Design, synthesis, and biological evaluation of a new series of biphenyl/bibenzyl derivatives functioning as dual inhibitors of acetylcholinesterase and butyrylcholinesterase

Article abstract of DOI:10.3390/molecules22010172

Alzheimer's disease (AD), the most common form of dementia in adults, is a progressive neurodegenerative disorder of the brain characterized by loss of memory and steady deterioration of cognition. Here, a series of symmetrical molecules containing biphenyl/bibenzyl scaffolds (12-36) were designed, synthesized, and evaluated for their ability to inhibit both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). A biological evaluation showed that most of these biphenyl derivatives were potent AChE and BuChE inhibitors. Among them, compound 15 displayed the greatest ability to inhibit BuChE (IC₅₀ = 0.74 μM) and was also a good AChE inhibitor (IC₅₀ = 1.18 μM). Compound 19 was not only a potent AChE inhibitor (IC₅₀ = 0.096 μM), but also a mild BuChE inhibitor (IC₅₀ =1.25 μM). Overall, these results suggested that compound 19 may be a promising agent in the treatment of AD.

Full text of DOI:10.3390/molecules22010172

molecules
Article
Design, Synthesis, and Biological Evaluation of a
New Series of Biphenyl/Bibenzyl
Derivatives Functioning as Dual Inhibitors of
Acetylcholinesterase and Butyrylcholinesterase
Dong-mei Wang 1, Bo Feng 1, Hui Fu 2, Ai-lin Liu 1, Lin Wang 1, Guan-hua Du 1 and Song Wu 1,
*
1
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines & Ministry of Health Key
Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical
Sciences & Peking Union Medical College, Beijing 100050, China; wangdmchina@imm.ac.cn (D.-m.W.);
fengbo@imm.ac.cn (B.F.); liuailin@imm.ac.cn (A.-l.L.); wlin@imm.ac.cn (L.W.); dugh@imm.ac.cn (G.-h.D.)
Beijing Institutes For Drug Control, Beijing 100050, China; Fuhuifree@sina.com
2
*
Correspondence: ws@imm.ac.cn; Tel.: +86-10-8316-3542
Academic Editors: Diego Muñoz-Torrero and Derek J. McPhee
Received: 19 October 2016; Accepted: 16 January 2017; Published: 20 January 2017
Abstract: Alzheimer’s disease (AD), the most common form of dementia in adults, is a progressive
neurodegenerative disorder of the brain characterized by loss of memory and steady deterioration of
cognition. Here, a series of symmetrical molecules containing biphenyl/bibenzyl scaffolds (12
were designed, synthesized, and evaluated for their ability to inhibit both cetylcholin sterase (AChE)
and butyrylcholin sterase (BuChE). A biological evaluation showed that most of these biphenyl
derivatives were potent AChE and BuChE inhibitors. Among them, compound 15 displayed the
greatest ability to inhibit BuChE (IC50 = 0.74 M) and was also a good AChE inhibitor (IC50 = 1.18
M). Compound 19 was not only a potent AChE inhibitor (IC50 = 0.096 M), but also a mild BuChE
M). Overall, these results suggested that compound 19 may be a promising
–36)
a
e
e
µ
µ
µ
inhibitor (IC50 =1.25
µ
agent in the treatment of AD.
Keywords: Alzheimer’s disease; biphenyl/bibenzyl derivatives; acetylcholinesterase inhibitors;
butyrylcholinesterase inhibitors; molecular docking
1. Introduction
Alzheimer’s disease (AD), the most common form of dementia in adults, is a progressive
neurodegenerative disorder of the brain characterized by loss of memory and steady deterioration of
cognition. This disease currently affects more than 30 million people worldwide [
exact pathophysiology of AD remains unclear. Several findings indicate that amyloid-
tau protein aggregation [ ], oxidative stress [ ], and a low level of acetylcholine in the brain play
important roles in the pathophysiology of the disease [ ]. At present, no drug is available to decrease,
reverse, or stop the pathological process of AD. Although several research strategies have been
envisaged recently, the current therapeutic option is limited to only four AChE inhibitors [ ], namely,
1]. However, the
β
plaques [ ],
2
3
4,5
6
7,8
donepezil, rivastigmine, galantamine, and tacrine (now discontinued), and one N-methyl-D-aspartate
receptor antagonist, memantine [9].
However, two types of cholinesterase (ChE) enzymes have been found in the central nervous
system, AChE and BuChE. Studies have indicated that BuChE catalyzes the hydrolysis of acetylcholine
in the brains of normal people, as well as patients with Alzheimer’s disease (AD) [10]. While ACh
activity decreases to 10%–15% of normal values in certain brain regions in patients with mild to severe
AD, BuChE levels remain unchanged, or even increase, with disease progression [11]. Therefore, mixed
Molecules 2017, 22, 172; doi:10.3390/molecules22010172
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AChE/BuChE inhibitors may result in higher efficacy for treatment of AD. Rivastigmine, used in AD
treatment, is a potent inhibitor of both enzymes [12]. Selective BuChE inhibitors cause an elevation of
acetylcholine, augment long-term potentiation, and enhance learning. They have also been shown to
decrease amyloid-β in a rodent model of AD [13]. Therefore, dual AChE/BuChE inhibitors may have
better clinical efficacy without remarkable adverse effects.
To find new ChE inhibitors, structure-based virtual screening and high throughput
screening were used in the present study to screen the Institute of Meteria Medica’s library
of compounds, which contains more than 10,000 molecules. A small symmetrical molecule,
0
4,4 -bi-(
β-dimethylamino-propinoyl)-biphenyl (
1, Figure 1), was discovered to be a novel dual inhibitor
of AChE and BuChE, with a concentration required for 50% inhibition of the maximal response in vitro
(its IC50 value) of 0.75 µM for AChE and 0.19 µM for BuChE.
Figure 1. Chemical Structure of 1.
The biphenyl scaffold is a privileged structure [14] and it exists in 4.3% of the molecules in
commercially available drugs [15]. However, only limited studies have examined these types of
compounds for their anti-AChE and anti-BuChE activities. Thus, to find potential anti-AD agents, we
report here on the synthesis of a series of derivatives and analogues of compound 1. The chemical
modifications included the following: (i) replacing the dimethylamino group with appropriate
secondary amines and heterocyclic amines; (ii) changing the length of the linkers that connect the
biphenyl carbonyl core with an amine moiety; (iii) alternating the rigid biphenyl core with a flexible
bibenzyl; and (iv) reducing the carbonyl group to a hydroxyl group.
2. Results and Discussion
2.1. Chemistry
The synthetic route for these target compounds (12
–
36) is outlined in Scheme 1. Commercially
reacted with Cl(CH2)nCOCl (n = 1–4) in the presence of AlCl3 in dry
11) with yields of 71%–82%. The target compounds (12 33
11) with appropriate secondary
available biphenyl
CS2 at 40–50 C to generate intermediates (
2 or bibenzyl 3
◦
4
–
–
)
were synthesized by substitution of chlorine in the intermediates (4–
alkyl amines or heterocyclic amines. The reaction was carried out in CH3CN with K2CO3 and KI.
The yields were 52%–65%. Reduction of compounds 19, 20, and 30 with sodium borohydride in THF
gave the desired alcohols (34
compounds, but two asymmetrical carbon centers can be produced during the reduction process
to generate compounds 34 36. Thus, the final products may have existed as three isomers, namely,
–36) with yields of 52%–57%. Diketones (19, 20, and 30) are symmetrical
–
distereomeric mixtures of a meso form (R,S/S,R), and a racemic mixture of two chiral compounds (R,R)
and (S,S). These isomers can be separated by chiral column chromatography. However, as our aim is
only to investigate whether the carbonyl groups in the molecules were crucial for biological activities,
the separation was not conducted. Twenty-one compounds were novel, and the other four compounds
have been previously reported [16], although neither the synthetic route nor the structure–activity
relationships were provided. The crude products were purified in the present study by column
chromatography on silica gel eluted with CH2Cl2 and MeOH. The purities of all compounds were
higher than 99%. The structures of the target compounds are shown in Table 1.

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Table 1. AChE and BuChE inhibition by target compounds 12–36.
IC50 µM ± SEM
Compounds a
12
m
n
R
BuChE c ± SEM
91.68 ± 15.68
AChE b ± SEM
d
0
1
d
d
13
14
15
0
0
0
1
1
2
71.68 ± 3.01
1.44 ± 0.23
0.74 ± 0.09
1.18 ± 0.29
0.88 ± 0.17
16
0
2
2.83 ± 0.50
17
18
19
20
21
0
0
0
0
0
2
2
2
2
2
0.71 ± 0.06
0.33 ± 0.06
0.096 ± 0.006
0.97 ± 0.08
1.43 ± 0.33
2.39 ± 0.75
1.16 ± 0.09
1.25 ± 0.15
0.72 ± 0.10
1.94 ± 0.07
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
0
0
0
2
2
2
2
2
2
2
2
2
0
0
2
2
3
4
1
1
1
1
2
2
2
3
4
2
2
2
1.61 ± 0.38
1.32 ± 0.43
0.82 ± 0.16
62.27 ± 14.46
74.54 ± 8.50
13.77 ± 0.81
5.9 ± 0.40
2.65 ± 0.11
9.11 ± 2.55
20.72 ± 5.44
66.51 ± 17.04
6.30 ± 1.60
1.21 ± 0.22
3.14 ± 0.24
6.38 ± 1.26
19.18 ± 4.78
1.64 ± 0.27
74.13 ± 11.53
d
2.62 ± 0.63
0.50 ± 0.06
3.53 ± 0.79
3.67 ± 0.92
0.19 ± 0.08
31.74 ± 10.24
13.78 ± 1.59
d
d
74.93 ± 35.56
25.94 ± 6.81
Donepezil
0.044
ISO-OMPA
0.72
a
b
SEM: Standard error of the mean. The structure of compounds 12
–
36 shown in Scheme 1. 50% inhibitory
c
concentration (means
(means
100 µM/L were inactive.
±
SEM of three experiments using AChE from rat brain). 50% inhibitory concentration
SEM of three experiments using BuChE from human serum). Concentrations of the compounds up to
d
±

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Scheme 1. Synthetic route for target compounds 12
–
36
.
Reagents, conditions, and yields:
(a) Cl(CH2)nCOCl, CS2; 40–50 ◦C, 4–8 h; 71%
−
82%; (b) secondary amines or heterocyclicamines,
◦
◦
CH3CN, K2CO3, KI; 50 C, 20 min; 52%−65%; (c) NaBH4, THF; 50 C, 6 h; 52%–57%.
2.2. Biological Evaluation and SAR Analysis
The abilities of the target compounds (12–36) to inhibit AChE (from rat brain) and BuChE (from
human serum) activities were evaluated in vitro using the spectrophotometric method of Ellman [16].
Donepezil and tetraisopropyl-pyrophosph-oramide (iso-OMPA) were used as reference compounds,
and each test was conducted in triplicate. The IC50 values are summarized in Table 1. We found
that most of the compounds showed good inhibition against both AChE and BuChE activities,
with IC50 values ranging from micromolar to sub-micromolar levels. Among these compounds
(
12
approximately half the potency of donepezil (IC50 = 0.044
inhibition of BuChE (IC50 = 1.25 M) which was higher than donepezil. These results indicated that
compound 19 was a potent dual inhibitor against AChE and BuChE. We also found that compound 15
exhibited the strongest inhibition against BuChE, with IC50 values of 0.74 M, which was as potent as
M); additionally, compound 15 was a potent AChE
–
36), compound 19 showed the strongest inhibition against AChE, with an IC50 value of 0.096
µM,
µM). Compound 19 also exhibited potent
µ
µ
the reference compound iso-OPMA (IC50 = 0.72
µ
inhibitor (IC50 = 1.18 µM).
For all compounds, the length of linker between biphenylcarbonyl or bibenzylcarbonyl and the
tertiary amine moiety played a significant role in their inhibitory activities against both AChE and
BuChE. The results indicated that a linker length of one carbon atom was not preferred. Compounds
with such linker length (12
three compounds displayed low potency for BuChE, with IC50 values of 1.44
–
14
,
25
–
28) showed no activity, except for compounds 14
,
25, and 28. These
M, and 6.30 M,
µM, 9.11
µ
µ
respectively. For the biphenyl derivatives, the biological assay showed that their AChE and BuChE
inhibitory activities decreased as the length of linker increased from one carbon atom to three or four
carbon atoms, although compound 24, containing a four-carbon linker, was only slightly less potent
(IC50 = 0.82 µM for AChE) than compound 19, with a two-carbon linker (IC50 = 0.096 µM for AChE).
These results suggested that a linker length of two carbon atoms was optimal for improving AChE
inhibitory activities in compounds with a biphenyl moiety.
By contrast, the linker length for the bibenzyl derivatives made little difference with respect to their
ability to inhibit AChE activity. For example, bibenzyl derivative 33
four-carbon linker, displayed similar inhibitory activity to that for compound 19
AChE), having a two-carbon linker. Furthermore, replacing the dimethylamine moiety in compound
with a heterocycloamine, such as piperidine, pyrrolidine, or azetidine, enhanced the inhibition,
especially for compound 19, which was eight-fold more potent than the lead compound ( ) for
(
IC50 = 0.19 µM for AChE), with a
(
IC50 = 0.096 µM for
1
1
inhibiting AChE, while its ability to inhibit BuChE activity was only mildly decreased. Compared with
biphenyl derivatives, the corresponding bibenzyl derivatives generally displayed less potent inhibition
against both AChE and BuChE. Finally, it appeared that the carbonyl groups in the molecules were
indispensable. Reducing compounds 19
, 20, and 30 to the corresponding alcohols (34–36) caused them
all to lose their ability to inhibit both AChE and BuChE.

Molecules 2017, 22, 172
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2.3. Molecular Docking
To further investigate 19, the most potent compound against AChE and BuChE, a molecular
docking study was performed, and the different types of interactions between 19 and AChE and
BuChE are shown in Figures 2 and 3. As shown in Figure 2, compound 19 was located on the long
tunnel of the active site interacting with some residues. To be more specific, the aromatic structure of
compound 19 adopted an appropriate orientation for its binding to the phenyl ring of TYR334 and
PHE330 via π-π stacking interaction. In addition to the hydrophobic interaction, we can also see two
key amino residues (PHE288 and ARG289) forming hydrogen bonds with the carbonyl group. All of
these results indicated that compound 19 could bind to AChE.
Figure 2. Three-dimensional docking model of compound 19 with AChE.
Figure 3. Three-dimensional docking model of compound 19 with BuChE.
Compare to AChE, BuChE displayed a strong hydrophobic interaction with compound 19. As
shown in Figure 3, the symmetric piperidine ring could insert into the hydrophobic cavities as well.
One interaction consisted of HIS438 and TRP82, and the other was formed by THR284, PHE329.
Furthermore, the aromatic structure of compound 19 adopted an appropriate orientation for its
binding to the phenyl ring of TYR332 via a π-π stacking interaction as well. These results illustrated
that compound 19 also can bind to BuChE.
3. Materials and Methods
3.1. Chemistry
Melting points were measured on a YRT-3 apparatus (Tianjin precision apparatus factory, Tian Jin,
China), uncorrected. 1H-NMR spectra were recorded on a Varain Mercury-300 & 400 instrument
(Varian, Salt Lake City, UT, USA) using tetramethylsilane as an internal standard, DMSO-d6 and CDCl3

Molecules 2017, 22, 172
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as solvents, chemical shifts (
δ) in ppm, coupling constants (J) in Hz. High-resolution mass spectra were
determined with Thermo Scientific Exactive Plus mass spectrometry (Thermo Scientific, Waltham, MA,
USA) with the ESI method. Column chromatography was carried out using 200–300 mesh silica gel
(Qingdao Marine Chemical Inc., Qingdao, China,). All chemicals and solvents were analytical reagent
grade and were used without further purification. All tested compounds were purified until the purity
≥99% detected by HPLC (Shimadzu Co., Kyoto, Japan) under a wavelength of 254 nm, NMR.
3.1.1. General Method for Synthesis of Friedel-Crafts Acylation Intermediates (4–11)
A mixture of commercial available biphenyl (2) or bibenzyl (3) (1.0 mmol) and AlCl3 (3.0 mmol)
was stirred in dry CS2 (60 mL), and followed by Cl(CH2)nCOCl (n = 1–4) (3.0 mmol) was added
drop-wise. The reaction mixture was stirred at 50–80 ◦C for 4–8 h. Then the reaction mixture was
poured into 200 mL ice water, and the solid compound was precipitated. After washing with cold
ethanol, the solid was dried to afford compounds (4–11) with the yield of 71%–82%.
3.1.2. General Method for Synthesis of the Target Compounds (12–33)
A mixture of secondary alkyl amines or heterocyclic amines (3.0 mmol), anhydrous K2CO3
◦
(4.0 mmol) and KI (1.0 mmol) was stirred in acetonitrile (40 mL) at 50 C for 30 min. Then the
Friedel-Crafts acylation intermediates (4–11) was added and stirred for 20 min. The reaction mixture
was filtered and concentrated. After washing with water, the mixture was filtered and subjected
to silica gel column chromatography with CH2Cl2:MeOH = 100:1 as an eluent to afford the target
compounds (12–33) with the yield of 52%–65%.
◦
1,10-([1,10-Biphenyl]-4,40-diyl)bis(2-(dimethylamino)ethanone) (12). Yield, 52%; m.p. 113.7–114.6 C;
1H-NMR (300 MHz, CDCl3)
13C-NMR (75 MHz, CDCl3):
δ 8.10 (d, J = 8.1, 4H), 7.70 (d, J = 8.1, 4H), 3.79 (s, 4H), 2.41 (s, 12H);
199.0, 144.4, 136.8, 129.0, 127.6, 49.5, 36.9; HRMS (ESI+) calculated for
δ
C20H25N2O2: 325.1916 [M + H]+, found: 325.1903.
1,10-([1,10-Biphenyl]-4,40-diyl)bis(2-(piperidin-1-yl)ethanone) (13). Yield, 55%; m.p. 114.3–115.4 ◦C;
1H-NMR (400 MHz, CDCl3)
δ 8.07 (d, J = 7.8, 4H), 7.64 (d, J = 7.8, 4H), 3.65 (s, 4H), 3.42 (m, 8H), 1.63
(m, 12H); 13C-NMR (75 MHz, CDCl3):
δ 196.3, 143.4, 136.5, 129.8, 127.1, 65.2, 54.7, 33.0, 22.5; HRMS
(ESI+) calculated for C26H33N2O2: 405.2542 [M + H]+, found: 405.2544.
1,10-([1,10-Biphenyl]-4,40-diyl)bis(2-morpholinoethanone) (14). Yield, 65%; m.p. 112.2–113.5 ◦C; 1H-NMR
(300 MHz, CDCl3)
J = 4.5, 8H); 13C-NMR (125 MHz, CDCl3):
(ESI+) calculated for C24H29N2O4: 409.2127 [M + H]+, found: 409.2123.
δ
8.04 (d, J = 8.4, 4H), 7.74 (d, J = 8.4, 4H), 3.78 (s, 4H), 3.72 (t, J = 4.5, 8H), 2.57 (t,
δ
195.8, 144.7, 135.5, 129.1, 127.7, 67.0, 65.0, 54.1; HRMS
4,40-Bis( -diethylamino-propinoyl)-biphenyl (15). Yield, 57%; m.p. 129.2–130.6 ◦C; 1H-NMR (400 MHz,
DMSO-d6) 8.15 (d, 4H, J = 8.4), 7.99 (d, 4H, J = 8.4), 3.71 (t, 4H, J = 6.9), 3.43 (t, 4H, J = 6.9), 3.22 (q, 8H,
J = 7.2), 1.270 (t, 12H, J = 7.2); 13C-NMR (75 MHz, DMSO-d6):
197.2, 144.2, 132.77, 129.0, 127.6, 47.6,
42.0, 33.9, 11.4; HRMS (ESI+) calculated for C26H27N2O2: 409.2855 [M + H]+, found: 409.2845.
β
δ
δ
4,40-Bis(
CDCl3)
β
-dipropylamino-propinoyl)-biphenyl (16). Yield, 52%; m.p. 131.0–132.6 ◦C; 1H-NMR (400 MHz,
δ 8.06 (d, 4H, J = 8.4), 7.71 (d, 4H, J = 8.4), 3.14 (t, 4H, J = 6.8), 2.94 (t, 4H, J = 6.8), 2.42 (t,
8H, J = 7.6), 1.47 (m, 8H), 0.871 (t, 12H, J = 7.6); 13C-NMR (75 MHz, CDCl3):
δ 199.7, 144.4, 136.8,
129.0, 127.6, 56.4, 49.5, 36.9, 20.6, 12.1; HRMS (ESI+) calculated for C30H45N2O2: 465.3481 [M + H]+,
found: 465.3479.
0
◦
4,4 -Bis(β
-azetidin-propinoyl)-biphenyl (17). Yield, 60%; m.p. 150.8–151.1 C; 1H-NMR (400 MHz, CDCl3)
δ
8.05 (d, 4H, J = 8.0), 7.71 (d, 4H, J = 8.0), 3.26 (t, 8H, J = 6.8), 3.05 (t, 4H, J = 7.2), 2.86 (t, 4H, J = 7.2),
2.09 (t, 4H, J = 6.8); 13C-NMR (75 MHz, CDCl3): δ 198.4, 144.3, 136.3, 128.8, 127.5, 55.3, 54.5, 37.0, 17.6;
HRMS (ESI+) calculated for C24H29N2O2: 377.2229 [M + H]+, found: 377.2218.

Molecules 2017, 22, 172
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◦
4,40-Bis(
β-pyrrolidin-propinoyl)-biphenyl (18). Yield, 62%; m.p. 151.2–152.1 C; H-NMR (300 MHz,
1
CDCl3,)
δ 8.06 (d, 4H, J = 8.1), 7.72 (d, 4H, J = 8.1), 3.28 (t, 4H, J = 7.2), 2.97 (t, 4H, J = 7.2), 2.62 (m,
8H), 1.824 (m, 8H); 13C-NMR (75 MHz, CDCl3): δ 198.4, 144.2, 136.3, 128.8, 127.5, 54.3, 51.0, 38.2, 23.5;
HRMS (ESI+) calculated for C26H33N2O2: 405.2542 [M + H]+, found: 405.2535.
4,40-Bis(
DMSO-d6)
4H, J = 7.2), 3.01 (t, 4H, J = 8.8 ), 1.87 (m, 4H), 1.71 (m, 4H), 1.41 (m, 4H); 13C-NMR (125 MHz, CDCl3):
196.3, 143.4, 135.5, 128.8, 127.4, 52.2, 51.1, 33.0, 22.5, 21.3; HRMS (ESI+) calculated for C28H37N2O2
[M + H]+: 433.2855, found: 433.2844.
β
-(piperidin-propinoyl)-biphenyl (19). Yield, 59%; m.p. 138.5–139.6 C; H-NMR (400 MHz,
8.15 (d, 4H, J = 8.0), 7.99 (d, 4H, J = 8.0), 3.66 (t, 4H, J = 7.2), 3.55 (t, 4H, J = 8.8), 3.44 (t,
◦
1
δ
δ
4,40-Bis(
CDCl3)
β
-(morpholino-propinoyl)-biphenyl (20). Yield, 58%; m.p. 140.1–141.0 ◦C; 1H-NMR (400 MHz,
8.06 (d, J = 8.0, 4H), 7.72 (d, J = 8.0, 4H), 3.76 (m, 8H), 3.22 (t, J = 7.2, 4H), 2.86 (t, J = 7.2,
δ
4H), 2.53 (m, 8H); 13C-NMR (125 MHz, CDCl3): δ 198.4, 144.6, 136.4, 129.0, 127.7, 66.9, 53.9, 53.7, 36.1;
HRMS (ESI+) calculated for C26H33N2O4: 437.2440 [M + H]+, found: 437.2431.
4,40-Bis(
8.05 (d, 4H, J = 8.4), 7.70 (d, 4H, J = 8.4), 3.23 (t, 4H, J = 7.6), 3.03 (t, 4H, J = 7.6), 2.74 (t, 8H, J = 5.6),
1.68 (m, 8H), 1.59 (m, 8H); 13C-NMR (75 MHz, CDCl3):
199.1, 144.4, 136.5, 128.9, 127.6, 55.6, 53.3, 37.0,
27.7, 27.1; HRMS (ESI+) calculated for C30H41N2O2: 461.3168 [M + H]+, found: 461.3158.
β
-azepan-propinoyl)-biphenyl (21). Yield, 52%; m.p. 109.2–111.5 ◦C; 1H-NMR (400 MHz, CDCl3)
δ
δ
4,40-Bis( -4-methylpiperazin-propinoyl)-biphenyl (22). Yield, 50%; m.p. 146.1–148.7 ◦C; 1H-NMR (400
MHz, CDCl3) 8.06 (d, 4H, J = 8.4), 7.73 (d, 4H, J = 8.4), 3.23 (t, 4H, J = 7.6), 2.89 (t, 4H, J = 7.6), 2.59 (m,
8H), 2.48 (m, 8H), 2.31 (s, 6H); 13C-NMR (75 MHz, CDCl3):
198.6, 144.3, 136.4, 128.7, 127.5, 55.1, 53.2,
53.1, 46.0, 36.4; HRMS (ESI+) calculated for C28H39N4O2 [M + H]+: 463.3073, found: 463.3067.
1,10-([1,10-Biphenyl]-4,40-diyl)bis(4-(piperidin-1-yl)butan-1-one) (23). Yield, 53%; m.p. 146.4–147.8 C;
1H-NMR (400 MHz, CDCl3)
8.12 (d, 4H, J = 8.0), 7.78 (d, 4H, J = 8.0), 3.09 (t, 4H, J = 7.2), 2.44 (m,
β
δ
δ
◦
δ
12H), 2.07 (m, 4H), 1.59 (m, 8H), 1.44 (m, 4H); 13C-NMR (125 MHz, CDCl3):
δ 199.7, 144.4, 136.6, 129.0,
127.6, 58.5, 54.5, 36.7, 25.7, 24.4, 21.5. HRMS (ESI+) calculated for C30H41N2O2 [M + H]+: 461.3168,
found: 461.3162.
◦
1,10-([1,10-Biphenyl]-4,40-diyl)bis(5-(piperidin-1-yl)pentan-1-one) (24). Yield, 48%; m.p. 115.7–117.8 C;
1H-NMR (400 MHz, CDCl3)
δ
8.05 (d, 4H, J = 8.4), 7.70 (d, 4H, J = 8.4), 3.03 (t, 4H, J = 7.2), 2.45 (m,
12H), 1.80 (m, 4H), 1.59 (m, 12H), 1.44 (m, 4H); 13C-NMR (125 MHz, CDCl3):
δ 199.7, 144.4, 136.6, 129.0,
127.6, 58.5, 54.5, 36.7, 25.7, 24.4, 21.5; HRMS (ESI+) calculated for C32H45N2O2 [M + H]+: 489.3481;
found, 489.3473.
1,10-(Ethane-1,2-diylbis(4,1-phenylene))bis(2-(dimethylamino)ethanone) (25). Yield, 58%; m.p. 104.2–106.9
◦C; 1H-NMR (300 MHZ, CDCl3)
δ 7.91 (d, 4H, J = 8.1), 7.22 (d, 4H, J = 8.1), 3.75 (s, 4H), 2.99 (s, 4H),
2.39 (s, 12H). 13C-NMR (125 MHz, CDCl3):
δ 196.2, 147.2, 134.2, 130.0, 128.9, 65.3, 45.9, 37.5; HRMS
(ESI+): calculated for C22H29N2O2 [M + H]+: 353.2229, found: 353.2217.
1,10-(Ethane-1,2-diylbis(4,1-phenylene))bis(2-(piperidin-1-yl)ethanone) (26). Yield, 52%; m.p. 96.7–98.6 ◦C;
1H-NMR (300 MHz, CDCl3)
δ 7.92 (d, 4H, J = 7.8), 7.21 (d, 4H, J = 7.8), 3.79 (s, 4H), 2.99 (s, 4H), 2.58 (m,
8H), 1.67 (m, 8H), 1.47 (m, 4H); 13C-NMR (125 MHz, CDCl3):
δ 196.7, 147.0, 134.6, 128.8, 128.6, 65.5,
55.1, 37.6, 26.0, 24.2; HRMS (ESI+) calculated for C28H37N2O2 [M + H]+, 433.2855, found: 433.2850.
1,10-(Ethane-1,2-diylbis(4,1-phenylene))bis(2-(4-methylpiperazin-1-yl)ethanone) (27). Yield, 54%; m.p.
◦
1
150.9–152.2 C; H-NMR (300 MHz, CDCl3)
δ
7.91 (d, 4H, J = 8.1), 7.223 (d, 4H, J = 8.1), 3.79 (s,
195.9, 147.1,
4H), 2.99 (s, 4H), 2.64 (m, 8H), 2.530 (m, 8H) 2.31 (s, 6H); 13C-NMR (125 MHz, CDCl3):
134.3, 128.9, 128.5, 64.4, 55.0, 53.4, 46.0, 37.5; HRMS (ESI+) calculated for C28H39N4O2 [M + H]+:
δ
463.3073, found: 463.3066.
0
◦
1,1 -(Ethane-1,2-diylbis(4,1-phenylene))bis(5-morpholinopentan-1-one) (28). Yield, 52%; m.p. 160.1–162.6 C;
1H-NMR (300 MHz, CDCl3)
7.91 (d, 4H, J = 8.4), 7.23 (d, 4H, J = 8.4), 3.79 (m, 12H), 3.01 (s, 4H), 2.63
δ

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(m, 8H); 13C-NMR (125 MHz, CDCl3):
δ
195.8, 147.2, 134.3, 128.9, 128.5, 67.0, 64.8, 54.1, 37.5; HRMS
(ESI+) calculated for C28H33N2O4 [M + H]+: 437.2440, found: 437.2434.
0
1,1 -(Ethane-1,2-diylbis(4,1-phenylene))bis(3-(dimethylamino)propan-1-one) (29). Yield, 56%; m.p. 94.5–95.6
◦C; 1H-NMR (400 MHz, CDCl3)
δ 7.91 (d, 4H, J = 8.0 Hz), 7.25 (d, 4H, J = 8.0), 3.14 (t, 4H, J = 7.2), 2.99
(s, 4H), 2.77 (t, 4H, J = 7.2), 2.24 (s, 12H); 13C-NMR (125 MHz, CDCl3):
δ 198.6, 147.0, 135.1, 129.0, 128.5,
54.4, 45.6, 37.5, 36.8; HRMS (ESI+) calculated for C24H33N2O2 [M + H]+: 381.2545, found: 381.2542.
0
1,1 -(Ethane-1,2-diylbis(4,1-phenylene))bis(3-(piperidin-1-yl)propan-1-one) (30). Yield, 52%; m.p. 103.5–104.8
◦C; 1H-NMR (400 MHz, CDCl3)
δ
7.86 (d, J = 8.2, 4H), 7.21 (d, J = 8.2, 4H), 3.10 (m, 4H), 2.99 (s, 4H),
2.79 (t, J = 7.5, 4H), 2.45 (s, 8H), 1.60 (m, 8H), 1.44 (d, J = 4.8, 4H); 13C-NMR (125 MHz, CDCl3):
δ
199.0, 146.9, 135.2, 129.0, 128.5, 54.8, 54.0, 37.5, 36.3, 26.0, 24.3; HRMS (ESI+) calculated for C30H40N2O2
[M + H]+: 461.3168, found: 461.3162.
0
◦
1,1 -(Ethane-1,2-diylbis(4,1-phenylene))bis(3-morpholinopropan-1-one) (31). Yield, 56%; m.p. 105.2–106.6 C;
1H-NMR (400 MHz, CDCl3)
7.86 (d, J = 8.2, 4H), 7.22 (d, J = 8.1, 4H), 3.75–3.66 (m, 8H), 3.17 (t, J = 7.3
δ
,
4H), 3.00 (s, 4H), 2.84 (t, J = 7.3, 4H), 2.53 (s, 8H); 13C-NMR (125 MHz, CDCl3):
129.0, 128.5, 67.1, 53.9 53.8, 37.5, 36.1; HRMS (ESI+) calculated for C28H36N2O4 [M + H]+: 465.2753,
found: 465.2744.
δ 198.7, 147.0, 135.2,
0
1,1 -(Ethane-1,2-diylbis(4,1-phenylene))bis(4-(piperidin-1-yl)butan-1-one) (32). Yield, 53%; m.p. 145.2–146.6
◦C; 1H-NMR (400 MHz, CDCl3)
δ
7.87 (d, 4H, J = 8.4), 7.21 (d, 4H, J = 8.4), 2.99 (s, 4H), 2.95 (t, 4H,
J = 7.2), 2.32 (m, 12H), 1.86 (m, 4H), 1.48 (m, 8H), 1.35 (m, 4H); 13C-NMR (125 MHz, CDCl3):
δ
199.9,
146.7, 135.5, 129.0, 128.5, 58.7, 54.6, 37.5, 36.6, 25.9, 24.5, 21.6; HRMS (ESI+) calculated for C32H45N2O2
[M + H]+, 488.3481, found: 488.3475.
0
1,1 -(Ethane-1,2-diylbis(4,1-phenylene))bis(5-(piperidin-1-yl)pentan-1-one) (33). Yield, 68%; m.p. 146.1–147.5
◦C; 1H-NMR (300 MHz, CDCl3)
δ
7.87 (d, 4H, J = 8.1 Hz), 7.21 (d, 4H, J = 8.1), 3.00 (s, 4H), 2.97 (t,
4H, J = 7.2), 2.36 (m, 12H), 1.74 (m, 4H), 1.60 (m, 12H), 1.44 (m, 4H); 13C-NMR (125 MHz, CDCl3):
δ
200.1, 146.7, 135.3, 128.9, 128.5, 59.2, 54.7, 38.5, 37.5, 26.6, 25.9, 24.5, 22.6; HRMS (ESI+) calculated for
C34H49N2O2 [M + H]+: 517.3794, found: 517.3787.
3.1.3. General Method for Synthesis of Target Compounds (34–36)
To a stirred solution of compounds (19, 20 and 30) (1.0 mmol) in anhydrous THF (30 mL), sodium
borohydride (3.0 mmol) was added with stirring. The reaction mixture was stirred at 50 ◦C for 6 h.
Then the reaction mixture was filtered and quenched by methanol. After the concentrated, the residue
was subjected to silica gel column chromatography with DCM:MeOH = 100:1 as an eluent to afford the
target alcohols (34–36) with the yield of 52%–57%.
◦
1,10-([1,10-Biphenyl]-4,40-diyl)bis(3-(piperidin-1-yl)propan-1-ol) (34). Yield, 52%; m.p. 139.5–141.6 C;
1H-NMR (400 MHz, CDCl3)
δ
7.57 (d, 4H, J = 8.4), 7.43 (d, 4H, J = 8.4), 4.98 (t, 2H, J = 5.6), 2.58 (m, 8H),
2.43 (m, 4H), 1.87 (m, 4H), 1.64 (m, 8H), 1.06 (m, 4H); 13C-NMR (75 MHz, CDCl3):
δ 144.1, 139.6, 126.8,
125.9, 75.5, 57.8, 54.7, 33.6, 26.1, 24.3; HRMS (ESI+) calculated for C28H41N2O2 [M + H]+: 437.3168,
found: 437.3162.
0
0
0
◦
1,1 -([1,1 -Biphenyl]-4,4 -diyl)bis(3-morpholinopropan-1-ol) (35). Yield, 57%; m.p. 131.2–133.6 C; 1H-NMR
(400 MHz, CDCl3) 7.57 (d, 4H, J = 7.6), 7.43 (d, 4H, J = 7.6), 6.40 (brs, 2H), 4.96 (brs, 2H), 3.75 (m,
δ
8H), 2.65 (m, 8H), 2.49 (m, 4H), 1.89 (m, 4H, J = 4.4 ); 13C-NMR (75 MHz, CDCl3):
δ 143.6, 139.5, 126.7,
125.8, 75.0, 66.8, 57.3, 53.5, 33.3; HRMS (ESI+) calculated for C28H37N2O4 [M + H]+: 440.2753, found:
440.2743.
1,10-(Ethane-1,2-diylbis(4,1-phenylene))bis(3-(piperidin-1-yl)propan-1-ol) (36). Yield, 52%; m.p. 141.5–142.6
◦C; 1H-NMR (400 MHz, CDCl3):
δ
7.28 (d, 4H, J = 8.4), 7.15 (d, 4H, J = 8.4), 4.90 (t, 2H, J = 5.6) 2.89 (s,
4H), 2.57 (m, 8H), 2.41 (m, 4H), 1.83 (m, 4H), 1.62 (m, 8H), 1.46 (m, 4H); 13C-NMR (75 MHz, CDCl3):
δ

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142.7, 140.3, 128.2, 125.5, 75.5, 57.7, 54.6, 37.6, 33.6, 26.0, 24.2; HRMS (ESI+) calculated for C30H45N2O2
[M + H]+: 465.3481, found: 465.3462.
3.2. Biological Evaluation
All of the assay of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were performed
according to the method described by Ellman et al. [17] using the rat AChE purified from the rat brain
and human BuChE purified by human serum. In an enzymatic reaction medium contained different
concentration of tested compounds and AChE suspended in the phosphate buffer solution at pH
7.2. After 30 min at room temperature, acetylthiocholine iodide and 5.50-dithiobis-2-nitrobenzoic acid
(DNTB) were added and the reaction mixture incubated at 37 ◦C for 60 min. After the initiation of
enzymatic reaction, absorbance of each tested compound mixture of colored end-product was measured
by using the UV-VIS spectrophotometer at 412 nm. The parallel control experiment was carried without
tested compounds in the reaction mixture. For BuChE inhibitory assay, the procedure described above
was followed except for the use of enzyme and substrate, instead of which butyrylcholinesterase and
butyrylthiocholine chloride were used, respectively. Assays were done with a blank containing all
compounds except AChE or BuChE in order to account for nonenzymatic reactions. Each inhibitory
test was conducted in triplicate. Donepezil was used as a positive control of AChE and ISO-OMPA
was used as a positive control of BuChE, respectively. Each concentration was assayed in triplicate and
data were expressed as the mean ± SEM.
3.3. Molecular Docking
To further investigate the interaction of the most active compound 19 for AChE and BuChE, a
molecular docking study was performed by utilizing the Gold 3.0.1 software package (Cambridge
Crystallographic Data Center, Cambridge, UK). The X-ray crystal structure of the AChE (PDB code:
1EVE) and BuChE (PDB code: 4BDS) were obtained from protein data bank. Discovery Studio and
Pymol program were also used to prepare 2D and 3D schematic diagrams of docking model to exhibit
different interaction types between 19 and AChE/BuChE.
4. Conclusions
In summary, a new class of diphenyl/dibenzyl derivatives (12–36) has been designed, synthesized,
and determined to function as novel dual inhibitors of AChE and BuChE. Most of these compounds
possessed moderate to high AChE and BuChE inhibitory activities in vitro. Compound 19 showed the
most potent AChE inhibitory activity, in the sub-micromolar range (IC50 = 0.096 µM), and moderate
BuChE inhibition, with an IC50 value of 1.25
against BuChE, with an IC50 value of 0.74
µ
M. Compound 15 exhibited the most potent inhibition
M, which was as potent as the reference compound
µ
iso-OPMA. Our molecular modeling study confirmed that compound 19 is capable of binding to the
active-site cavity of both AChE and BuchE. Among the tested compounds, compound 19 is considered
to be a promising dual inhibitor against AChE and BuChE and, thus, a therapeutic candidate in AD.
Acknowledgments: This work was supported by National Natural Science Foundation of China (for young
scientists) (No. 21302228).
Author Contributions: Design, Synthesis, experimental work and writing were performed by Dong-mei Wang,
Hui Fu, Lin Wang. Bo Feng performed molecular docking and revision of the first draft of paper. Biological
Evaluation were performed by Ai-lin Liu and Guan-hua Du. Song Wu supervised the whole project. All authors
reviewed and approved the final version.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 12–33 are available from the authors.
©
2017 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).