Multifunctional tacrine-trolox hybrids for the treatment of Alzheimer's disease with cholinergic, antioxidant, neuroprotective and hepatoprotective properties

Article abstract of DOI:10.1016/j.ejmech.2015.01.058

Combining tacrine with trolox in a single molecule, novel multifunctional hybrids have been designed and synthesized. All these hybrids showed ChE inhibitory activity in nanomolar range and strong antioxidant activity close to the parent compound trolox. Among them, compound 6d was the most potent inhibitor against AChE (IC₅₀ value of 9.8 nM for eeAChE and 23.5 nM for hAChE), and it was also a strong inhibitor to BuChE (IC₅₀ value of 22.2 nM for eqBuChE and 20.5 nM for hBuChE). Molecular modeling and kinetic studies suggested that 6d was a mixed-type inhibitor, binding simultaneously to CAS and PAS of AChE. In vivo hepatotoxicity assays indicated that 6d was much less toxic than tacrine. In addition, it showed neuroprotective effect and good ability to penetrate the BBB. Overall, all these results highlighted 6d a promising multifunctional agent for AD treatment.

Full text of DOI:10.1016/j.ejmech.2015.01.058

European Journal of Medicinal Chemistry 93 (2015) 42e50
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Multifunctional tacrineetrolox hybrids for the treatment of
Alzheimer's disease with cholinergic, antioxidant, neuroprotective
and hepatoprotective properties
Sai-Sai Xie 1, Jin-Shuai Lan 1, Xiao-Bing Wang, Neng Jiang, Ge Dong, Zhong-Rui Li,
Kelvin D.G. Wang, Ping-Ping Guo, Ling-Yi Kong*
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing
210009, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Combining tacrine with trolox in a single molecule, novel multifunctional hybrids have been designed
and synthesized. All these hybrids showed ChE inhibitory activity in nanomolar range and strong anti-
oxidant activity close to the parent compound trolox. Among them, compound 6d was the most potent
inhibitor against AChE (IC50 value of 9.8 nM for eeAChE and 23.5 nM for hAChE), and it was also a strong
inhibitor to BuChE (IC50 value of 22.2 nM for eqBuChE and 20.5 nM for hBuChE). Molecular modeling and
kinetic studies suggested that 6d was a mixed-type inhibitor, binding simultaneously to CAS and PAS of
AChE. In vivo hepatotoxicity assays indicated that 6d was much less toxic than tacrine. In addition, it
showed neuroprotective effect and good ability to penetrate the BBB. Overall, all these results highlighted
6d a promising multifunctional agent for AD treatment.
Received 26 November 2014
Received in revised form
30 December 2014
Accepted 27 January 2015
Available online 28 January 2015
Keywords:
Alzheimer's disease
Tacrine
© 2015 Elsevier Masson SAS. All rights reserved.
Trolox
Cholinesterase
Antioxidant
Hepatotoxicity
1. Introduction
neurodegenerative AD cascade [6,7]. Since AChE inhibitors are still
clinical effective for improving symptoms of AD, the common
Alzheimer's disease (AD) is the most common neurodegenera-
tive disease characterized by progressive memory loss, decline in
language skills and other cognitive impairments [1,2]. Despite
considerable efforts devoted to investigate the pathophysiology of
AD, the causes and mechanisms of the disease are still not fully
understood [3]. Currently, the predominant class of therapeutic
agent in AD treatment is acetylcholinesterase (AChE) inhibitors,
which can increase the cholinergic neurotransmission in the syn-
aptic cleft by inhibiting degradation of acetylcholine (ACh) [4].
However, due to the multifactorial nature of AD, modulation of such
a single target can only enable a palliative treatment instead of
slowing or halting the neurodegeneration [5]. Thus, a more
appropriate strategy to face this disease is proposed by develop-
ment of multifunctional molecules that can simultaneously
modulate different targets or mechanisms involved in the
approach for designing multifunctional molecules is to modify a
known AChE inhibitor, providing it with additional activity for AD
treatment [8].
Tacrine (Fig. 1), the first AChE inhibitor approved for the treat-
ment of AD, emerge as a popular structure in design of multi-
functional molecules in recent years, because it can inhibit AChE in
high potency and have a low molecular weight suitable for modi-
fication [9,10]. A lot of multifunctional molecules by connecting
tacrine with another fragment having additional activities have
been designed and synthesized [10e14]. However, due to the
serious hepatotoxicity of tacrine, further use of these compounds
was limited [15]. To date, the exact mechanisms of tacrine-induced
hepatotoxicity are largely unknown [16]. However, evidences
indicated that oxidative stress seemed to play a role. It was
demonstrated that tacrine could decrease intracellular glutathione
concentration, which lead to the generation of reactive oxygen
species (ROS) and lipid peroxidation in the human liver cell [17].
Meanwhile, several studies also proved that tacrine-induced hep-
atotoxicity could be prevented by antioxidants [18]. .
* Corresponding author.
E-mail address: cpu_lykong@126.com (L.-Y. Kong).
These authors contributed equally.
1
http://dx.doi.org/10.1016/j.ejmech.2015.01.058
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

S.-S. Xie et al. / European Journal of Medicinal Chemistry 93 (2015) 42e50
43
2. Results and discussion
2.1. Chemistry
The synthesis of target and intermediate compounds is repre-
sented in Scheme 1. As a previously described protocol [34,35],
heating anthranilic acid with cyclohexanone in the presence of
POCl3 for 2 h gave 9-chloro-1, 2, 3, 4-tetrahydroacridine 3. The
obtained 3 was then engaged with different aliphatic diamines to
produce the intermediate 9-aminoalkylamino-1, 2, 3, 4-
tetrahydroacridines 4aeg [12,34]. Finally, activation of trolox with
1, 10-carbonyldiimidazole (CDI) and subsequent coupling to 4aeg
afforded target compounds 6aeg.
2.2. In vitro inhibition of ChEs
Fig. 1. Structures of trolox, tacrine and tacrineetrolox hybrids.
There are two types of cholinesterases (ChEs) in human body,
namely, acetylcholinesterase (AChE) and butyrylcholinesterase
(BuChE). In normal conditions, AChE hydrolyzes about 80% of
acetylcholine while BuChE plays only a supportive role [36].
However, recent studies indicate that, as AD progresses, the activity
of AChE gradually decreases while that of BuChE significantly in-
creases. In later stage of the disease, BuChE can compensate for the
deficit of AChE in brain [37]. Therefore, simultaneous inhibition of
both AChE and BuChE would be more valuable for AD treatment.
The ChE inhibitory activity of 6aeg was measured by Ellman's
assay using tacrine as reference compound [38]. Because of their
lower cost and the high degree of sequence identity to the human
enzymes, acetylcholinesterase (AChE) from electric eel and butyr-
ylcholinesterase (BuChE) from equine serum were initially used.
The results shown in Table 1 indicate that all compounds are potent
inhibitors of both ChEs. Similar to the tacrine, most of them showed
more potent inhibitory activity for BuChE than for AChE. The length
of the alkyl spacer between trolox and tacrine moiety could
significantly influence the AChE inhibitory activity. Compound 6d
with a six-carbon spacer exhibited the most potent AChE inhibitory
activity with IC50 value of 9.8 nM, which was 11-fold stronger than
that of tacrine. Lengthening or shortening the length of alkyl spacer
reduced AChE inhibition. In contrast, the length of the alkyl spacer
did not show a clear trend for BuChE inhibition. Compound 6b with
a four-carbon spacer gave the most potent BuChE inhibitory activity
(IC50 ¼ 15.6 nM). However, the long spacer length was in general
not beneficial for BuChE inhibition (e.g., 6f: IC50 ¼ 150.5 nM; 6g:
IC50 ¼ 262.8 nM).
In fact, besides the ability to conquer the hepatotoxicity of
tacrine, antioxidants are also beneficial for AD treatment [19].
Compared with other tissues, the brain is more sensitive to free
radical damage [20]. The antioxidant system in brain progressively
decays during aging. Especially in AD brain, it decline more rapidly
than normal. An increasing body of evidence indicates that the
oxidative damage occurs in earliest stage of AD pathogenesis and
promotes the formation of other pathological hallmarks of the
disease, such as amyloid plaques and neurofibrillary tangles [21].
Therefore, given all the above facts, researchers hypothesized that
combining tacrine with an antioxidant fragment might afford more
effective multifunctional molecules, which could not only inhibit
AChE and reduce the hepatotoxicity of tacrine but also exhibit
neuroprotective effect by decreasing the oxidant damage in brain.
To test this hypothesis, some tacrineeantioxidant hybrids have
been synthesized such as tacrineesilibinin hybrid [22], tacri-
neeferulic acid hybrid [23] and mercaptoetacrine hybrids [24]. All
these compounds showed improved pharmacological properties
and low hepatotoxicity compared to tacrine. Driven by these pos-
itive results, recently we focused our work on searching new
tacrineeantioxidant hybrids.
Trolox (Fig.1) is a powerful antioxidant widely used in biological
or biochemical applications to reduce oxidative stress [25].
Numerous reports indicate that trolox can protect liver from the
damage induced by chemical insults both in vitro and in vivo
[26e28]. In addition, it showed neuroprotective effect through
scavenging ROS and attenuating the neurotoxicity mediated by A
b
and H2O2 on hippocampal neurons [29,30]. Furthermore, as a lipid-
soluble analog of trolox, Vitamin E has been already used to prevent
the tacrine-induced hepatotoxicity in clinic and delay the AD pro-
gression in patients with moderately severe AD [18,31]. Therefore,
all these results suggested that trolox might be a useful antioxidant
fragment for designing multifunctional tacrineeantioxidant
hybrids.
In this study, a series of new multifunctional compounds by
hybridizing tacrine with trolox have been designed and synthe-
sized (Fig. 1). Tacrine was selected to inhibit AChE, and trolox was
used to overcome the hepatotoxicity of tacrine and prevent oxidant
damage in brain. The AChE has a nearly 20 Å deep narrow gorge
which consists of two binding sites: a catalytic active site (CAS) at
the bottom of the gorge and a peripheral anionic site (PAS) near the
entry of the gorge. Considering compounds binding simultaneously
to PAS and CAS were more promising for AD treatment, thus,
alkylene linkers of different lengths were used to tether these two
fragments [32,33]. Here, we report the design, synthesis and bio-
logical evaluation of a series of new tacrineetrolox hybrids as
multifunctional agents for the treatment of AD.
All compounds having potent inhibitory activity for ChEs of
animal origin were then tested on human ChEs with aim to better
evaluate them and the results are summarized in Table 1. From the
table, it can be seen that all compounds are also potent inhibitors of
human ChEs. For BuChE inhibition, most of these compounds
showed improved inhibitory activity for hBuChE compared to
eqBuChE. Compound 6b displayed the highest inhibitory activity
for hBuChE with the IC50 value of 5.2 nM. However, no significant
change between hAChE and eeAChE was observed. Although
compound 6d exhibited the inhibitory activity for hAChE was
slightly lower than for eeAChE (9.8 nM versus 23.5 nM), it was still
the most potent inhibitor of hAChE. Such an activity change made
6d more balanced for both ChE inhibition (IC50 ¼ 23.5 nM for
hAChE; IC50 ¼ 20.5 nM for hBuChE), which would be more bene-
ficial for AD treatment.
2.3. In vitro antioxidant activities
The antioxidant activities of target compounds 6aeg were
evaluated using a radical scavenging assay (DPPH assay) with trolox
as reference compound (Table 1) [39]. The results indicated that all

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S.-S. Xie et al. / European Journal of Medicinal Chemistry 93 (2015) 42e50
Scheme 1. Synthesis of tacrineetrolox hybrids. Reagents and conditions: (a) POCl3, reflux, 4 h; (b) NH2(CH2)nNH2, KI, 1-pentanol, reflux, 12 h; (c) 1, 10-carbonyldiimidazole (CDI), r.t.
overnight.
compounds retained the antioxidant activity of the parent com-
pound trolox. Connecting the trolox with tacrine moiety and vari-
ation of linker length between the two fragments did not
significantly affect the antioxidant activity of trolox, which indi-
cated that hybridizing these two scaffolds was rational. After all
these biological evaluation, 6d was chosen as the most promising
compound for further study because of its strong and balanced
inhibition for both ChEs and antioxidant activity close to trolox.
in PC 12 cells. H2O2 was used as toxic insult to induce oxidative
damage, and the parent compound trolox was selected as positive
control. After treating the cells with different concentrations of
compound 6d for 2 h, 100 mM of H2O2 was added and incubated
for 24 h. Cell viability was measured by MTT assay [41]. As re-
ported in Fig. 4A, 6d could significantly inhibit cell death at
3.125
showed decreased cell viability as the concentration increased. At
12.5 M, the compound even exhibited much lower viability than
mM and 6.25 mM. However, to our surprise, compound
m
that of H2O2 only treated group. These results made us to test
potential cytotoxicity of 6d on PC 12 cells. However, it can be seen
from Fig. 4B that the compound do not lead to cell death at
2.4. Kinetic study of AChE inhibition
To investigate whether 6d is a dual binding site inhibitor, an
enzyme kinetic study was performed. By plotting the 1/velocity
against 1/[substrate], a set of LineweavereBurk double reciprocal
plots were constructed and shown in Fig. 2. Form the figure, it can
be seen that both increasing slopes and intercepts with increasing
inhibitor concentrations, which suggested that 6d was a mixed-
type inhibitor for AChE. On basis of the previous reports [35,40],
this result meant that compound 6d might be a dual binding site
inhibitor of AChE.
concentration ranging from 1.563 to 12.5 mM. After studying the
previous reports [24,42], we found that, in fact, similar phenom-
ena were also observed in reported compounds and marketed
drugs (e.g., mercapro-tacrine derivatives, galanthamine and
donepezil). The increased cell death at high concentration might
arise from additional interactions between compound and other
biological targets.
2.7. In vivo hepatotoxicity study
2.5. Molecular docking study with AChE
To determine whether introduction of trolox could reduce
hepatotoxicity, compound 6d was selected for the assay with male
rats [24,43]. After treating rats with highest tolerated dose of
In order to further demonstrate the dual-site binding mode and
get insight into the interaction mechanism of 6d with AChE, mo-
lecular docking study based on X-ray crystal structure of recom-
binant human acetylcholinesterase in complex with donepezil
(hAChE, PDB code 4EY7) was carried out using Molecular Operating
Environment (MOE) software. As shown in Fig. 3, trolox moiety was
tacrine (6 mmol/100 g body weight) or equimolar dose of 6d, the
aspartate aminotransferase (AST) and alanine aminotransferase
(ALT) activities in serum obtained at different times (0, 24, 36 and
72 h) were measured. As shown in Fig. 5, tacrine caused signifi-
cant hepatotoxicity, showing increased activity of both AST and
ALT at 24 and 36 h, while compound 6d did not alter these pa-
rameters from 0 to 72 h, which suggested that 6d had little
hepatotoxicity.
For histological study, the Fig. 6B indicated that, after tacrine
administration, an increase in the number of inflammatory cells in
portal fields and a large area of necrosis and distinct fatty
degeneration of the hepatocytes were observed. In contrast, only
minor morphological changes were found after the treatment with
compound 6d (Fig. 6C). All these results strongly demonstrated
that introduction of trolox could reduce the hepatotoxicity of
tacrine.
located into PAS of AChE through a pep stacking interaction with
Trp 286 (3.55 Å). The tacrine moiety occupied the CAS and stacked
against the indole ring of Trp 86 with ring to ring distance of 4.53 Å.
After analyzing the docking results and taking the kinetic study into
consideration, we could confirm that compound 6d was a dual
binding site inhibitor that could interact simultaneously with PAS
and CAS of AChE.
2.6. In vitro neuroprotection study
Since 6d was a potent antioxidant comparable to the trolox, we
investigated its neuroprotective capacity against oxidative stress

S.-S. Xie et al. / European Journal of Medicinal Chemistry 93 (2015) 42e50
45
Table 1
Inhibitory activities of eeAChE, eqBuChE, hAChE, hBuChE and DPPH.
Compd.
IC50 (nM)a
AChEb
SId
IC50 (nM)a
hAChEe
SIg
IC50
(
m
M)a
BuChEc
hBuChEf
DPPH
6a
6b
6c
6d
6e
6f
6g
Tacrine
Trolox
149.7 2.4
558.4 9.7
249.5 2.3
9.8 0.7
92.4 1.4
228.1 2.1
386.9 4.2
112.1 4.2
41.3 1.9
15.6 1.4
35.4 2.1
22.2 1.3
61.7 2.2
150.5 7.3
262.8 5.4
35.2 4.2
0.28
0.03
0.14
2.27
0.67
0.66
0.68
0.31
283.8 4.5
535.0 10.1
215.3 2.4
23.5 1.7
143.7 3.6
226.5 7.1
453.3 6.2
435.1 5.9
56.3 2.5
5.2 1.1
0.20
0.01
0.05
0.87
0.13
0.15
0.24
0.05
30.4 2.3
44.2 2.1
40.7 1.8
48.7 2.8
46.9 2.2
47.3 1.9
43.2 2.2
10.2 1.8
20.5 1.2
19.2 1.9
34.7 1.1
110.1 3.1
23.2 2.3
35.6 3.2
a
Results are the mean of three independent experiments (n ¼ 3) SD.
AChE (EC 3.1.1.7) from electric eel.
BuChE (EC 3.1.1.8) from horse serum.
AChE selectivity index ¼ IC50(BuChE)/IC50(AChE).
AChE (EC 3.1.1.7) from human erythrocytes.
BuChE (EC 3.1.1.8) from human serum.
hAChE selectivity index ¼ IC50(hBuChE)/IC50(hAChE).
b
c
d
e
f
g
nanomolar range, and also gave strong antioxidant activity close to
parent compound trolox. Among these compounds, 6d possessing
the highest inhibitory activity for eeAChE and hAChE as well as
strong inhibition for eqBuChE and hBuChE stood out as the most
promising compound for further study. Kinetic and molecular
docking studies revealed that 6d was a mixed-type inhibitor that
could simultaneously interact with PAS and CAS of AChE. In vivo
hepatotoxicity assays indicated that 6d did not show signs of
hepatotoxicity, which was much safer than tacrine. In addition, it
displayed neuroprotective effect against H2O2-induced PC 12 cell
injury and good ability to penetrate the CNS. Overall, all these re-
sults qualified 6d as a potential multifunctional agent for the
treatment of AD.
Fig. 2. Kinetic study on the mechanism of eeAChE inhibition by compound 6d. Over-
laid LineweavereBurk reciprocal plots of AChE initial velocity at increasing substrate
concentration (0.05e0.50 mM) in the absence of inhibitor and in the presences of
different concentrations of 6d are shown.
4. Experimental section
4.1. Chemistry
All chemicals (reagent grade) used were purchased from Sino
pharm Chemical Reagent Co., Ltd. (China). Reaction progress was
monitored using analytical thin layer chromatography (TLC) on
precoated silica gel GF254 (Qingdao Haiyang Chemical Plant, Qing-
Dao, China) plates and the spots were detected under UV light
(254 nm). IR (KBr-disc) spectra were recorded by Bruker Tensor 27
spectrometer. 1H NMR and 13C NMR spectra were measured on a
Bruker ACF-500 spectrometer at 25 ꢂC and referenced to TMS.
2.8. In vitro bloodebrain barrier permeation study
A success CNS drug should be able to cross the BloodeBrain
Barrier (BBB) [44]. Thus, to determine whether compound 6d could
penetrate the BBB, a parallel artificial membrane permeation assay
(PAMPA) of bloodebrain barrier was used. This model described by
Di et al. was widely used to predict BBB permeation with high
success [45]. The in vitro permeabilities (Pe) of compound 6d and
reference drugs were determined through a lipid extract of porcine
brain with a mixture of PBS and EtOH (70:30). Assay validation was
performed by comparing experimental permeabilities of 10 refer-
ence drugs with their reported values (Supporting information,
Table S1), which gave a good linear correlation: Pe (exp.) ¼ 0.8597
Pe (Bibl.) e 0.276 (R2 ¼ 0.9726). From this equation and considering
the limits established by Di et al. for BBB permeation, we estab-
lished that compound with Pe value above 3.16 ꢀ 10ꢁ6 cm sꢁ1could
penetrate into the CNS. Compound 6d showed a Pe value of
8.87 ꢀ 10ꢁ6 cm sꢁ1, indicating that it could easily cross the BBB.
Chemical shifts are reported in ppm (d) using the residual solvent
line as internal standard. Splitting patterns are designed as s,
singlet; d, doublet; t, triplet; m, multiplet. Mass spectra were ob-
tained on a MS Agilent 1100 Series LC/MSD Trap mass spectrometer
(ESI-MS) and a Mariner ESI-TOF spectrometer (HRESIMS), respec-
tively. Column chromatography was performed on silica gel
(90e150 mm; Qingdao Marine Chemical Inc.). Compounds 3 and 4
were prepared following previously published methods
[12,34,35,46].
4.1.1. General procedures for the preparation of compounds 6aeg
A solution of 5 (2.0 mmol) and 1, 10-carbonyldiimidazole
(2.2 mmol) in 25 mL of anhydrous THF was stirred at room tem-
perature for 1 h. The 4 (2.0 mmol) was added to the solution, and
stirring was continued overnight. The reaction mixture was diluted
with H2O and extracted with EtOAc. The organic extracts were
combined, washed with brine, and dried with anhydrous Na2SO4,
and the solvent was evaporated in vacuo to give the crude product,
3. Conclusion
In conclusion, a series of novel tacrineetrolox hybrids have been
designed and synthesized as multifunctional agents for the treat-
ment of AD. The biological screening results indicated that all of
them showed potent ChE inhibitory activity with IC50 value in

46
S.-S. Xie et al. / European Journal of Medicinal Chemistry 93 (2015) 42e50
Fig. 3. (A) 3D docking model of compound 6d with AChE. Atom colors: yellowecarbon atoms of 6d, cyanecarbon atoms of residues of AChE, dark blueenitrogen atoms, redeoxygen
atoms. (B) 2D schematic diagram of docking model of compound 6d with AChE. The figure was prepared using the ligand interactions application in MOE. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
which was purified by silica gel chromatography with
CH2Cl2eMeOH ¼ 20:1 as an eluent to afford corresponding target
compound as yellow oil.
4.1.1.2. 6-Hydroxy-2, 5, 7, 8-tetramethyl-N-(4-((1, 2, 3, 4-
tetrahydroacridin-9-yl)amino)butyl) chroman-2-carboxamide (6b).
Intermediate 4b was reacted with 6-hydroxy-2, 5, 7, 8-
tetramethylchroman-2-carboxylic acid following the general pro-
cedure to give the desired product 6b with a yield of 60%. IR (KBr)
n
4.1.1.1. 6-Hydroxy-2,5,7,8-tetramethyl-N-(2-((1, 2, 3, 4-
tetrahydroacridin-9-yl)amino)ethyl)chroman-2-carboxamide (6a).
Intermediate 4a was reacted with 6-hydroxy-2, 5, 7, 8-
tetramethylchroman-2-carboxylic acid following the general pro-
3425, 2930, 1647, 1589, 1522, 1441, 1257, 1088, 756, 678 cmꢁ1; ESI/
MS m/z: 502.2 [MþH]þ; 1H NMR (500 MHz, DMSO)
d 8.18 (d,
J ¼ 8.5 Hz, 1H), 7.76 (d, J ¼ 8.5 Hz, 1H), 7.64 (t, J ¼ 7.5 Hz, 1H), 7.41 (t,
J ¼ 7.5 Hz, 1H), 7.24 (t, J ¼ 6.0 Hz, 1H), 6.27 (s, 1H), 3.50 (t, J ¼ 6.5 Hz,
2H), 3.11e3.00 (m, 2H), 2.93 (t, J ¼ 5.5 Hz, 2H), 2.66 (t, J ¼ 5.5 Hz,
2H), 2.47 (d, J ¼ 6.0 Hz, 1H), 2.42e2.33 (m, 1H), 2.12e2.07 (m, 1H),
2.05 (d, J ¼ 2.0 Hz, 6H), 1.96 (s, 3H), 1.86e1.75 (m, 4H), 1.72e1.64 (m,
1H), 1.49e1.34 (m, 4H), 1.32 (s, 3H). 13C NMR (125 MHz, DMSO)
cedure to give the desired product 6a with a yield of 64%. IR (KBr)
n
3422, 3235, 2932, 1701, 1586, 1488, 1427, 1260, 767, 705, 678 cmꢁ1
;
ESI/MS m/z: 474.2 [MþH]þ; 1H NMR (500 MHz, DMSO)
d 8.18 (d,
J ¼ 8.5 Hz, 1H), 7.75 (d, J ¼ 8.5 Hz, 1H), 7.66e7.57 (m, 2H), 7.39 (t,
J ¼ 7.6 Hz, 1H), 6.18 (s, 1H), 3.64 (s, 2H), 3.48e3.32 (m, 2H), 2.91 (t,
J ¼ 5.9 Hz, 2H), 2.71e2.54 (m, 2H), 2.48e2.41 (m,1H), 2.37e2.28 (m,
1H), 2.07e1.98 (m, 7H), 1.93 (s, 3H), 1.72e1.65 (m, 4H), 1.72e1.63
d
173.73, 164.01, 155.54, 152.83, 146.35, 144.34, 130.20, 125.17,
124.42, 124.24, 123.12, 121.63, 120.69, 118.91, 117.58, 114.54, 77.68,
47.80, 38.52, 31.79, 29.98, 27.93, 26.92, 25.06, 24.42, 22.72, 22.07,
20.55, 13.19, 12.48, 12.21. HRMS: calcd for C31H40N3O3 [MþH]þ
502.3064, found 502.3060.
(m, 1H), 1.24 (s, 3H). 13C NMR (125 MHz, DMSO)
d 174.82, 163.93,
155.84, 152.79, 146.35, 144.17, 130.02, 125.55, 124.32, 124.25, 123.12,
121.65, 120.60, 118.92, 117.48, 114.72, 77.63, 55.36, 48.47, 32.01,
29.95, 24.99, 24.01, 22.73, 22.14, 20.39, 13.20, 12.46, 12.19. HRMS:
calcd for C29H36N3O3 [MþH]þ 474.2751, found 474.2755.

S.-S. Xie et al. / European Journal of Medicinal Chemistry 93 (2015) 42e50
47
Fig. 4. (A) Protective effects of compound 6d on H2O2-induced cell death in PC 12 cells. Cells were incubated with different concentration of 6d for 2 h and then treated with 100 mM
H2O2 for 24 h. Trolox was used as positive control. (B) Effects of various concentrations of compound 6d on cell viability in PC 12 cells after treatment for 48 h. Cell viability was
measured by MTT assay. Data were shown as mean SD of three independent experiments (n ¼ 6). P### < 0.001 compared to control, P** < 0.01, P*** < 0.001 compared to H2O2-
treated cells.
4.1.1.3. 6-Hydroxy-2, 5, 7, 8-tetramethyl-N-(5-((1, 2, 3, 4-
tetrahydroacridin-9-yl) amino) pentyl)chroman-2-carboxamide (6c).
Intermediate 4c was reacted with 6-hydroxy-2, 5, 7, 8-
tetramethylchroman-2-carboxylic acid following the general pro-
cedure to give the desired product 6c with a yield of 66%. IR (KBr)
n
3425, 2930, 2860, 1651, 1589, 1523, 1449, 1348, 1256, 1088, 756,
678 cmꢁ1; ESI/MS m/z: 516.3 [MþH]þ; 1H NMR (500 MHz, DMSO)
d
8.18 (d, J ¼ 8.5 Hz, 1H), 7.75 (d, J ¼ 8.5 Hz, 1H), 7.63 (t, J ¼ 7.5 Hz,
1H), 7.46e7.31 (m, 1H), 7.18 (t, J ¼ 6.0 Hz, 1H), 6.13 (s, 1H), 3.47 (t,
J ¼ 7.0 Hz, 2H), 3.12e2.97 (m, 2H), 2.92 (t, J ¼ 6.0 Hz, 2H), 2.67 (t,
J ¼ 6.0 Hz, 2H), 2.47 (d, J ¼ 6.0 Hz, 1H), 2.42e2.35 (m, 1H), 2.17e2.09
(m, 1H), 2.05 (d, J ¼ 8.0 Hz, 6H), 1.96 (s, 3H), 1.85e1.76 (m, 4H),
1.72e1.66 (m, 1H), 1.58e1.48 (m, 2H), 1.38e1.27 (m, 5H), 1.15e1.07
(m, 2H). 13C NMR (125 MHz, DMSO)
d 173.63, 163.85, 155.75, 152.82,
146.63, 144.53, 130.13, 125.27, 124.29, 124.34, 123.31, 121.39, 120.74,
118.83, 117.69, 114.57, 77.69, 48.23, 38.66, 31.72, 30.68, 29.61, 29.58,
26.52, 26.21, 25.02, 24.58, 22.22, 20.57, 13.25, 12.39, 12.31. HRMS:
calcd for C32H42N3O3 [MþH]þ 516.3221, found 516.3225.
4.1.1.4. 6-Hydroxy-2, 5, 7, 8-tetramethyl-N-(6-((1, 2, 3, 4-
tetrahydroacridin-9-yl)amino)hexyl) chroman-2-carboxamide (6d).
Intermediate 4d was reacted with 6-hydroxy-2, 5, 7, 8-
tetramethylchroman-2-carboxylic acid following the general pro-
cedure to give the desired product 6b with a yield of 61%. IR (KBr)
n
3428, 2928, 2857, 1648, 1589, 1524, 1452, 1415, 1256, 1088, 757,
678 cmꢁ1; ESI/MS m/z: 530.3 [MþH]þ; 1H NMR (500 MHz, DMSO)
d
8.21 (s, 1H), 7.76 (d, J ¼ 8.5 Hz, 1H), 7.65 (t, J ¼ 7.5 Hz, 1H), 7.42 (t,
Fig. 5. The AST and ALT activity after administration of equimolar doses (6
mmol/
100 g b. wt.) of tacrine or compound 6d. Results are expressed as mean SD (n ¼ 8e9,
t test, statistical significant difference compared to control values before administra-
tion, P* ꢃ 0.05, P** ꢃ 0.01, P*** ꢃ 0.001).
J ¼ 7.5 Hz, 1H), 7.15 (t, J ¼ 6.0 Hz, 1H), 6.27 (s, 1H), 3.52 (t, J ¼ 7.0 Hz,
2H), 3.07 (dq, J ¼ 13.0, 6.5 Hz, 1H), 3.01e2.95 (m, 1H), 2.93 (t,
J ¼ 6.0 Hz, 2H), 2.69 (d, J ¼ 6.0 Hz, 2H), 2.47 (t, J ¼ 6.0 Hz, 1H),
2.42e2.34 (m, 1H), 2.19e2.10 (m, 1H), 2.07 (s, 3H), 2.04 (s, 3H), 1.96
(s, 3H), 1.87e1.75 (m, 4H), 1.72e1.64 (m, 1H), 1.57e1.47 (m, 2H), 1.35
(s, 3H), 1.31e1.26 (m, 2H), 1.23e1.16 (m, 2H), 1.07e0.99 (m, 2H). 13
C
Fig. 6. Histomorphological appearance of livers of male rats after treatment with the solvent only (control) (A) and 24 h after administration of tacrine (B) and 6d (C). H&E, original
magnification: ꢀ200.

48
S.-S. Xie et al. / European Journal of Medicinal Chemistry 93 (2015) 42e50
NMR (125 MHz, DMSO)
d
173.58, 163.91, 155.57, 152.87, 146.34,
calcd for C37H52N3O3 [MþH]þ 586.4003, found 586.4002.
144.37, 130.17, 125.22, 124.37, 124.29, 123.12, 121.56, 120.72, 118.93,
117.61, 114.55, 77.74, 48.18, 38.61, 31.84, 30.74, 29.97, 29.34, 26.33,
26.07, 25.05, 24.69, 22.73, 22.11, 20.60, 13.19, 12.47, 12.22. HRMS:
calcd for C33H44N3O3 [MþH]þ 530.3377, found 530.3379.
4.1.2. In vitro inhibition experiments of ChEs
Acetylcholinesterase (AChE, E.C. 3.1.1.7) from electric eel and
human erythrocytes, butyrylcholinesterase (BuChE, E.C. 3.1.1.8)
from equine serum and human serum, S-butyrylthiocholine iodide
(BTCI), acetylthiocholine iodide (ATCI), 5, 50-dithiobis-(2-
nitrobenzoic acid) (Ellman's reagent, DTNB) and tarcine hydro-
chloride were purchased from SigmaeAldrich (St. Louis, MO, USA).
The inhibitory activities of test compounds 6aeg was evaluated by
Ellman's method [38]. The compounds were dissolved in DMSO and
diluted with the buffer solution (50 mM TriseHCl, pH ¼ 8.0, 0.1 M
NaCl, 0.02 M MgCl2$6H2O) to yield corresponding test concentra-
4.1.1.5. 6-Hydroxy-2, 5, 7, 8-tetramethyl-N-(7-((1, 2, 3, 4-
tetrahydroacridin-9-yl)amino)heptyl)chroman-2-carboxamide (6e).
Intermediate 4e was reacted with 6-hydroxy-2, 5, 7, 8-
tetramethylchroman-2-carboxylic acid following the general pro-
cedure to give the desired product 6e with a yield of 58%. IR (KBr)
n
3425, 2930, 2856, 1649, 1588, 1524, 1447, 1349, 1256, 1088, 756,
678 cmꢁ1; ESI/MS m/z: 544.3 [MþH]þ; 1H NMR (500 MHz, DMSO)
d
8.23 (d, J ¼ 8.5 Hz, 1H), 7.78 (d, J ¼ 8.5 Hz, 1H), 7.67 (t, J ¼ 7.5 Hz,
tions (DMSO less than 0.01%). In each well of the plate, 160
1.5 mM DTNB, 50 L of AChE (0.22 U/mL eeAChE or 0.05 U/mL
hAChE) or 50 L of BuChE (0.12 U/mL eqBuChE or 0.024 U/mL
hBuChE) were incubated with 10 L of different concentrations of
test compounds (0.001e100
M) at 37 ꢂC for 6 min. After this
period, acetylthiocholine iodide (15 mM) or S-butyrylthiocholine
iodide (15 mM) as the substrate (30 L) was added and the absor-
mL of
1H), 7.44 (t, J ¼ 7.5 Hz, 1H), 7.14 (t, J ¼ 6.0 Hz, 1H), 6.46 (s, 1H), 3.58
(t, J ¼ 7.0 Hz, 2H), 3.11e3.03 (m, 1H), 3.01e2.95 (m, 1H), 2.94 (t,
J ¼ 6.0 Hz, 2H), 2.68 (d, J ¼ 5.5 Hz, 2H), 2.47 (d, J ¼ 6.0 Hz, 1H),
2.43e2.34 (m, 1H), 2.20e2.12 (m, 1H), 2.08 (s, 3H), 2.05 (s, 3H),1.97
(s, 3H), 1.82 (s, 4H), 1.73e1.65 (m, 1H), 1.62e1.54 (m, 2H), 1.35 (s,
3H), 1.30e1.12 (m, 6H), 1.06e0.96 (m, 2H). 13C NMR (125 MHz,
m
m
m
m
m
DMSO)
d
173.42, 163.63, 155.27, 153.37, 146.41, 144.27, 130.36,
bance was measured with a wavelength of 405 nm at different time
intervals (0, 60, 120, and 180 s). IC50 values were calculated as
concentration of compound that produces 50% enzyme activity
inhibition, using the Graph Pad Prism 4.03 software (San Diego, CA,
124.72, 124.59, 124.61, 123.27, 121.57, 120.72, 118.52, 117.71, 114.38,
77.69, 48.24, 38.68, 31.61, 30.74, 29.91, 29.58, 29.13, 29.05, 26.59,
26.41, 25.07, 22.71, 22.05, 20.68, 13.26, 12.25, 12.28. HRMS: calcd for
C
34H46N3O3 [MþH]þ 544.3534, found 544.3536.
USA). Results are expressed as the mean
different experiments performed in triplicate.
SD of at least three
4.1.1.6. 6-Hydroxy-2, 5, 7, 8-tetramethyl-N-(8-((1, 2, 3, 4-
tetrahydroacridin-9-yl)amino)octyl) chroman-2-carboxamide (6f).
Intermediate 4f was reacted with 6-hydroxy-2, 5, 7, 8-
tetramethylchroman-2-carboxylic acid following the general pro-
4.1.3. In vitro evaluation of antioxidant activity
The antioxidant activities of compounds 6aeg were evaluated
by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free-radical scavenging
assay according to the method of Lee et al. with slightly modifica-
cedure to give the desired product 6f with a yield of 63%. IR (KBr)
n
3428, 2927, 2854, 1652, 1589, 1524, 1451, 1415, 1256, 1088, 756,
tions [39]. Briefly, 95
added in a 96-well plate containing 5
m
L of DPPH radical solution (300
mM) was
678 cmꢁ1; ESI/MS m/z: 558.3 [MþH]þ; 1H NMR (500 MHz, DMSO)
mL of different concentrations
d
8.23 (d, J ¼ 8.5 Hz, 1H), 7.78 (d, J ¼ 8.5 Hz, 1H), 7.67 (t, J ¼ 7.5 Hz,
of test compound dissolved in MeOH, and incubated for 30 min at
37 ꢂC in the dark. The absorbance of each well was measured at
517 nm using a microplate reader. The IC50 values of test com-
pounds were determined using the Graph Pad Prism 4.03 software
(San Diego, CA, USA). Results are expressed as the mean SD of at
least three different experiments performed in triplicate.
1H), 7.44 (t, J ¼ 7.5 Hz, 1H), 7.13 (t, J ¼ 6.0 Hz, 1H), 6.45 (s, 1H), 3.59
(t, J ¼ 7.0 Hz, 2H), 3.45 (q, J ¼ 7.0 Hz, 1H), 3.07 (td, J ¼ 13.0, 6.5 Hz,
1H), 3.01e2.95 (m, 1H), 2.93 (d, J ¼ 5.5 Hz, 2H), 2.68 (d, J ¼ 5.5 Hz,
2H), 2.43e2.34 (m, 1H), 2.20e2.13 (m, 1H), 2.08 (s, 3H), 2.08 (s, 3H),
1.97 (s, 3H), 1.82 (s, 4H), 1.73e1.65 (m, 1H), 1.64e1.58 (m, 2H), 1.36
(s, 3H), 1.31e1.20 (m, 4H), 1.19e1.09 (m, 4H), 1.03e0.96 (m, 2H). 13
C
NMR (125 MHz, DMSO)
d
173.57, 163.95, 155.21, 153.22, 146.38,
4.1.4. Kinetic study of AChE inhibition
144.35, 130.42, 124.76, 124.45, 124.38, 123.08, 121.52, 120.69, 118.69,
117.62, 114.27, 77.76, 48.19, 38.72, 31.53, 30.73, 29.98, 29.37, 29.04,
29.00, 26.61, 26.28, 25.00, 24.74, 22.66, 22.00, 20.60, 13.18, 12.45,
12.21. HRMS: calcd for C35H48N3O3 [MþH]þ 558.3690, found
558.3691.
The kinetic study of AChE was performed by Ellman's method
with three different concentrations (12.5, 6.25 and 3.125 nM) of
compound 6d. LineweavereBurk reciprocal plots were constructed
by plotting 1/velocity against 1/[substrate] at varying concentra-
tions of the substrate acetylthiocholine (0.05e0.5 mM). The plots
were assessed by a weighted least-squares analysis that assumed
the variance of velocity (v) to be a constant percentage of v for the
entire data set. Data analysis was performed with Graph Pad Prism
4.03 software (San Diego, CA, USA).
4.1.1.7. 6-Hydroxy-2, 5, 7, 8-tetramethyl-N-(10-((1, 2, 3, 4-
tetrahydroacridin-9-yl)amino)decyl)chroman-2-carboxamide (6g).
Intermediate 4g was reacted with 6-hydroxy-2, 5, 7, 8-
tetramethylchroman-2-carboxylic acid following the general pro-
cedure to give the desired product 6g with a yield of 65%. IR (KBr)
n
4.1.5. Docking study
3428, 2927, 2856, 1652, 1589, 1524, 1451, 1413, 1256, 1088, 758,
Molecular docking studies were performed using Molecular
Operating Environment (MOE) software version 2008.10 (Chemical
Compouting Group, Montreal, Canada). The X-ray crystal structure
of recombinant human acetylcholinesterase in complex with
donepezil (hAChE, PDB code 4EY7) was obtained from the Protein
Data Base (PDB). Hydrogens and partial charges were added using
protonate 3D application in MOE. The compound 6d was con-
structed using the MOE builder module and energy minimized
using Merck Molecular force field (MMFF94x, RMSD gradient:
678 cmꢁ1; ESI/MS m/z: 586.3 [MþH]þ; 1H NMR (500 MHz, DMSO)
d
8.24 (d, J ¼ 8.5 Hz, 1H), 7.78 (d, J ¼ 8.5 Hz, 1H), 7.67 (t, J ¼ 7.5 Hz,
1H), 7.45 (dd, J ¼ 11.5, 4.0 Hz, 1H), 7.16 (t, J ¼ 6.0 Hz, 1H), 6.37 (s, 1H),
3.60 (t, J ¼ 7.0 Hz, 2H), 3.12e3.06 (m, 1H), 3.03e2.97 (m,1H), 2.95 (t,
J ¼ 5.8 Hz, 2H), 2.71 (d, J ¼ 5.8 Hz, 2H), 2.46e2.35 (m, 2H), 2.23e2.15
(m, 2H), 2.10 (s, 3H), 2.08 (s, 3H), 2.00 (s, 3H), 1.88e1.78 (m, 4H),
1.75e1.67 (m, 1H), 1.65e1.58 (m, 2H), 1.38 (s, 3H), 1.35e1.08 (m,
10H), 1.07e0.96 (m, 3H). 13C NMR (125 MHz, DMSO)
d 173.59,
164.03, 155.07, 152.84, 146.38, 144.34, 130.54, 125.29, 124.49,
124.42, 123.07, 121.52, 120.67, 118.83, 117.61, 114.19, 77.76, 48.14,
38.73, 30.71, 29.97, 29.38, 29.29, 29.25, 29.07, 26.63, 26.36, 24.99,
24.75, 22.75, 22.63, 22.16, 21.91, 20.60, 13.19, 12.46, 12.22. HRMS:
0.05 kcal molꢁ1 ꢁ1) [47]. The MOE Dock application was used for
Å
docking 6d into the active site of the protein. The poses were
generated by the Triangle Matcher placement method and then
were rescored using ASE scoring function. The Forcefiled was

S.-S. Xie et al. / European Journal of Medicinal Chemistry 93 (2015) 42e50
49
selected as the refinement method. The retained best poses were
visually inspected and the interactions with binding pocket resi-
dues were analyzed.
Molecular Devices, Sunnyvale, CA, USA). Each sample was analyzed
at least three independent runs in four wells, and the results are
given as the means SD. A plot of the experimental Pe values of 10
standard drugs versus their bibliographic values provided a good
linear correlation, Pe (exp.) ¼ 0.8597 Pe (Bibl.) e 0.276 (R2 ¼ 0.9726)
(Supporting information, Fig. S1).
4.1.6. Cell culture and measurement of cell viability
PC 12 cells was obtained from the Cell Bank of the Chinese
Academy of Sciences (Shanghai, China) and grown in RPMI-1640
medium containing 10% (v/v) foetal bovine serum, 100 U peni-
cillin/mL and 100 mg streptomycin/mL under 5% CO2 at 37 ꢂC. The
culture media were changed every other day. After pretreatment
Acknowledgments
This research work was financially supported by Program for
Changjiang Scholars and Innovative Research Team in University
(IRT1193), A Project Funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions (PAPD).
with different concentrations of test compound (1.563e12.5
for 2 h, PC 12 cells were incubated with 100 M of H2O2 for 24 h.
The cell viability was evaluated using MTT assay [41]. Briefly, 10
of 5 mg/ml MTT was added to each well and incubated for 4 h at
37 ꢂC. Then, 200
L of dimethylsulfoxide (DMSO) was added to
mM)
m
mL
m
Appendix A. Supplementary data
dissolve the dark blue formazan crystals formed in intact cells, and
the absorbance at 570 nm was detected by a microplate reader. PC
12 cells were cultured without test compound or H2O2 as control
group and the results were expressed by percentage of control.
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2015.01.
058. These data include MOL files and InChiKeys of the most
important compounds described in this article.
4.1.7. In vivo hepatotoxicity study
Investigations were performed on adult male Sprague Dawley
(SD) rat [24,43]. Rats were housed in a room that was automatically
maintained on a 12-h light/dark cycle at 25 ꢂC and proper humidity.
Rats were given food and tap water ad libitum. Tacrine hydro-
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