Design, synthesis and evaluation of novel tacrine-multialkoxybenzene hybrids as multi-targeted compounds against Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2016.03.077

A series of benzoates (or phenylacetates or cinnamates) - tacrine hybrids (7a-o) were designed, synthesized and evaluated as multi-potent anti-Alzheimer drug candidates. The screening results showed that most of them exhibited a significant ability to inhibit ChEs, certain selectivity for AChE over BuChE and strong potency inhibitory of self-induced β-amyloid (Aβ) aggregation. All IC₅₀ values of biological activity were at the nanomolar range. Especially, compound 7c displayed the greatest ability to inhibit AChE with an IC₅₀ value of 5.63 nM and the highest selectivity with ratio of BuChE/AChE value of 64.6. Moreover, it also exhibited a potent inhibitory of Aβ aggregation with an IC₅₀ value of 51.81 nM. A Lineweaver-Burk plot and molecular modeling study showed that compound 7c targeted both the CAS and PAS of ChEs. A structure-activity relationship analysis suggested that the electron density of aromatic ring which was linked with tacrine through acetyl group played a significant role in determining the inhibitory activity.

Full text of DOI:10.1016/j.ejmech.2016.03.077

Accepted Manuscript
Design, synthesis and evaluation of novel tacrine-multialkoxybenzene hybrids as
multi-targeted compounds against Alzheimer's disease
Chao Zhang, Qiao-Yi du, Lang-Di Chen, Wen-Hao Wu, Si-Yan Liao, Li-Hong Yu, Xin-
Tong Liang
PII:
S0223-5234(16)30260-4
10.1016/j.ejmech.2016.03.077
EJMECH 8502
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 December 2015
Revised Date: 24 March 2016
Accepted Date: 25 March 2016
Please cite this article as: C. Zhang, Q.-Y. du, L.-D. Chen, W.-H. Wu, S.-Y. Liao, L.-H. Yu, X.-T.
Liang, Design, synthesis and evaluation of novel tacrine-multialkoxybenzene hybrids as multi-targeted
compounds against Alzheimer's disease, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.03.077.
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ACCEPTED MANUSCRIPT

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Design, synthesis and evaluation of novel
tacrine-multialkoxybenzene hybrids as multi-targeted
compounds against Alzheimer’s disease
a
a
a
a
Chao Zhang a, *, Qiao-Yi Du , Lang-Di Chen , Wen-Hao Wu , Si-Yan Liao ,
Li-Hong Yu a and Xin-Tong Liang a
a, * School of Pharmaceutical Sciences, Guangzhou Medical University, Xinzao, Panyu
District, Guangzhou, 511436, P. R. China
Abstract: A series of benzoates (or phenylacetates or cinnamates) – tacrine hybrids
(7a-o) were designed, synthesized and evaluated as multi-potent anti-Alzheimer drug
candidates. The screening results showed that most of them exhibited a significant
ability to inhibit ChEs, certain selectivity for AChE over BuChE and strong potency
inhibitory of self-induced β-amyloid (Aβ) aggregation. All IC50 values of biological
activity were at the nanomolar range. Especially, compound 7c displayed the greatest
ability to inhibit AChE with an IC50 value of 5.63 nM and the highest selectivity with
ratio of BuChE/AChE value of 64.6. Moreover, it also exhibited a potent inhibitory of
Aβ aggregation with an IC50 value of 51.81 nM. A Lineweaver-Burk plot and
molecular modeling study showed that compound 7c targeted both the CAS and PAS
of ChEs. A structure-activity relationship analysis suggested that the electron density
of aromatic ring which was linked with tacrine through acetyl group played a
significant role in determining the inhibitory activity.
Keywords: Tacrine, Multi-target-directed ligands, Alzheimer’s disease,
Cholinesterase inhibitors, Self-induced Aβ aggregation.
1. Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative brain disorder that is
characterized by dementia, cognitive impairment, memory loss, severe behavioral
abnormalities and ultimately death [1,2]. It is estimated that more than 18 million
people presently suffer from AD, and the number of patients is expected to sharply
increase to 114 million by 2050[3]. The vast number of people requiring constant care
and related services will severely strain medical, monetary and human resources.
Consequently, the developing treatments that slow or halt the disease progression
have become imperative. Despite enormous efforts devoted to research in AD and
many factors have been found to be implicated in AD over past decades, its etiology
and pathogenesis remain unclear. To date AD is thought to be a complex,
multifactorial syndrome with many related molecular lesions contributing to its
pathogenesis[4-8]. Several factors including low levels of acetylcholine (ACh),

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amyloid-β(Aβ) deposits, oxidative stress and dyshomeostasis of biometals have been
considered to play definitive roles in its etiology, and several hypotheses based on
these factors have been proposed to explain the mechanism of AD development [9,10]
.
[4,5,11]
One of the AD hypothesis is the cholinergic hypothesis
which has become
the leading strategy for the development of cholinesterase inhibitors(ChEIs) aimed to
increasing of levels of acetylcholine through inhibition of cholinesterases (ChEs)
[12-14], since the cognitive and memory deterioration of AD is due to the low level of
choline, especially ACh in the brain. The most prevailing hypothesis, the amyloid
hypothesis posits that an increased production of β-amyloid peptide and its
aggregation and accumulation in a brain lead to a neuronal cell death, cause the Aβ
soluble oligomers and the assembly of Aβ aggregates into fibrils are toxic to
neurons[6,14]. Another hypothesis was reported, it was suggested that oxidative stress is
involved in the early stage of the pathologic cascade and represents a key factor to
initiate the aggregation of β-amyloid and tau-protein hyperphosphorylation[15]
.
Accordingly, many antioxidants that specifically scavenge oxygen radicals were
observed to be able to attenuate the syndrome of AD and prevent the progression of
the disease[16]. In addition, another AD hypothesis called metal hypothesis indicates
that the metals(Fe, Cu and Zn) also play a role in pathogenesis of AD [17]. It was
observed that during disease progression from moderate to severe AD, the metals
progressively accumulate in AD patients [18]. The abnormal accumulation of metals is
closely associated with the formation of Aβ plaques and neurofibrillary tangles (NFT),
the hallmarks in brain of AD patients [19, 20]. Besides, the abnormally high levels of Cu
and Fe in brain catalyze the production of reactive oxygen species (ROS), which
further elicit oxidative stress contributing to the AD pathogenesis [20-22]. Thus,
lowering the concentration of metals in brain by chelating metals represents another
rational therapeutic approach for halting AD pathogenesis.
Because of the multifactorial nature of AD as above mentioned, AD treating
remains a challenge for the pharmaceutical community and no effective drug is
currently available. There have been many approaches to potential therapies, most
treatment strategies have aimed at cholinergic neurotransmission and β-amyloid
peptide. However, cholinesterase inhibitors were the first and, to date, the only class
of drugs in the market that showed some results in the treatment of AD (such as
tacrine, donepezil, rivastigmine and galantamine, Fig. 1). As the traditional ‘one
molecule, one target’ paradigm, the so-called magic bullets, can generally only offer
limited and transient benefits, these drugs that modulate such a single target could
only improve symptoms or enable a palliative treatment instead of curing or
preventing the neurodegeneration, and exert limited effects on most patients[23]. It is
unlikely that a unitary mechanism of action will provide a comprehensive therapeutic
approach to such multifaceted neurodegenerativedisease. Thus, efficient therapy is
more likely to be achieved by drugs that incorporate several pharmacological effects
into a single molecular entity. This concept has been rationalized in the field of
neurodegenerative diseases affording the multitarget-directed ligands (MTDLs)
design strategy[24-27]. The strategy holds that single molecules being properly
combined two or more distinct pharmacological properties and then able to act at

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different targets in the neurodegenerative process, can achieve greater effectiveness
compared to single-targeted drugs for investigating AD. Hence many studies have
been devoted to the search for multifunctional agents that simultaneously inhibit
cholinesterase, decrease Aβ levels and antioxidant[28, 29]
.
Figure 1. Chemical structure of reported AChE inhibitors
Tacrine was the first approved cholinesterase inhibitor by the FDA for the treatment
of AD[30]. Although, its side effects, its modification is still of great interest. Recent
studies have demonstrated that homo- and hetero-dimers can improve the biological
profile of tacrine and even overcome some of its side effects[31]. Nevertheless, some of
tacrine derivatives as hybrid potential drugs, have been developed to improve its
activity[29], including tacrine-8-hydroxyquinoline[32], tacrine-fluorobenzoic acid[33] and
tacrine-multialkoxybenzene hybrids[34]. Moreover, some studies have suggested that
electron-rich aromatics would bind to the peripheral binding site, which have been
confirmed in a comparison of aromatics with differing electron density[35]. Therefore
development of tacrinehybrids that combines the ChEs inhibition, for the
enhancement of the cholinergic neurotransmission, with reduction the Aβ fibril
self-aggregation and antioxidant by conjugating with other active groups are
attractive[36]. In order to further explore the anti-Alzheimer potential of the
electron-rich aromatics based on MTDLs strategy, we herein reported a series of
novel multifunctional compounds by conjugating a tacrine and electron-rich aromatics
including benzoates, phenylacetates, cinnamates and acetylindole. A selection of these
compounds were synthesized and evaluated for their in vitro activity as inhibitors of
AChE, BuChE and self-induced Aβ aggregation activity.
2. Results and discussion
2.1. Chemistry
The tacrine-benzoates (or phenylacetates or cinnamates or acetylindole) hybrids
were synthesized as shown in Scheme 1. Tacrine 4 was firstly prepared according to
the literature with a total yield of 68.1% (see supporting information, scheme s1)[36]. It
was then treated with bromoacetyl bromide in the presence of Et3N to yield
bromoacetylated intermediate 5 in a yield of 82.3%. Compound 5 was finally reacted
with corresponding carboxylic acid derivatives 6a-6o to afford the desired products
7a-7n.

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R1
R2
R3
O
R1
HO
Br
or
or
R2
HN
NH2
O
O
6k-6n
a
HO
R3
+
n
R4
O
N
N
NH
Br
O
Br
5
6a-6j
4
HO
6o
R1
R2
R3
O
O
R1
R2
O
O
HN
N
HN
n
O
R4
O
R4
b
R3
N
7a, R1=R3=R4=H, R2=OMe, n=1
7b, R1=R2=OMe, R3=R4=H, n=1
7c, R2=R3=R4=OMe, R1=H, n=1
7d, R1=R3=R4=H, R2=OH, n=1
7e, R1=R3=R4=H, R2=Cl, n=1
7f, R1=R3=OMe, R2=R4=H, n=0
7g, R1=R2=R3=OMe, R4=H, n=0
7h, R2=R3=R4=OMe, R1=H, n=0
7k, R1=R4=H, R2=R3=OMe
7l, R1=R2=R3=OMe, R4=H
7m, R1=OH, R2=OMe, R3=R4=H
7n, R1=R3=R4=H, R2=Cl
O
O
HN
NH
7o =
O
7i, R1=OMe, R2=OH, R3=R4=H, n=0
7j, R1=R4=H, R2=R3=OH, n=0
N
Scheme 1.Synthesis of tacrine-electron-rich aromatics hybrids. Reagents and conditions: (a) Et3N,
reflux; (b) DMF,K2CO3, rt.
2.2. In vitro inhibition studies on AChE and BuChE
To determine the potential of the target compounds 7a-7o for the treatment of AD,
their acetylcholinesterase (AChE, from electric eel) and butyrylcholinesterase
(BuChE, from equine serum) inhibitory activities were determined by the method of
Ellman et al[37], using tacrine as a reference compound. The IC50 values of all test
compounds for ChEs (AChE and BuChE) and the affinity ratios were summarized as
shown in Table 1.The results showed that most of the tested compounds (7a-7o) had
significant ChEs inhibitory activity with IC50 values ranging from sub-micromolar to
low nanomolar range. All synthesized compounds showed certain selectivity for
AChE over BuChE and the ratio of BuChE/AChE affinity ranged from 1.2- to 64.6-
fold. The compounds showed higher inhibitory potential against AChE that would
possess higher BuChE/AChE affinity ratios.
Some of these new compounds have much more capability to inhibit AChE but
all of these compounds unfortunatly showed weaker inhibitory activity for BuChE
when compared to the reference drug, tacrine. Among these, compound 7c, with a
trimethoxyl phenylacetate group, showed the most potent inhibition for AChE with an

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IC50 value of 5.63 nM, and the potency was 13-times stronger than the reference
compound tacrine with IC50 value of 73.36 nM. The inhibition for BuChE with an
IC50 value of 364 nM was weaker than AChE and the highest ratio of BuChE/AChE
was also observed with affinity ratio value of 64.6. This result indicated that
compound 7c showed the highest selectivity for AChE over BuChE. Furthermore, a
structure-activity relationship analysis suggested that the electron density of aromatic
ring which was linked with tacrine through acetyl group played a significant role in
determining the inhibitory activity for AChE in present work. When the aromatic ring
bore more electron-donating substituents among the homogeneous class of
compounds, the AChE inhibitory activity was increased. For instance, the
homogeneous class of compounds 7a and 7b (n=1) with only one and two methoxy
substituents respectively, exhibited relatively low AChE inhibitory activities
compared with 7c. Compound 7g, which had trimethoxylbenzoate substituent similar
to 7c, appeared to be the third strong inhibitor with IC50 value of 8.46 nM. A similar
result was attached by compound 7o with an electron-rich indolyl moiety and it was a
strong inhibitor with IC50 value of 8.13 nM. Compound 7g and 7o also exhibited the
2nd and 3rd high selectivity with affinity ratio values were 38.5 and 35.7 respectively.
However, some decreased inhibitory activities for AChE were afforded from
compounds 7b, 7f and 7k with two methoxy substituents on the aromatic ring and all
of them showed weak selectivity with affinity ratio values were 8.5, 14.3 and 9.8
respectively. In contrast, the weakest two inhibitory activities for AChE were obtained
from compounds 7e and 7n, with IC50 values of 182.8 nM and 213.3 nM respectively.
Both of them had a chloro group as electron-withdrawing substituent. The results
revealed that the inhibitory activity for AChE could be drastic decreased with an
electron-deficient moiety. In addition, it was observed that there was a slightly
reduction of inhibitory activity for AChE of compound 7l compared to 7c and 7g.
Despite that the same electron-rich aromatic ring was afforded, the IC50 value for
AChE of compound 7l was raised to 16.88 nM. The possible reason of the result
could be that the additional vinyl group enlarged the conjugated regions in the same
substituents of aromatic ring, which meant the electron density of phenyl group was
averaged and thus led to an electron-deficient effect. Furthermore, the linker length
between tacrine and phenyl moiety affected the AChE inhibitory activity and
selectivity as well. It was suggested that phenylacetate group (7c) was preferred to
benzoate group (7g). Finally, the AChE inhibitory activities of all hydroxyl substituted
derivatives were less than methoxy substituted derivatives, which indicated that
methoxy moiety was superior to hydroxyl for inhibited AChE. The reason for this
result could be hyperconjugation effect of methoxy group, which led to more potency
electron-donating ability of methoxy group compared with hydroxyl group.
Table 1. Inhibition of ChEs activity, affinity ratios and inhibition of Aβ(1-42) self-induced
aggregation
a
a
Compds IC50
for IC50
for Affinity
BuChE (nM) ratios b
319.3±0.05 12.6
Aβ(1-42) aggregationc
inhibition (nM)
71.28±0.04
AChE (nM)
25.26±0.75
7a

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65.07±0.23
5.63±0.98
73.96±2.44
182.8±0.16
15.69±1.19
8.46±0.51
15.46±0.56
70.06±0.37
132.85±0.90
16.22±0.85
16.88±0.47
74.87±0.57
213.34±0.38
8.13±4.72
554.1±0.05
364±0.05
8.5
64.6
3.2
22.54±0.01
51.81±0.05
45.29±0.03
77.45±0.03
55.86±0.04
30.74±0.03
65.50±0.03
37.36±0.05
17.36±0.03
49.14±0.02
45.88±0.02
46.12±0.04
51.77±0.02
38.96±0.01
12.21±0.02
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
238.8±0.03
218.2±0.06
223.5±0.04
329.3±0.05
254.8±0.05
319±0.06
344.4±0.06
159.2±0.04
298.9 ±0.03
355 ±0.01
1.2
14.3
38.5
16.4
4.5
2.6
9.8
17.9
4.7
2.5
35.7
0.20
541.7 ±0.05
291.7 ±0.05
14.45 ±0.04
73.36±0.22
Tacrine
aAChE from electric eel, BuChE from equine serum; IC50, inhibitor concentration (means±SEM
of three experiments) for 50% inactivation of AChE.
b Affinity ratios = IC50 (BuChE)/IC50 (AChE)
cInhibition of self-mediated Aβ(1-42) aggregation, the thioflavin-T fluorescence method was used,
the mean±SD of at least three independent experiments and the measurements were carried out in
the presence of 5, 10, 20, 50, 100 ꢀM compounds respectively.
2.3. Kinetic characterization of ChEs inhibition

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Figure 2. Lineweaver-Burk plot for compound 7c with AChE and BuChE, respectively.
Because compounds 7c was the best AChE inhibitor and a better BuChE inhibitor,
it was selected for kinetic measurements by using graphical analysis of steady-state
inhibition data in order to gain information about the mode of inhibition and binding
of the novel inhibitors. As shown in Fig. 2, The Lineweaver-Burk plots for AChE
showed reversible and a mixed type inhibition, which indicated that compounds 7c
could bind to both catalytic active site (CAS) and the peripheral anionic site (PAS) of
AChE simultaneously. It had been reported that some amino acids in PAS of AChE
could promote Aβ fibrillation, and an inhibitor of PAS, propidium, was able to block
this activity [39,40]. Therefore, the inhibitors which could bind to both CAS and PAS of
AChE might have dual inhibition for both AChE activity and AChE induced Aβ
aggregation. A similar interaction was found between 7c and BuChE by means of
Lineweaver-Burk plot.
2.4. Inhibition of self-mediated Aβ(1-42) aggregation
All synthesized compounds were also tested for their ability to inhibit self-mediated
aggregation of Aβ(1-42) by using a thioflavin T fluorescence method[38]. Tacrine was
used as reference compound. From the results summarized in Table 1, it could be seen
that these hybrids apparently prevented the self-mediated Aβ aggregation with IC50
ranging from 17.36 nM to 71.28 nM and showed a little less potencies relative to that
of tacrine (12.21 nM). Compounds 7b, 7c, 7g and 7o having the high inhibitory
activity for AChE also exhibited the good potency on inhibition of self-induced Aβ
(1-42) aggregation with IC50 values of 22.54, 51.81 nM, 30.74 nM and 38.96 nM
respectively, which were weaker than that of reference compound tacrine.
Unfortunately, 7c as the most potent AChE inhibitor only showed relatively moderate
inhibitory potency, with an IC50 value of 51.81 nM. In contrast, compound 7j with a

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hyroxylbenzoate group unexpectedly showed the most potency with an IC50 value of
17.36 nM among all these compounds. These results indicated that hydroxyl moiety
was superior to alkoxyl for inhibitory activity of Aβ (1-42) self-aggregation compared
to inhibitory activity of AChE. Besides, different from AChE inhibition, the electron
density of aromatic ring which was linked with tacrine through acetyl group and the
linker length between tacrine and phenyl moiety were not important factors for
inhibition of Aβ (1-42) self-aggregation. The relationship between them and the
inhibitory activity was not clear. For instance, compounds 7e and 7n with chloro
phenylacetate and chloro cinnamate as electron-withdrawing substituents, exhibited
relatively low inhibitory activities with IC50 values of 77.45 and 51.77 nM,
respectively. However, there were no significant changes in the inhibitory activities
compared with the homogeneous class of compounds 7a and 7k with IC50 values of
71.28 and 49.14 nM, respectively.
2.5. Inhibition of Aβ(1-42) fibril formation monitored by transmission electron
microscopy (TEM)
To further confirm the ability of synthesized compounds and tacrine in inhibiting
Aβ(1-42) aggregation, the inhibitory activities of compounds 7b, 7c and tacrine were
monitored by using TEM[41]. As shown in Fig. 3, after 24 h of incubation at 37oC,
Aβ(1-42) alone aggregated into well-defined Aβ fibrils and amorphous deposits were
observed for the sample of Aβ(1-42) alone (Fig. 3b). In contrast, no obvious Aβ fibril
was observed in the presence of tacrine and compound 7b (Fig. 3c, d) under identical
conditions. Meanwhile, when compound 7c was incubated with Aβ(1-42) under
identical conditions, few short fibrils were found (Fig. 3e). These TEM experimental
results were in agreement with the results of ThT studies, which further strongly
proved that compound 7b and tacrine could inhibit and slow down the Aβ(1-42)
fibrils formation. Unfortunately, tacrine was the most potency inhibitory activity
compared to other synthesized compounds.
(a) Aβ alone (0h)
(b) Aβ alone (24h)

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(c) Aβ+ Tacrine (24h)
(d) Aβ+ 7b (24h)
(e) Aβ+ 7c (24h)
Figure 3. TEM image analysis of Aβ(1-42) aggregation. (a) Aβ(1-42) alone (20 ꢀM) was
o
o
incubated at 37 C for 0 h (a) Aβ(1-42) alone (20 ꢀM) was incubated at 37 C for 24 h; (c)
o
Aβ(1-42) (20 ꢀM) and tacrine (50 ꢀM) were incubated at 37 C for 24 h; (c) Aβ(1-42) (20 ꢀM)
o
and 7b (50 ꢀM) were incubated at 37 C for 24 h; (d) Aβ(1-42) (20 ꢀM) and 7c (50 ꢀM) were
incubated at 37 oC for 24 h
2.6. Molecular modeling study
Table 2. The Docking Cscore of 7c and AchE/ BchE respectively
Compound
Cscore for AChE
8.00ꢀcrash:-1.59ꢁ 6.90(crash:-2.63)
Cscore for BChE
Ratio
12.6
7c

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Figure 4. Molecular docking models of compound 7c inside active site (3 Å) of AChE (A) and

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BuChE (B).
To investigate the interaction mode of compound 7c with TcAChE (PDB code:
1ACJ) and hBuChE(PDB codeꢂ4BDS), molecular modeling was carried out by
Sybyl_8.1.1 package with Surflex-Dock program as shown in Fig. 4. Their Cscores
were calculated as shown in Table 2. As expected that AChE exhibited higher Cscore
(8.00) while BuChE (6.90) which were consistent with the experimental result.
Furthermore, Ser122 in AChE, and Tyr332 in BuChE were considered as the
important residues which were reasonable for the binding mode of 7c and AChE/
BuChE respectively. Meanwhile, there were several differences in the pocket between
AChE and BuChE. Such as Trp84, Asp72, Tyr121, Gly118, Phe288, in AChEꢃand
Asp70, Thr120 Val331, GLY116, ILE69, TYR332 in BuChE, and so on. These key
amino residues around the binding pocket of AChE/BuChE, can influence charge in
the active pocket conformation. Which indicated that, there was conformation
difference of the same binding ligand. In another aspect, different substituted group in
the main frame will get different selectivity of AChE/ BuChE. It was claimed that
electron contributing group in R1, R2, R3 and R4 are facilited for AChE selectivity.
3. Conclusion
In summary, a series of tacrine - benzoates (or phenylacetates or cinnamates)
hybrids have been designed, synthesized and evaluated as novel multi-potent
anti-Alzheimer drug candidates. The results showed that most of these synthesized
compounds had high ChEs and self-induced β-amyloid (Aβ) aggregation inhibitory
potency in the nanomolar range in vitro, certain selectivity for AChE over BuChE
with the ratio of BuChE/AChE selectivity ranged from 0.34- to 64.6- fold. Especially
compound 7c, with a trimethoxyl phenylacetate group, showed the most potent
inhibition for AChE with an IC50 value of 5.63 nM, which was 13-times stronger than
the reference compound tacrine and the highest selectivity (AChE / BuChE = 64.6). A
Lineweaver-Burk plot and molecular modeling study showed that 7c targeted both the
CAS and PAS of ChEs. In inhibition of Aβ aggregation assay, compound 7j exhibited
the most potency inhibitory of self-induced Aβ aggregation. The electron density of
aromatic ring which was linked with tacrine through acetyl group and the linker
length between tacrine and phenyl moiety were important factors for inhibition of
AChE but were less important for Aβ (1-42) self-aggregation. Altogether, the
multifunctional effects of the new hybrids qualified them as potential anti-AD drug
candidates, and compound 7c might be considered as a promising lead compound for
further research.
4. Experimental section
4.1. Chemistry
1
The H NMR and 13C NMR spectra were recorded using TMS as the internal
standard on a Bruker Avnace III 400 spectrometer at 400 MHz and 100 MHz,

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respectively. Coupling constants are given in Hz. High-resolution mass (HRMS)
spectra were obtained with Shimazu LCMS-IT-TOF mass spectrometer. Mass
spectra were recorded on a Agilent 6330 Ion Trap Mass Spectrometer. Reaction
progress was monitored using analytical thin layer chromatography (TLC) on
precoated silica gel GF254 (Qingdao Haiyang Chemical Plant, Qingdao, China) plates
and the spots were detected under UV light (254 nm). Flash column chromatography
was performed with silica gel (200-300 mesh) purchased from Qingdao Haiyang
Chemical Co. Ltd or alumina from Sinopharm Chemical Reagent Co. Ltd. The purity
of all compounds used for biological evaluation was confirmed to be higher than 95%
through analytical HPLC performed with Agilent 1260 HPLC System (methol/water
as eluent with a YMC ODS-A column, detected at UV 256 nM).
4.2. Synthesis of intermediate 5
A solution of bromoacetyl bromide (0.5 mL) in CH2Cl2 (10 mL) was added
dropwise to an ice-cold solution of Et3N (2.5 mmol, 1.8 mL) and tacrine 4 (1.0 g, 5.0
mmol) in CH2Cl2 (30 mL). After completed addition the solution was stirred for 2 h at
room temperature. When the reaction was completed, it was diluted with CH2Cl2,
washed with water, followed by brine solution. The organic layer was dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The obtained residue
was purified bysilica gel chromatography using CH2Cl2/MeOH (100:1) as eluent to
give compound 5 as a white solid. Yield: 82.3%. 1H NMR (DMSO, 400MHz) δ(ppm):
10.14 (s, 1H), 8.04 (d, J = 8.3Hz, 1H), 7.80 (d, J = 8.5Hz, 1H), 7.66 (t, J = 7.6Hz, 1H),
7.52 (t, J = 7.7Hz, 1H), 4.20 (s, 2H), 3.18 (t, J = 6.5Hz, 2H), 2.85 (t, J = 6.4Hz, 2H),
1.95 – 1.74 (m, 4H). MS m/z(M+H)+: 320.17.
4.3. General procedures for the preparation of 7a-7o
A solution of 5 (5.0mmol) in DMF (10 mL) was added dropwise to an ice-cold
solution of a mixture of 6 (5.0mmol) and anhydrous potassium carbonate (5.0 mmol)
in DMF (20 mL). After completed addition the solution was stirred for 2.5 h at room
temperature. When the reaction was completed, it was diluted with H2O, extracted
with EtOAc three times (50 mL x 3), washed with water, followed by brine solution.
The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced
pressure. The obtained residue was purified by silica gel chromatography.
4.3.1 2-oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl
2-(4-methoxyphenyl)acetate (7a)
Compound 5 was treated with 2-(4-methoxyphenyl) acetic acid (6a) according to
1
general procedure to give the desired product 7a as a white solid. Yield: 48.9%. H
NMR (DMSO, 400MHz) δ(ppm): 10.11 (s, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.86 (d, J =
8.3 Hz, 1H), 7.67 (t, J = 8.3 Hz, 1H), 7.50 (t, J = 8.1 Hz, 1H), 7.24 (d, J = 8.7 Hz, 2H),
6.90 (d, J = 8.7 Hz, 2H), 4.89 (s, 2H), 3.77 (s, 2H), 3.74 (s, 3H), 3.05 (t, J = 6.5 Hz,
13
2H), 2.74 (t, J = 6.3 Hz, 2H), 1.95 – 1.74 (m, 4H). C NMR (100 MHz, DMSO)

ACCEPTED MANUSCRIPT
δ(ppm): 171.92, 166.50, 159.82, 158.67, 146.76, 139.23, 131.00, 130.85, 129.21,
128.61, 128.00, 126.47, 126.06, 124.53, 123.74, 114.22, 114.10, 63.21, 55.49, 33.84,
25.16, 22.86, 22.43. ESI-HRMS Calcd. for C24H24N2O4 [M+H]+: 405.1809; found:
405.1799.
4.3.2 2-oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl
2-(3,4-dimethoxyphenyl) acetate (7b)
Compound 5 was treated with 2-(3,4-dimethoxyphenyl) acetic acid (6b) according
to general procedure to give the desired product 7b as a white solid. Yield: 50.2%. 1H
NMR (400 MHz, DMSO) δ(ppm) :10.14 (s, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.85 (d, J =
8.2 Hz, 1H), 7.66 (t, J = 7.2 Hz, 1H), 7.49 (t, J = 7.2 Hz, 1H), 6.92 (dd, J = 8.2, 1.8
Hz, 1H), 6.88 (s, 1H), 6.83 (dd, J = 8.2, 1.8 Hz, 1H), 4.89 (s, 2H), 3.76 (s, 2H), 3.72 (s,
3H), 3.71 (s, 3H), 3.04 (t, J = 6.4 Hz, 2H), 2.73 (t, J = 6.3 Hz, 2H), 1.93 – 1.72 (m,
13
4H). C NMR (100 MHz, DMSO) δ(ppm): 171.83, 166.51, 159.83, 148.96, 148.22,
146.78, 139.22, 129.20, 128.62, 128.00, 126.87, 126.04, 124.53, 123.73, 121.96,
113.65, 112.13, 63.22, 55.93, 55.83, 33.85, 25.17, 22.86, 22.43. ESI-HRMS Calcd. for
C25H26N2O5 [M+H]+: 435.1914; found: 435.1922.
4.3.3 2-oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl
2-(3,4,5-trimethoxyphenyl) acetate (7c)
Compound 5 was treated with 2-(3,4,5-trimethoxyphenyl)acetic acid (6c)
according to general procedure to give the desired product 7c as a white solid. Yield:
1
45.9%. H NMR (DMSO, 400MHz) δ(ppm): 10.13 (s, 1H), 7.90 (d, J = 8.3 Hz, 1H),
7.87 (d, J = 8.4 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 6.66 (s, 2H),
4.92 (s, 2H), 3.78 (s, 3H), 3.74 (s, 3H), 3.64 (s, 3H), 3.05 (t, J = 6.4 Hz, 2H), 2.75 (t, J
13
= 6.3 Hz, 2H), 1.96 – 1.73 (m, 4H). C NMR (100 MHz, DMSO) δ(ppm): 171.56,
166.49, 159.83, 153.16, 146.78, 139.20, 136.74, 130.17, 129.21, 128.63, 127.99,
126.03, 124.51, 123.72, 107.18, 63.28, 60.41, 56.21, 33.84, 25.18, 22.87, 22.43.
ESI-HRMS Calcd. for C26H28N2O6 [M+H]+: 465.2020; found: 465.2023.
4.3.4 2-oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl
2-(4-hydroxyphenyl)acetate (7d)
Compound 5 was treated with 2-(4-hydroxyphenyl)acetic acid (6d) according to
1
general procedure to give the desired product 7d as a white solid. Yield: 55.1%. H
NMR (400 MHz, DMSO) δ(ppm): 10.13 (s, 1H), 9.35 (s, 1H), 7.90 (d, J = 8.3 Hz,
1H), 7.85 (d, J = 8.1 Hz, 1H), 7.6 (t, J = 7.5 Hz, 1H), 7.50 (t, J = 7.5 Hz, 1H), 7.10 (d,
J = 8.4 Hz, 2H), 6.71 (d, J = 8.5 Hz, 2H), 4.87 (s, 2H), 3.70 (s, 2H), 3.04 (t, J = 6.4
Hz, 2H), 2.73 (t, J = 6.3 Hz, 2H), 1.84 (m, 4H). 13C NMR (100 MHz, DMSO) δ(ppm):
172.04, 166.52, 159.83, 156.74, 146.77, 139.23, 130.90, 129.19, 128.61, 128.01,
126.05, 124.65, 124.53, 123.74, 115.57, 63.19, 33.85, 25.16, 22.86, 22.43.
ESI-HRMS Calcd. for C23H22N2O4 [M+H]+: 391.1652; found: 391.1657.
4.3.5 2-oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl
2-(4-chlorophenyl)acetate (7e)

ACCEPTED MANUSCRIPT
Compound 5 was treated with 2-(4-chlorophenyl)acetic acid (6e) according to
general procedure to give the desired product 7e as a white solid. Yield: 47.7%.
1HNMR (DMSO, 400MHz) δ(ppm): 10.16 (s, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.85 (d, J
= 8.2 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H), 7.50 (t, J = 7.5 Hz, 1H), 7.40 (d, J = 8.5 Hz,
2H), 7.35 (d, J = 8.5 Hz, 2H), 4.91 (s, 2H), 3.87 (s, 2H), 3.04 (t, J = 6.4 Hz, 2H), 2.73
(t, J = 6.3 Hz, 2H), 1.95 - 1.73 (m, 4H). 13C NMR (DMSO, 100MHz) δ(ppm): 171.37,
166.39, 159.81, 146.76, 139.17, 133.66, 132.09, 131.90, 129.19, 128.74, 128.62,
127.99, 126.05, 124.50, 123.70, 63.33, 33.84, 25.16, 22.85, 22.42. ESI-HRMS Calcd.
for C23H21N2O3C l [M+H]+: 409.1313; found: 409.1311.
4.3.6 2-oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl
3,5-dimethoxybenzoate (7f)
Compound 5 was treated with 3,5-dimethoxybenzoic acid (6f) according to general
1
procedure to give the desired product 7f as a white solid. Yield: 42.4%. H NMR
(DMSO, 400MHz) δ(ppm): 10.25 (s, 1H), 7.92 (t, J = 9.1 Hz, 2H), 7.67 (t, J = 7.0 Hz,
1H), 7.53 (t, J = 7.2 Hz, 1H), 7.20 (d, J = 2.3 Hz, 2H), 6.83 (t, J = 2.3 Hz, 1H), 5.13 (s,
2H), 3.82 (s, 6H), 3.05 (t, J = 6.3 Hz, 2H), 2.80 (t, J = 6.2 Hz, 2H), 1.95 – 1.73 (m,
13
4H). C NMR (DMSO, 100MHz) δ(ppm): 166.37, 165.75, 160.96, 159.83, 146.78,
139.25, 131.58, 129.20, 128.63, 127.95, 126.07, 124.51, 123.78, 107.56, 106.04,
63.80, 56.03, 33.85, 25.21, 22.87, 22.45. ESI-HRMS Calcd. for C24H24N2O5 [M+H]+:
421.1715; found: 421.1761.
4.3.7 2-oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl
3,4,5-trimethoxybenzoate (7g).
Compound 5 was treated with 3,4,5-trimethoxybenzoic acid (6g) according to
1
general procedure to give the desired product 7g as a white solid. Yield: 57.8%. H
NMR (DMSO, 400MHz) δ(ppm): 10.24 (s, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.91 (d, J =
8.4 Hz, 1H), 7.67 (t, J = 8.2 Hz, 1H), 7.53 (t, J = 8.1 Hz, 1H), 7.36 (s, 2H), 5.13 (s,
2H), 3.86 (s, 6H), 3.76 (s, 3H), 3.05 (t, J = 6.4 Hz, 2H), 2.80 (t, J = 6.3 Hz, 2H), 1.95
– 1.75 (m, 4H). 13C NMR (DMSO, 100MHz) δ(ppm): 166.44, 165.65, 159.84, 153.24,
146.78, 142.43, 139.25, 129.20, 128.63, 127.93, 126.06, 124.68, 124.51, 123.78,
107.23, 63.71, 60.67, 56.50, 33.85, 25.21, 22.87, 22.46. ESI-HRMS Calcd. for
C25H26N2O6 [M+H]+: 451.1864; found: 451.1868.
4.3.8 2-oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl
2,3,4-trimethoxybenzoate (7h)
Compound 5 was treated with 2,3,4-trimethoxybenzoic acid (6h) according to
1
general procedure to give the desired product 7h as a white solid. Yield: 45.2%. H
NMR (CDCl3, 400MHz) δ(ppm): δ 8.85 (s, 1H), 8.03 (d, J = 8.5 Hz, 1H), 7.82 (d, J =
8.3 Hz, 1H), 7.71 (d, J = 8.8 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H),
6.80 (d, J = 8.9 Hz, 1H), 5.14 (s, 2H), 3.94 (s, 3H), 3.93 (s, 3H), 3.81 (s, 3H), 3.18 (t,
13
J = 6.3 Hz, 2H), 2.87 (t, J = 6.0 Hz, 2H), 1.93 – 1.72 (m, 4H). C NMR (DMSO,
100MHz) δ(ppm): 166.64, 164.76, 159.83, 157.68, 154.67, 146.78, 142.86, 139.30,
129.18, 128.61, 127.93, 127.39, 126.03, 124.50, 123.83, 117.13, 108.20, 63.34, 62.17,

ACCEPTED MANUSCRIPT
61.06, 56.57, 33.85, 25.23, 22.87, 22.45. ESI-HRMS Calcd. for C25H26N2O6 [M+H]+:
451.1864; found: 451.1868.
4.3.9 2-oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl
3-hydroxy-4-methoxybenzoate (7i)
Compound 5 was treated with 3-hydroxy-4-methoxybenzoic acid (6i) according
to general procedure to give the desired product 7i as a white solid. Yield: 42.6%.
1HNMR (DMSO, 400MHz) δ(ppm): 10.23 (s, 1H), 10.08 (s, 1H), 7.91 (t, J = 8.3, 2H),
7.67 (t, J = 8.2 Hz, 1H), 7.60 (dd, J = 8.3, 1.9 Hz, 1H), 7.56 – 7.49 (m, 2H), 6.91 (d, J
= 8.3 Hz, 1H), 5.07 (s, 2H), 3.84 (s, 3H), 3.04 (t, J = 6.3 Hz, 2H), 2.79 (t, J = 6.2 Hz,
2H), 1.93 – 1.73 (m, 4H). 13C NMR (DMSO, 100MHz) δ(ppm): 166.72, 165.88,
159.83, 152.32, 147.83, 146.77, 139.34, 129.18, 128.61, 127.96, 126.05, 124.55,
124.48, 123.82, 120.38, 115.68, 113.24, 63.34, 56.10, 33.85, 25.21, 22.87, 22.46.
ESI-HRMS Calcd. for C23H22N2O5 [M+H]+: 407.1601; found: 407.1601.
4.3.10 2-oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl 3,4-hydroxybenzoate
(7j)
Compound 5 was treated with 3, 4-dihydroxybenzoic acid (6j) according to
1
general procedure to give the desired product 7j as a white solid. Yield: 40.4%. H
NMR (DMSO, 400MHz) δ(ppm): 10.26 (s, 1H), 9.71 (s, 2H), 7.91 (t, J = 7.7 Hz, 2H),
7.67 (t, J = 7.6 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 6.93 (d, J = 2.2 Hz, 2H), 6.48 (t, J =
2.2 Hz, 1H), 5.08 (s, 2H), 3.04 (t, J = 6.3 Hz, 2H), 2.78 (t, J = 6.2 Hz, 2H), 1.95 –
13
1.72 (m, 4H). C NMR (DMSO, 100MHz) δ(ppm): 166.51, 166.15, 159.83, 159.02,
146.76, 139.32, 131.31, 129.20, 128.59, 127.95, 126.05, 124.51, 123.80, 107.93,
63.53, 33.84, 25.22, 22.87, 22.45. ESI-HRMS Calcd. for C22H20N2O5 [M+H]+:
393.1445; found: 393.1451.
4.3.11 (E)-2-oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl
3-(3,4-dimethoxyphenyl)acrylate (7k)
Compound 5 was treated with (E)-3-(3,4-dimethoxyphenyl)acrylic acid (6k)
according to general procedure to give the desired product 7k as a pale yellow solid.
1
Yield: 48.6%. H NMR (DMSO, 400MHz) δ(ppm): 10.21 (s, 1H), 7.91 (dd, J = 8.4,
2.6 Hz, 2H), 7.72 (d, J = 16.0 Hz, 1H), 7.67 (t, J = 7.7 Hz, 1H), 7.52 (t, J = 7.5 Hz,
1H), 7.40 (d, J = 1.6 Hz, 1H), 7.29 (dd, J = 8.3, 1.6 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H),
6.70 (d, J = 16.0 Hz, 1H), 4.99 (s, 2H), 3.81 (s, 3H), 3.80 (s, 3H), 3.05 (t, J = 6.4 Hz,
13
2H), 2.78 (t, J = 6.2 Hz, 2H), 1.96 – 1.73 (m, 4H). C NMR (DMSO, 100MHz)
δ(ppm): 166.79, 166.73, 159.83, 151.60, 149.44, 146.77, 146.06, 139.34, 129.19,
128.60, 128.00, 127.23, 126.04, 124.55, 123.80, 123.67, 115.38, 111.96, 110.82, 62.95,
56.07, 56.02, 33.84, 25.20, 22.87, 22.46. ESI-HRMS Calcd. for C26H26N2O5 [M+H]+:
447.1914; found: 447.1918.
4.3.12 (E)-2-oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl
3-(3,4,5-trimethoxyphenyl)acrylate (7l)
Compound 5 was treated with (E)-3-(3,4,5-trimethoxyphenyl)acrylic acid (6l)

ACCEPTED MANUSCRIPT
according to general procedure to give the desired product 7l as a pale yellow solid.
Yield: 43.1%.1H NMR (DMSO, 400MHz) δ(ppm): 10.23 (s, 1H), 7.91 (dd, J = 8.2,
3.8 Hz, 2H), 7.73 (d, J = 16.0 Hz, 1H), 7.67 (t, J = 7.5 Hz, 1H), 7.52 (t, J = 7.5 Hz,
1H), 7.13 (s, 2H), 6.81 (d, J = 16.0 Hz, 1H), 5.00 (s, 2H), 3.82 (s, 6H), 3.70 (s, 3H),
13
3.05 (t, J = 6.3 Hz, 2H), 2.78 (t, J = 6.1 Hz, 2H), 1.95 – 1.75 (m, 4H). C NMR
(DMSO, 100MHz) δ(ppm): 166.64, 159.84, 153.56, 146.78, 146.06, 140.02, 139.31,
130.03, 129.19, 128.62, 127.99, 126.04, 124.54, 123.79, 117.24, 106.50, 63.03, 60.57,
56.50, 33.85, 25.21, 22.88, 22.46. ESI-HRMS Calcd. for C27H28N2O6 [M+H]+:
477.2020; found: 477.2014.
4.3.13 (E)-2-oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl
3-(3-hydroxy-4-methoxyphenyl)acrylate (7m)
Compound 5 was treated with (E)-3-(3-hydroxy-4-methoxyphenyl)acrylic acid
(6m) according to general procedure to give the desired product 7m as a pale yellow
solid. Yield: 52.6%. 1H NMR (DMSO, 400MHz) δ(ppm): 10.19 (s, 1H), 9.69 (s, 1H),
7.90 (d, J = 8.5 Hz, 2H), 7.68 (d, J = 15.8 Hz, 1H), 7.67 – 7.64 (m, 1H), 7.52 (t, J =
7.5 Hz, 1H), 7.37 (d, J = 1.5 Hz, 1H), 7.17 (dd, J = 8.2, 1.5 Hz, 1H), 6.81 (d, J = 8.2
Hz, 1H), 6.62 (d, J = 15.9 Hz, 1H), 4.97 (s, 2H), 3.82 (s, 3H), 3.05 (t, J = 6.3 Hz, 2H),
13
2.78 (t, J = 6.2 Hz, 2H), 1.93 – 1.75 (m, 4H). C NMR (DMSO, 100MHz) δ(ppm):
166.89, 166.78, 159.83, 150.01, 148.41, 146.78, 146.41, 139.34, 129.18, 128.61,
128.00, 126.03, 125.98, 124.56, 123.82, 116.00, 114.29, 111.72, 62.90, 56.15, 33.85,
25.20, 22.88, 22.46. ESI-HRMS Calcd. for C25H24N2O5 [M+H]+: 433.1758; found:
433.1755.
4.3.14 (E)-2-oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl
3-(4-chlorophenyl)acrylate (7n)
Compound 5 was treated with (E) -3-(4-chlorophenyl)acrylic acid (6n) according
to general procedure to give the desired product 7n as a white solid. Yield: 49.5%.
1H NMR (DMSO, 400MHz) δ(ppm): 10.18 (s, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.84 –
7.73 (m, 3H), 7.67 (t, J = 7.7 Hz, 1H), 7.55 – 7.47 (m, 3H), 6.81 (d, J = 16.0 Hz, 1H),
5.00 (s, 2H), 3.04 (t, J = 6.3 Hz, 2H), 2.78 (t, J = 6.2 Hz, 2H), 1.95 – 1.75 (m, 4H).
13C NMR (DMSO, 100MHz) δ(ppm): 166.56, 166.36, 159.82, 144.42, 135.66, 133.39,
130.69, 129.50, 129.25, 128.55, 128.00, 126.09, 124.52, 123.79, 118.79, 63.14, 33.80,
25.19, 22.85, 22.44. ESI-HRMS Calcd. for C24H21N2O3Cl [M+H]+: 421.1313; found:
421.1305.
4.3.15 2-oxo-2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl
2-(1H-indol-3-yl)acetate (7o)
Compound 5 was treated with 2-(1H-indol-3-yl)acetic acid (6o) according to
general procedure to give the desired product 7o as a white solid. Yield: 43.5%, 1H
NMR (DMSO, 400MHz) δ(ppm): 10.97 (s, 1H), 10.12 (s, 1H), 7.89 (dd, J = 8.3, 7.7
Hz 2H), 7.67 (t, J = 7.6 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H),
7.37 (d, J = 8.1 Hz, 1H), 7.32 (d, J = 2.3 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 6.98 (t, J =

ACCEPTED MANUSCRIPT
7.5 Hz, 1H), 4.90 (s, 2H), 3.93 (s, 2H), 3.05 (t, J = 6.5 Hz, 2H), 2.75 (t, J = 6.3 Hz,
2H), 1.97 – 1.70 (m, 4H). 13C NMR (DMSO, 100MHz) δ(ppm): 172.01, 166.65,
159.83, 146.75, 139.28, 136.56, 129.21, 128.59, 128.03, 127.60, 126.06, 124.73,
124.54, 123.77, 121.57, 119.08, 118.96, 111.88, 107.17, 63.17, 33.84, 30.85, 25.18,
13
22.87, 22.44. C NMR(DMSO, 100MHz) δ(ppm): 172.01, 166.65, 159.83, 146.75,
139.28, 136.56, 129.21, 128.59, 128.03, 127.60, 126.06, 124.73, 124.54, 123.77,
121.57, 119.08, 118.96, 111.88, 107.17, 63.17, 33.84, 30.85, 25.18, 22.87, 22.44.
ESI-HRMS Calcd. for C25H23NO3 [M+H]+: 414.1812; found: 414.1799.
4.4. Biological activity
4.4.1. In vitro inhibition studies on AChE and BuChE
Acetylcholinesterase
(AChE,
E.C.
3.1.1.7,
from
electric
eel),
butyrylcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum), 5, 5’-dithiobis-
(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), acetylthiocholinechloride (ATC),
butylthiocholine chloride (BTC), and tacrine hydrochloride were purchased from
Sigma Aldrich. Tacrine and synthesized derivatives were dissolved in DMSO (5‰)
and fetal bovine serum(1‰), and then diluted in 0.1 M NaH2PO4/Na2HPO4 buffer (pH
7.4) to provide a final concentration range.
All the assays were under 0.1 M NaH2PO4/Na2HPO4 buffer, pH 7.4,using a
THERMO Enzyme-labeled Instrument. Enzyme solutions were prepared to give 2.0
units/mL in 2 mL aliquots. The assay medium contained phosphate buffer, pH 7.4 (1
mL), 30 ꢀL of 0.01 M DTNB, 10 ꢀL of enzyme, and 30 ꢀL of 0.01 M substrate
(acetylthiocholine chloride). The substrate was added to the assay medium containing
enzyme, buffer, and DTNB with inhibitor after 20 min of incubation time. The
activity was determined by measuring the increase in absorbance at 410 nm at 1 min
intervals at 37oC. Calculations were performed according to the method of the
equation in Ellman et al. Each concentration was assayed in triplicate.
In vitro BuChE assays were carried out using a similar method as described
above.
4.4.2. Inhibition of self-mediated Aβ(1-42) aggregation
In order to investigate the self-mediated Aβ(1-42) aggregation, a thioflavin-T
fluorescence assay was performed. Experiments were performed by incubating the
peptides in 10 mM phosphate buffer (pH 7.4) at 37oC for 24 h (final Aβ(1-42) 20 ꢀM)
with and without the tested compounds at different concentrations (5, 10, 20, 50 ,100
ꢀM). After incubation, the samples were diluted to a final volume of 150 ꢀL with 50
mM glycine-NaOH buffer (pH 8.5) containing 5 ꢀM Thioflavin T. Fluorescence
signal was measured (excitation wavelength 450 nm, emission wavelength 485 nm
and slit widths set to 5 nm) on a Varioskan Flash Multimode Reader (Thermo
Scientific). The percentage of inhibition on aggregation was calculated by the
following expression: (1- IFi/IFc)*100% in which IFi and IFc were the fluorescence

ACCEPTED MANUSCRIPT
intensities obtained for absorbance in the presence and absence of inhibitors,
respectively, after subtracting the background fluorescence of the 5 ꢀM Thioflavin T
solution. Each measurement was run in triplicate.
4.4.3. Transmission electron microscopy (TEM) assay
Microscopy images were captured on HITACHI H-7650 transmission electron
microscope. Aβ(1-42) peptide (Sigma) stock was diluted with 10 mM phosphate
buffer (pH 7.4) at 4oC to 100 ꢀM before use. For the inhibition of self-induced
Aβ(1-42) aggregation experiment, it was incubated in the presence and absence of
compounds and tacrine at 37oC for 24 h. Then, the 250 ꢀM tested compound was
added and incubated at 37oC for another 24 h. The final concentrations of Aβ(1-42)
was 20 ꢀM and the compounds were 50 ꢀM. Aliquots (5 uL) of the samples were
placed on a carboncoated copper/rhodium grid for 2 min at room temperature. Each
grid was stained with phosphomolybdic acid solution (3%, 5 uL) for 2 min. Excess
staining solution was removed and the specimen was transferred for imaging with
transmission electron microscopy.
4.5. Molecular modeling
For the docking studies, Surflex-Dock in Sybyl 8.1.1 was used. The structures of
the inhibitors were drawn in the Sybyl package with standard bond lengths and angles,
and minimized using the conjugate gradient method. The Gasteiger-Huckel charge
was applied for the minimization process, with a distance-dependent dielectric
function. A preliminary docking study was carried out using the crystal structure of
TcAChE (PDB code: 1ACJ) and hBuChE(PDB codeꢂ4BDS), The structure was
polished as follows: all water molecules were removed from the crystal structure and
the ligand was extracted. The AChE and BuChE proteins were then analyzed using the
Protein Structure Preparation Tool in Sybyl. After adding hydrogens, the side-chain
amides were also fixed and two bumping amino acids were adjusted. Stage
minimization was also applied with the AMBER FF99 force field. Then the Protomol
was generated.
Acknowledgements
We thank the Foundation of Educational Commission of Guangdong Province
of China (Nos. 2013KJCX0151, 2014KTSCX096 ) for financial support of this study.
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15 novel tacrine derivatives were synthesized as multifunctional anti-AD agents.
Most of compounds showed excellent inhibition of AChE, BuChE and Aβ aggregation.
Most of compounds showed certain selectivity for AChE over BuChE.
The electron density of phenylacetate is the key to inhibitory activity of AChE.
Compound 7c was the best compound as out of the synthesized compounds.