMS:
calcd
for
C21H27N2O
[M + H]+ 323.2118, found 323.2116.
N-(1-benzylpiperidin-4-yl)-4-chlorobenzamide (w2). Yield 73%, N-(2-(1-benzylpiperidin-4-yl)ethyl)-2-chlorobenzamide
pale white solid, m.p. 192–194 ꢀC; IR (KBr) ꢁ 3340, 2924, 1631, (w7). Yield 70%, white solid, m.p. 145–147 ꢀC; IR (KBr) ꢁ
1
1544, 1453, 1070, 803, 757, 706 cmꢁ1
;
1H NMR (500 MHz, 3276, 2918, 1640, 1553, 1452, 763, 733, 709 cmꢁ1; H NMR
DMSO) ꢀ 8.32 (d, J ¼ 7.6 Hz, 1H), 7.88 (d, J ¼ 8.1 Hz, 2H), 7.54 (500 MHz, DMSO) ꢀ 8.37 (t, J ¼ 5.2 Hz, 1H), 7.50 (d, J ¼ 7.9 Hz,
(d, J ¼ 8.1 Hz, 2H), 7.38–7.30 (m, 4H), 7.27 (t, J ¼ 6.8 Hz, 1H), 1H), 7.46–7.42 (m, 1H), 7.39 (d, J ¼ 4.2 Hz, 2H), 7.35–7.29 (m,
3.83–3.70 (m, 1H), 3.49 (s, 2H), 2.84 (d, J ¼ 11.5 Hz, 2H), 2.03 (t, 4H), 7.25 (t, J ¼ 6.9 Hz, 1H), 3.45 (s, 2H), 3.27 (dd, J ¼ 13.0,
J ¼ 14.4 Hz, 2H), 1.79 (d, J ¼ 11.1 Hz, 2H), 1.59 (qd, J ¼ 12.1, 6.9 Hz, 2H), 2.80 (d, J ¼ 11.3 Hz, 2H), 1.90 (t, J ¼ 10.9 Hz, 2H),
3.2 Hz, 2H). 13C NMR (125 MHz, DMSO) ꢀ 165.03, 139.13, 1.67 (d, J ¼ 12.0 Hz, 2H), 1.46 (q, J ¼ 6.9 Hz, 2H), 1.42–1.34 (m,
136.28, 133.97, 129.69, 129.19, 128.69, 128.62, 127.31, 62.60, 1H), 1.17 (qd, J ¼ 12.3, 3.4 Hz, 2H). 13C NMR (125 MHz,
52.69, 47.54, 31.93. ESI-MS m/z: 329.13 [M + H]+; HRMS: calcd DMSO) ꢀ 166.65, 139.10, 137.78, 131.02, 130.27, 130.00,
for C19H21ClN2O [M + H]+ 329.1415, found 329.1416.
129.26, 129.19, 128.56, 127.54, 127.25, 63.01, 53.78, 37.06,
36.04, 33.26, 32.27. ESI-MS m/z: 357.16 [M + H]+; HRMS: calcd
for C21H26ClN2O [M + H]+ 357.1728, found 357.1727.
N-(1-benzylpiperidin-4-yl)-5-chloropicolinamide
(w3). Yield
65%, yellow solid, m.p. 74–76 ꢀC; IR (KBr) ꢁ 3315, 2945,
2789, 1656, 1527, 1469, 1079, 771, 707, 652 cmꢁ1; H NMR N-(2-(1-benzylpiperidin-4-yl)ethyl)-2-(2-bromophenyl)acetamide
1
(500 MHz, DMSO) ꢀ 8.70 (d, J ¼ 2.2 Hz, 1H), 8.59 (d, J ¼ 8.4 Hz, (w8). Yield 58%, yellow solid, m.p(0).83–85 ꢀC; IR (KBr) ꢁ
1H), 8.13 (dd, J ¼ 8.4, 2.4 Hz, 1H), 8.05 (d, J ¼ 8.4 Hz, 1H), 7.39– 3124, 2921, 1646, 1546, 1448, 1057, 755, 660 cmꢁ1; H NMR
1
7.29 (m, 4H), 7.26 (t, J ¼ 6.8 Hz, 1H), 3.89–3.70 (m, 1H), 3.48 (s, (500 MHz, DMSO) ꢀ 7.98 (t, J ¼ 5.1 Hz, 1H), 7.59 (d, J ¼ 8.1 Hz,
2H), 2.81 (d, J ¼ 11.6 Hz, 2H), 2.07 (t, J ¼ 11.0 Hz, 2H), 1.77 (d, 1H), 7.37–7.28 (m, 6H), 7.26 (t, J ¼ 6.9 Hz, 1H), 7.22–7.17 (m,
J ¼ 10.9 Hz, 2H), 1.66 (qd, J ¼ 12.1, 3.5 Hz, 2H). 13C NMR 1H), 3.58 (s, 2H), 3.46 (s, 2H), 3.12 (dd, J ¼ 12.7, 6.8 Hz, 2H),
(125 MHz, DMSO) ꢀ 162.95, 148.94, 147.47, 138.04, 134.36, 2.80 (d, J ¼ 11.0 Hz, 2H), 1.91 (t, J ¼ 11.0 Hz, 2H), 1.63 (d,
129.96, 129.64, 128.72, 127.67, 123.89, 62.26, 52.36, 46.91, J ¼ 12.2 Hz, 2H), 1.37 (dd, J ¼ 13.6, 6.8 Hz, 2H), 1.32–1.29 (m,
31.22. ESI-MS m/z: 330.11 [M + H]+; HRMS: calcd for 1H), 1.14 (qd, J ¼ 12.4 Hz, 3.5 Hz, 2H). 13C NMR (125 MHz,
C18H21ClN3O [M + H]+ 330.1368, found 330.1365.
DMSO) ꢀ 169.30, 138.53, 135.69, 132.71, 132.32, 129.44,
129.08, 128.57, 128.02, 127.37, 122.17, 62.87, 53.59, 42.88,
36.79, 36.05, 33.05, 31.99. ESI-MS m/z: 415.08 [M + H]+;
HRMS: calcd for C22H27BrN2O [M + H]+ 415.1380, found
415.1378.
N-(1-benzylpiperidin-4-yl)nicotinamide (w4). Yield 75%, yellow
solid, m.p. 133–135 ꢀC; IR (KBr) ꢁ 3294, 2917, 2793, 1632, 1551,
1063, 742, 701, 660 cmꢁ1; 1H NMR (500 MHz, DMSO) ꢀ 9.01 (s,
1H), 8.71 (d, J ¼ 3.6 Hz, 1H), 8.46 (d, J ¼ 7.5 Hz, 1H), 8.19 (d,
J ¼ 7.9 Hz, 1H), 7.50 (dd, J ¼ 7.9, 4.8 Hz, 1H), 7.37–7.30 (m, 5H),
7.27 (t, J ¼ 6.4 Hz, 1H), 3.81 (qd, J ¼ 11.7, 5.7 Hz, 1H), 3.49 (s,
2H), 2.84 (d, J ¼ 11.7 Hz, 2H), 2.05 (t, J ¼ 10.9 Hz, 2H), 1.82 (d,
J ¼ 10.9 Hz, 2H), 1.60 (qd, J ¼ 12.1, 3.5 Hz, 2H). 13C NMR
(125 MHz, DMSO) ꢀ 164.78, 152.16, 148.84, 139.01, 135.52,
130.67, 129.26, 128.64, 127.36, 123.86, 62.58, 52.60, 47.53, 31.84.
ESI-MS m/z: 296.10 [M + H]+; HRMS: calcd for C18H22N3O
[M + H]+ 296.1757, found 296.1755.
2-benzoyl-N-(2-(1-benzylpiperidin-4-yl)ethyl)benzamide
(w9). Yield 65%, white solid, m.p. 144–146 ꢀC; IR (KBr) ꢁ
3294, 2963, 1678, 1261, 800, 698 cmꢁ1; 1H NMR (500 MHz,
DMSO) ꢀ 7.72 (d, J ¼ 7.0 Hz, 1H), 7.59–7.49 (m, 2H), 7.39–7.29
(m, 7H), 7.29–7.21 (m, 4H), 3.41 (s, 2H), 3.40–3.35 (m, 2H), 2.71
(d, J ¼ 2.4 Hz, 2H), 1.84 (dd, J ¼ 19.7, 9.2 Hz, 2H), 1.60 (d,
J ¼ 12.4 Hz, 1H), 1.48 (d, J ¼ 12.4 Hz, 1H), 1.44–1.32 (m, 1H),
1.32–1.21 (m, 1H), 1.15 (dd, J ¼ 6.9, 3.6 Hz, 1H), 1.00 (qd,
J ¼ 11.7, 3.1 Hz, 2H). 13C NMR (125 MHz, DMSO) ꢀ 167.11,
150.09, 140.72, 138.85, 132.84, 131.15, 129.61, 129.29, 128.87,
N-(1-benzylpiperidin-4-yl)-5-chlorothiophene-2-carboxamide
(w5). Yield 78%, pale white, solid m.p. 174–176 ꢀC; IR (KBr) ꢁ 128.56, 128.51, 127.29, 126.32, 123.16, 122.80, 62.88, 53.52,
3299, 2924, 1613, 1554, 1454, 742, 701 cmꢁ1 1H NMR 37.08, 35.26, 33.44, 32.16. ESI-MS m/z: 427.20 [M + H]+;
;
(500 MHz, DMSO)
ꢀ
8.34 (d, J ¼ 7.6 Hz, 1H), 7.68 (d, HRMS: calcd for C28H31N2O2 [M + H]+ 427.2380, found
J ¼ 4.0 Hz, 1H), 7.37–7.30 (m, 4H), 7.26 (t, J ¼ 6.8 Hz, 1H), 427.2379.
7.18 (d, J ¼ 4.0 Hz, 1H), 3.78–3.66 (m, 1H), 3.48 (s, 2H), 2.83 (d,
J ¼ 11.4 Hz, 2H), 2.03 (t, J ¼ 11.4 Hz, 2H), 1.79 (d, J ¼ 10.9 Hz,
N-(2-(1-benzylpiperidin-4-yl)ethyl)-3-nitrobenzamide
2H), 1.57 (qd, J ¼ 12.1, 3.4 Hz, 2H). 13C NMR (125 MHz,
DMSO) ꢀ 160.12, 139.54, 138.65, 133.43, 129.38, 128.64,
128.43, 128.36, 127.42, 62.50, 52.49, 47.57, 31.70. ESI-MS
m/z: 335.07 [M + H]+; HRMS: calcd for C17H20ClN2OS
[M + H]+ 335.09, found 335.0977.
(w10). Yield 75%, yellow solid, m.p. 125–127 ꢀC; IR (KBr) ꢁ
3353, 2923, 1638, 1530, 1350, 821, 724 cmꢁ1 1H NMR
;
(500 MHz, DMSO) ꢀ 8.81 (t, J ¼ 5.3 Hz, 1H), 8.68 (s, 1H), 8.39
(dd, J ¼ 8.0, 1.7 Hz, 1H), 8.30 (d, J ¼ 8.0 Hz, 1H), 7.79 (t,
J ¼ 8.0 Hz, 1H), 7.36–7.27 (m, 4H), 7.25 (t, J ¼ 7.0 Hz, 1H), 3.45
(s, 2H), 3.35 (dd, J ¼ 11.8, 4.5 Hz, 2H), 2.80 (d, J ¼ 11.0 Hz, 2H),
N-(2-(1-benzylpiperidin-4-yl)ethyl)benzamide (w6). Yield 58%, 1.91 (t, J ¼ 11.0 Hz, 2H), 1.67 (t, J ¼ 15.3 Hz, 2H), 1.51 (dd,
yellow solid, m.p. 94–96 ꢀC; IR (KBr) ꢁ 3314, 2924, 1631, 1535, J ¼ 14.2, 7.0 Hz, 2H), 1.36–1.32 (m, 1H), 1.19 (qd, J ¼12.2,
1
1490, 1435, 802, 774, 695 cmꢁ1; H NMR (500 MHz, DMSO) ꢀ 3.5 Hz, 2H). 13C NMR (125 MHz, DMSO) ꢀ 164.40, 148.28,

4
F. Li et al.
J Enzyme Inhib Med Chem, Early Online: 1–13
139.53, 136.53, 134.05, 130.54 (s), 129.22, 128.55, 127.24, 1H), 8.18–8.14 (m, 2H), 8.10 (d, J ¼ 8.0 Hz, 1H), 7.89 (dd,
126.14, 122.32, 62.97, 53.70, 37.65, 36.17, 33.44, 32.31. ESI-MS J ¼ 11.2, 4.1 Hz, 1H), 7.74 (t, J ¼ 7.5 Hz, 1H), 7.35–7.27 (m, 4H),
m/z: 368.19 [M + H]+; HRMS: calcd for C21H26N3O3 7.25 (t, J ¼ 6.8 Hz, 1H), 3.42 (dd, J ¼ 15.6, 8.2 Hz, 4H), 2.80 (d,
[M + H]+ 368.1969, found 368.1970.
J ¼ 11.0 Hz, 2H), 1.91 (t, J ¼ 11.0 Hz, 2H), 1.72 (d, J ¼ 12.1 Hz,
2H), 1.55 (dd, J ¼ 14.2, 7.0 Hz, 2H), 1.37–1.29 (m, 1H), 1.17 (qd,
J ¼ 12.1, 3.3 Hz, 2H). 13C NMR (125 MHz, DMSO) ꢀ 164.40,
150.78, 146.48, 138.28, 130.94, 129.94, 129.65, 129.33, 129.23,
128.58, 128.53, 128.46, 127.35, 119.09, 62.85, 53.62, 37.20,
36.45, 33.42, 32.18. ESI-MS m/z: 374.21 [M + H]+; HRMS: calcd
for C24H28N3O [M + H]+ 374.2227, found 374.2226.
N-(2-(1-benzylpiperidin-4-yl)ethyl)-4-nitrobenzamide
(w11). Yield 73%, yellow solid, m.p. 113–115 ꢀC; IR (KBr) ꢁ
3330, 2928, 1644, 1597, 1542, 1518, 741, 726, 698 cmꢁ1; H
1
NMR (500 MHz, DMSO) ꢀ 8.76 (t, J ¼ 5.2 Hz, 1H), 8.32 (d,
J ¼ 8.7 Hz, 2H), 8.07 (d, J ¼ 8.7 Hz, 2H), 7.35–7.27 (m, 4H), 7.25
(t, J ¼ 6.9 Hz, 1H), 3.45 (s, 2H), 3.34 (d, J ¼ 11.5 Hz, 2H), 2.80 (d,
J ¼ 11.0 Hz, 2H), 1.91 (t, J ¼ 11.0 Hz, 2H), 1.68 (d, J ¼ 12.0 Hz, N-(2-(1-benzylpiperidin-4-yl)ethyl)nicotinamide (w16). Yield
2H), 1.50 (dd, J ¼ 14.1, 7.0 Hz, 2H), 1.34–1.31 (m, 1H), 1.18 (qd, 65%, yellow solid, m.p. 64–66 ꢀC; IR (KBr) ꢁ 3306, 2925,
J ¼ 12.2, 3.5 Hz, 2H). 13C NMR (126 MHz, DMSO) ꢀ 164.99, 1636, 1548, 1311, 731, 707, 663 cmꢁ1
;
1H NMR (500 MHz,
149.40, 140.81, 139.06, 129.24, 129.09, 128.55, 127.24, 123.95, DMSO) ꢀ 9.00 (s, 1H), 8.71 (dd, J ¼ 4.8, 1.4 Hz, 1H), 8.61 (t,
62.98, 53.69, 37.65, 36.13, 33.46, 32.28. ESI-MS m/z: 368.18 J ¼ 5.2 Hz, 1H), 8.18 (dt, J ¼ 7.9, 1.8 Hz, 1H), 7.51 (dd, J ¼ 7.9,
[M + H]+; HRMS: calcd for C21H26N3O3 [M + H]+ 368.1969, 4.8 Hz, 1H), 7.35–7.28 (m, 5H), 7.25 (t, J ¼ 6.9 Hz, 1H), 3.44 (s,
found 368.1968.
2H), 3.31 (d, J ¼ 6.9 Hz, 2H), 2.79 (d, J ¼ 11.9 Hz, 2H), 1.91 (t,
J ¼ 10.8 Hz, 2H), 1.68 (d, J ¼ 11.9 Hz, 2H), 1.49 (dd, J ¼ 14.2,
7.0 Hz, 2H), 1.35–1.30 (m, 1H), 1.18 (qd, J ¼ 12.2, 3.5 Hz, 2H).
13C NMR (125 MHz, DMSO) ꢀ 165.14, 152.13, 148.73, 135.34,
129.28, 128.56, 127.29, 123.89, 62.92, 53.67, 37.42, 36.20, 33.42,
32.24. ESI-MS m/z: 324.18 [M + H]+; HRMS: calcd for
C20H26N3O [M + H]+ 324.2070, found 324.2068.
N-(2-(1-benzylpiperidin-4-yl)ethyl)-4-methoxybenzamide
(w12). Yield 75%, yellow solid, m.p. 125–127 ꢀC; IR (KBr) ꢁ
3293, 2918, 1627, 1558, 1509, 1255, 843, 736, 673 cmꢁ1; H
1
NMR (500 MHz, DMSO) ꢀ 8.26 (t, J ¼ 5.3 Hz, 1H), 7.82 (d,
J ¼ 8.8 Hz, 2H), 7.35–7.28 (m, 5H), 7.25 (t, J ¼ 6.9 Hz, 1H), 6.99
(d, J ¼ 8.8 Hz, 2H), 3.82 (s, 3H), 3.44 (s, 2H), 3.28 (dd, J ¼ 13.4,
6.6 Hz, 2H), 2.79 (d, J ¼ 11.3 Hz, 2H), 1.90 (t, J ¼ 10.9 Hz, 2H), N-(2-(1-benzylpiperidin-4-yl)ethyl)isonicotinamide (w17). Yield
1.68 (d, J ¼ 11.7 Hz, 2H), 1.47 (dd, J ¼ 14.1, 6.9 Hz, 2H), 1.32– 78%, yellow oil; IR (KBr) ꢁ 3294, 2925, 1648, 1551, 1451, 1309,
1.28 (m, 1H), 1.17 (qd, J ¼ 12.3, 3.4 Hz, 2H). 13C NMR 1065, 742, 701, 660 cmꢁ1; 1H NMR (500 MHz, DMSO) ꢀ 8.73 (d,
(125 MHz, DMSO) ꢀ 166.13, 161.90, 139.04, 129.38, 129.27, J ¼ 5.8 Hz, 1H), 7.75 (d, J ¼ 5.8 Hz, 1H), 7.35–7.27 (m, 4H), 7.25
128.56, 127.43, 127.26, 113.91, 62.98, 55.80, 53.72, 37.33, 36.43, (t, J ¼ 6.9 Hz, 1H), 7.03 (s, 1H), 3.44 (s, 2H), 3.33–3.29 (m, 2H),
33.49, 32.31. ESI-MS m/z: 353.20 [M + H]+; HRMS: calcd for 2.79 (d, J ¼ 11.3 Hz, 2H), 1.96–1.86 (m, 2H), 1.66 (t, J ¼ 14.0 Hz,
C22H29N2O [M + H]+ 353.2224, found 353.2222.
1H), 1.49 (dd, J ¼ 14.0, 6.9 Hz, 1H), 1.34–1.28 (m, 1H), 1.18 (qd,
J ¼ 12.1, 3.5 Hz, 2H). 13C NMR (125 MHz, DMSO) ꢀ 165.04,
150.63, 142.07, 138.88, 129.29, 128.57, 127.30, 121.65,
62.91, 53.64, 37.50, 36.09, 33.40, 32.20. ESI-MS m/z: 324.18
[M + H]+; HRMS: calcd for C20H26N3O [M + H]+ 324.2070, found
324.2071.
N-(2-(1-benzylpiperidin-4-yl)ethyl)-4-methylbenzamide
(w13). Yield 77%, yellow solid m.p. 85–92 ꢀC; IR (KBr) ꢁ 3321,
2925, 1633, 1550, 1453, 1311, 730, 697 cmꢁ1 1H NMR
;
(500 MHz, DMSO)
ꢀ
8.31 (t, J ¼ 5.4 Hz, 1H), 7.75 (d,
J ¼ 8.1 Hz, 2H), 7.35–7.29 (m, 4H), 7.25–7.23 (m, 3H), 3.45 (s,
2H), 3.29 (dd, J ¼ 13.4, 6.9 Hz, 2H), 2.79 (d, J ¼ 11.0 Hz, 2H),
2.36 (s, 3H), 1.92 (t, J ¼ 11.0 Hz, 2H), 1.68 (d, J ¼ 11.9 Hz, 2H),
1.47 (dd, J ¼ 14.2, 6.9 Hz, 2H), 1.33–1.29 (m, 1H), 1.17 (qd,
J ¼ 12.2, 3.4 Hz, 2H). 13C NMR (125 MHz, DMSO) ꢀ 166.40,
141.21, 139.07, 132.44, 129.23, 129.18, 128.55, 127.59, 127.25,
62.95, 53.72, 37.32, 36.40, 33.49, 32.33, 21.38. ESI-MS m/z:
N-(2-(1-benzylpiperidin-4-yl)ethyl)-5-chloropicolinamide
(w18). Yield 66%, yellow solid, m.p. 65–69 ꢀC; IR (KBr) ꢁ 3415,
3301, 2930, 2855, 1663, 1533, 1455, 1110, 790, 732, 684 cmꢁ1; 1H
NMR (500 MHz, DMSO) ꢀ 8.80 (t, J ¼ 5.8 Hz, 1H), 8.70 (d,
J ¼ 2.2 Hz, 1H), 8.13 (dd, J ¼ 8.4, 2.4 Hz, 1H), 8.04 (d, J ¼ 8.4 Hz,
1H), 7.38–7.28 (m, 4H), 7.26 (t, J ¼ 6.9 Hz, 1H), 3.47 (s, 2H), 3.34–
3.30 (m, 2H), 2.80 (d, J ¼ 8.7 Hz, 2H), 1.92 (s, 2H), 1.69 (d,
J ¼ 12.3 Hz, 2H), 1.49 (dd, J ¼ 14.0, 6.9 Hz, 2H), 1.33–1.28 (m,
1H), 1.17 (qd, J ¼ 12.3, 3.4 Hz, 2H). 13C NMR (125 MHz, DMSO)
ꢀ 163.38, 149.15, 147.47, 138.84, 138.84, 137.98, 134.24, 129.30,
128.56, 127.30, 123.80, 62.89, 53.64, 37.10, 36.29, 33.36, 32.18.
ESI-MS m/z: 358.15 [M + H]+; HRMS: calcd for C20H25ClN3O
[M + H]+ 358.1681, found 358.1680.
337.21
[M + H]+;
HRMS:
calcd
for
C22H29N2O
[M + H]+ 337.2274, found 337.2273.
N-(2-(1-benzylpiperidin-4-yl)ethyl)-4-chlorobenzamide
(w14). Yield 88%, yellow solid, m.p. 122–124 ꢀC; IR (KBr) ꢁ
3305, 2914, 1632, 1543, 845, 743, 701 cmꢁ1
;
1H NMR
(500 MHz, DMSO)
ꢀ
8.49 (t, J ¼ 5.3 Hz, 1H), 7.86 (d,
J ¼ 8.5 Hz, 2H), 7.54 (d, J ¼ 8.5 Hz, 2H), 7.37–7.27 (m, 4H),
7.25 (t, J ¼ 6.7 Hz, 1H), 3.44 (s, 2H), 3.30 (dd, J ¼ 13.4, 6.7 Hz,
2H), 2.79 (d, J ¼ 11.0 Hz, 2H), 1.91 (t, J ¼ 11.0 Hz, 2H), 1.68 (d,
J ¼ 11.9 Hz, 2H), 1.48 (dd, J ¼ 14.1, 7.0 Hz, 2H), 1.32–1.29 (m,
1H), 1.17 (qd, J ¼ 12.2, 3.4 Hz, 2H). 13C NMR (125 MHz,
DMSO) ꢀ 165.59, 136.32, 135.07, 133.89, 129.53, 129.41,
128.80, 128.61, 127.43, 62.77, 53.59, 37.44, 36.19, 33.31,
32.08. ESI-MS m/z: 357.16 [M + H]+; HRMS: calcd for
C21H26ClN2O [M + H]+ 357.1728, found 357.1727.
N-(2-(1-benzylpiperidin-4-yl)ethyl)-5-fluoropicolinamide
(w19). Yield 55%, yellow solid, m.p. 81–83 ꢀC; IR (KBr) ꢁ
1
3306, 2928, 1661, 1530, 1469, 1227, 732, 682 cmꢁ1; H NMR
(500 MHz, DMSO) ꢀ 8.71 (t, J ¼ 5.7 Hz, 1H), 8.64 (d, J ¼ 2.7 Hz,
1H), 8.11 (dd, J ¼ 8.7, 4.7 Hz, 1H), 7.90 (d, J ¼ 8.7 Hz, 1H), 7.34–
7.28 (m, 4H), 7.24 (t, J ¼ 6.9 Hz, 1H), 3.44 (s, 2H), 3.38–3.33 (m,
2H), 2.78 (d, J ¼ 11.2 Hz, 2H), 1.89 (t, J ¼ 11.2 Hz, 2H), 1.68 (d,
J ¼ 11.2 Hz, 2H), 1.49 (dd, J ¼ 14.0, 6.9 Hz, 2H), 1.31–1.26 (m,
1H), 1.16 (qd, J ¼ 12.2, 3.4 Hz, 2H). 13C NMR (125 MHz,
DMSO) ꢀ 163.27, 160.04, 147.37, 139.03, 137.15, 129.24,
128.54, 127.24, 124.85, 124.43, 62.96, 53.68, 37.08, 36.36,
33.42, 32.26. ESI-MS m/z: 342.18 [M + H]+; HRMS: calcd for
C20H25FN3O [M + H]+ 342.1976, found 342.1974.
N-(2-(1-benzylpiperidin-4-yl)ethyl)quinoline-2-carboxamide
(w15). Yield 85%, yellow solid, m.p. 95–97 ꢀC; IR (KBr) ꢁ
3382, 2938, 1667, 1522, 1496, 781, 737, 697 cmꢁ1; H NMR
1
(500 MHz, DMSO) ꢀ 8.90 (t, J ¼ 5.9 Hz, 1H), 8.57 (d, J ¼ 8.5 Hz,

DOI: 10.1080/14756366.2016.1201814
Donepezil-based agents
5
N-(2-(1-benzylpiperidin-4-yl)ethyl)-6-methylpicolinamide
Sigma-Aldrich (St. Louis, MO). The capacity of the test
(w20). Yield 65%, yellow oil; IR (KBr) ꢁ 3389, 2923, 1673, compounds (w1–23) to inhibit AChE and BuChE activities were
1593, 1526, 1452, 740, 699 cmꢁ1; 1H NMR (500 MHz, DMSO) ꢀ assessed by Ellman’s method. Stock solution of test compounds
8.58 (t, J ¼ 5.8 Hz, 1H), 7.90–7.80 (m, 2H), 7.45 (d, J ¼ 7.3 Hz, was dissolved in a minimum volume of DMSO (1%) and was
1H), 7.36–7.27 (m, 4H), 7.25 (t, J ¼ 6.9 Hz, 1H), 3.43 (s, 2H), diluted using the buffer solution (50 mM Tris-HCl, pH¼ 8.0,
3.35 (d, J ¼ 13.7 Hz, 2H), 2.78 (t, J ¼ 11.3 Hz, 2H), 2.57 (s, 3H), 0.1 M NaCl, 0.02 M MgCl2ꢂ6H2O). In 96-well plates, 160 mL of
1.90 (t, J ¼ 11.3 Hz, 2H), 1.69 (d, J ¼ 11.9 Hz, 2H), 1.49 (dd, 1.5 mM DTNB, 50 mL of AChE (0.22 U/mL prepared in 50 mM
J ¼ 14.2, 6.9 Hz, 2H), 1.31–1.23 (m, 1H), 1.17 (qd, J ¼ 12.1, Tris-HCl, pH ¼8.0, 0.1% w/v bovine serum albumin, BSA) or
3.3 Hz, 2H). 13C NMR (125 MHz, DMSO) ꢀ 164.36, 157.57, 50 mL of BuChE (0.12 U/mL prepared in 50 mM Tris-HCl,
149.84, 138.32, 129.34, 128.58, 127.31, 126.37, 119.36, 62.93, pH ¼ 8.0, 0.1% w/v BSA) were incubated with 10 mL of various
53.64, 36.97, 36.45, 33.42, 32.16, 24.32. ESI-MS m/z: 338.22 concentrations of test compounds (0.001–100 mM) at 37 ꢀC for
[M + H]+; HRMS: calcd for C21H28N3O [M + H]+ 338.2227, 6 min followed by the addition of the substrates (30 mL)
found 338.2226.
acetylthiocholine iodide (15 mM) or S-butyrylthiocholine iodide
(15 mM) and the absorbance was measured at different time
intervals (0, 60, 120, and 180 s) at a wavelength of 405 nm. The
concentration of compound producing 50% of enzyme activity
inhibition (IC50) was calculated by nonlinear regression analysis
of the response-concentration (log) curve, using the Graph-Pad
Prism program package (Graph Pad Software, San Diego, CA).
Results are expressed as the mean SD of at least three different
experiments performed in triplicate.
N-(2-(1-benzylpiperidin-4-yl)ethyl)-5-chlorothiophene-2-carboxa-
mide (w21). Yield 80%, yellow solid, m.p. 115–117 ꢀC; IR
(KBr) ꢁ 3299, 2929, 1661, 1531, 1453, 1108, 730, 682 cmꢁ1; 1H
NMR (500 MHz, DMSO) ꢀ 8.53 (t, J ¼ 5.4 Hz, 1H), 7.63 (d,
J ¼ 4.0 Hz, 1H), 7.35–7.28 (m, 4H), 7.25 (dd, J ¼ 9.3, 4.4 Hz, 1H),
7.18 (d, J ¼ 4.0 Hz, 1H), 3.47 (s, 2H), 3.26 (dd, J ¼ 13.4, 6.7 Hz,
2H), 2.80 (d, J ¼ 11.1 Hz, 2H), 1.93 (t, J ¼ 11.1 Hz, 2H), 1.67 (d,
J ¼ 12.1 Hz, 2H), 1.46 (dd, J ¼ 14.2, 7.0 Hz, 2H), 1.31–1.29 (m,
1H), 1.17 (qd, J ¼ 12.3, 3.5 Hz, 2H). 13C NMR (125 MHz,
DMSO) d 160.40, 139.90, 133.07, 129.27, 128.57, 128.43,
128.08, 127.31, 62.87, 53.64, 37.30, 36.28, 33.36, 32.21. ESI-
MS m/z: 362.12 [M + H]+; HRMS: calcd for C19H24ClN2OS
[M + H]+ 363.1292, found 363.1293.
Inhibitory activity against hMAO-A and hMAO-B
Monoamine Oxidases (hMAO-A, hMAO-B, E.C. 1.4.3.4),
p-tyramine, Amplex Red and horseradish peroxidase (E.C.
1.11.1.7) were purchased from Sigma-Aldrich. Firstly, MAOs
activity were adjusted to obtain in our experimental conditions the
same reaction velocity in the presence of both isoforms (i.e., to
oxidize (in the control group) the same concentration of substrate:
165 pmol of p-tyramine/min(hMAO-A: 1.1 mg protein; specific
activity: 150 nmol of p-tyramine oxidized to p-hydroxyphenyla-
cetaldehyde/min/mg protein; hMAO-B: 7.5 mg protein; specific
activity: 22 nmol of p-tyramine transformed/min/mg protein).
Then compounds were dissolved in DMSO (10 mM) and diluted
in 0.05 M KH2PO4/K2HPO4 buffer (pH ¼ 7.4) to the desired final
concentration. All the compounds are soluble at the tested
concentration. Test drugs (20 mL) and MAO (80 mL) were
incubated at 37 ꢀC for 15 min in a flat-black-bottom 96-well
microtest plate in dark. The reaction was started by adding
200 mM Amplex Red reagent, 2 U/mL horseradish peroxidase, and
2 mM p-tyramine for hMAO at 37 ꢀC for 20 min. The production
of H2O2 and consequently, of resorufin, was quantified at 37 ꢀC in
a SpectraMax Paradigm (Molecular Devices, Sunnyvale, CA)
multi-mode detection platform reader based on the fluorescence
generated (excitation, 545 nm; emission, 590 nm). The specific
fluorescence emission was calculated after subtraction of the
background activity. The background activity was determined
from wells containing all components except the MAO isoforms,
which were replaced by a sodium phosphate buffer solution
(0.05 mM, pH 7.4). The percent inhibition was calculated by the
following expression: (1 ꢁ IFi/IFc) ꢃ 100 in which IFi and IFc are
the Fuorescence intensities obtained for hMAO in the presence
and absence of inhibitors after subtracting the respective
background.
N-(2-(1-benzylpiperidin-4-yl)ethyl)-5-bromothiophene-2-carboxa-
mide (w22). Yield 59%, yellow solid, m.p. 124–126 ꢀC; IR
(KBr) ꢁ 3279, 2924, 1617, 1564, 1421, 1304, 736, 694 cmꢁ1; 1H
NMR (500 MHz, DMSO) ꢀ 8.50 (t, J ¼ 5.4 Hz, 1H), 7.58 (d,
J ¼ 4.0 Hz, 1H), 7.35–7.28 (m, 4H), 7.28 (d, J ¼ 4.0 Hz, 1H), 7.25
(t, J ¼ 6.9 Hz, 1H), 3.44 (s, 2H), 3.25 (dd, J ¼ 13.5, 6.6 Hz, 2H),
2.79 (d, J ¼ 10.9 Hz, 2H), 1.90 (t, J ¼ 10.9 Hz, 2H), 1.66 (d,
J ¼ 11.7 Hz, 2H), 1.46 (dd, J ¼ 14.2, 6.9 Hz, 2H), 1.36–1.29 (m,
1H), 1.17 (qd, J ¼12.3, 3.3 Hz, 2H). 13C NMR (125 MHz, DMSO)
ꢀ 160.40, 142.53, 139.03, 131.86, 129.24, 128.91, 128.55, 127.25,
116.81, 62.96, 53.67, 37.30, 36.27, 33.41, 32.26. ESI-MS m/z:
407.07 [M + H]+; HRMS: calcd for C19H24BrN2OS
[M + H]+ 407.0787, found 407.0786.
N-(2-(1-benzylpiperidin-4-yl)ethyl)-5-methylthiophene-2-carbox-
amide (w23). Yield 66%, yellow solid, m.p. 107–110 ꢀC; IR
(KBr) ꢁ 3281, 2923, 1616, 1562, 1449, 1308, 736, 694 cmꢁ1; 1H
NMR (500 MHz, DMSO) ꢀ 8.27 (t, J ¼ 5.4 Hz, 1H), 7.53 (d,
J ¼ 3.5 Hz, 1H), 7.37–7.28 (m, 4H), 7.25 (t, J ¼ 6.9 Hz, 1H), 6.83
(d, J ¼ 3.5 Hz, 1H), 3.45 (s, 2H), 3.24 (dd, J ¼ 13.4, 6.6 Hz, 2H),
2.79 (d, J ¼ 11.0 Hz, 2H), 2.46 (s, 3H), 1.91 (t, J ¼ 11.0 Hz, 2H),
1.67 (d, J ¼ 11.9 Hz, 2H), 1.45 (dd, J ¼ 14.2, 6.9 Hz, 2H), 1.33–
1.28 (m, 1H), 1.17 (qd, J ¼ 12.2, 3.4 Hz, 2H). 13C NMR
(125 MHz, DMSO) ꢀ 161.42, 144.60, 139.07, 138.21, 129.22,
128.55, 128.36, 127.24, 126.68, 62.96, 53.70, 37.19, 36.46, 33.44,
32.32, 15.56. ESI-MS m/z: 343.17 [M + H]+; HRMS: calcd for
C20H27N2OS [M + H]+ 343.1839, found 343.1838.
Kinetic study of AChE inbition
Biological activity
To obtain of the mechanism of action w18, reciprocal plots of
1/velocity versus 1/substrate were constructed at different
concentrations of the substrate thiocholine iodide 0.05–0.5 mM
by using Ellman’s method. Three concentrations of w18 were
selected for the studies: 0.440, 0.220 and 0.110 mM for the kinetic
analysis of AChE inhibition. The plots were assessed by a
weighted least-squares analysis that assumed the variance of
velocity (v) to be a constant percentage of v for the entire data set.
Slopes of these reciprocal plots were then plotted against the
Inhibitory activity against AChE and BuChE
Acetylcholinesterase (eeAChE, E.C. 3.1.1.7, from electric eel
and hAChE, EC 3.1.1.7, from human erythrocyes), butyrylcho-
linesterase (BuChE, E.C. 3.1.1.8, from equine serum), 5,50-
dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), S-
butyrylthiocholine iodide (BTCI), acetylthiocholine iodide
(ATCI), and donepezil hydrochloride were purchased from

6
F. Li et al.
J Enzyme Inhib Med Chem, Early Online: 1–13
concentration of w18 in a weighted analysis and Ki was (50 mM) was mixed with growing amounts of metal ions (10–
determined as the intercept on the negative x-axis. Data analysis 80 mM).
was performed with GraphPad Prism 4.03 software (GraphPad
Software Inc., San Diego, CA).
In vitro BBB permeation assay
Brain penetration of compounds was evaluated using a parallel
artificial membrane permeation assay (PAMPA). Commercial
Reversibility and kinetic studies of hMAO-B inhibition
To determine whether the inhibition of hMAO-B by the drugs were purchased from Sigma and Alfa Aesar. The porcine
donepezil-like compounds were reversible or irreversible, the brain lipid (PBL) was obtained from Avanti Polar Lipids. The
time-dependence of inhibition of the selected inhibitor w18 and donor microplate (PVDF membrane, pore size 0.45 mm) and the
reference compound pargyline were examined. Compounds were acceptor microplate were both from Millipore (St. Charles, MO).
allowed to pre-incubate with recombinant human hMAO-B for The 96-well UV plate (COSTAR@) was from Corning
various periods of time (0, 15, 30, 60 min) at 37 ꢀC in potassium Incorporated. The acceptor 96-well microplate was filled with
phosphate buffer (0.05 mM, pH 7.4). The concentrations of the 300 mL of PBS:EtOH (7:3), and the filter membrane was
compounds were about twofold the measured IC50 values for the impregnated with 4 mL of PBL in dodecane (20 mg/mL).
inhibition of hMAO-B. The reactions were subsequently diluted Compounds were dissolved in DMSO at 5 mg/mL and diluted
two-fold to yield a final enzyme concentration of 0.015 mg mLꢁ1 50-fold in PBS/EtOH (7:3) to achieve a concentration of 100 mg/
and concentrations of the inhibitors that are about equal to the mL, 200 mL of which was added to the donor wells. The acceptor
IC50 values. The reactions were incubated at 37 ꢀC for a further filter plate was carefully placed on the donor plate to form a
15 min. All measurements were carried out in triplicate and are sandwich, which was left undisturbed for 16 h at 25 ꢀC. After
expressed as mean SD.
incubation, the donor plate was carefully removed and the
Then, the type of hMAO-B inhibition was determined by concentration of compound in the acceptor wells was determined
constructing a set of Lineweaver–Burk plots. Six different using a UV plate reader (Flexsta-tion@ 3). Every sample was
concentrations of the substrate p-tyramine (0.05, 0.1, 0.25, 0.33, analyzed at five wavelengths, in four wells, in at least three
0.5, and 1.0 mM) was applied, and the initial catalytic rates of independent runs, and the results are given as the mean standard
hMAO-B were measured in the absence and in the presence deviation. In each experiment, 9 quality control standards of
of three different concentrations (6.28, 3.14, and 1.57 mM) of known BBB permeability were included to validate the analysis
compound w18. The assay conditions and measurements were set.
similar to the IC50 determination. The plots were assessed by a
weighted leastsquares analysis that assumed the variance of Rat pheochromocytoma (PC12) cell toxicity
velocity (v) to be a constant percentage of v for the entire data set.
The toxicity effect of compounds on the rat pheochromocytoma
Slopes of these reciprocal plots were then plotted against the
(PC12) cells was examined. The PC12 cells were routinely grown
concentration of w18 in a weighted analysis. Data analysis was
at 37 ꢀC in a humidified incubator with 5% CO2 in Dulbecco’s
performed with GraphPad Prism 4.03 software (San Diego, CA).
modified Eagle’s medium (DMEM) supplemented with 10%
bovine calf serum, 100 units/mL penicillin, and 100 units/mL of
Molecular modeling studies of w18 with ChEs and hMAO-B
streptomycin. Cells were subcultured in 96-well plates at a
Molecular modeling calculations and docking studies were seeding density of 10 000 cells per well and allowed to adhere
performed using Molecular Operating Environment (MOE) and grow. When cells reached the required confluence, they
software version 2008.10 (Chemical Computing Group, were placed into serum-free medium and treated with compound
Montreal, Canada). The X-ray crystallographic structure of w18. Twenty-four hours later the survival of cells was determined
AChE in complexed with donepezil (PDB code 1EVE), by MTT assay. Briefly, after incubation with 20 mL of MTT
hBuChE (PDB code 1P0I) and human MAO-B in complexed at 37 ꢀC for 4 h, living cells containing MTT formazan crystals
with 7-(3-chlorobenzyloxy)-4-formylcoumarin (PDB code 2V60) were solubilized in 200 mL DMSO. The absorbance of each well
were obtained from the Protein Data Bank. All water molecules in was measured using a microculture plate reader with a test
PDB files were removed and hydrogen atoms were subsequently wavelength of 570 nm and a reference wavelength of 630 nm.
added to the protein. The compound w18 was built using the Results are expressed as the mean SD of three independent
builder interface of the MOE program and energy minimized experiments.
using MMFF94x forcefield. Then the w18 was docked into the
active site of the protein by the ‘‘Triangle Matcher’’ method,
which generated poses by aligning the ligand triplet of atoms with
Results and discussion
Chemistry
the triplet of alpha spheres in cavities of tight atomic packing. The
Dock scoring in MOE software was done using ASE scoring
function and forcefield was selected as the refinement method.
The best 10 poses of molecules were retained and scored. After
docking, the geometry of resulting complex was studied using the
MOE’s pose viewer utility.
The synthetic route for target compounds is shown in Scheme 1.
Activation of different carboxylic acid compounds with 1,10-
carbonyldiimidazole (CDI) and subsequent coupling to 1-benzyl-
piperidin-4-amine or 2-(1-benzylpiperidin-4-yl) ethanamine
afforded target compounds w1–23 in good yields46–48
.
Structures of all synthesized compounds were characterized by
1H and 13C nuclear magnetic resonance (NMR) spectroscopy (see
Supplementary data).
Spectrophotometric measurement of complex with Cu2+
The study of metal chelation was performed in methanol at 298 K
using UV–Vis spectrophotometer (SHIMADZU UV-2450PC)
with wavelength ranging from 200 to 500 nm. The difference
Inhibitory activity against AChE and BuChE
UV–Vis spectra due to complex formation was obtained by To determine the potential interest of the new donepezil-like
numerical subtraction of the spectra of the metal ions alone and compounds for the treatment of AD, the ChEs inhibitory activities
the compound alone (at the same concentration used in the were assayed by the method of Ellmam et al. using donepezil and
mixture) from the spectra of the mixture. A fixed amount of w18 galanthamine as reference compounds49. The AChE inhibitory

DOI: 10.1080/14756366.2016.1201814
Donepezil-based agents
7
O
(i)
N
N
OH
H
N
R
R
+
H2N
n
n
O
w1, 2, 6, 7, 9, 10, 11, 12, 13, 14
1a-j
2a-b
O
N
N
N
N
(i)
(i)
R
H
N
OH
R
+
H2N
n
n
O
1k-n
2a-b
w3, 18, 19, 20
O
S
S
N
N
R
OH
H
N
+
R
H2N
n
n
Ow5, 21, 22, 23
1o-q
2a-b
O
N
H
N
(i)
N
R
+
OH
H2N
n
R
n
O
1r-u
2a-b
w4, 8, 15, 16, 17
1a R = H
1l R = 5-Cl
1m R = 5-F
1n R = 6-CH3
2a n = 0
2b n = 2
w1 R = H, n = 0
w13 R = 4-CH3, n = 2
w14 R = 4-Cl, n = 2
1b R = 2-Cl
w2 R = 3-Cl, n = 0
w3 R = 3-Cl, n = 0
1c R = 2-NO2
w15 R = 2-quinoline, n = 2
1d
R = 2-Ph CO
1o
R = 5-Cl
w4
R = 3-pyridine, n = 0
w16
R = 3-pyridine, n = 2
1e R = 3-NO2
1f R = 3-Cl
1p R = 5-Br
w5 R =5-Cl, n = 0
w6 R = H, n = 2
w7 R = 2-Cl, n = 2
w17 R = 4-pyridine, n = 2
w18 R = 5-Cl, n = 2
w19 R = 5-F, n = 2
1q R = 5-CH3
1g R = 4-NO2
1h R = 4-OCH3
1r R = 2-quinoline
1s R = 2-Br PhCH2
w8 R = 2-Br PhCH2, n = 2
w9
w20 R = 6-CH3, n = 2
1i
R = 4-CH3
1t
R = 3-pyridine
1u R = 4-pyridine
R = 2-Ph CO, n = 2
w21
R = 5-Cl, n = 2
1J R = 4-Cl
1k R = 3-Cl
w10 R = 3-NO2, n = 2
w11 R = 4-NO2, n = 2
w12 R = 4-OCH3, n = 2
w22 R = 5-Br, n = 2
w23 R = 5-CH3, n = 2
Scheme 1. Reagents and conditions: (i) 1,10-carbonyldiimidazole (CDI), CH2Cl2, r.t. overnight.
was tested against the Electrophorus electricus enzyme on the 4-position of phenyl ring (w11, w14), showed inhibitory
(eeAChE), and the inhibition of BuChE was carried out using activities for both ChEs better than those of compound w6. On
the equine serum enzyme (eqBuChE). The IC50 values of all the contrary, incorporating the benzoyl group in the 2-position of
tested compounds and their slectivity index for AChE over phenyl ring (w9) showed a decreased ChEs inhibitory activities
BuChE are summarized in Table 1.
(IC50 ¼52.2 mM for AChE; IC50 ¼3.57 mM for BuChE). This
From the table, it can be seen that most of our target might be attributed to the steric hindrance of the benzoyl group.
compounds showed potent inhibitory activity to both ChEs with Moreover, different substituents were introduced to the 5- or 6-
IC50 values ranging from micromolar to nanomolar. The length position of pyridine ring, most of them showed good inhibitory
of linker of the new compounds has great influences on the activity against AChE. Especially, the inhibitory activities of
inhibitory activities. From the IC50 values, compounds w6–23 w18 against AChE and BuChE (IC50 ¼0.220 mM for AChE;
with N-ethylcarboxamide linkage exhibited a higher activity than IC50 ¼1.23 mM for BuChE) were 12-fold and 10-fold more
those w1–5 with carboxamide linkage. This suggested that the potent, respectively, than those of reference compound galantha-
suitable linker length seemed to be 2 carbon atoms for ChEs mine (IC50 ¼2.67 mM for AChE; IC50 ¼12.7 mM for BuChE). To
inhibition. Then, the introduction of substituents with different extend the series of our compounds, compounds w21–23 with
sizes to phenyl ring and pyridine ring were planned. Among thiophene moiety were synthesized. As the trend with w21–23
compounds w6–20, w11 (IC50 ¼0.123 mM) showed the highest for AChE inhibition, w21–23 were also sensitive to the size of
inhibitory activity against AChE, which was 20 times stronger substituents at 5-position of thiophene ring. For example, the
than that of reference compound galanthamine (IC50 ¼2.67 mM), AChE inhibitory activity of w21 (IC50 ¼0.269 mM) for was 6-
and 3 times less than that of donepzil (IC50 ¼0.035 mM). fold more potent than that of w23 (IC50 ¼1.55 mM). However,
Compound w8 exhibited strongest inhibition against BuChE BuChE inhibitory activities of w21–23 seemed to be the
with IC50 value of 0.323 mM, which was 7 times more potent opposite trend compared with the AChE inhibitory activities,
than that of donepezil (IC50 ¼2.32 mM), and showed the highest inhibitory activity of w22 for BuChE (IC50 ¼0.208 mM) was 13-
selectivity with a selectivity index of 0.0392. Compared with the fold more potent than that of w21 (IC50 ¼2.75 mM). This
unsubstituted compound w6 (IC50 ¼1.51 mM for AChE; suggested the thiophene ring also might be favorable for ChEs
IC50 ¼1.82 mM for BuChE), introduction of Cl or NO2 group inhibition.

8
F. Li et al.
J Enzyme Inhib Med Chem, Early Online: 1–13
Table 1. Cholinesterases and human recombinant MAO isoforms inhibitory activities of tested compounds and reference compounds.
a
a
IC50 (mM)
Selectivity index
IC50 (mM)
Selectivity index
Compound
R
n
eeAChE
eqBuChE
eqBuChE/eeAChE
hMAO-A
hMAO-B
hMAO-A/hMAO-B
w1
w2
w3
w4
w5
w6
w7
w8
w9
w10
w11
w12
w13
w14
w15
w16
w17
w18
w19
w20
w21
w22
w23
Donepezil
Galantamine
Lazabemide
Iproniazide
Ph
3-Cl Ph
3-Cl-2-pyridine
3-Pyridine
5-Cl-2-thiophene
Ph
0
0
0
0
0
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
–
–
–
–
76.6 4.5
80.7 4.0
9.95 0.12
54.2 2.9
23.6 1.5
1.51 0.22
1.86 0.09
8.23 0.34
52.2 1.3
0.298 0.031 0.918 0.080
0.123 0.022 0.570 0.035
0.662 0.029
1.45 0.07
70.4 3.2
Nb
58.7 2.7
N
0.92
41.24
5.90
41.85
44.34
1.21
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
–
–
–
–
–
–
N
1.82 0.08
2.49 0.14
0.323 0.033
3.57 0.14
2-Cl Ph
1.34
–
42.14
–
43.53
0.331
–
1.06
2.5
0.504
–
2-Br PhCH2
2-(PhCO) Ph
3-NO2 Ph
4-NO2 Ph
4-OCH3 Ph
4-CH3 Ph
4-Cl Ph
2-quinoline
3-pyridine
4-pyridine
5-Cl-2-pyridine
5-F-2-pyridine
6-CH3-2-pyridine
5-Cl-2-thiophene
5-Br-2-thiophene
5-CH3-2-thiophene
–
0.0392
0.0684
3.08
4.63
2.03
1.14
1.64
3.54
17.2
15.9
5.6
15.3
8.39
10.2
46.8 2.2
N
26.2 1.5
68.3 4.0
N
94.1 4.9
9.27 1.3
48.6 3.3
N
N
92.4 7.1
22.6 1.8
N
N
23.2 1.8
24.5 2.3
N
1.26 0.11
1.65 0.08
0.378 0.015 0.620 0.07
0.174 0.009 0.601 0.06
0.791 0.045
0.340 0.031
0.220 0.006
0.502 0.011
0.434 0.019
0.269 0.017
0.768 0.037 0.208 0.009
1.55 0.013 0.370 0.021
0.035 0.003
13.6 0.6
5.40 0.27
1.23 0.10
3.85 0.27
3.64 0.18
2.75 0.13
N
13.4 0.9
N
–
4.27
–
3.14 027
N
2.53 1.4
11.5 0.9
9.47 0.54
N
N
N
76.4 5.4
96.2 4.8
N
439.5
6.64
10.2
–
0.271
0.239
2.32 0.10
12.7 0.3
nt
nt
nt
nt
–
–
–
–
–
–
–
2.67 0.18
ntc
nt
nt
nt
–
–
nt
6.7 0.4
0.105 0.008
7.69 0.23
aIC50: 50% inhibitory concentration (means SD of three experiments).
bInactive at 100 mM (highest concentration tested), at higher concentrations the compounds precipitate.
cnt ¼ not tested.
Inhibitory activity against MAOs
and w22, compound w21 (IC50 ¼76.4 mM for MAO-A;
IC50 ¼11.5 mM for MAO-B, SI ¼ 6.64) and w22 (IC50 ¼96 mM
for MAO-A; IC50 ¼9.47 mM for MAO-B, SI ¼ 10.2) were potent
for MAO-A and MAO-B, and good selective inhibitors toward
MAO-B. Finally, we found that, no matter introducing the Cl
group to 4-position of phenyl ring, 5-position of pyridine or
thiophene ring, respectively, all of them showed good inhibition
for both MAO-A and B.
For all target compounds, the MAO inhibitory activities were
measured, and lazabemine and iproniazide were used as reference
compounds. The corresponding IC50 values and MAO selectivity
ratios are also shown in Table 1. Based on the screening data, it
could be seen that only a part of the tested compounds could
effectively inhibit MAO-A or MAO-B. Among the synthesized
compounds, w20 was the most potent and selective inhibitor against
MAO-B (IC50 ¼2.53 mM, SI439.5), which is nearly 3 times
stronger than that of iproniazide. Compound w18 (IC50 ¼13.4 mM
Inhibitory activity against hAChE
for MAO-A; IC50 ¼3.14 mM for MAO-B) with 5-Cl substituent at Based on the results of ChEs and MAOs inhibitory activity,
pyridine ring exhibited the most potent MAO-A and MAO-B. From compounds w10, 11, 14, 15, 18, 21, 22 showed good inhibition.
the IC50 values, compounds w1–5 with carboxamide linkage However, their ChE inhibitory activity was initially tested on
showed no activity, which suggested that the suitable linker length enzymes of animal origin due to the lower cost. To better evaluate
seemed to be 2 carbon atoms for MAOs inhibition.
them, their inhibitory activity was retested on human AChE and
Compared to no substituted compound w6 (no activity at the results are summarized in Table 2. Most of the tested
100 mM), introduction of different sizes to the 2-position of phenyl compounds gave IC50 values in nanomolar, which were slightly
ring also showed no activity at 100 mM with exception of w8 less potent inhibition for hAChE than for eeAChE. However,
(IC50 ¼46.8 mM for MAO-B). Furthermore, among w6–14, com- compound w22 exhibited the inhibitory activity for hAChE
pounds with 3- and 4-position electron-withdrawing substitutions (IC50 ¼0.363 mM) was 2 times stronger than for eeAChE
of phenyl ring were more potent inhibition for MAOs than those (IC50 ¼0.768 mM). Among them, we chose compound w18,
with 2-position substitution. Replacement of Cl group (w18) with which showed balanced potential to inhibit ChEs (eeAChE:
F group (w19) in 5-position of pyridine ring presented a total loss IC50 ¼0.220 mM;
eqBuChE:
IC50 ¼1.23 mM;
hAChE:
of inhibitory activity for both MAO-A and B. In addition, the IC50 ¼0.454 mM) and MAOs (MAO-A: IC50 ¼13.4 mM; MAO-
electronic properties of substitutions at 5-position of thiophene B: IC50 ¼3.14 mM) as a promising multi-targeted inhibitor for
ring also affected the MAOs inhibitory activity. Compared to w21 further study.

DOI: 10.1080/14756366.2016.1201814
Donepezil-based agents
9
Table 2. Inhibition of human AChE activity.
a
Compound
R
n
IC50 (mM) hAChEb
w10
w11
w14
w15
w18
w21
w22
Donepezil
3-NO2 Ph
4-NO2 Ph
4-Cl Ph
2-quinoline
5-Cl-2-pyridine
5-Cl-2-thiophene
5-Br-2-thiophene
–
2
2
2
2
2
2
2
–
0.544 0.023
0.325 0.009
0.621 0.036
0.527 0.010
0.454 0.023
0.412 0.017
0.363 0.027
0.029 0.002
aIC50: 50% inhibitory concentration (means SD of three experiments).
bAChE from human erythrocytes.
Figure 3. Reversibility studies of hMAO-B inhibition by compound w18.
Compound w18 and pargyline were preincubated for various periods of
time (0–60 min) with hMAO-B at concentrations equal to twofold the
IC50 values for the inhibition of the enzyme. After dilution to
concentrations of w18 and pargyline equal to IC50, the inhibitory rates
were recorded.
for different concentrations of w18 were linear and intersected at
the y-axis. This behavior indicated that compound w18 acted as a
competitive MAO-B inhibitor, and this result further proved that
w18 was reversible MAO-B inhibitor.
Figure 2. Kinetic study on the mechanism of eeAChE inhibition by
compound w18. Overlaid Lineweaver–Burk reciprocal plots of AChE
initial velocity at increasing substrate concentration (0.05–0.50 mM) in
the absence of inhibitor and in the presence of w18 are shown. Lines were
derived from a weighted least-squares analysis of the data points.
Molecular modeling studies of ChEs
To further study the interaction mode of compound w18 for ChEs,
molecular docking study was performed using software package
MOE 2008.10. The X-ray crystal structure of the TcAChE
Kinetic study of AChE
Kinetic study of compound w18 was further examined to complex with donepezil (PDB code: 1EVE) was applied to build
investigate the AChE inhibitory mechanism. Graphical analysis the starting model of AChE. As shown in Figure 5(A) and (C), the
of the Lineweaver–Burk reciprocal plots (Figure 2) indicated that N-benzylpiperidine moiety of w18 was oriented towards the CAS
both increasing slopes and intercepts with increasing inhibitor of AChE, via ꢂ-cation interaction with the quaternary nitrogen of
˚
concentrations. This pattern suggested w18 is a mixed-type of piperidine ring from Tyr341 with the distance of 4.26 A. Besides,
inhibition and this revealed that it could interact simultaneously its benzene ring could interact with Tyr337 via ꢂ–ꢂ stacking
˚
with dual sites (PAS and CAS) of AChE. Replots of the slope interaction with the distance of 4.08 A. The 5-chloropicolinamide
versus concentration of w18 gave an estimate of competitive moiety interacted with the pridine ring from Trp286 of the PAS
˚
inhibition constant, Ki, of 0.220 mM.
via ꢂ–ꢂ stacking interaction with the distance of 4.35 A. All these
results indicated that compound w18 was a dual binding site
(DBS) AChE inhibitor in agreement with the kinetic study, which
demonstrated the rationality of our molecular design.
Kinetic study for MAO-B
From the point of view of AD treatment, reversible inhibitors of
MAO-B have significant advantages over the irreversible inhibi-
tors. Therefore, to examine whether w18 was reversible or
irreversible MAO-B inhibitor, the time dependencies of inhibition
were evaluated with an irreversible inhibitor, pargyline, as
reference compound50. Compound w18 was preincubated for
various time periods (0–60 min) with human MAO-B at a
concentration of 6.28 mM. This concentration of the inhibitor
are twofold the measured IC50 value for the inhibition of MAO-B.
Since the crystal structure of BuChE from equine serum has
not been reported and the sequence of equine BuChE is highly
similar to human BuChE, the crystal structure of hBuChE (PDB
code: 1P0I) was used in the docking study. As shown in Figure
5(B) and (D), the pyridine ring of w18 stacked against the Trp82
˚
through a ꢂ–ꢂ interaction with the distance of 2.76 A at the CAS.
Molecular modeling studies of MAO-B
As shown in Figure 3, we could observe that w18 was reversible To evaluate the binding mode of compound w18 with MAO-B,
MAO-B inhibitors as evidenced by the time-dependent decrease docking studies were employed with MOE 2008.10, based on the
of their inhibitory activity. In contrast, after treatment of MAO-B protein crystal structure of MAO-B (2V60). The 3D and 2D
with pargyline, the enzyme inhibitory activity was increased.
images of binding are illustrated in Figure 6. It can be seen from
Compound w18 was also used to further investigate the mode Figure 6(A) and (B), the N-benzylpiperidine moiety of w18 was
of MAO-B inhibition. The type of MAO-B inhibition was located within the substrate cavity of the enzyme, in close
determined by the Michaelis–Menten kinetic experiments51. In proximity of the flavin adenine dinucleotide (FAD) cofactor, and
this study, four different concentrations of w18 (0, 1.57, 3.14 and the ꢂ–ꢂ stacking interaction was seen between its benzene moiety
˚
6.28 mM) were selected and five different concentrations of p- with Tyr398 with the distance of 4.59 A. Besides, the N-
tyramine (0.05–1 mM) were used as substrate. The overlaid benzylpiperidine moiety via ꢂ–cation interaction with the quater-
reciprocal Lineweaver–Burk plots (Figure 4) showed that all plots nary nitrogen of piperidine ring from Gln206 had the distance of

10 F. Li et al.
J Enzyme Inhib Med Chem, Early Online: 1–13
˚
1.75 A. Finally, the 5-chloropicolinamide moiety of w18 occupied differential spectra, indicated that there was an interaction
the hydrophobic pocket in the entrance cavity, formed by Pro104, between Cu2+ and compound w18. These observations indicated
Ser200, Leu171, Tyr326, Phe103, Pro102, Ile199 and Ile316.
that our compounds could effectively chelate Cu2+, and thereby
could serve as metal chelators in treating AD.
Metal chelating effect
In vitro BBB permeation assay
The complexation ability of compound w18 for Cu2+ in methanol
was studies by using UV–Vis spectrometry with wavelength Because the first requirement for successful CNS drugs is to reach
ranging from 200 to 500 nm52,53. In Figure 7(A), UV–Vis spectra their therapeutic targets in brain, screening for the BBB penetra-
of w18 at increasing Cu2+ concentration were shown. The tion is of particular importance54. To determine whether the
decrease in absorbance (at about 375 nm peak in Figure 7B), present compounds could penetrate into the brain, we used a
which could be better estimated by an inspection of the parallel artificial membrane permeation assay for BBB (PAMPA-
BBB), which was described by Di et al.55. Assay validation was
performed by comparing experimental permeability of 9 com-
mercial drugs with reported values (Table 3). A plot of
experimental data versus bibliographic values gave a good
linear correlation, Pe (exp) ¼ 0.85 Pe (bibl.) ꢁ 0.13 (R2 ¼0.98).
From this equation-and taking into account the limits established
by Di et al. for BBB permeation, we established that molecules
with permeability values over 3.3 ꢃ 10ꢁ6 cm sꢁ1 would be able to
cross the BBB. Three compounds (w14, w18 and w21) that
exhibited good activities against ChEs and MAOs were chosen as
the tested compounds. The results summarized in Table 4
indicated that all of them showed higher Pe values than 3.3,
which suggested they were able to cross the BBB and target the
enzyme in the central nervous system.
Figure 4. Kinetic study on the mechanism of hMAO-B inhibition by w18.
Overlaid Lineweaver–Burk reciprocal plots of hMAO-B initial velocity at
Rat pheochromocytoma (PC12) cell toxicity
increasing p-tyramine concentration (0.05–1 mM) in the absence of The compound w18 was selected as the candidate to further study
inhibitor and in the presence of w18 are shown. Lines were derived from a
weighted least-squares analysis of the data points.
the potential toxicity effect on the rat pheochromocytoma (PC12)
cells. After incubating the cells to compound w18 for 24 h, the
Figure 5. (A) 3D docking model of compound w18 with TcAChE. (B) 3D docking model of compound w18 with hBuChE. (C) 2D schematic diagram
of docking model of compound w18 with TcAChE. (D) 2D schematic diagram of docking model of compound w18 with hBuChE. The figure was
prepared using the ligand interactions application in MOE.

DOI: 10.1080/14756366.2016.1201814
Donepezil-based agents 11
Figure 6. Molecule docking of compound w18 with hMAO-B generated with MOE: (A) The 2D picture of binding was depicted; (B) The 3D picture of
binding was depicted.
Figure 7. (A) UV–Vis (200–500 nm) absorp-
tion spectra of compound w18 (50 mM) in
methanol after addition of ascending amounts
of CuCl2 (10–80 mM). (B) The differential
spectra due to w18-Cu2+ complex formation
obtained by numerical subtraction from the
above spectra of those of Cu2+ and w18 at the
corresponding concentrations.

12 F. Li et al.
J Enzyme Inhib Med Chem, Early Online: 1–13
Table 3. Permeability (Pe ꢃ 10 ꢁ 6 cm s ꢁ 1) in the PAMPA-
stronger to inhibit ChEs than w18, w20 is more activity to interact
with MAOs. But w18 was the most attractive compound with
balanced bioactivity, which is able to inhibit ChEs (eeAChE:
BBB assay for 9 commercial drugs, used in the experiment
validation.
IC50 ¼0.220 mM;
eqBuChE:
IC50 ¼1.23 mM;
hAChE:
Commercial drugs
Bibliographya
Experimentb
IC50 ¼0.454 mM) and MAOs (MAO-B: IC50 ¼3.14 mM; MAO-
A: IC50 ¼13.4 mM). Meanwhile, compound w18 could penetrate
the BBB and showed low cell toxicity on rat pheochromocytoma
(PC12) cells in vitro. Altogether, the multifunctional ligand w18
endowed with balanced ChEs and MAOs inhibiting activities
might be a promising anti-AD candidate for further research.
Testosterone
Verapamil
b-Estradiol
Progesterone
Clonidine
Corticosterone
Piroxicam
Hydrocortisone
Dopamine
17
16
12
9.3
5.3
5.1
2.5
1.9
0.2
15.6 1.03
13.2 0.85
8.90 0.64
6.62 0.43
4.74 0.26
4.92 0.42
1.91 0.17
1.57 0.12
0.15 0.01
Declaration of interest
The authors declare no conflicts of interest.
aTaken from Ref55.
We gratefully acknowledge the financial support by the National
Natural Science Foundation of China (81573313), the program for
Changjiang Scholars and Innovative Research Team in University
(IRT_15R63), and the Priority Academic Program Development of
Jiangsu Higher Education Institutions (PAPD).
bData are the mean SD of three independent experiments.
Table 4. Permeability results (Pe ꢃ 10 ꢁ 6 cm s ꢁ 1
)
from the PAMPA-BBB assay for selected
compounds with their predicted penetration into
the CNS.
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DOI: 10.1080/14756366.2016.1201814
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