Design, synthesis and evaluation of a novel metal chelator as multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bioorg.2019.03.064

A series of compounds following the lead compounds including deferasirox and tacrine were designed, synthesized and evaluated as multifunctional agents against Alzheimer's disease (AD). In vitro studies showed that most synthesized compounds exhibited good multifunctional activities in inhibiting acetylcholinesterase (bAChE), and chelating metal ions. Especially, compound TD_e demonstrated significant metal chelating property, a moderate acetylcholinesterase (AChE) inhibitory activity and an antioxidant activity. Results from the molecular modeling indicated that TD compounds were mixed-type inhibitor, binding simultaneously to the catalytic anionic site (CAS) and the peripheral anionic site (PAS) of TcAChE. Moreover, TD_e showed a low cytotoxicity but a good protective activity against the injury caused by H₂O₂. These results suggest that TD compounds might be considered as attractive multi-target cholinesterase inhibitor and will play important roles in the treatment of AD.

Full text of DOI:10.1016/j.bioorg.2019.03.064

Accepted Manuscript
Design, synthesis and evaluation of a novel metal chelator as multifunctional
agents for the treatment of Alzheimer’s disease
Yingying Wang, Yue Yang, Kwon Ho Hong, Yao Ning, Ping Yu, Jinghui Ren,
Min Ji, Jin Cai
PII:
S0045-2068(18)31264-1
DOI:
https://doi.org/10.1016/j.bioorg.2019.03.064
YBIOO 2890
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 November 2018
18 February 2019
23 March 2019
Please cite this article as: Y. Wang, Y. Yang, K. Ho Hong, Y. Ning, P. Yu, J. Ren, M. Ji, J. Cai, Design, synthesis
and evaluation of a novel metal chelator as multifunctional agents for the treatment of Alzheimer’s disease,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.03.064
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design, synthesis and evaluation of a novel metal chelator as
multifunctional agents for the treatment of Alzheimer’s disease
Yingying Wanga, Yue Yanga, Kwon Ho Hongb, Yao Ninga, Ping Yua, Jinghui Rena, Min Jic,d,
Jin Caia,d*
aSchool of Chemistry and Chemical Engineering, Southeast University, Nanjing, Jiangsu, 211189, P.R. China
bDepartment of Medicinal Chemistry and the Institute for Therapeutics Discovery and Development, University of Minnesota, Minneapolis, MN
55414, USA
cSchool of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, P.R. China
dSuzhou Key Laboratory of Biomaterials and Technologies & Collaborative Innovation Center of Suzhou Nano Science and Technology, Suzhou
215123, P.R. China
Abstract: A series of compounds following the lead compounds including deferasirox and tacrine were designed, synthesized
and evaluated as multifunctional agents against Alzheimer’s disease (AD). In vitro studies showed that most synthesized
compounds exhibited good multifunctional activities in inhibiting acetylcholinesterase (bAChE), and chelating metal ions.
Especially, compound TDe demonstrated significant metal chelating property, a moderate acetylcholinesterase (AChE) inhibitory
activity and an antioxidant activity. Results from the molecular modeling indicated that TD compounds were mixed-type
inhibitor, binding simultaneously to the catalytic anionic site (CAS) and the peripheral anionic site (PAS) of TcAChE. Moreover,
TDe showed a low cytotoxicity but a good protective activity against the injury caused by H2O2. These results suggest that TD
compounds might be considered as attractive multi-target cholinesterase inhibitor and will play important roles in the treatment
of AD.
Keywords: Alzheimer's disease; Multifunctional agents; Acetylcholinesterase inhibitors; Metal chelator; Tacrine; Deferasirox
1. Introduction
Alzheimer’s disease (AD), first reported in 1906, is rapidly becoming a major threat to
healthcare in our society. With more than 48 million people are affected by AD, the number is
estimated to show unprecedented growth and increase to spread 131.5 million by 2050 [1, 2]. AD
is a neurodegenerative disorder clinically characterized with cognitive impairment, the loss of
language skills, and ultimately results in complete dependencies and the deaths of the patients
with AD [3, 4]. Faced with the daunting public health problem of our time, understanding,
treatment and prevention of AD are urgent.
Although it has been more than 100 years since AD was first reported, there are many
unanswered questions [5]. Due to the etiopathogenesis of AD involving many targets and
signaling pathways, various hypotheses have arisen including acetylcholine hypothesis, amyloid
deposition hypothesis, oxidative stress hypothesis and metal ion homeostasis imbalance
hypothesis [6-8]. Currently, the main therapeutic approach in the treatment of patients with AD is
to enhance the cholinergic activity in the brain. Acetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE) are involved in cholinergic signaling. Based on cholinergic
*
Corresponding authors.
E-mail address: caijin@seu.edu.cn (J. Cai).

hypothesis, the decrease in AChE levels results in a series of symptoms such as memory loss and
cognitive impairment and the clinical restoration of cholinergic function is believed to alleviate
AD symptoms [9, 10]. Acetylcholinesterase inhibitors can inhibit the activity of
acetylcholinesterase, thereby regulating the content of acetylcholine in the brain to achieve the
goal in alleviating AD symptoms [11]. The drugs approved by FDA include one
N-methyl-D-aspartate (NMDA) receptor antagonist and four acetylcholinesterase inhibitors,
namely tacrine, donepezil, rivastigmine and galantamine [12, 13]. These agents can compensate
for the depletion of acetylcholine in the brain, or prevent aberrant neuronal stimulation.
Nevertheless, these drugs modestly alleviate the symptoms but cannot prevent, halt or reverse the
progression of AD.
Abnormal protein folding and aggregation in the brain are another important key contributing
factor in the pathology of AD, and can cause neuronal damage, synaptic abnormalities and organ
dysfunction [14, 15]. Converging lines of evidences suggest that Amyloid-β (Aβ) tends to bunch
up into small clusters and finally into the amyloid plaques, and is an important biomarker in AD.
Aβ, a peptide containing 39~43 amino acid residues, has two main isoforms, Aβ (1-40) and Aβ
(1-42). Aβ (1-42) shows stronger neuronal cytotoxicity and tends to aggregate more rapidly than
Aβ (1-40) [16]. The potential therapy for AD consists of preventing the formation and
accumulation of Aβ. It has been shown, that abnormally high levels of Cu+/2+, Fe2+/3+ and/or Zn2+
lead to promote of Aβ peptide formation and generate reactive oxygen species (ROS) in AD
patients [17]. Therefore, metal chelators have the ability to attenuate amyloid precursor protein
(APP) expression, Aβ aggregates and neurofibrillary tangle (NFT) formation [18]. Therefore,
lowering the high concentration of such metal ions in the brain by chelating the metals is an
approach for the treatment of AD.
Deferasirox (DEF) (Fig. 1) is a bis-hydroxy-triazole tridentate iron chelator that is strongly
chelating iron ions, non-toxic, and highly safe [19]. In clinic, deferasirox was used to reduce the
iron overload caused by blood transfusion [20]. However, there was no effect on reducing iron ion
content in the brain [21]. Deferasirox smoothly passes through the blood-brain barrier following
binding to lactoferrin. Accordingly, we have prepared a new type of iron chelator DBP that can
chelate metal ions only under oxidative stress conditions. In order to mitigate the damaging effects
of metals while preserving their beneficial properties, DBP can be activated in the presence of
H2O2, revealing phenolic hydroxyl groups and forming the deferasirox as shown in Fig. 1.
In view of the multiple pathogeneses of AD, the development of anti-AD drugs is moving
from traditional single-target drugs to novel drugs aiming at multiple targets simultaneously
including acetylcholinesterase, metal ions and free radicals which are involved in the progression
of AD [22]. Tacrine (Fig. 1) is an acting anticholinesterase and was the first cholinesterase
inhibitor approved by FDA for the treatment of AD [23]. Although tacrine was withdrawn due to
its hepatotoxicity, its ideal pharmacokinetic properties can tolerate significant structural
modifications and still maintain the inhibitory activity against AChE. In recent years, tacrine has
been widely used as a lead for the development of new multifunctional agents, including
tacrine-deferiprone hybrids designed by Chand Karam, et al [24-26]. In search of new anti-AD
drugs, we adopted promising drug development approaches based on multi-target directed ligands
(MTLs) and then reasonably combined tacrine and deferasirox to achieve new compounds TD.
These hybrids were anticipated to manifest AChE inhibitory activity with metal chelation and
antioxidant properties [27].

Fig. 1. Design strategy for novel compounds.
2. Results and discussion
2.1. Chemistry
Based on the reported synthetic routes of boric acid ester compounds, we designed a new
synthetic route for DBP as shown in Scheme 1. DBP was prepared from DEF via the two-step
process, the triflation of phenols followed by the formation of boronate esters [28].
The key intermediates 4a–e were synthesized as shown in Scheme 2. Compound 3 were
reacted with diamines with different lengths in 1-pentanol to afford the intermediates 4a-e [29, 30].
Then, DEF and corresponding 4a-e were coupled in the presence of EDCI and HOBt in DCM at
room temperature to give TDa-e, respectively. Target compounds were confirmed through 1H NMR,
13C NMR and IR.
Scheme 1. (a) DIPEA, Tf2O, DCM, -20 °C, 3 h, 85%; (b) KOAc, PdCl2(dppf), DMSO, 80 °C, 24 h, 33%.

Scheme 2. (a) POCl3, 3 h, 180 °C; (b) H2N(CH2)nNH2 (3-4 equiv), 1-pentanol, 160 °C; (c) EDCI, HOBT, Et3N.
Table 1.
Compound inhibition of bAChE activity, antioxidant activity and ClogP value prediction.
IC50b (μM)
Antioxidant
activityd(%)
Compound
na
CLogP
bAChEc
TDa
TDb
4
5
6
7
8
13.5±0.82
17.8±0.21
15.4±0.27
16.1±0.36
13.7±0.21
14.1±0.33
-
32±0.23
34±0.56
39±0.42
49±0.31
56±0.69
-
6.996
6.909
7.315
7.297
7.265
-
TDc
TDd
TDe
Tacrine
DEF
23±0.52
3.688
a carbon length.
b d All values are expressed as mean ± SEM from three independent experiments.
c bAChE from bovine serum.
2.2. Metal-chelating properties of DBP
The chelating effect of DBP under oxidative stress conditions was studied with a UV-vis
spectrometer and the results are shown in Fig. 2a. The peak in the absorption spectrum of DBP is
at 304 nm. When FeCl3 was added, the UV-vis absorption spectrum of DBP showed no obvious
change. Conversely, addition of H2O2 to solution of DBP induces a significant change in spectrum,

which suggests that boronate esters moieties of DBP is replaced with hydroxyl groups converting
DBP to DEF after the addition of H2O2 [31]. In the presence of H2O2 and0.5 equiv of FeCl3, the
spectrum of DBP showed a red shift (304 nm to 315 nm) indicating formation of the 1:2
[Fe(DEF)2]3- complex [32]. The observations indicated that deferasirox borate ester compound has
a metal chelating activity, DBP has no affinity for metal ions unless it is under oxidative stress
condition and DEF serves as a metal chelator.
Fig. 2. The UV-vis spectra of compounds in methanol; (a) DBP (50 μM) alone or in the presence of FeCl3 (0.5
equiv), 1.5 mM H2O2 and FeCl3+ H2O2. (b) DEF (50 μM) alone or in the presence of FeCl3 (0.5 equiv) and CuCl2
(1 equiv), (c) TAC (40 μM) alone or in the presence of FeCl3 (0.5 equiv) and CuCl2 (1 equiv), (d) TDe (30 μM )
alone or in the presence of FeCl3 (0.5 equiv) and CuCl2 (1 equiv).
2.3. Pharmacological activity of TD compounds
2.3.1. Cholinesterase inhibitory activity
The inhibitory activities of hybrid TD compounds against AChE from bovine serum (bAChE)
were determined by the ELISA method [33]. Bovine serum AChE shows a high degree of
homology with the corresponding human enzymes and it has more availability from commercial
sources, making it a reasonable alternative [34]. For comparison purpose, tacrine was used as a
reference compound. The IC50 values of all test compounds were summarized in Table 1. As
shown in Table 1, all new target compounds displayed inhibitory activities against bAChE with
micromolar IC50 values, which were virtually the same inhibitory activities as that of tacrine. The
introduction of the DEF scaffold to tacrine with linkers with various lengths did not alter the
potency of tacrine. Among the target compounds, TDa (IC50 = 13.5 μM) and TDe (IC50 = 13.7 μM)
showed the slightly better potent inhibitory activity for bAChE.

a
SER-81
b
TRP-84
HIS-440
ASP-72
TYP-70
PHE-330
TYR-334
TRP-279
c
Fig. 3. Docking models of the TDa in Torpedo californica AChE (PDB code: 2CMF). The dashed lines
represent the interactions between the AChE and the ligand: (a) 3D docking image of compound TDa with AChE,
(b) interactions between the binding sites of compounds TDa and AChE, (c) docking compound TDa in the surface
model of AChE.
2.3.2. Molecular modeling study
To further study the possible binding modes of TD derivatives in AChE, a molecular docking
study with Torpedo californica variant of AChE (TcAChE) (PDB ID: 2CMF) was performed using
Glide [35]. As shown in Fig. 3, the tacrine moiety of TDa occupies the catalytic active site (CAS)

of TcAChE, forming π-π stacking interactions with Phe 330 and Trp 84. The N atom of quinoline
ring also forms a water-mediated H-bond with His 440. The long alkyl linker is located in the
mid-gorge region of TcAChE and has no interactions with the amino acid residues. The
deferasirox moiety of TDa occupies the peripheral anionic site (PAS) of TcAChE, via a π-π
interaction with Trp 279 and the hydroxyl via H-bonding interactions with Trp 279 and Tyr 334.
The docking results indicate that new TD compounds could simultaneously bind to the CAS and
PAS of TcAChE, demonstrating the rationale of our design. The new tacrine hybrids inhibitory
activities for TcAChE were no significantly improvement, as compared with tacrine, which might
be due to their chain length and steric hindrance, similarly to previous report for the same linker in
other tacrine hybrids [36].
2.3.3. Cytotoxicity and effect on H2O2-induced oxidative cell damage in PC12
To examine the cytotoxicity of TD compounds, cell viability was assayed using the rat
pheochromocytoma cell line PC12 [37, 38], and tested using the 3-(4,5- dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium (MTT) assays [39]. PC12 cells were treated with TDa, TDb, TDc, TDd and
TDe at three different concentrations (3, 5 and 10 μM) for 24 h. H2O2 (150 μM) was used to
induce oxidative damage, and DEF and tacrine were included as reference compounds. As shown
in Fig. 4, the treatment of P12 cells with compound DEF did not show any change in the cell
viability compared to the control treated with only H2O2. Tacrine was potent in protecting cell
damage manifesting the cell viability of 92.6% at 10 μM, but it did not exert the cell protective
activity against the injury caused by H2O2. At 10 μM concentration, TDa or TDb manifested a
decrease of cell viability (21.4% and 29.4%, respectively). The viability of cells treated with TDa
or TDb further decreased to 9.1% and 21.4%, respectively, when H2O2 was added. Compound TDe
exhibited a good protective activity for PC12 cells damaged by oxidative stress at 10 μM with cell
viability of 64.4% and 68.1% in the presence and absence of H2O2, respectively. These results
showed that cytotoxicity was significantly reduced as the length of the alkyl linker increased and
this result deserved a further study.
Fig. 4. Effect of TDa, TDb, TDc, TDd and TDe on H2O2-induced oxidative cell damage in PC12 cells.
2.3.4. DPPH free radical scavenging activity
To further study, free radical scavenging activities of TD compounds was evaluated using the

DPPH assay [40]. As shown in Table 1, it was observed that TDe exhibited more profound radical
scavenging effect and TDc, TDd were found to exhibit moderate antioxidant activity. The radical
scavenging capacity of new compounds results from the hydroxyl group of DEF and the length of
linker [26].
2.3.5. In vitro blood–brain barrier permeation assay
For successful central nervous system (CNS) drugs, it is important that the compounds are
able to cross the blood–brain barrier (BBB). The ‘rule of 5’ predicts that good absorption or
permeation is more likely when there are less than 5 H-bond donors, 10 H-bond acceptors, the
molecular weight (MW) is less than 500 and the calculated LogP (CLogP) is less than 5 (or
MLogP<4.15) [41]. To evaluate the potential of these hybrids to cross the BBB, we calculated
CLogP values using the ChemDraw 17.0. From Table 1, the CLogP value of five compounds (TDa,
TDb, TDc, TDd and TDe) were all greater than 5, indicating that TD compounds might be difficult
to penetrate the BBB. Therefore, we consider using nanoparticles or liposomes as carriers to
enable drugs to cross the blood-brain barrier in the future.
2.3.6. Metal-chelating properties of compound TDe
As compound TDe showed a favorable antioxidant activity and a bAChE inhibitory activity
with IC50 of 13.7 μM, its metal-chelating ability was investigated. The chelating abilities of
compounds DEF, TAC and TDe with 0.5 equiv of Fe3+ and 1 equiv of Cu2+ were studied using the
UV-vis spectrometric method are shown in Fig. 2b, Fig. 2c, Fig. 2d (respectively). When FeCl3 or
of CuCl2 was added, the UV-vis spectrum of TAC showed no obvious change, indicating that
TAC did not chelate metal ions. Upon the addition of FeCl3 to DEF, a red shift of the maximum
absorption from 295 nm to 315 nm, suggesting the formation of the [Fe(DEF)2]3- complex. The
results were similar to a previous report for UV-vis spectra of DEF [32]. When CuCl2 was added,
a significant change could be observed, which was similar to reported DEF-Cu2+ complex [42].
Similarly, the addition of Fe3+ or Cu2+ to TDe resulted in the shifts from 305 nm to 324 nm (for
Fe3+) or 338 nm (for Cu2+). The complexation ability of TDe might be ascribed to DEF moiety of
the compound.
3. Conclusions
In conclusion, we designed and synthetized tacrine-deferasirox compounds as multifunctional
agents against AD. The new hybrids combined the capacities of TcAChE inhibition of tacrine and
chelate metal ability of deferasirox. In all new compounds, TDa and TDe displayed the inhibitory
activity against bAChE with IC50 values of 13.5 μM and 13.7 μM, respectively, which were
virtually the same inhibitory activities as that of tacrine. Molecular modeling studies indicated that
new TD compounds could simultaneously occupy the CAS and PAS of TcAChE. Moreover, TDe
exhibited the low cytotoxicity and effectively cell protective activity for the injury caused by H2O2.
Furthermore, TDe was found a more profound radical scavenging effect than DEF and was able to
bind Fe3+ and Cu2+ to form chelate metal complexes. In addition, new compound DBP could serve
as a metal chelator under an oxidative stress condition. It suggests that DBP will facilitate
management metal burden at locations of disease without stimulating widespread metal
redistribution. Moreover, such multifunctional properties indicated that TDe as a novel multi-target

cholinesterase inhibitor and TD compounds could be considered as promising compounds for the
further study in development of anti-AD drugs.
4. Experimental section
4.1. Chemistry
All chemicals (reagent grade) were obtained from commercial suppliers and used without
further purification. Reaction progress was monitored by thin-layer chromatography (TLC) using
silica gel GF254 plates from Qingdao Haiyang Chemical Plant (China) and the spots were detected
under UV light (254 nm). Melting points were measured on RY-1 melting-point instrument (China)
and are uncorrected. Column chromatography using silica gel (200-300 mesh) purchased from
1
Qingdao Haiyang Chemical Plant (China). H NMR and 13C NMR spectra were measured on a
Bruker Avance DPX-300/500 spectrometer at 25 °C and referenced to tetramethylsilane (TMS) by
China Pharmaceutical University Analytical Testing Center. IR (FI-IR) spectra were recorded on a
Perkin-Elmer spectrometer by Suzhou Southeast Pharmaceutical Co., Ltd. (China).
4.1.1. 4-(3,5-bis(2-(((trifluoromethyl)sulfonyl)oxy)phenyl)-1H-1,2,4-triazol-1-yl)benzoic acid
(DOF)
To a stirred solution of DEF (0.73 g, 1 mmol) and a few drops of DMF in dry CH2Cl2 at
-20 °C was added dropwise N,N-diisopropylethylamine (0.006 mmol) and
trifluoromethanesulfonic anhydride (0.006 mmol) in 40 minutes. The reaction mixture was stirred
for 4 h at room temperature and quenched in ice-cold water, filtered and washed with water, brine
and dried (Na2SO4). Evaporation of the solvent give (DOF) as a yellow oil which was used in the
1
next step without further purification. H NMR (500 MHz, DMSO-d6) δ: 8.26-8.15 (m, 4H),
7.69-7.54 (m, 4H), 7.14-7.01 (m, 4H); MS: [M-H]- m/z calcd 635.9975 for C23H13F6N3O8S2, found
636.9975.
4.1.2.
4-(3,5-bis(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1H-1,2,4-triazol-1-yl)benzoic
acid (DBP)
A mixture of DOF, potassium acetate (0.54 g, 5.46 mmol), bis(pinacolato)diboron (0.26 g,
5.46 mmol) and catalytic PdCl2(dppf) in DMSO was heated at 80 °C under N2 atmosphere for 24
h. The mixture was cooled to RT, diluted with ethyl acetate and washed with water, brine and
dried (Na2SO4). The residue was purified by silica gel chromatography to yield the desired (DBP)
1
as a yellow solid (0.19 g, 33% yield). mp 178.2-179.5 °C; H NMR (300 MHz, CD3OD) δ: 1.25
(m, 21H), 2.25 (m, 3H), 6.92 (m, 4H), 7.39 (m, 4H), 8.08 (m, 4H); 13C NMR (75 MHz, DMSO-d6)
δ:116.27, 154.95, 151.15, 150.12, 148.59, 138.36, 133.34, 132.34, 130.33, 129.96, 128.99, 128.60,
128.55, 126.40, 126.36, 126.32, 123.74, 117.98, 83.90, 25.07; IR (KBr, cm-1): 3412.9, 3365.9
(C=C), 2977.9 (C-H), 2736.1 (N=N), 1686, 1606.3, 1561.5, 1532.5, 1481, 1446.1, 974.9, 959.9,
849.6; MS: [M+H]+ m/z calcd 594.2941 for C33H37B2N3O6, found 594.2943.
4.1.3. 9-chloro-1,2,3,4-tetrahydroacridine (3)
To an ice-cooled solution of anthranilic acid (3.2 g, 3 mmol) and cyclohexanone (2.65 mL,

27 mmol) was added dropwise POCl3 with a constant pressure dropping funnel. Then, the reaction
was heated at reflux for 3 h. The solvent was reduced in vacuum and the residue was dissolved in
ethyl acetate and washed with 1N K2CO3 solution, brine and dried (Na2SO4). The residue was
purified by silica gel chromatography to yield the desired (3) as a yellow solid (4.6g, 91% yield).
mp 67.2-69.2 °C.
4.1.4. General procedure for the synthesis of intermediates 4a-e
A mixture of compound 3 (1.0 g, 3.61 mmol) and different amines (13.8 mmol) in 1-pentanol
(5 mL) was stirred at 160 °C for 36 h until TLC revealed completion of the reaction. The mixture
was diluted with DCM, filtered and the filtrate was washed with water, 1N NaOH solution, brine
and dried (Na2SO4). The crude was reduced in vacuum and the residue purified by silica gel
chromatography to yield the desired (4a-e), respectively.
1
4.1.4.1. N1-(1,2,3,4-tetrahydroacridin-9-yl)butane-1,4-diamine (4a) Brown oil; 71% yield; H
NMR (300 MHz, CDCl3) δ: 1.08 (s, 2H), 1.28 (d, J = 7.4 Hz, 2H), 1.45 (d, J = 7.4 Hz, 2H),
2.49-2.54 (m, 4H), 2.87 (t, J = 6.0 Hz, 2H), 3.25 (t, J = 7.1 Hz, 2H), 3.89-3.95 (m, 4H), 7.13 (t, J =
7.6 Hz, 1H), 7.35 (t, J = 7.6 Hz, 1H), 7.75 (t, J = 8.4 Hz, 2H); MS: [M-H]- m/z calcd 268.1819 for
C17H23N3, found 268.1910.
1
4.1.4.2. N1-(1,2,3,4-tetrahydroacridin-9-yl)pentane-1,5-diamine (4b) Brown oil; 89% yield; H
NMR (300 MHz, CDCl3) δ: 1.26-1.33 (m, 2H), 1.35-1.42 (m, 2H), 1.54 (d, 2H, J = 7.3 Hz), 1.79
(d, J = 3.3 Hz, 4H), 2.24 (s, 2H), 2.53 (s, 2H), 2.61 (s, 2H), 2.91(s, 2H), 3.24 (t, 2H, J = 7.2 Hz),
3.87 (s, 1H), 7.13 (dd, J = 9.0, 2.2 Hz, 1H), 7.76 (d, J = 9.0 Hz, 1H), 7.77 (d, J = 2.2 Hz, 1H); MS:
[M+H]+ m/z calcd 284.2121 for C18H25N3, found 284.2012.
1
4.1.4.3. N1-(1,2,3,4-tetrahydroacridin-9-yl)hexane-1,6-diamine (4c) Yellow oil; 56% yield; H
NMR (300 MHz, CDCl3) δ: 1.35-1.39 (m, 6H), 1.59-1.64 (m, 4H), 1.85-1.96 (m, 4H), 2.61-2.73
(m, 4H), 3.01-3.67 (m, 2H), 3.41 (t, J = 7.1 Hz, 2H), 3.89 (s, 1H), 7.28 (t, J = 7.6 Hz, 1H), 7.49 (t,
J = 7.6 Hz, 1H), 7.85 (d, J = 8.6 Hz, 1H), 7.90 (d, J = 8.5 Hz, 1H); MS: [M+H]+ m/z calcd
298.2277 for C19H27N3, found 298.2272.
1
4.1.4.4. N1-(1,2,3,4-tetrahydroacridin-9-yl)heptane-1,7-diamine (4d) Brown oil; 71% yield; H
NMR (300 MHz, CD3OD) δ: 1.24-1.47 (m, 8H), 1.52 (d, J = 7.2 Hz, 2H), 1.64 (d, J = 7.2 Hz, 2H),
1.86-1.90 (m, 4H), 2.70-2.78 (m, 6H), 2.93-2.95 (m, 2H), 3.55 (t, J = 7.2 Hz, 2H), 7.34(ddd, J =
1.1, 7.2, 8.7 Hz, 1H), 7.56 (d, J = 6.9 Hz, 1H), 7.74 (dd, J = 0.8, 8.7 Hz, 1H), 8.10 (dd, J = 0.8, 8.6
Hz, 1H); MS: [M+H]+ m/z calcd 312.2434 for C20H29N3, found 312.2442.
1
4.1.4.5. N1-(1,2,3,4-tetrahydroacridin-9-yl)octane-1,8-diamine (4e) Brown oil; 76% yield; H
NMR (300 MHz, CD3OD) δ: 1.31 (s, 8H), 1.55-1.69 (m, 4H), 1.88-1.91 (m, 4H), 2.71 (s, 2H),
2.83 (t, J = 7.6 Hz, 2H), 2.98 (s, 2H), 3.67 (t, J = 7.2 Hz, 2H), 7.42 (ddd, J = 1.4, 7.0, 8.4 Hz, 1H),
7.64 (d, J = 6.9 Hz, 1H), 7.76 (d, J = 8.6 Hz, 1H), 8.18 (d, J = 8.5 Hz, 1H); MS: [M+H]+ m/z calcd
326.2590 for C21H31N3, found 326.2597.
4.1.5. General procedure for the synthesis of TDa-e
To an ice-cooled solution of DEF (0.373 g, 1 mmol), HOBT (0.2 g 1.5 mmol) and EDCI (0.2

g, 1.5 mmol) in DMF was stirred for 1 h. Then, compound 4a-e was added and the mixture was
stirred at room temperature for 6 h until TLC revealed completion of the reaction. The mixture
was diluted with DCM, filtered and the filtrate was washed with water, brine and dried (Na2SO4).
The crude was reduced in vacuum and the residue purified by silica gel chromatography to yield
the desired (TDa-e), respectively.
4.1.5.1.
4-(3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl)-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)b
1
utyl)benzamide (TDa) Yellow solid; 33% yield; mp 147.3-148.5 °C; H NMR (300 MHz,
DMSO-d6) δ: 1.05 (t, J = 5.73 Hz, 2H), 1.59 (t, J = 8.94 Hz, 4H), 2.71 (d, J = 5.73 Hz, 2H), 2.88
(d, J = 5.19 Hz, 2H), 3.22-3.28 (m, 2H), 3.46-3.52 (m, 2H), 5.75 (s, 1H), 6.88 (d, J = 8.22 Hz, 1H),
6.95-7.04 (m, 3H), 7.35 (t, J = 7.95 Hz, 3H), 7.49-7.57 (m, 4H), 7.70 (d, J = 8.34 Hz, 1H), 7.84 (d,
J = 8.43 Hz, 2H), 8.05 (d, J = 7.47 Hz, 1H), 8.14 (d, J = 8.58 Hz, 2H), 8.50 (t, J = 5.04, 1H), 10.20
(s, 1H), 10.79 (s, 1H); 13C NMR (75 MHz, DMSO-d6) δ: 168.55, 162.63, 158.14, 157.92, 157.60,
154.33, 150.50, 148.11, 147.71, 132.70, 132.47, 131.58, 129.88, 129.69, 129.02, 128.25, 125.45,
125.38, 124.76, 124.30, 123.25, 120.79, 120.00, 117.82, 117.27, 116.23, 114.59, 111.25, 43.82,
37.78, 32.69, 27.54, 27.14, 26.04, 22.78, 22.02; IR (KBr, cm-1): 3425.5, 3373.9 (C=N), 3059.1
(O-H), 2927.9 (C-H), 2534.2 (N=N), 1650, 1608.4, 1572.3 (C=C), 1510.1, 1462.4, 858.3, 754.3;
MS: [M+H]+ m/z calcd 625.2921 for C38H36N6O3, found 625.2927.
4.1.5.2.
4-(3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl)-N-(5-((1,2,3,4-tetrahydroacridin-9-yl)amino)p
entyl)benzamide (TDb) Yellow solid; 59% yield; mp 140.1-141.2 °C. 1H NMR (300 MHz, CDCl3)
δ: 1.49 (t, J = 7.05 Hz, 2H), 1.63-1.74 (m, 4H), 1.88 (s, 4H), 2.68 (s, 2H), 2.96 (s, 2H), 3.44-3.54
(m, 4H), 3.99 (d, J = 5.88 Hz, 1H), 6.34 (s, 1H), 6.71 (t, J = 7.56 Hz, 1H), 6.99-7.09 (m, 4H),
7.26-7.39 (m, 4H), 7.48-7.56 (m, 3H), 7.79-7.94 (m, 4H), 8.13 (d, J = 7.71 Hz, 1H); 13C NMR (75
MHz, DMSO-d6) δ: 167.89, 162.63, 158.14, 157.92, 157.60, 154.33, 150.50, 148.11, 144.28,
132.70, 132.57, 131.19, 129.87, 129.69, 129.02, 128.25, 125.45, 125.38, 124.76, 124.31, 123.25,
120.79, 120.00, 117.82, 117.21, 116.23, 112.50, 111.24, 43.90, 36.67, 32.69, 29.80, 29.18, 24.68,
24.14, 22.94, 22.02; IR (KBr, cm-1): 3270.3 (C=N), 3190.4, 3055.7 (O-H), 2929.3 (C=H), 2857.2,
2547.7 (N=N), 2491, 1643.5, 1609.5, 1583.6, 1504, 1462.2, 855, 830.5, 754.9; MS: [M+H]+ m/z
calcd 639.3078 for C39H38N6O3, found 639.3071.
4.1.5.3.
4-(3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)h
exyl)benzamide (TDc) Yellow solid; 50% yield; mp 120.3-122.2 °C; 1H NMR (300 MHz, CDCl3)
δ: 0.83-0.89 (m, 3H), 1.43 (s, 5H), 1.88 (s, 4H), 2.67 (s, 2H), 2.98 (s, 2H), 3.44-3.52 (m, 4H), 4.13
(s, 1H), 6.40 (s, 1H), 6.69 (t, J = 7.53 Hz, 1H), 6.99-7.09 (m, 4H), 7.30-7.39 (m, 3H), 7.49-7.56
(m, 3H), 7.83-7.97 (m, 4H), 8.13 (d, J = 7.62 Hz, 1H); 13C NMR (75 MHz, DMSO-d6) δ: 168.13,
162.63, 160.41, 156.96, 156.79, 154.33, 150.29, 148.91, 145.79, 132.63, 132.54, 131.48, 129.94,
129.69, 129.11, 128.15, 125.59, 125.38, 124.81,, 124.46, 123.18, 120.79, 119.84, 117.82, 117.61,
116.23, 114.59, 112.26, 44.00, 37.38, 32.69, 30.64, 29.82, 27.11, 24.68, 22.41, 20.60; IR (KBr,
cm-1): 3270.3, 3192.3, 3055.6, 2967.9, 2930.1 (C-H), 2547.8, 2491, 1642.5, 1609.6, 1563.6,
1504.1, 1462.2, 855.2, 830.5, 754.8; MS: [M+H]+ m/z calcd 653.3234 for C40H40N6O3, found
653.3237.

4.1.5.4.
4-(3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl)-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)h
eptyl)benzamide (TDd) Yellow solid; 57% yield; mp 117.1-118.5 °C. 1H NMR (300 MHz, CDCl3)
δ: 1.26-1.36 (m, 6H), 1.56-1.64 (m, 4H), 1.89 (s, 4H), 2.69 (s, 4H), 2.99 (s, 2H), 3.40-3.51 (m,
4H), 4.00 (s, 1H), 6.35 (s, 1H), 6.70 (t, J = 7.65 Hz, 1H), 7.00-7.08 (m, 4H), 7.29-7.39 (m, 3H),
7.49-7.59 (m, 3H), 7.80 (d, J = 8.10 Hz, 1H), 7.94 (t, J = 8.10 Hz, 3H), 8.14 (d, J = 7.74 Hz, 1H);
13C NMR (75 MHz, DMSO-d6) δ: 169.97, 163.73, 160.55, 157.92, 157.75, 154.14, 150.46, 148.26,
144.28, 132.73, 132.38, 131.58, 130.14, 129.75, 128.87, 128.03, 126.80, 126.59, 124.96, 124.30,
122.11, 120.88, 120.30, 117.82, 117.27, 116.23, 114.15, 111.44, 45.10, 36.36, 32.93, 31.05, 29.55,
28.49, 26.91, 26.75, 24.78, 23.07, 21.32; IR (KBr, cm-1): 3352.1, 3271.1, 3060.9, 2926, 2853.7,
2488, 1920, 1738.4, 1608.9, 1581.2, 1504.5, 1463.2, 854.7, 830.7, 752.4; MS: [M+H]+ m/z calcd
667.3391 for C41H42N6O3, found 667.3395.
4.1.5.5.
4-(3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl)-N-(8-((1,2,3,4-tetrahydroacridin-9-yl)amino)o
ctyl)benzamide (TDe) Yellow solid; 60% yield; mp 112.7-113.5 °C; 1H NMR (300 MHz, CDCl3) δ:
1.06 (t, J = 7.11 Hz, 6H), 1.57-1.66 (m, 5H), 1.89 (s, 4H), 2.54-2.61 (m, 3H), 2.69 (s, 2H), 3.00 (s,
2H), 3.43-3.50 (m, 4H), 4.02 (s, 1H), 6.39 (s, 1H), 6.70 (t, J = 7.59 Hz, 1H), 7.00-7.08 (m, 4H),
7.33-7.7.39 (m, 3H), 7.49-7.59 (m, 3H), 7.80 (d, J = 8.34 Hz, 1H), 7.94 (d, J = 8.07 Hz, 3H), 8.14
(d, J = 7.65 Hz, 1H); 13C NMR (75 MHz, DMSO-d6) δ: 169.10, 162.65, 158.57, 157.68, 157.61,
154.33, 151.01, 148.62, 145.23, 132.47, 132.33, 131.58, 129.92, 129.53, 128.87, 128.25, 125.45,
124.12, 123.07, 122.89, 119.82, 117.62, 117.13, 115.55, 114.06, 111.64, 44.22, 37.17, 32.69, 30.64,
29.82, 27.47, 26.91, 24.68, 22.94, 22.34; IR (KBr, cm-1): 3276.2, 3190.9, 3057.9, 2929.8, 2855.9,
2547.3, 1913.5, 1830.8, 1609.9, 1504.2, 1462.5, 855.2, 830.6, 754.9; MS: [M+H]+ m/z calcd
681.3547 for C42H44N6O3, found 681.3577.
4.2. Biological evaluation
4.2.1. Metal binding studies
The study of metal chelation was performed in methanol at 298 K using UV-vis
spectrophotometer (Shanghai Csoif Co., Ltd) with wavelength ranging from 200 to 500 nm. To
investigate whether DBP, TDe and DEF could chelate metals, the UV absorption tested in the
absence or presence of metal ion. Stock solutions of DBP (2.5 mM), DEF (2.5 mM), TAC (2 mM)
and TDe (1.5 mM) were eventually diluted 50 times with methanol. Metal salt solutions of FeCl3
(10 mM) and CuCl2 (10 mM) were prepared in water. The solutions were incubated for 1 h with
0.5 equiv of Fe3+, 1 equiv of Cu2+ or 2 mM H2O2 before UV-Vis spectra were recorded.
4.2.2. Inhibition experiments of bAChE
AChE activities were measured by ELISA method with using AChE from bovine serum
(bAChE) [43]. Double antibody sandwich assay used to determine the activities of bAChE by the
microplate was coated with purified bAchE antibody to prepare a solid phase antibody, and AchE
was sequentially added to the microplate, followed by HRP-labeled AChE. Combines to form an
antibody-antigen-enzyme-labeled antibody complex, which is thoroughly washed and TMB was
added and turned from blue to yellow under the action of acid. Follow the cattle (AChE) ELISA

kit instructions (Shanghai Du Yi biology Co., Ltd.), on the enzyme label coating plate, 40 μL of
biological reference preparation (120 U/L, 80 U/L, 40 U/L, 20 U/L and 10 U/L) was incubated
with different test compounds (10 μL) at 37 °C for 30 min followed by the enzyme standard
reagent (50 μL) and the absorbance was measured at a wavelength of 450 nm in 15 min. The
concentration of compound producing 50% of enzyme activity inhibition (IC50) was calculated by
linear regression analysis using the Origin 8.0 program package. Tacrine were purchased from
Shanghai Zhen Zhi Biological Technology Co., Ltd. positive drug and all samples were assayed in
triplicate.
4.2.3. Molecular modeling study
Molecular modeling calculations and docking studies were performed using Schrodinger's
software Glide-Docking. The X-ray crystallographic structures of Torpedo californica AChE
complexed with bis (5)-tacrine (PDB code 2CMF) was obtained from the Protein Data Bank [35].
All water molecules in PDB files were removed and hydrogen atoms were subsequently added to
the protein. The compound TDa was built and performed geometry optimization by molecular
mechanics. Then, the TDa was docked into the active site of the protein by using Glide 6.7
program with default parameters [44, 45]. Docking accuracy is Standard Precision (SP) and the
dock scoring in Glide software was done using Glide score function. After docking, the geometry
of resulting complex was studied using the Glide’s pose viewer.
4.2.4. Effect on H2O2-induced oxidative cell damage in PC12 cells
The rat pheochromocytoma PC12 cells were routinely grown at 37 °C in a humidified
incubator with 5% CO2 and were plated at a density of 3×103 cells/well on 96-well plates in 100
μL of DMEM. Compounds TDa, TDb, TDc, TDe and TDe were dissolved with 150 μL DMSO first,
and then diluted with PBS. The cells were pre-incubated with compounds for 24 h before H2O2
(150 μM) was added. The cells were treated without H2O2 for 3 h, and cell viability was
determined after replaced with fresh DMEM medium. The cells were treated with 50 μL MTT for
4 h at 37 °C. The absorption was measured by a well plate reader at 490 nm.
4.2.5. DPPH scavenging activity
DPPH radical scavenger solution in methanol showed a maximum absorption wavelength at
517 nm (Fig. S1). The test compound was formulated into10-1, 10-2, 10-3, 10-4, 10-5 and 10-6 M
solution, then 2.5 mL of DPPH solution was added separately. The mixture treated for 30 min at
room temperature, the absorbance of the solution with different concentrations at 517 nm was
obtained. The antioxidant activity was computed according to equation shown below:
AA%  ADPPH  Asample / ADPPH
AA% indicates the antioxidant activity of a compound, ADPPH indicates the absorbance of
pure DPPH methanol solution, and Asample indicates the absorbance of a tested compound.
4.2.6. In vitro blood–brain barrier permeation prediction
To evaluate the potential for these hybrids to cross the BBB, we predict CLogP values by
ChemDraw 17.0. The CLogP values of compound was studied in the chemical properties window.

Acknowledgments
This work was supported by Jiangsu Natural Science Foundation (BK20161438), the
Southeast University's Basic Research Business Fee Innovation Foundation (3207048416).
References
[1] H. Tang, H. T. Zhao, S. M. Zhong, Z. Y. Wang, Z. F. Chen, H. Liang, Novel oxoisoaporphine-based inhibitors of acetyl-and
butyrylcholinesterase and acetylcholinesterase-induced β-amyloid aggregation, Bioorganic & medicinal chemistry letters 22(6)
(2012) 2257-2261.
[2] Y. Rook, K. U. Schmidtke, F. Gaube, D. Schepmann, B. Wuensch, J. Heilmann, J. Lehmann, T. Winckler, Bivalent
β-Carbolines as Potential Multitarget Anti-Alzheimer Agents, Journal of Medicinal Chemistry 53(9) (2010) 3611-3617.
[3] K. Bettens, K. Sleegers, C. Van Broeckhoven, Current status on Alzheimer disease molecular genetics: from past, to present,
to future, Human molecular genetics 19(R1) (2010) R4-R11.
[4] A. Wimo, L. Jonsson, J. Bond, M. Prince, B. Winblad, I. Alzheimer Dis, The worldwide economic impact of dementia 2010,
Alzheimers & Dementia 9(1) (2013) 1-11.
[5] E. D. Roberson, L. Mucke, 100 years and counting: prospects for defeating Alzheimer's disease, science 314(5800) (2006)
781-784.
[6] C. X. Gong, K. Iqbal, Hyperphosphorylation of microtubule-associated protein tau: a promising therapeutic target for
Alzheimer disease, Current medicinal chemistry 15(23) (2008) 2321-2328.
[7] F. M. LaFerla, K. N. Green, S. Oddo, Intracellular amyloid-β in Alzheimer's disease, Nat. Rev. Neurosci. 8(7) (2007)
499-509.
[8] E. Scarpini, P. Scheltens, H. Feldman, Treatment of Alzheimer's disease: current status and new perspectives, Lancet Neurol.
2(9) (2003) 539-547.
[9] Q. Xie, H. Wang, Z. Xia, M. Lu, W. Zhang, X. Wang, W. Fu, Y. Tang, W. Sheng, W. Li, W. Zhou, X. Zhu, Z. Qiu, H. Chen,
Bis-(-)-nor-meptazinols as novel nanomolar cholinesterase inhibitors with high inhibitory potency on amyloid-β aggregation,
Journal of Medicinal Chemistry 51(7) (2008) 2027-2036.
[10] J. S. Lan, J. W. Hou, Y. Liu, Y. Ding, Y. Zhang, L. Li, T. Zhang, Design, synthesis and evaluation of novel cinnamic acid
derivatives bearing N-benzyl pyridinium moiety as multifunctional cholinesterase inhibitors for Alzheimer's disease, Journal
of Enzyme Inhibition and Medicinal Chemistry 32(1) (2017) 776-788.
[11] D. Munoz-Torrero, Acetylcholinesterase inhibitors as disease-modifying therapies for Alzheimer's disease, Current
Medicinal Chemistry 15(24) (2008).
[12] N. Herrmann, S. A. Chau, I. Kircanski, K. L. Lanctot, Current and Emerging Drug Treatment Options for Alzheimer's
Disease A Systematic Review, Drugs 71(15) (2011) 2031-2065.
[13] D. Wilkinson, Y. Wirth, C. Goebel, Memantine in Patients with Moderate to Severe Alzheimer's Disease: Meta-Analyses
Using Realistic Definitions of Response, Dementia and Geriatric Cognitive Disorders 37(1-2) (2014) 71-85.
[14] L. D. Estrada, C. Soto, Disrupting β-amyloid aggregation for Alzheimer disease treatment, Current Topics in Medicinal
Chemistry 7(1) (2007) 115-126.
[15] R. L. Ildefonso, C. G. A. Laura, E. J. C. Maria, Protein misfolding and neurodegeneration, Rev. Mex. Neurocienc. 16(1)
(2015) 51-72.
[16] S. Paul, S. Planque, Y. Nishiyama, Beneficial Catalytic Immunity to Aβ Peptide, Rejuvenation Research 13(2-3) (2010)
179-187.
[17] M. Isabel Fernandez-Bachiller, C. Perez, G.C. Gonzalez-Munoz, S. Conde, M.G. Lopez, M. Villarroya, A.G. Garcia, M.
Isabel Rodriguez-Franco, Novel Tacrine-8-Hydroxyquinoline Hybrids as Multifunctional Agents for the Treatment of
Alzheimer's Disease, with Neuroprotective, Cholinergic, Antioxidant, and Copper-Complexing Properties, Journal of

Medicinal Chemistry 53(13) (2010) 4927-4937.
[18] A. Rauk, Why is the amyloid β peptide of Alzheimer's disease neurotoxic?, Dalton Transactions (10) (2008) 1273-1282.
[19] G. J. M. Bruin, T. Faller, H. Wiegand, A. Schweitzer, H. Nick, J. Schneider, K.O. Boernsen, F. Waldmeier, Pharmacokinetics,
Distribution, Metabolism, and Excretion of Deferasirox and Its Iron Complex in Rats, Drug Metab. Dispos. 36(12) (2008)
2523-2538.
[20] J. L. Liu, Y. G. Fan, Z. S. Yang, Z. Y. Wang, C. Guo, Iron and Alzheimer's Disease: From Pathogenesis to Therapeutic
Implications, Frontiers in Neuroscience 12 (2018).
[21] G. Kamalinia, F. Khodagholi, F. Atyabi, M. Amini, F. Shaerzadeh, M. Sharifzadeh, R. Dinarvand, Enhanced Brain Delivery
of Deferasirox-Lactoferrin Conjugates for Iron Chelation Therapy in Neurodegenerative Disorders: In Vitro and in Vivo
Studies, Molecular Pharmaceutics 10(12) (2013) 4418-4431.
[22] S. Y. Li, N. Jiang, S. S. Xie, K. D. G. Wang, X. B. Wang, L. Y. Kong, Design, synthesis and evaluation of novel tacrine-rhein
hybrids as multifunctional agents for the treatment of Alzheimer's disease, Organic & Biomolecular Chemistry 12(5) (2014)
801-814.
[23] P. B. Watkins, H. J. Zimmerman, M. J. Knapp, S. I. Gracon, K. W. Lewis, Hepatotoxic effects of tacrine administration in
patients with Alzheimer's disease, Jama 271(13) (1994) 992-998.
[24] M. I. Fernandez-Bachiller, C. Perez, L. Monjas, J. Rademann, M.I. Rodriguez-Franco, New Tacrine-4-Oxo-4H-chromene
Hybrids as Multifunctional Agents for the Treatment of Alzheimer's Disease, with Cholinergic, Antioxidant, and
β-Amyloid-Reducing Properties, Journal of Medicinal Chemistry 55(3) (2012) 1303-1317.
[25] X. Chen, K. Zenger, A. Lupp, B. Kling, J. Heilmann, C. Fleck, B. Kraus, M. Decker, Tacrine-Silibinin Codrug Shows
Neuro- and Hepato protective Effects in Vitro and Pro-Cognitive and Hepatoprotective Effects in Vivo, Journal of Medicinal
Chemistry 55(11) (2012) 5231-5242.
[26] K. Chand, Rajeshwari, E. Candeias, S.M. Cardoso, S. Chaves, M. Amelia Santos, Tacrine-deferiprone hybrids as
multi-target-directed metal chelators against Alzheimer's disease: a two-in-one drug, Metallomics 10(10) (2018) 1460-1475.
[27] M.A. Santos, K. Chand, S. Chaves, Recent progress in multifunctional metal chelators as potential drugs for Alzheimer's
disease, Coordination Chemistry Reviews 327 (2016) 287-303.
[28] M. M. Khodaei, E. Nazari, Tf2O-Mediated Direct and Regiospecific para-Acylation of Phenols with Carboxylic Acids, Bull.
Korean Chem. Soc. 32(5) (2011) 1784-1786.
[29] Y. F. Han, C. P. L. Li, E. Chow, H. Wang, Y. P. Pang, P. R. Carlier, Dual-site binding of bivalent 4-aminopyridine- and
4-aminoquinoline-based AChE inhibitors: Contribution of the hydrophobic alkylene tether to monomer and dimer affinities,
Bioorganic & Medicinal Chemistry 7(11) (1999) 2569-2575.
[30] W. F. Patton, J. Coleman, Y. Xiang, P. Pande, Z. Li, Autophagy and phospholipidosis pathway assays, Google Patents, 2016.
[31] M. G. Dickens, K. J. Franz, A Prochelator Activated by Hydrogen Peroxide Prevents Metal-Induced Amyloid β Aggregation,
Chembiochem 11(1) (2010) 59-62.
[32] U. Heinz, K. Hegetschweiler, P. Acklin, B. Faller, R. Lattmann, H.P. Schnebli, 4- 3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl
benzoic acid: A novel efficient and selective iron(III) complexing agent, Angewandte Chemie-International Edition 38(17)
(1999) 2568-2570.
[33] E. Engvall, P. Perlmann, Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G,
Immunochemistry 8(9) (1971) 871-874.
[34] B.P. Doctor, T.C. Chapman, C.E. Christner, C.D. Deal, D.M.D.L. Hoz, M.K. Gentry, R.A. Ogert, R.S. Rush, K.K. Smyth,
A.D. Wolfe, Complete amino acid sequence of fetal bovine serum acetylcholinesterase and its comparison in various regions
with other cholinesterases, Febs Letters 266(1) (1990) 123-127.
[35] E. H. Rydberg, B. Brumshtein, H. M. Greenblatt, D. M. Wong, D. Shaya, L. D. Williams, P. R. Carlier, Y. P. Pang, I. Silman,
J. L. Sussman, Complexes of alkylene-linked tacrine dimers with Torpedo californica acetylcholinesterase: Binding of
bis(5)-tacrine produces a dramatic rearrangement in the active-site gorge, Journal of Medicinal Chemistry 49(18) (2006)

5491-5500.
[36] J. S. Lan, S. S. Xie, S. Y. Li, L. F. Pan, X. B. Wang, L. Y. Kong, Design, synthesis and evaluation of novel
tacrine-(β-carboline) hybrids as multifunctional agents for the treatment of Alzheimer’s disease, Bioorganic & medicinal
chemistry 22(21) (2014) 6089-6104.
[37] B. C. Magliaro, C. J. Saldanha, Clozapine protects PC-12 cells from death due to oxidative stress induced by hydrogen
peroxide via a cell-type specific mechanism involving inhibition of extracellular signal-regulated kinase phosphorylation,
Brain Research 1283 (2009) 14-24.
[38] L. A. Greene, A. S. Tischler, Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which
respond to nerve growth factor, Proceedings of the National Academy of Sciences 73(7) (1976) 2424-2428.
[39] M. Isabel Fernandez-Bachiller, C. Perez, N. Eugenia Campillo, J. Antonio Paez, G. Cristina Gonzalez-Munoz, P. Usan, E.
Garcia-Palomero, M. G. Lopez, M. Villarroya, A. G. Garcia, A. Martinez, M. Isabel Rodriguez-Franco, Tacrine-Melatonin
Hybrids as Multifunctional Agents for Alzheimer's Disease, with Cholinergic, Antioxidant, and Neuroprotective Properties,
Chemmedchem 4(5) (2009) 828-841.
[40] M. S. J. N. Blois, Antioxidant determinations by the use of a stable free radical, Nature 181(4617) (1958) 1199.
[41] C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Experimental and computational approaches to estimate solubility
and permeability in drug discovery and development settings, Adv. Drug Deliv. Rev. 64 (2012) 4-17.
[42] S. Steinhauser, U. Heinz, J. Sander, K. Hegetschweiler, Complex formation of 2,6-bis-(2 '-hydroxyphenyl)pyridine with
Al-III, Fe-III and Cu-II, Zeitschrift Fur Anorganische Und Allgemeine Chemie 630(12) (2004) 1829-1838.
[43] E. Engvall, K. Jonsson, P. Perlmann, Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen,
immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes, Biochimica et Biophysica Acta
(BBA)-Protein Structure 251(3) (1971) 427-434.
[44] Glide, Version 6.7, Schrödinger, LLC, New York, NY, 2015.
[45] R.A. Friesner, J.L. Banks, R.B. Murphy, T.A. Halgren, J.J. Klicic, D.T. Mainz, M.P. Repasky, E.H. Knoll, M. Shelley, J.K.
Perry, D.E. Shaw, P. Francis, P.S. Shenkin, Glide: A new approach for rapid, accurate docking and scoring. 1. Method and
assessment of docking accuracy, Journal of Medicinal Chemistry 47(7) (2004) 1739-1749.

Structure of TD compound and docking models of the TDa in TcAChE
(PDB code: 2CMF).

Highlights
 Synthesis of a new series of metal chelators as multifunctional agents for the
treatment of Alzheimer’s disease.
 Docking simulations were applied the active compound.
 Cytotoxicity evaluation on H2O2-induced oxidative cell damage in PC12.
 The compounds demonstrated significant metal chelating property.
 TD compounds indicated their moderate acetylcholinesterase inhibitory
activity.