The development of 2-acetylphenol-donepezil hybrids as multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmcl.2019.126625

A series of 2-acetylphenol-donepezil hybrids was designed and synthesized based on multi-target-directed ligands strategy. The biological activities were evaluated by AChE/BChE inhibition and MAO-A/MAO-B inhibition. The results revealed that the tertiary

Full text of DOI:10.1016/j.bmcl.2019.126625

Bioorganic&MedicinalChemistryLetters29(2019)126625
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The development of 2-acetylphenol-donepezil hybrids as multifunctional
agents for the treatment of Alzheimer’s disease
Gaofeng Zhua,d, Keren Wangb,d, Jian Shib, Pengfei Zhangc, Dan Yangb, Xiaotian Fanb, Ziyi Zhangb,
⁎
Wenmin Liub, Zhipei Sangb,
a State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550014, China
b College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang 473061, China
c School of Medicine, Henan University Minsheng College, Kaifeng 475000, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of 2-acetylphenol-donepezil hybrids was designed and synthesized based on multi-target-directed li-
gands strategy. The biological activities were evaluated by AChE/BChE inhibition and MAO-A/MAO-B inhibi-
tion. The results revealed that the tertiary amines and methylene chain length significantly affected the eeAChE
inhibitory potency, in particular, compound TM-14 showed the best eeAChE inhibitory activity with IC50 value
of 2.9 μM, in addition, both kinetic analysis of AChE inhibition and docking study displayed that TM-14 could
simultaneously bind to the catalytic active site and peripheral anionic site of AChE. Moreover, compound TM-14
was a selective metal chelator and could form 1:1 TM-14-Cu2+ complex. The structure-active-relationship also
indicated that the O-alkylamine fragment remarkably decreased hMAO-B inhibitory activity, compound TM-2
exhibited potent hMAO-B inhibitory activity (IC50 = 6.8 μM), which was supported by the molecular docking
study. More interestingly, compounds TM-14 and TM-2 could cross the blood-brain barrier in vitro. Therefore,
the structure-active-relationship of 2-acetylphenol-donepezil hybrids could encourage the development of
multifunction agents with selective AChE inhibition or selective MAO-B inhibition for the treatment of
Alzheimer’s disease.
Alzheimer’s disease
2-Acetylphenol-donepezil hybrids
Structure-active-relationship
eeAChE inhibition
hMAO-B inhibition
Alzheimer’s disease (AD) is a devastating neurodegenerative disease
and the most common cause of dementia, affecting nearly 36 million
people today and the number will be approximately triple to 131 mil-
lion by 2050.1 Up to now, the etiology of AD is not fully understood, but
several factor including cholinergic dysfunction, amyloid-β (Aβ) de-
posits, tau protein aggregation, and metal ion disorder are considered
to be important roles in the pathophysiology of AD.2 Therefore, there
are several acetylcholinesterase inhibitors (AChEIs), named donepezil,
rivastigmine and galantamine, have been approved by Food and Drug
Administration (FDA) for the treatment of AD by increasing acet-
ylcholine (ACh) level. However, these single target drugs could tem-
porarily delay the progression of cognitive decline in AD, their effects
are modest. Moreover, AChE inhibition for long time lead to undesir-
able side effects, such as gastrointestinal disturbances, bradycardia, and
excessive salivation, which are associated with peripheral cholinergic
receptors.3–5
Due to the multi-factors pathogenesis of AD, the multi-target-di-
rected ligands (MTDLs), one single molecule could hit different targets
involved in the cascade of AD pathological events, have been con-
sidered as an appropriate strategy to achieve better therapeutic efficacy
for AD. Further, the MTDLs strategy has been applied and developed by
many researchers.6–8 It is gratified that several MTDLs candidate drugs
with disease modifying potential are now in the pipeline and have
reached testing stage in clinical trials, such as Latrepirdine hydro-
chloride (Phase III), Eltoprazine hydrochloride (Phase II), Bexarotene
(Phase II), Bryostatin 1 (Phase II), Ladostigil tartrate (Phase II), Indole-
3-propionic acid (Phase I), Telmisartan (Phase I) and so on.9
In our previous work, novel 2-acetylphenol-O-alkylamine deriva-
tives were designed and synthesized as multi-function agents for the
treatment of AD by combining 2-acetylphenol analogs DDDT-2d with
Abbreviations: AD, Alzheimer’s disease; Aβ, β-amyloid; AChEIs, acetylcholinesterase inhibitors; FDA, Food and Drug Administration; ACh, acetylcholine; MTDLs,
multi-target-directed ligands; BChE, butyrylcholinesterase; EeAChE, Electrophorus electricus AChE; eqBuChE, equine serum BuChE; CAS, catalytic active site; PAS,
peripheral anionic site; MAO-B, monoamine oxidase B; MAO-A, monoamine oxidase A; PAMPA-BBB, parallel artificial membrane assay for the blood-brain barrier
⁎ Corresponding author.
E-mail address: sangzhipei@126.com (Z. Sang).
d These authors contributed equally.
https://doi.org/10.1016/j.bmcl.2019.126625
Received 25 May 2019; Received in revised form 15 August 2019; Accepted 16 August 2019
Availableonline19August2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.

G. Zhu, et al.
Bioorganic&MedicinalChemistryLetters29(2019)126625
Fig. 1. Design strategy for the 2-acetylphenol-donepezil hybrids.
scutellarein-O-alkylamine derivatives EJMC-16d, the lead compound
obtained provided significant data for the development of anti-AD
drugs.10 As we all known, donepezil is a selective AChE inhibitor ap-
proved by FDA to treat mild-to-moderate AD, and the benzylpiperidine
fragment is the key pharmacophore.11 In addition, the methylene chain
length is also an important factor on AChE and butyrylcholinesterase
(BChE) inhibitory activity based on our preliminary work.12–16 There-
fore, a series of novel 2-acetylphenol-donepezil hybrids was designed
and synthesized (Fig. 1), the tertiary amine groups focused on benzyl-
piperidine fragment, as well as its modified derivatives, such as ben-
zylpiperazine, 4-phenylpiperidine, 1,2,3,4-tetrahydroisoquinoline, 6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline, N-ethylbenzylamine and
diethylamine, and the methylene chain length including n = 2, 3, 4, 5,
6, 9, 10 and 12. Furthermore, the biological activities were evaluated
by AChE/BuChE inhibition and MAO-A/MAO-B inhibition. Hoping the
structure-active-relationship would give important clues for developing
multi-function agents to treat AD.
donepezil hybrids were evaluated using the modified Ellman method.17
The eeAChE was from electric eel and eqBuChE was from equine serum.
The positive compound donepezil and compound 1 were also tested as
control purpose. As shown in Table 1, the target compounds TM-
1 ∼ TM-30 displayed significant eeAChE inhibitory activity with IC50
values ranging from 35.2 μM to 6.6 μM, implying that introduction of O-
alkylamine fragment could increase AChE inhibitory activity, which
was consist with our design strategy. In addition, these target com-
pounds TM-1 ∼ TM-30 indicated weak eqBuChE inhibitory potency, so
the target compounds were selective AChE inhibitors, as similar with
donepezil.
According to the screening data in Table 1, the tertiary amine
fragment NR1R2 and methylene chain length were focused on to explore
the structure-active-relationship of AChE inhibitory activity. Firstly, the
tertiary amine fragment remarkably affected AChE inhibitory potency,
the general tendency was N-ethylbenzylamine > 1,2,3,4-tetra-
hydroisoquinoline > benzylpiperazine > benzylpiperidine > 6,7-di-
methoxy-1,2,3,4-tetrahydroisoquinoline > 4-phenylpiperidine >
diethylamine. For example, when the methylene chain length was
three, compound TM-5 with benzylpiperidine fragment showed potent
AChE inhibitory activity with IC50 value of 11.7 μM, replacing benzyl-
piperidine of TM-5 with 4-phenylpiperidine to get compound TM-6, the
AChE inhibitory activity slightly decreased to 13.4 μM, and then ex-
The synthetic route of target compounds TM-1 ∼ TM-30 was de-
scribed in Scheme 1. Briefly, 2,4-dihydroxyacetophenone 1 as raw
material was treated with excessive amounts of 1,2-dibromoethane
(2a), 1,3-dibromopropane (2b), 1,4-dibromobutane (2c), 1,5-di-
bromopentane (2d), 1,6-dibromohexane (2e), 1,9-dibromononane (2f),
1,10-dibromodecane (2 g) or 1,12-dibromododecane (2 h) in the pre-
sence of K2CO3 in CH3CN at 65 °C to get the bromine alkoxy inter-
mediates 3a ∼ 3 h. Finally, the intermediates 3a ∼ 3 h were treated
with secondary amines (A ∼ G) in the presence of K2CO3 in anhydrous
CH3CN at 65 °C to obtain the desired products TM-1 ∼ TM-30.
The cholinesterase inhibitory activities of 30 2-acetylphenol-
changing
4-phenylpiperidine
of
TM-6
with
1,2,3,4-tetra-
hydroisoquinoline to obtain TM-7, which showed better AChE in-
hibitory activity (IC50 = 8.9 μM) than compounds TM-5 and TM-6. And
further, adding methoxy groups into the 1,2,3,4-tetrahydroisoquinoline
of TM-7 to get compound TM-8, the AChE inhibitory potency decreased
Scheme 1. Synthesis of target compounds TM-1 ∼ TM-30. Reagents and conditions: (i) Br(CH2)nBr (2a ∼ 2 h), K2CO3, CH3CN, 65 °C, 6–8 h; (ii) K2CO3, NR1R2 (A ∼ G)
anhydrous CH3CN, 65 °C, 6–10 h.
2

G. Zhu, et al.
Bioorganic&MedicinalChemistryLetters29(2019)126625
Table 1
eeAChE and eqBuChE inhibitory activities, MAO-A and MAO-B inhibitory potencies of target compounds and control compounds.
Comp.
n
NR1R2
IC50 (μM)a
SIf
IC50 (μM)a
MAO-Ad
n.a.j
eeAChEb
eqBuChEc
MAO-Be
22.8
TM-1
TM-2
2
2
16.5
6.6
0.82
0.19
0.22
n.a.h
–
–
0.05
0.44
N
11.2
n.a.h
n.a.h
0.03%g
26.1
n.a.j
n.a.j
0.07
10.2
13.8
11.7
N
N
N
TM-3
TM-4
2
2
18.9
35.2
–
–
0.31
0.20
0.39
N
TM-5
3
11.7
0.18
0.21
63.1
0.44
0.18
5.4
n.a.j
18.7
0.42
N
N
TM-6
TM-7
3
3
13.4
8.9
29.4
n.a.h
2.2
–
24.7
n.a.j
0.09
19.8
16.7
0.13
0.28
0.19
0.27
N
j
TM-8
3
10.1
57.9
0.99
0.18
5.7
8.1
0.01%
13.8
0.16
O
O
N
TM-9
3
3
7.2
0.13
0.56
18.7
n.a.h
2.6
–
n.a.j
n.a.j
14.9
16.1
0.57
0.19
N
TM-10
26.7
N
N
N
TM-11
TM-12
4
4
8.9
5.9
0.17
0.19
0.89
n.a.h
47.5
–
n.a.j
6.3
13.5
12.8
0.26
0.05
j
0.81
0.91
8.1
0.02%
TM-13
4
15.9
56.4
3.5
n.a.j
15.7
0.86
O
O
N
TM-14
TM-15
4
4
2.9
0.03
0.19
34.6
n.a.h
0.11
11.9
–
n.a.j
n.a.j
16.6
22.2
0.43
0.29
N
20.9
N
N
N
N
N
N
TM-16
TM-17
TM-18
TM-19
TM-20
TM-21
5
5
5
6
6
6
8.2
7.5
5.2
7.1
9.2
16.3
0.29
0.17
0.12
0.34
0.15
n.a.h
n.a.h
37.2
66.9
32.6
n.a.h
–
n.a.j
n.a.j
n.a.j
22.1
21.2
n.a.j
20.8
15.2
29.8
15.8
26.9
22.1
0.22
0.36
0.33
0.24
0.36
0.15
–
0.64
0.49
0.76
7.1
9.4
3.5
–
0.17
0.04
0.11
N
N
N
N
N
TM-22
TM-23
TM-24
TM-25
TM-26
TM-27
9
8.5
0.71
0.15
n.a.h
19.8
n.a.h
13.8
15.1
0.8
–
n.a.j
19.7
n.a.j
n.a.j
19.8
n.a.j
27.9
21.6
19.4
21.6
17.3
14.2
0.31
0.22
0.31
0.28
0.16
0.11
9
6.7
0.24
2.9
–
0.17
0.04
N
N
9
11.6
9.2
0.15
g
g
10
10
10
0.17
0.49
0.06%
0.02%
–
6.9
–
N
N
g
18.4
0.24
0.01%
–
(continued on next page)
3

G. Zhu, et al.
Bioorganic&MedicinalChemistryLetters29(2019)126625
Table 1 (continued)
Comp.
n
NR1R2
IC50 (μM)a
SIf
IC50 (μM)a
MAO-Ad
n.a.j
eeAChEb
eqBuChEc
MAO-Be
23.4
TM-28
TM-29
TM-30
10
12
12
6.5
9.5
7.7
0.18
0.42
0.59
50.7
n.a.h
38.9
0.31
7.8
–
0.41
0.39
0.64
N
N
N
n.a.j
n.a.j
25.8
33.1
0.67
0.02
5.0
1
n.a.h
0.019
n.t.i
n.a.h
4.76
n.t.i
n.t.i
n.t.i
n.t.i
n.t.i
donepezil
rasagiline
iproniazid
0.0003
251
–
n.t.i
0.63
6.18
0.01
0.07
0.036
1.78
0.002
0.01
n.t.i
–
aIC50 values represent the concentration of inhibitor required to decrease enzyme activity by 50% and are the mean of three independent experiments, each
performed in triplicate (SD = standard deviation). bFrom electrophorus electricus. cFrom equine serum. dFrom recombinant human MAO-A. eFrom recombinant human
f
MAO-B. SI (selectivity index) = IC50(eqBuChE)/IC50 (EeAChE). gThe inhibition percent ratio of compounds for eqBuChE at a concentration of 50 μM in the assay
conditions. hn.a. = no active. Compounds defined “no active” means percent inhibition less than 5.0% at a concentration of 50 μM in the assay conditions. in.t. = no
j
test. n.a. = no active. Compounds defined “no active” means percent inhibition less than 10.0% at a concentration of 10 μM in the assay conditions.
to 10.1 μM. Moreover, ring opening of 1,2,3,4-tetrahydroisoquinoline
of TM-7 to get compound TM-9 with N-ethylbenzylamine, the AChE
inhibitory activity increased to 7.2 μM. And further removing benzene
ring of N-ethylbenzylamine in TM-9 to gain compound TM-10 with
diethylamine, the AChE inhibitory activity sharply decreased to
26.7 μM. Besides, the compounds TM-2, TM-23 and TM-26 with ben-
zylpiperazine fragment showed significant AChE inhibitory activity,
while exhibited lower inhibitory activity than compound TM-28 with
N-ethylbenzylamine. The similar phenomenon were also observed, such
as TM-14 < TM-12 < TM-11 < TM-13 < TM-15; TM-18 < TM-
17 < TM-16; TM-19 < TM-20 < TM-21; TM-23 < TM-22 < TM-
24; TM-28 < TM-26 < TM-25 < TM-27; TM-30 < TM-29. Parti-
cularly, compound TM-14 with N-ethylbenzylamine fragment exhibited
the best AChE inhibitory activity with IC50 value of 2.9 μM. Secondly,
the methylene chain length also influenced AChE inhibitory activity, in
general, the AChE inhibitory activity enhanced as methylene chain
increased, but the inflection point was presented in appropriate me-
thylene chain. For example, when the tertiary amine was benzylpiper-
idine, the optimal methylene chain was 6, TM-1 (n = 2) > TM-5
(n = 3) > TM-11 (n = 4) > TM-16 (n = 5) > TM-19 (n = 6)
< TM-22 (n = 9) < TM-25 (n = 10) < TM-29 (n = 12); when the
Fig. 2. Steady state inhibition by compound TM-14 of the AChE hydrolysis of
ACh. The plots show mixed-type AChE inhibition for compound TM-14.
intramolecular hydrogen bonding, the carbonyl group interacted with
Arg289 via one intermolecular hydrogen bonding, and the hydroxyl
group interacted with Phe288 and Arg289 via one intermolecular hy-
drogen bonding, respectively. In addition, the N atom of N-ethylben-
zylamine interacted with Tyr121 via one intermolecular hydrogen
bonding. Moreover, the benzene ring of N-ethylbenzylamine could in-
teract with Asp70 via σ-π interaction. Meanwhile, the potential hy-
drophobic interactions could be observed between the TM-14 and re-
sidues Tyr121, Tyr334, Phe288, Phe331, Arg289, Asp72, Tyr70 and
Phe330. Therefore, the molecular docking of TM-14 provided reason-
ably explanation for its potent AChE inhibitory activity.
tertiary
amine was N-ethylbenzylamine and 1,2,3,4-tetra-
hydroisoquinoline, the optimal methylene chain was 4, TM-3
(n = 2) > TM-9 (n = 3) > TM-14 (n = 4) < TM-18 (n = 5) < TM-
28
(n = 10) < TM-30
(n = 12),
TM-3
(n = 3) > TM-4
(n = 4) < TM-17 (n = 5) < TM-20 (n = 6) < TM-24 (n = 9).
However, when the tertiary amine was diethylamine, the AChE in-
hibitory potency gradually strengthened as methylene increased, TM-4
(n = 2) > TM-10 (n = 3) > TM-15 (n = 4) > TM-21 (n = 6), and
the optimal methylene was six. Therefore, compound TM-14 was a
potent selective AChE inhibitor (IC50 = 2.9 μM), and the selective index
was 11.9.
The metal chelation ability of TM-14 was tested by UV–visual
spectrometry using Cu2+, Fe2+, Zn2+ and Al3+ 18
As shown in Fig. 4,
.
the electronic spectra of TM-14 presented a red shift (the peak at
315 nm shifted to 359 nm) after adding CuCl2. However, the electronic
spectra of TM-14 displayed no obvious change after adding FeSO4,
ZnCl2 and AlCl3. Therefore, TM-14 would be a selective metal chelator.
The molar ratio method was performed to test the stoichiometry of
the TM-14-Cu2+ complex by compound TM-14 with ascending
amounts of CuCl2. As shown in Fig. 5, the absorbance linearly increased
initially and then plateaued. The two straight lines intersected at a mole
fraction of 0.97, meaning a 1:1 stoichiometry for the complex TM-14-
The further kinetic study of compound TM-14 was performed to
explore the inhibition mechanism of AChE.13 As shown in Fig. 2, the
Lineweaver–Burk plots displayed that both inhibitions had rising slopes
and increasing intercepts at higher concentration, indicating a mixed-
type inhibition.
The docking study was performed to explore possible mechanism of
AChE (PDB code: 1EVE) (x: 2.023, y: 63.295, z: 67.062) with compound
TM-14 using AutoDock 4.2 package with Discovery Studio 2.5.13,16 As
shown in Fig. 3, TM-14 occupied the entire enzymatic catalytic site
(CAS), the mid-gorge sites and the peripheral site (PAS), and could si-
multaneously bind to both the CAS and the PAS. The 2-hydroxy and
carbonyl group at the 2,4-dihydroxyacetophenone nucleus formed one
Cu2+
.
Moreover, the inhibitory activities against huMAOs for target
compounds TM-1 ∼ TM-30 were tested by fluorimetric assay.19 Rasa-
giline and iproniazid were also tested as control drugs. The results were
shown in Table 1, all the target compounds showed moderate hMAO-B
inhibitory activity with IC50 values ranging from 10.2 μM to 33.1 μM,
4

G. Zhu, et al.
Bioorganic&MedicinalChemistryLetters29(2019)126625
Fig. 4. The UV spectrum of compound TM-14 (37.5 μM, in methanol) alone or
in the presence of CuCl2, FeSO4, ZnCl2 and AlCl3 (37.5 μM, in methanol).
Fig. 5. Determination of the stoichiometry of complex-Cu2+ was used molar
ratio method by titrating the methanol solution of compound TM-14 with as-
cending amounts of CuCl2. The final concentration of tested compound was
37.5 μM, and the final concentration of Cu2+ ranged from 3.75 to 93.75 μM.
negative effect on hMAO-B inhibitory activity. Especially, the com-
pound TM-2 with benzylpiperazine and n = 2 exhibited the best
hMAO-B inhibitory activity with IC50 value of 10.2 μM. Furthermore, all
the target compounds displayed weak hMAO-A inhibitory activity,
implying that the target compounds were selective hMAO-B inhibitors,
which was beneficial for the treatment of AD.
In order to explore mechanism of TM-2 with MAO-B, docking study
was performed based on the X-ray crystal structures of human MAO-B
(PDB code: 2V60) (x: 14.846, y: 128.673, z: 24.971).20 As shown in
Fig. 6, the 2-hydroxyacetophenone nucleus was located in the huMAO-B
binding pocket, 2-hydroxy and carbonyl group formed one in-
tramolecular hydrogen bonding. In addition, the carbonyl group could
interact with the enzymatic cofactor FAD via intermolecular hydrogen
bonding. Moreover, the benzene of 2-hydroxyacetophenone nucleus
could interact with Tyr398 via one π-π interaction. Meanwhile, some
hydrophobic interactions were observed between TM-2 and Trp119,
Ile316, Ile199, Phe103, Ile198, Leu171, Ieu167, Phe343, Gln206 and
Tyr398. Therefore, the above interactions observed could explain the
potent MAO-B inhibitory activity for TM-2.
Fig. 3. (A) Representation of compound TM-14 (green stick) interacted with
residues in the binding site of TcAChE (PDB code: 1EVE). (B) 3D docking model
of compound TM-14 with TcAChE. (C) 2D schematic diagram of docking model
of compound TM-14 with TcAChE.
and indicated lower hMAO-B inhibitory potency than the referenced
compounds. It meant that the O-alkylamine fragment sharply decreased
hMAO-B inhibitory activity. In addition, the tertiary amine groups and
methylene chain length led to slightly influence on hMAO-B inhibitory
potency. In general, when the methylene chain was 2, the tertiary
amine inhibition tendency was benzylpiperazine > diethylamine >
N-ethylbenzylamine > benzylpiperidine, such as TM-2 < TM-
4 < TM-3 < TM-1; when the methylene chain was 4, 1,2,3,4-tetra-
hydroisoquinoline > benzylpiperidine > 6,7-dimethoxy-1,2,3,4-tet-
rahydroisoquinoline > N-ethylbenzylamine > diethylamine. Mean-
while, it appeared that the compounds with n = 2 showed better
hMAO-B inhibitory activity than the other compounds (n = 3, 4, 5, 6, 9,
10, 12), indicating that the extended methylene chain produced
To test the brain penetration of compounds TM-14 and TM-2, a
parallel artificial membrane assay for the blood- brain barrier (PAMPA-
BBB) was performed.21,22 The assay was validated by comparing the
permeability of 11 commercial drugs with reported values (Table 2). A
good linear correlation, Pe (exp) = 0.9163Pe (bibl.) − 0.2247
(r2 = 0.9558) was produced (Fig. 7). Based on this equation, and con-
sidering the limit conditions established by Di et al., we determined that
compound with permeability above 3.44 × 10−6 cm/s could cross the
blood–brain barrier (Table 3). According to the measured permeability
(Table 4), TM-14 and TM-2 could cross the BBB in vitro.
5

G. Zhu, et al.
Bioorganic&MedicinalChemistryLetters29(2019)126625
Fig. 6. Compound TM-2 (green stick) interacting with residues in the binding site of MAO-B (PDB code: 2V60), highlighting the protein residues that participate in
the main interactions with the inhibitor.
Table 2
Table 3
Permeability (Pe × 10−6 cm/s) in the PAMPA-BBB assay for 11 commercial
Ranges of permeability of PAMPA-BBB assays (Pe × 10−6 cm/s).
drugs used in the experiment validation.
Commercial drugs
Compounds of high BBB permeation (CNS +)
Compounds of uncertain BBB permeation (CNS +/−)
Compounds of low BBB permeation (CNS −)
Pe > 3.44
b
Bibla
PBS:EtOH(70:30)
3.44 > Pe > 1.61
Pe < 1.61
Verapamil
Oxazepam
Diazepam
Clonidine
16
16.90
9.60
10
16
11.86
5.10
Table 4
5.3
13
Permeability Pe (×10−6 cm/s) in the PAMPA-BBB assay of the selected com-
pounds TM-2 and TM-14 and the predictive penetration in the CNS.
Imipramine
Testosterone
Caffeine
10.10
16.30
1.28
17
1.3
0.9
2.5
0.1
0.12
Compounda
Pe (×10−6 cm/s)b
Prediction
Enoxacine
Piroxicam
Norfloxacin
Theophylline
0.47
0.72
TM-14
TM-2
15.13
17.37
0.29
0.83
CNS +
CNS +
0.42
0.10
a
Compounds TM-14 and TM-2 was dissolved in DMSO at 5 mg/mL and
a
b
Taken from Ref. 21.
diluted with PBS/EtOH (70:30). The final concentration of the compound was
Data are the mean
SD of three independent experiments.
100 μg/mL.
b
Values are expressed as the mean
SD of three independent experiments.
Briefly, a series of 2-acetylphenol-donepezil hybrids was developed
as multi-function agents. The structure-active-relationship showed that
both tertiary amines groups and methylene chain length remarkably
affected AChE inhibitory activity, in particular, compound TM-14
showed the best eeAChE inhibitory potency with IC50 value of 2.9 μM,
in addition, both the kinetic analysis and docking study exhibited that
TM-14 was a mixed-type inhibitor, and could simultaneously bind to
both CAS and PAS of AChE. And compound TM-14 served as a selective
metal chelator. Moreover, all the target compounds displayed moderate
hMAO-B inhibitory activity, the extended methylene chain decreased
hMAO-B inhibitory potency, and the tertiary amine did not produce
positive effect, compound TM-2 showed good hMAO-B inhibitory ac-
tivity with IC50 value of 10.2 μM. Furthermore, compounds TM-2 and
Fig. 7. Linear correlation between experimental and reported permeability of commercial drugs using the PAMPA-BBB assay. Pe(exp) = 0.9163, Pe(bibl.) −0.2247
(r2 = 0.9558).
6

G. Zhu, et al.
Bioorganic&MedicinalChemistryLetters29(2019)126625
TM-14 could cross BBB in vitro. Therefore, the structure-active-re-
lationship of 2-acetylphenol-donepezil hybrids encouraged the dis-
covery of selective AChE inhibitors or selective hMAO-B inhibitors, and
improved the development of multifunction agents for the treatment of
AD.
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ligands and other therapeutic strategies in the search of a real solution for
Alzheimer's disease. Curr Neuropharmacol. 2014;12(1):2–36.
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Declaration of Competing Interest
The authors declare no competing financial interest.
Acknowledgments
9. http://integrity.thomson-pharma.com.
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acetyl-5-O-(amino-alkyl)phenol derivatives as multifunctional agents for the treat-
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This work was supported in part by the Foundation and Frontier
Projects of Nanyang Science and Technology Bureau (2017JCQY020
and 2017 JCQY021). The Special Project of Nanyang Normal University
(SYKF2018081 and 2019QN001). The Key Scientific Research Project
of Colleges and Universities in Henan Province (NO.20A350006).
International Training of High-level Talents in Henan Province in 2019
(NO.17) (Henan Foreign Experts Bureau). Graduate Innovation Fund
Project of Nanyang Normal University (NO.02). The Social
Development of Technical Supporting Foundation of Guizhou Province
(No. [2017]2839).
13. Sang Z, Qiang X, Li Y, et al. Design, synthesis and evaluation of scutellarein-O-al-
kylamines as multifunctional agents for the treatment of Alzheimer’s disease. Eur J
Med Chem. 2015;94:348–366.
14. Sang Z, Wang K, Han X, Cao M, Tan Z, Liu W. Design, synthesis, and evaluation of
novel ferulic acid derivatives as multi-target-directed ligands for the treatment of
Alzheimer’s disease. ACS Chem Neurosci. 2019;10(2):1008–1024.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2019.126625.
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