Investigation of multi-target-directed ligands (MTDLs) with butyrylcholinesterase (BuChE) and indoleamine 2,3-dioxygenase 1 (IDO1) inhibition: The design, synthesis of miconazole analogues targeting Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2018.02.014

In our endeavor towards the development of potent multi-target ligands for the treatment of Alzheimer's disease, miconazole was identified to show BuChE-IDO1 dual-target inhibitory effects. Morris water maze test indicated that miconazole obviously ameliorated the cognitive function impaired by scopolamine. Furthermore, it showed good safety in primary hepatotoxicity evaluation. Based on these results, we designed, synthesized, and evaluated a series of miconazole derivatives as BuChE-IDO1 dual-target inhibitors. Out of the 12 compounds, 5i and 5j exhibited the best potency in enzymatic evaluation, thus were selected for subsequent behavioral study, in which the two compounds exerted much improved effect than tacrine. Meanwhile, 5i and 5j displayed no apparent hepatotoxicity. The results suggest that miconazole analogue offers an attractive starting point for further development of new BuChE-IDO1 dual-target inhibitors against Alzheimer's disease.

Full text of DOI:10.1016/j.bmc.2018.02.014

Accepted Manuscript
Investigation of multi-target-directed ligands (MTDLs) with butyrylcholines-
terase (BuChE) and indoleamine 2,3-dioxygenase 1 (IDO1) inhibition: the
design, synthesis of miconazole analogues targeting Alzheimer’s disease
Xin Lu, Si-yu He, Qi Li, Hongyu Yang, Xueyang Jiang, Hongzhi Lin, Yao Chen,
Wei Qu, Feng Feng, Yaoyao Bian, You Zhou, Haopeng Sun
PII:
S0968-0896(17)32257-5
https://doi.org/10.1016/j.bmc.2018.02.014
BMC 14200
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 November 2017
3 February 2018
12 February 2018
Please cite this article as: Lu, X., He, S-y., Li, Q., Yang, H., Jiang, X., Lin, H., Chen, Y., Qu, W., Feng, F., Bian,
Y., Zhou, Y., Sun, H., Investigation of multi-target-directed ligands (MTDLs) with butyrylcholinesterase (BuChE)
and indoleamine 2,3-dioxygenase 1 (IDO1) inhibition: the design, synthesis of miconazole analogues targeting
Alzheimer’s disease, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.02.014
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Investigation of multi-target-directed ligands (MTDLs) with
butyrylcholinesterase (BuChE) and indoleamine 2,3-dioxygenase 1
(IDO1) inhibition: the design, synthesis of miconazole analogues
targeting Alzheimer’s disease
a
Xin Lu a, Si-yu He a, Qi Li , Hongyu Yang a, Xueyang Jiang b, Hongzhi Lin a, Yao
Chen c, Wei Qu b, Feng Feng b, Yaoyao Bian d, You Zhou e,*, Haopeng Sun a,*
a
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing,
210009, China;
b
Key Laboratory of Biomedical Functional Materials, School of Science, China
Pharmaceutical University, Nanjing 211198, China
c
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023,
China;
d School of Nursing, Nanjing University of Chinese Medicine, Nanjing, 210023, China
e College of Biotechnology, Southwest University, Chongqing, 400715, China
* Corresponding author
sunhaopeng@163.com (Haopeng Sun), Tel: 86-25-13951934235 ;
zhouy701005@swu.edu.cn (You Zhou), Tel: 86-25-15123233780 ;
Abstract : In our endeavor towards the development of potent multi-target ligands for
the treatment of Alzheimer's disease, miconazole was identified to show
BuChE-IDO1 dual-target inhibitory effects. Morris water maze test indicated that
miconazole obviously ameliorated the cognitive function impaired by scopolamine.
Furthermore, it showed good safety in primary hepatotoxicity evaluation. Based on
1

these results, we designed, synthesized, and evaluated a series of miconazole
derivatives as BuChE-IDO1 dual-target inhibitors. Out of the 12 compounds, 5i and
5j exhibited the best potency in enzymatic evaluation, thus were selected for
subsequent behavioral study, in which the two compounds exerted much improved
effect than tacrine. Meanwhile, 5i and 5j displayed no apparent hepatotoxicity. The
results suggest that miconazole analogue offers an attractive starting point for further
development of new BuChE-IDO1 dual-target inhibitors against Alzheimer’s disease.
Keywords Alzheimer's disease；Miconazole derivatives; Multifunctional agents;
Butyrylcholinesterase (BuChE); Indoleamine 2,3-dioxygenase 1 (IDO1)
1. Intriduction
Alzheimer’s disease (AD), characterized by a decline in memory or other
cognitive skills, is currently prevailing cause of dementia in the elderly.[1] Currently
approved AD treatments only provide temporary and modest improvement in
cognitive functions through cholinergic and anti-glutamatergic mechanisms, but they
cannot significantly modify the course and reverse the progress of the disease.[2, 3]
Thus, it is urgent to provide disease-modifying therapies to fight the upcoming AD
epidemic.[4]
Given that the complexity nature of AD and the involvement of multi-systems in
its progression, traditional drugs that only act on a specific target to produce the
desired clinical effects might not be valid.[5] Thus, multi-target-directed ligands
(MTDLs) strategy that incorporates several pharmacological effects into a single
molecular entity is more likely to achieve desired effects and has been rationalized in
the field of neurodegenerative diseases.[6-9] Many studies have been devoted to the
search for multifunctional agents concerning different systems such as cholinesterases,
secretases, monoamine oxidase-A/B (MAO-A/B), cannabinoid receptor type 1/2
(CB1/2), H3 receptors, Tau, glycogen synthase kinase 3ꢀ (GSK3ꢀ), protein involved
oxidative stress etc.[10-14]
Cholinesterases (ChEs), including acetylcholinesterase (AChE, EC 3.1.1.7) and
butyrylcholinesterase (BuChE, EC 3.1.1.8), play important roles in cholinergic
transmission by hydrolyzing the neurotransmitter acetylcholine (ACh).[15] Though
2

AChE inhibitors (AChEIs) have been investigated strongly by many researchers, the
therapeutic effect of AChEIs is limited, while peripheral side effects also hinder the
long-term application in clinical trials.[16] With the course of AD development, the
AChE level in the brain decreases progressively, while BuChE level remains the same
or even increases up to 165% of the normal levels.[17] Moreover, BuChE undertakes
the hydrolysis of ACh in the AChE deficient brain in the advanced stage. Therefore,
BuChE is a proposed drug target for treatment of the advanced stage of AD.[18]
Selective BuChE inhibition is potentially advantageous because it may circumvent the
classical cholinergic toxicity.[19] Several lines of evidence also indicate that an
increased BuChE activity plays an important role in Aꢀ aggregation during the early
stages of senile plaque formation.[20] Likewise, selective BuChE inhibitors have been
demonstrated to reduce ꢀ-amyloid precursor (APP) secretion and ꢀ-amyloid (Aꢀ) in
vitro and in vivo.[21] Up to date, no specific BuChE inhibitors have been approved
for AD treatment. The discovery of safe, effective and specific BuChE inhibitors that
are capable to ameliorate the symptoms of AD with much reduced peripheral side
effects represents a significant advancement.[22]
Over the past two decades, neuroinflammation has emerged as an integral
process in the pathogenesis of AD. [23, 24] In the brains of AD patients, an increased
amount of activated microglia and astrocytes as well as significantly higher levels of
tumor necrosis factor-alpha (TNF-ꢁ) and proinflammatory cytokines, namely
interleukin-1 (IL-1), interleukin-6 (IL-6), have been observed and are currently
considered as an early and continuous features of the AD brain.[25, 26] While the
mechanism underlying neuroinflammation-induced AD pathology remains not fully
elucidated, increasing evidences suggest that the kynurenine pathway (KP) may play a
crucial role.[27] Studies have shown that the level of tryptophan of AD patients in
serum is lower than normal while the concentration of kynurenine is higher.
Furthermore, the ratio of kynurenine/tryptophan concentration is negatively correlated
with cognitive ability.[28] Indoleamine 2,3-dioxygenase 1 (IDO1), the rate-limiting
enzyme of KP, is driven by proinflammatory cytokines.[29] The activation of IDO1 is
3

considered as a pathogenic factor of amyloid-beta (Aꢀ)-related inflammation in
AD.[30] IDO1 and quinoline acid (QUIN) which is regarded as a neurotoxin express
the highest at microglia and astrocytes in the hippocampus of AD patients, namely
they specifically co-localized with pathological hallmarks of AD, such as senile
plaques and neurofibrillary tangles.[31, 32] Thus, the role of IDO1 in the pathogenesis
of AD is mainly focused on neuronal inflammation and activation of glial cells.
Moreover, several studies have revealed that the induction of IDO1 is related to
the down-regulation of brain-derived neurotrophic factor (BNDF) in the prefrontal
cortex and hippocampus in a mouse model of inflammation-associated depression.[26,
33] Researchers have reported that the expression of many neurotrophic factors are
markedly reduced in the brain of AD patients and AD mouse models.[34]
Neurotrophic factors are important for driving neurogenesis, synaptic plasticity and
neuronal survival, which are all processes affected by the pathogenesis of AD.[35, 36]
Therefore, a neurotrophic deficiency could be an additional mechanism implicated
with IDO1 activation by pro-inflammatory cytokines in AD brain.[30] That effectively
inhibiting the over-activation of IDO1 can prevent the accumulation of harmful
intermediates in KP, thereby reducing the overall oxidative stress, cytotoxicity, tau
protein phosphorylation, is expected to become a new strategy for the treatment of AD.
Based on the results mentioned above, we hypothesize that a compound with dual
BuChE-IDO1 inhibitory activity would benefit the treatment of AD, especially the
progressed AD. Our previous studies have identified that miconazole can act as a
potential ChEs inhibitor (slightly more potent on BuChE).[37] Moreover, Rohrig et al.
discovered miconazole as a potential IDO1 inhibitor.[38] Miconazole was first
approved by FDA in 1993 as an antifungal agent. As an approved drug, it possesses
acceptable drug-like property and safety. Inspired by the “old drug new use” strategy,
we proposed to identify new BuChE-IDO1 dual inhibitors starting from miconazole.
Herein, twelve miconazole analogues were designed, synthesized. Their inhibitory
activities on both BuChE and IDO1 were in vitro evaluated. More importantly,
miconazole and its derivatives 5i and 5j remarkably ameliorated the cognitive
4

disorder in the Morris water maze test in vivo, which provides important supportive
data to our hypothesis. As far as our concern, it is the first time to report the activity of
miconazole and its derivatives as potential anti-AD agents. Additionally, it is also the
first attempt to design BuChE-IDO1 dual inhibitors, which can enlarge the
understanding of new therapeutic methods on AD treatment, thus providing a new
example for “old drug new use” strategy.
2. Results and discussion
2.1. Chemistry
The synthesis of miconazole analogues were systematically represented in
Scheme 1, in which 1-(2,4-dichlorophenyl)ethan-1-one (1) was used as the starting
material. ꢁ-Bromination of 1 using Br2 afforded 2.[39] N-Alkylation of imidazole
with 2 in the presence of K2CO3 in tetrahydrofuran (THF) at room temperature (RT)
resulted in the intermediates 3, which was then reacted with sodium borohydride in
refluxing methanol to give 4.[40] In sequence, the intermediate 4 was reacted with the
corresponding benzyl chloride or benzyl bromide in the presence of NaH in
N,N-Dimethylformamide (DMF) at RT to obtain the final miconazole derivatives 5a ~
5l.[41] All the target compounds were purified by chromatography and structurally
confirmed by spectroscopic data as described in experimental section.
Scheme 1. Synthetic route of miconazole analogues 5a ~ 5l. Regents and conditions:
(i) Br2, CHCl3, 0 then RT; (ii) imidazole, K2CO3, THF, RT; (iii) NaBH4, MeOH, 0
then reflux; (iv) NaH/DMF, substituted benzyl chloride or benzyl bromide, 0 then
RT.
5

2.2. Pharmacology
2.2.1 Evaluation the activity and cytotoxicity of miconazole in vivo
Though miconazole has been verified to be an inhibitor of BuChE/IDO1,
whether it possesses the potential to treat AD is not clear. We first examined it by
Morris water maze test following the previous method.[42] Tacrine (15 mg/kg) was
used as a positive control. As shown in figure 1A, miconazole revealed a
dose-dependent effect on learning behavior. The medium dose group exhibited equal
effect vs. tacrine group (p > 0.05) while the high dose group is much potent in
recovering the cognitive disorder in the tested mice (p < 0.0001). However the low
dose group did not show apparent improvement of learning behavior. From the
trajectories of the mice (figure 1B), it could be easily observed that the mice in high
dose and medium dose groups escaped more directly with a shorter distance to target.
These results exhibited that miconazole indeed ameliorated the cognitive function
impaired by scopolamine, thus inspired us to evaluate the safety of miconazole in vivo.
The hepatotoxicity studies were evaluated according to the method previously
described.[43] After being treated with tacrine, three different doses of miconazole
(10 mg/kg, 50 mg/kg, and 100 mg/kg), the heparinized serum of the mice was
obtained after different time points (8h, 22h, and 36h) and the levels of aspartate
transaminase (AST) and alanine aminotransferase (ALT) were determined (Figure S1).
In comparison to the control, none of these groups caused hepatotoxicity, as indicated
by the activity of AST and ALT. Even at the dose of 100 mg/kg, miconazole still
showed good safety. Based on these results, we thought that miconazole was a
BuChE-IDO1 dual-target inhibitor exerting therapeutic effect on AD. Subsequent
modifications and structure-activity relationship (SAR) studies were performed in an
attempt to discover more potent compounds.
6

Figure 1. (A) Effects of oral administration of tacrine(15 mg/kg), miconazole with
three dose: low dose (10 mg/kg), medium dose (50 mg/kg) and high dose (100 mg/kg)
on scopolamine-induced cognitive impairment in ICR mice determined by Morris
water maze test. Data are presented as the mean SEM (n=6; ns. = no significance,
*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs tacrine group). (B) The
trajectories of mice in control (a), model (b), tacrine (c), high dose (d), medium dose
(e) and low dose (f) group in the Morris water maze test. Low dose: 10 mg/kg,
medium dose: 50 mg/kg, high dose: 100 mg/kg.
2.2.2. Evaluation of target compounds on the activity of ChEs and IDO1
To study the dual-target profile of the miconazole derivatives, target compounds
were first evaluated on ChEs. The inhibitory activities of 5a ~ 5l against AChE and
BuChE were measured according to a modified Ellman assay.[44] The potency data of
the compounds were listed in table 1, expressed as IC50 values. Compared with
miconazole, the analogues showed IC50 values on BuChE ranging from 8.25 to 46.32
µM (table 1). Moreover, they exhibited less potency against AChE, as IC50 values
7

ranging from 17.32 to 176.2 µM. Most of target compounds displayed selectivity on
BuChE inhibition potency (the ratio IC50 (AChE)/IC50 (BuChE) varied from 1.29 to
9.85), except compound 5d. Among these 5j and 5k showed the best selectivity (SI =
9.9 and 9.2, respectively). For SAR on ChEs, electron-donating groups improved the
potency better than electron withdrawing groups. The synthesized derivatives were
tested in vitro for their IDO1 inhibitory activities using miconazole as the reference
drug. The inhibitory activities were determined by a fluorimetric method with
kynuramine as a common substrate and recombinant human IDO1 as the enzyme
sources.[45] As shown in table 1, three synthesized target compounds (5b, 5f, 5i)
displayed higher inhibitory activities than miconazole. Both spatial and electronic
factors contributed to IDO1 inhibitory. 2,6-di-F substituent group (5k) showed poor
activities on both targets. We inferred that the F atom not only reduced the electron
cloud density of benzene ring, but also failed to form extra contacts with surrounding
amino acid residues. Among all the target compounds, 5l exhibited the best inhibitory
potency on IDO1 (IC50 = 1.6
0.3 µM). Considering the impact of the
para-substituents (5a ~ 5e, 5i) on the IDO1 IC50 value, we found 4-OCH3 > 4-Cl >
4-CH3 > 4-F > 4-t-Bu > 4-Br, indicating that modification in this position was
tolerated.
Table 1. Inhibitory activities of AChE, BuChE and IDO1 by miconazole, 5a ~ 5l.
Values are expressed as mean SEM.
SI a
IDO1 IC50(µM)
Comp.
R
AChE IC50 (µM) BuChE IC50 (µM)
4-F
4-Cl
2.3
1.3
1.5
0.5
2.5
2.1
2.0
1.6
5a
5b
5c
5d
5e
5f
33.0 7.9
22.3 5.6
19.5 5.2
24.3 4.9
26.9 4.5
30.7 7.6
37.5 7.0
17.3 4.2
14.8 3.0
17.4 2.6
13.1 4.4
46.3 13.4
10.9 2.3
14.6 4.2
18.4 2.8
11.2 3.6
4.0 0.7
1.1 0.2
5.7 1.0
4.4 0.9
2.0 0.3
1.2 0.3
2.2 0.5
2.2 0.4
4-Br
4-t-Bu
4-CH3
3-CH3
2-CH3
3,4-di-CH3
5g
5h
8

4-OCH3
3-OCH3
2,6-di-F
H
4.4
9.9
9.2
4.1
3.1
5i
73.1 14.4
81.3 15.9
176.2 77.5
73.7 19.8
21.4 3.5
16.5 3.8
8.3 1.6
19.2 3.2
18.2 3.5
6.8 1.4
1.0 0.2
2.8 0.4
4.2 1.0
1.6 0.3
1.4 0.2
5j
5k
5l
2,4-di-Cl
Miconazole
a SI = AChE IC50 /BuChE IC50
2.2.3. Behavioral studies
To determine whether the treatment of 5i and 5j improve the spatial memory in
the scopolamine-induced mice, we performed the Morris water maze tests. The spatial
memory defects of the mice were significantly shown by a longer latency and distance
to the target (figure2 and figure 3). Subsequently, we found that the latency of mice
showed no significant differences between the treatment with tacrine group (15 mg/kg,
positive group, 25.9 s) and model group (40.5 s), while treatment with 5i (15 mg/kg,
13.4 s) and 5j (15 mg/kg, 19.7 s) showed a shorter latency (p < 0.001, p < 0.01
respectively) and observably reversed the cognitive deficit induced by scopolamine. It
indicated that 5i and 5j had better performance at the same dose than tacrine. Besides,
5i exhibited slightly better effect over 5j.
Figure 2. Effects of oral administration of tacrine (15 mg/kg), 5i and 5j (15 mg/kg) on
scopolamine-induced cognitive impairment in ICR mice determined by Morris water
maze test. Latency to targets (left) and distance to targets (right) were shown in the
figure. Data are presented as the mean SEM (n=6; *p < 0.05, **p < 0.01, ***p <
0.001, ****p < 0.0001 vs the model group; ns. = no significance).
9

Figure 3. The trajectories of mice in control (A), scopolamine-induced cognitive
model (B), tacrine (C), 5i (D), 5j (E) group in the Morris water maze test.
2.2.4 Hepatotoxicity test in vivo
In the present study, 5i and 5j were investigated by hepatotoxicity assay as well.
The detailed procedure was the same as hepatotoxicity test of miconazole described
above. The experiment were carried out by oral administration of 5i and 5j (15 mg/kg)
on mice to investigate the influence on ALT and AST. In comparison to the control,
both compounds possessed safety by evidently increasing of neither ALT nor AST at
8h, 22h and 36h (figure 4).
For histological studies, liver tissues taken from the animals were stained with
hematoxylin and eosin (HE). Paraffin sections were observed 30h after administration
of 5i (15 mg/kg) and 5j (15 mg/kg). Neither pericentral necrosis nor distinct fatty
degeneration of the hepatocytes of the surrounding intermediate and periportal zones
were discovered (figure 5), indicating that the compounds did not trigger acute
hepatoxicity.
10

Figure 4. Determination of ALT (left) and AST (right) in serum after the
administration of 5i (15 mg/kg)and 5j (15 mg/kg) on mice. Data are presented as the
mean SEM, n = 3.
Figure 5. Histomorphological appearance of livers of the mice, treatment with the
solvent only: control (A), model (B) or 30 h after administration of 5i (C), 5j (D).
2.2.5 Molecular docking
A molecular modeling study using CDOCKER within Discovery Studio (DS,
BioVIA) was performed to explore the binding modes of compound 5i and 5j with the
enzyme BuChE. The crystal structure of the highly selective BuChE inhibitor
11

N-((1-(2,3-dihydro-1H-inden-2-yl)piperidin-3-yl)methyl)-N-(2-methoxyethyl)-2-napt
hamide-BuChE complex was taken as a model. The proposed binding model of 5i and
5j with the key residues in the gorge site were shown in figure 6. Compounds 5i and
5j were found to form similar conformation and interactions in the cavity. They
located in outer space and were incapable to interact with key residues such as Tyr82.
Specially, 5i formed H-bond with Asp70 and contacted with Trp332 and Phe329
through ꢂ-ꢂ stacking interaction. Besides, it interacted with Gly116 and Ala328
through amide–ꢂstacking and ꢂ-alkyl interactions. Compound 5j formed H-bond with
Asn83 and contacted with Tyr332, Ala328, phe329 and His438 through ꢂ-ꢂ and
ꢂ-alkyl stacking. Both 5i and 5j formed amide-ꢂ interaction with Gly116 and ꢂ-ꢃ
interaction with Tyr332. It should be noted that the additional interactions with 5j may
be responsible for the better inhibitory than 5i.
For IDO1, the co-crystal structure of the NLG919-IDO1 complex (PDB id:
5EK3) was chosen for molecular docking. The proposed binding model of 5i and 5j
with the key residues in the cavity were shown in figure 7. It can be observed that the
nitrogen atom of the imidazole did not accord to ferrous ion of heme which was seen
as critical to IDO1 inhibitory. Besides, compounds 5i and 5j occupied two pockets of
IDO1and formed the same interactions with corresponding residues. The moiety of
methoxy substituted phenyl positioned in pocket A and interacted with Phe163
through ꢂ-ꢂ interaction while with Ala264 through ꢂ-alkyl stacking interaction.
Imidazole located in pocket B and interacted through ꢂ-alkyl stacking and ꢂ-ꢃ with
Arg231 and Leu384. The 2,4-dichlorophenyl pointed to the solvent area and only
established week interactions with Ser263, Phe291 and Ala376. The -CDOCKER
SCORE of compounds 5i and 5j were 35.0938 and 34.7362 respectively; and the
difference of score is corresponding to their activities.
To better analyze the binding mode, kinetics studies of miconazole, 5i and 5j
were performed. The results suggested that miconazole was a non-competitive BuChE
inhibitor (figure S2). The result was consistent with our previous study. However,
when we carried out the kinetics study for 5i and 5j (for both compounds, n > 6), we
12

did not get consistent results. About a half of the times, the kinetic studies indicated
an uncompetitive binding mode of 5i and 5j to BuChE (figure S3), while the rest of
the times we obtain an abnormal result that the intersect located in the first quadrant
of the lineweaver-burk plot (figure S4). These results surprised us, we therefore
speculated that the compounds can bind to BuChE in a different pattern compared to
miconazole. Maybe there existed two or more binding modes for the two compounds,
such as non-competitive or uncompetitive types, leading to the different outcomes of
the intermolecular interaction. In the following research, we will perform more
experiments to clarify the binding mode of the synthesized compounds reported in the
manuscript. Considering that the binding mode was still not fully elucidated, we
supplied the current data of the kinetics studies in supporting information as
references for the reviewers and the readers.
Figure 6. Binding mode prediction of 5i (A) and 5j (B) with BuChE (PDB id: 4TPK).
Compounds are shown in paleturquoise stick mode; key residues are shown in spring
green line modes. ꢂ-ꢂ stacking is depicted in orange dot line; amide-ꢂ stacking are in
blue dot line; ꢂ-alkyl is in pink dot line; ꢂ-ꢃ is in purple dot line and H-bond is in
green dot line.
13

Figure 7. Binding mode prediction of 5i (A) and 5j (B) with IDO1 (PDB id: 5EK3).
Compounds a shown in paleturquoise stick mode, key residues are shown in spring
green line modes and heme is in gery. ꢂ-ꢂ stacking is depicted in magenta dot line;
ꢂ-anion is in orange dot line; ꢂ-alkyl is in pink dot line; ꢂ-ꢃ is in red dot line; alkyl is
in purple dot line and H-bond is in blue dot line.
3. Conclusion
Drug repurposing is a rational and time-saving strategy in drug development.[46]
Prompted from this strategy, in the present study, we identified that miconazole,
which inhibited both BuChE and IDO1, exerted promising therapeutic effects on
cognitive impairment mice model was conducive in AD model mice. Considering the
important role of the two targets in the development of AD, herein we proposed a new
MTDLs design strategy that combining BuChE and IDO1 inhibitory effects in a
single molecule. Starting from miconazole, we synthetized and evaluated 12
derivatives. Compound 5i (BuChE IC50 =16.5 µM, SI = 4.4, IDO1 IC50 = 1.0 µM), 5j
(BuChE IC50 = 8.3µM, SI = 9.9, IDO1 IC50 = 2.8 µM) were the most potent
compounds. Behavior studies on mice by Morris water maze test demonstrated that 5i
and 5j possessed the abilities to ameliorate the behavioral impairment. Additionally,
5i and 5j did not show acute hepatotoxicity. In summary, the present study is the first
time to show the potential of miconazole derivatives as new therapeutic agents on AD,
it also indicates that designing BuChE-IDO1 dual-target inhibitors may provide a new
strategy for AD treatment.
4. Experimental section
14

4.1. Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers
1
and used without further purification. H and 13C NMR spectra were recorded using
TMS as the internal standard in CDCl3 or DMSO-d6 with a Bruker spectrometer at
300 MHz, respectively. Coupling constants were given in Hz. Multiplicities are given
as s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet), and br (broad
signal). MS spectra data were obtained on an Agilent-6210 LC-MS spectrometer. All
the reactions were monitored by thin-layer chromatography (TLC) using silica gel
GF254 plates from Qingdao Haiyang Chemical Co. Ltd. (China) with an UV lamp
(254 nm). Column chromatography using silica gel (200-300 mesh) purchased from
Qingdao Haiyang Chemical Co. Ltd. (China).
4.1.1 2-bromo-1-(2,4-dichlorophenyl)ethan-1-one (2)
To a cold solution of 1 (10 mmol) in chloroform (25 ml), bromine (12 mmol) in
chloroform (10 ml) was gradually added in 30 min with continuous stirring. The
temperature of the reaction mixture was maintained under 0 °C. After the addition
was complete, the reaction mixture was slowly heated to the room temperature, and
stirred for another 60 min until the evolution of hydrogen bromide gas ceased. The
solvent was removed under reduced pressure, and the residue obtained was
recrystallized from ethanol to afford the pure phenacyl bromide. Yield: 88%. Mp
1
29 °C ~ 30 °C. H NMR (300 MHz, CDCl3) δ 7.56 (d, J = 8.4 Hz, 1H), 7.48 (d, J =
1.9 Hz, 1H), 7.36 (dd, J1 = 8.4, J2 = 2.0 Hz, 1H), 4.49 (s, 2H).
4.1.2 1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-one (3)
To a mixture of 2 (1 mmol, 1eq) in DMF (3ml), imidazole (3mmol, 3eq), and
anhydrous K2CO3 (3mmol, 3eq) were added. The reaction mixture was stirred at room
temperature for 2-3 hours. Dilute the mixture with H2O (10ml) and extract the
reaction mixture with EtOAc (15ml, twice). The combined organic phase were
sequentially washed with H2O (10ml) and brine (10ml). The organic extracts were
dried over Na2SO4 and purified by silica gel column chromatography to obtain
1
product. Yield: 78%. Mp 52 °C ~ 54 °C. H NMR (300 MHz, DMSO) δ 7.94 (d, J =
15

8.4 Hz, 1H), 7.80 (d, J = 2.0 Hz, 1H), 7.64 (dd, J1 = 8.4, J2 = 2.0 Hz, 1H), 7.59 (s, 1H),
5.57 (s, 2H).
4.1.1.3 1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-ol (4)
A solution of 3 (1 mmol, 1eq) in methanol (4ml) were added to a suspension of
sodium borohydride (2 mmol, 1eq) in anhydrous methanol (20ml) while maintaining
the temperature below 0 °C. The resulting mixture was stirred at room temperature for
1 hour and then reflux the mixture for 1 hour until 3 disappeared. The reaction
mixture was cooled to room temperature and water was added. The product was
extracted with ethyl acetate (10 ml × 3). The combined organic phase was dried over
Na2SO4 and purified by flash chromatography using Hexane/EtOAc (1:1) as the eluent.
1
Yield: 88%. Mp 130 °C ~ 132 °C. H NMR (300 MHz, DMSO) δ 7.59 (d, J = 1.7 Hz,
1H), 7.45 (d, J = 7.5 Hz, 2H), 7.41 (dd, J1 = 7.5 Hz, J2 = 1.8 Hz, 1H), 7.03 (s, 1H),
6.83 (s, 1H), 6.05 (s, 1H), 5.06 (dd, J1 = 7.1, J2 = 3.4 Hz, 1H), 4.16 (dd, J1 = 14.1, J2 =
3.4 Hz, 1H), 4.03 (dd, J1 = 14.1 Hz, J2 = 7.1Hz, 1H).
4.1.1.4 General procedure for the synthesis of 5a ~ 5l
To a suspension of sodium hydride (1.1 mmol) in DMF (5 mL), a solution of
alcohol 4 (1 mmol) in DMF (10 mL) was treated at -5 °C, the resulting mixture was
stirred under a nitrogen atmosphere at room temperature for 1.5 h. Then the mixture
was cooled at 0 °C and a solution of the substituted benzyl chloride or benzyl bromide
in DMF (5 mL) was added. The resulting mixture was stirred again for another 1 h at
room temperature. After adding water (20 mL) to quenching the action, the aqueous
phase was extracted with ethyl acetate (3 × 25 mL), the organic phase was washed
with water (150 mL), dried over Na2SO4. 5a ~ 5l were afford by column
chromatography.
4.1.1.5 1-(2-(2,4-dichlorophenyl)-2-((4-fluorobenzyl)oxy)ethyl)-1H-imidazole (5a)
Light yellow oil, 65.2% yield. 1H NMR (300 MHz, CDCl3) δ 7.46 (s, 1H), 7.44
(d, J = 1.8 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.29 (dd, J1 = 8.4, J2 = 1.8 Hz, 1H), 7.15
– 7.07 (m, 2H), 7.06 – 6.95 (m, 3H), 6.89 (s, 1H), 4.95 (dd, J1 = 7.7, J2 = 2.7 Hz, 1H),
4.43 (d, J = 11.5 Hz, 1H), 4.20 (d, J = 2.9 Hz, 1H), 4.17 – 4.14 (m, 1H), 4.02 (dd, J1 =
16

14.5, J2 = 7.7 Hz, 1H). 13C NMR (300 MHz, CDCl3): δ 164.12, 137.87, 134.92,
134.00, 133.35, 132.64, 132.59, 129.64, 129.54, 128.51, 128.00, 115.63, 115.35,
76.69, 70.84, 51.39. ESI: m/z [M + H]+, calcd. for C18H15Cl2FN2O:365.0545; found
365.0620.
4.1.1.6 1-(2-((4-chlorobenzyl)oxy)-2-(2,4-dichlorophenyl)ethyl)-1H-imidazole (5b)
Light yellow solid, 62.4% yield. Mp 82 °C ~ 84 °C. 1H NMR (300 MHz, CDCl3)
δ 7.46 (s, 1H), 7.44 (s, 1H), 7.32 (s, 1H), 7.30 (s, 2H), 7.27 (s, 1H), 7.08 (s, 2H), 7.04
(d, J = 3.9 Hz, 3H), 6.89 (s, 1H), 4.95 (dd, J1 = 7.6, J2 = 2.5 Hz, 1H), 4.43 (d, J = 11.8
Hz, 1H), 4.21 (s, 1H), 4.16 (d, J = 3.6 Hz, 1H), 4.03 (dd, J1 = 14.5, J2 = 7.6 Hz, 1H).
13C NMR (300 MHz, CDCl3): δ 137.84, 135.34, 134.95, 133.87, 133.81, 133.35,
129.65, 129.07, 129.03, 128.73, 128.47, 128.01, 119.83, 76.80, 70.71, 51.35. ESI: m/z
[M + H]+, calcd. for C18H15Cl3N2O:381.0250; found 381.0323.
4.1.1.7 1-(2-((4-bromobenzyl)oxy)-2-(2,4-dichlorophenyl)ethyl)-1H-imidazole (5c)
White solid, 68.4% yield. Mp 91 °C ~ 94 °C. 1H NMR (300 MHz, CDCl3) δ 7.48
(s, 1H), 7.45 (d, J = 3.4 Hz, 1H), 7.33 (d, J = 4.6 Hz, 1H), 7.05 (d, J = 5.4 Hz, 1H),
7.01 (s, 1H), 6.91 (s, 1H), 4.97 (dd, J1 = 7.6, J2 = 2.4 Hz, 1H), 4.43 (d, J = 11.8 Hz,
13
1H), 4.22 (s, 1H), 4.18 (s, 1H), 4.05 (dd, J1 = 14.5, J2 = 7.6 Hz, 1H). C NMR (300
MHz, CDCl3): δ 137.91, 135.86, 134.98, 133.89, 133.36, 131.71, 129.68, 129.33,
129.29, 128.46, 128.03, 122.01, 119.81, 76.89, 70.77, 51.36. ESI: m/z [M + H]+, calcd.
for C18H15Cl2BrN2O:424.9745; found 424.9820.
4.1.1.8
1-(2-((4-(tert-butyl)benzyl)oxy)-2-(2,4-dichlorophenyl)ethyl)-1H-imidazole
(5d)
Yellow oil, 57.1% yield. 1H NMR (300 MHz, CDCl3) δ 7.50 (s, 1H), 7.45 (s, 1H),
7.40 – 7.34 (m, 3H), 7.29 (d, J = 4.7 Hz, 1H), 7.14 (d, J = 8.1 Hz, 2H), 7.05 (s, 1H),
6.94 (s, 1H), 5.06 – 4.98 (m, 1H), 4.44 (d, J = 11.2 Hz, 1H), 4.23 (dd, J1 = 11.8, J2 =
6.9 Hz, 2H), 4.06 (dd, J = 14.3, 7.3 Hz, 1H), 1.34 (s, 9H). 13C NMR (300 MHz,
CDCl3): δ 151.12, 137.87, 134.79, 134.26, 133.87, 133.29, 129.57, 128.92, 128.62,
127.92, 127.64, 125.52, 119.93, 77.00, 71.61, 51.46, 34.61, 31.35. ESI: m/z [M + H]+,
calcd. for C22H24Cl2N2O:403.1266; found 403.1340.
17

4.1.1.9 1-(2-(2,4-dichlorophenyl)-2-((4-methylbenzyl)oxy)ethyl)-1H-imidazole (5e)
Yellow oil, 62.1% yield. 1H NMR (300 MHz, CDCl3) δ 7.45 (s, 1H), 7.43 (d, J =
1.9 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.14 (d, J = 7.8 Hz, 2H), 7.07 (s, 2H), 7.03 (d, J
= 5.1 Hz, 2H), 6.91 (s, 1H), 4.97 (dd, J1 = 7.4, J2 = 2.7 Hz, 1H), 4.42 (d, J = 11.3 Hz,
1H), 4.21 (d, J = 11.5 Hz, 1H), 4.19 – 4.13 (m, 1H), 4.02 (dd, J = J1 = 14.4, J2 = 7.4
Hz, 1H), 2.35 (s, 4H). 13C NMR (300 MHz, CDCl3): δ 137.88, 134.78, 134.24, 133.82,
133.33, 129.66, 129.57, 129.27, 128.94, 128.63, 127.92, 127.39, 119.95, 76.64, 71.48,
51.41, 21.23. ESI: m/z [M + H]+, calcd. for C19H18Cl2N2O:361.0796; found 361.0866.
4.1.1.10 1-(2-(2,4-dichlorophenyl)-2-((3-methylbenzyl)oxy)ethyl)-1H-imidazole (5f)
Light yellow oil, 58.2% yield. 1H NMR (300 MHz, CDCl3) δ 7.52 (s, 1H), 7.45
(s, 1H), 7.35 (t, J = 10.5 Hz, 2H), 7.21 (d, J = 7.5 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H),
7.04 (s, 1H), 6.96 (d, J = 7.0 Hz, 2H), 6.92 (s, 1H), 4.98 (dd, J1 = 7.5, J2 = 2.3 Hz, 1H),
4.46 (d, J = 11.5 Hz, 1H), 4.23 (s, 1H), 4.20 – 4.16 (m, 1H), 4.05 (dd, J1 = 14.4, J2 =
7.6 Hz, 1H), 2.34(s,3H). 13C NMR (300 MHz, CDCl3): δ 138.31, 137.77, 136.70,
134.84, 134.13, 133.35, 129.60, 128.85, 128.65, 128.60, 128.52, 128.48, 127.95,
124.89, 119.97, 76.64, 71.60, 51.49, 21.39. ESI: m/z [M + H]+, calcd. for
C19H18Cl2N2O:361.0796; found 361.0870.
4.1.1.11 1-(2-(2,4-dichlorophenyl)-2-((2-methylbenzyl)oxy)ethyl)-1H-imidazole (5g)
Light yellow oil, 56.7% yield. 1H NMR (300 MHz, CDCl3) δ 7.45 (d, J = 1.9 Hz,
1H), 7.43 (s, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 1.9 Hz, 1H), 7.22 (dd, J1 = 6.4,
J2 = 2.4 Hz, 1H), 7.17 (s, 1H), 7.17 – 7.12 (m, 2H), 7.01 (s, 1H), 6.89 (s, 1H), 5.01
(dd, J1 = 7.5, J2 = 2.7 Hz, 1H), 4.44 (d, J = 11.5 Hz, 1H), 4.26 (d, J = 11.5 Hz, 1H),
4.19 (dd, J1 = 14.5, J2 = 2.8 Hz, 1H), 4.03 (dd, J1 = 14.4, J2 = 7.5 Hz, 1H), 2.16 (s,
3H). 13C NMR (300 MHz, CDCl3): δ 137.82, 136.73, 134.85, 134.78, 134.18, 133.33,
130.39, 129.63, 129.08, 128.69, 128.56, 128.32, 127.96, 125.98, 119.79, 76.83, 69.87,
51.43, 18.67. ESI: m/z [M + H]+, calcd. for C19H18Cl2N2O:361.0796; found 361.0868.
4.1.1.12 1-(2-(2,4-dichlorophenyl)-2-((3,4-dimethylbenzyl)oxy)ethyl)-1H-imidazole
(5h)
1
Light yellow oil, 66.7% yield. H NMR (300 MHz, CDCl3) δ 7.50 (s, 1H), 7.45
18

(s, 1H), 7.36 (t, J = 11.3 Hz, 2H), 7.10 (d, J = 7.5 Hz, 1H), 7.05 (s, 1H), 6.91 (d, J =
8.1 Hz, 3H), 4.99 (dd, J1 = 7.5, J2 = 2.3 Hz, 1H), 4.43 (d, J = 11.2 Hz, 1H), 4.21 (s,
13
1H), 4.17 (s, 1H), 4.04 (dd, J1 = 14.4, J2 = 7.5 Hz, 1H), 2.27 (s, 3H), 2.26 (s, 3H). C
NMR (300 MHz, CDCl3): δ 137.84, 136.87, 136.58, 134.78, 134.27, 134.20, 133.35,
129.78, 129.57, 129.27, 128.81, 128.64, 127.93, 125.46, 119.96, 76.59, 71.53, 51.49,
19.77, 19.57. ESI: m/z [M + H]+, calcd. for C20H20Cl2N2O:375.0953; found 375.1029.
4.1.1.13
1-(2-(2,4-dichlorophenyl)-2-((4-methoxybenzyl)oxy)ethyl)-1H-imidazole
(5i)
1
Light yellow oil, 64.5% yield. H NMR (300 MHz, CDCl3) δ 7.45 (s, 1H), 7.43
(d, J = 2.0 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 2.0 Hz, 1H), 7.08 (d, J = 8.6
Hz, 2H), 7.02 (s, 1H), 6.88 (d, J = 10.5 Hz, 3H), 6.83 (s, 1H), 4.95 (dd, J1 = 7.5, J2 =
2.7 Hz, 1H), 4.40 (d, J = 11.2 Hz, 1H), 4.19 (d, J = 11.3 Hz, 1H), 4.14 (d, J = 2.7 Hz,
1H), 4.01 (dd, J1 = 14.4, J2 = 7.6 Hz, 1H), 3.81 (s, 3H). 13C NMR (300 MHz, CDCl3):
δ 159.50, 137.87, 134.78, 134.35, 133.33, 129.55, 129.45, 129.12, 128.94, 128.67,
127.90, 119.79, 113.97, 76.47, 71.32, 55.29, 51.43. ESI: m/z [M + H]+, calcd. for
C19H18Cl2N2O2:377.0745; found 377.0380.
4.1.1.14
1-(2-(2,4-dichlorophenyl)-2-((3-methoxybenzyl)oxy)ethyl)-1H-imidazole
(5j)
1
Light yellow oil, 61.8% yield. H NMR (300 MHz, CDCl3) δ 7.46 (s, 1H), 7.43
(d, J = 1.7 Hz, 1H), 7.34 (d, J = 8.3 Hz, 1H), 7.30 (d, J = 1.7 Hz, 1H), 7.22 (d, J = 7.7
Hz, 1H), 7.02 (s, 1H), 6.92 (s, 1H), 6.83 (d, J = 7.9 Hz, 1H), 6.74 (d, J = 7.6 Hz, 1H),
4.98 (dd, J1 = 7.4, J2 = 2.6 Hz, 1H), 4.44 (d, J = 11.7 Hz, 1H), 4.26 – 4.21 (m, 1H),
4.21 – 4.14 (m, 1H), 4.04 (dd, J1 = 14.5, J2 = 7.4 Hz, 1H), 3.79 (s, 3H). 13C NMR
(300 MHz, CDCl3): δ 159.80, 138.41, 137.89, 134.87, 134.16, 133.34, 129.59, 129.45,
129.17, 128.61, 127.93, 119.96, 119.84, 113.72, 113.10, 76.81, 71.51, 55.25, 51.39.
ESI: m/z [M + H]+, calcd. for C19H18Cl2N2O2:377.0745; found 377.0380.
4.1.1.15 1-(2-(2,4-dichlorophenyl)-2-((2,6-difluorobenzyl)oxy)ethyl)-1H-imidazole
(5k)
White solid, 67.8% yield. Mp 86 °C ~ 90 °C. 1H NMR (300 MHz, CDCl3) δ 7.44
19

(s, 1H), 7.40 (s, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 3.6 Hz, 2H), 6.98 (s, 1H),
6.89 (dd, J1 = 9.2, J2 = 6.1 Hz, 3H), 5.00 (dd, J1 = 6.9, J2 = 2.5 Hz, 1H), 4.55 (d, J =
11.0 Hz, 1H), 4.43 (d, J = 10.5 Hz, 1H), 4.18 (dd, J = 14.2, 2.9 Hz, 1H), 4.01 (dd, J1 =
14.4, J2 = 7.3 Hz, 1H). 13C NMR (300 MHz, CDCl3): δ 137.77, 134.80, 134.00,
133.17, 130.76, 129.48, 129.08, 128.50, 127.91, 127.85, 119.69, 111.54, 111.19, 77.57,
59.07, 51.36. ESI: m/z [M + H]+, calcd. for C18H14Cl2F2N2O:383.0451; found
383.0531.
4.1.1.16 1-(2-(benzyloxy)-2-(2,4-dichlorophenyl)ethyl)-1H-imidazole (5l)
Transparent oil, 66.1% yield. 1H NMR (300 MHz, CDCl3) δ 7.49 (s, 1H), 7.45 (s,
1H), 7.37 (d, J = 8.7 Hz, 2H), 7.33 (s, 3H), 7.18 (d, J = 7.3 Hz, 2H), 7.05 (s, 1H), 6.93
(s, 1H), 5.00 (dd, J1 = 7.5, J2 = 2.4 Hz, 1H), 4.49 (d, J = 11.5 Hz, 1H), 4.23 (dd, J1 =
14.2, J2 = 7.0 Hz, 2H), 4.06 (dd, J1 = 14.4, J2 = 7.4 Hz, 1H). 13C NMR (300 MHz,
CDCl3): δ 137.85, 136.84, 134.86, 134.11, 133.33, 129.61, 128.95, 128.60, 128.58,
128.09, 127.97, 127.77, 119.92, 76.79, 71.61, 51.44. ESI: m/z [M + H]+, calcd. for
C18H16Cl2N2O:347.0640; found 347.0716.
4.2. Biological assays
4.2.1 Enzyme inhibition of ChEs
Modified Ellman assay was performed to determine the cholinesterase inhibition
ability of the miconazole analogues using a Shimadzu 160 spectrophotometer. AChE
(EC 3.1.1.7, Type VI-S, from electric eel, C3389) and BuChE (EC 3.1.1.8, from
equine serum, C0663), 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB, D218200),
acetylthiocholine iodide (ATC, A5751), and butyrylthiocholine iodide (BTC, B3253)
were purchased from SigmaAldrich (St. Louis, MO, USA). Each compound was
prepared a dilution series of at least five different concentrations (normally 104 to 109
M). For measurement, a cuvette containing 100 µL of phosphate buffer, 10 µL of
AChE or BuChE, and 10 µL of the test compound solution was allowed to stand for 5
min before 10 µL of DTNB was added. After the addition of 20 µL of ATC or BTC,
the reaction solution was mixed immediately. Two minutes after substrate addition,
the absorption was determined at 25 at 412 nm. For the reference value, 10 µL of
20

water replaced the test compound solution. For determining the blank value, 10 µL of
additional water replaced the enzyme solution. The measurement for each
concentration was performed in triplicate. The inhibition curve was fitted by plotting
the percentage enzyme activity (100 % for the reference) versus the logarithm of the
test compound concentration. The IC50 values were calculated by GraphPad Prism 6,
and the data were shown as mean SEM.
4.2.2 Enzyme inhibition of IDO1
The reaction mixture (500 µL) contained potassium phosphate buffer at pH 6.5
(50 mM), ascorbic acid (40 mM), methylene blue (20 µM), bovine catalase (200
µg/mL), the substrate L-Trp (100 µM), the screening compound at varying
concentrations, DMSO (< 0.3%), and IDO1 (50 nM). After incubation at 37 for 30
min, the reaction was stopped by addition of trichloroacetic acid solution (200 µL, 30%
w/v), and were further incubated at 65 for 15 min to allow hydrolysis of the
enzymatic reaction product, NFK, to kynurenine. The reaction mixture was
centrifuged at 10000 rpm for 10 min. 100 µL supernatant from each well was
transferred to a new 96 well plate and mixed at equal volume with 2% (w/v)
p-dimethylaminobenzaldehyde (Ehrlich's reagent) in acetic acid. The yellow color
generated from the reaction with kynurenine was measured at 492 nm.
4.2.3 Behavioral studies
Behavioral studies were performed using the Morris water maze according to
method reported previously.[42] Adult male imprinting control region (ICR) mice
(8-10 weeks old, weight 20-25 g) were obtained from the Yangzhou University
Medical Center (Yangzhou, China). All animal handling and experimental protocols
were approved by the Institutional Animal Care and Use Committee of the China
Pharmaceutical University. Scopolamine hydrobromide was purchased from Aladdin
Reagents (S107418, Shanghai, China). Tacrine used as the positive control was
synthesized by our lab (purity > 95%).
Thirty-six mice were randomly separated into five subgroups (6 mice for each
group): (i) vehicle as a blank control group, (ii) scopolamine as a model group, (iii)
21

tacrine plus scopolamine as a positive control, and (iv) miconazole (10 mg/kg) plus
scopolamine as a test group, (v) miconazole (50 mg/kg) plus scopolamine as a test
group, (vi) miconazole (100 mg/kg) plus scopolamine as a test group. The mice for
model, tacrine, miconazole (10 mg/kg), miconazole (50 mg/kg) and miconazole (100
mg/kg) received intraperitoneal injections of scopolamine (1 mg/kg), while the blank
control group was injected with saline. Tacrine, miconazole (10 mg/kg), miconazole
(50 mg/kg) and miconazole (100 mg/kg) were orally administered (30 mg/kg) to mice
in groups (iii), (iv), (v) and (vi) 30 min before the ip administration of scopolamine or
saline.
A circular pool (120 cm in diameter, 60 cm height) which is fixed with an escape
platform (10 cm diameter) and filled to a depth of 40 cm fresh water (kept at 25 °C)
composes the water maze. It is placed in a lit room. After 5 days of learning and
memory training, a probe trial was performed on day 6. For the evaluation of
cognitive function, each mouse was individually trained on a visible-platform (labeled
by a small flag, 5 cm tall) for 2 days and followed by a hidden-platform version
(placed 1 cm below the surface of the water) of the water maze from day 3 to day 5.
All mice were subjected to 2 training trials daily, each of which lasted for 90s. The
time for each mouse to find the platform (a successful escape) was recorded. If a
mouse failed to reach the platform within 90 s, the test was terminated and the mouse
was carefully navigated to the platform by hands. Each mouse was kept on the
platform for 30s whether it succeeded or failed to reach the platform. On the last day
(day 6), a probe trial was given to the mice in which the platform was removed from
the pool and each mouse was allowed 90 s to search for the platform. The time the
animals spent to reach the missing platform location and the number of times they
crossed that location were recorded.
Data for the time of escape latency, the swimming trajectory, and the number of
visual platform crossings were recorded by Panl ab SMART 3.0 and analyzed by
GraphPad Prism 6 to evaluate the level of memory retention.
The experiment procedure of behavioral studies on 5i and 5j were just as told
22

above.
4.2.4 Hepatotoxicity studies.
Hepatotoxicity was evaluated according to the method previously described by
using adult male ICR mice (8-10 weeks old, weighing 20-25 g) obtained from the
Yangzhou University Medicine Centre (Yangzhou, China).[43] The test compounds
were dissolved in a sodium carboxymethyl cellulose (CMC-Na) solution (0.5 g
CMC-Na in 100 mL distilled water). Concentration of 3 mg/100g body wt. of 5i,
corresponding to 151.5 ꢄmol/kg body wt., was administered intragastrically (ig).
Equimolar dose of 5j to that of 5i was administered ig. 8, 22, and 36 h after the
administration. Heparinized serum was collected from the retrobulbar plexus and
subjected to hepatotoxicity evaluation. The activity of aspartate aminotransferase
(AST) and alanine aminotransferase (ALT), two indicators of liver damage, was
determined using corresponding assay kit (EF551 and EF550 for ALT, EH027 and
EF548 for AST, Wako, Japan). The data were processed by Biochemical Analyzer
(HITACHI 7020，Japan).
Mice were sacrificed 1 h after the collection of retrobulbar blood, and livers were
harvested for morphological studies by using immunohistochemical method. Two 3
mm sections of each liver extending from the hilus to the margin of the left lateral
lobe were isolated by Ultra-Thin Semiautomatic Microtome (Leica RM2245,
Germany) and immediately placed in 10% buffered formaldehyde, fixed for two days,
and embedded together in one paraffin block by using Paraffin Embedding Station
(Leica EG1150H，Germany). Subsequently, 100 ꢄm sections were prepared from
these paraffin blocks. They were deparaffinated and stained with hematoxylin and
eosin (HE) for histopathological examinations.
4.2.5 Molecular docking
The docking study was performed by CDOCKER module implemented in
Discovery Studio 3.0. The principle of CDOCKER can be briefly summarized as
follow: CDOCKER generated ligand “seeds” to populate the binding pocket. Each
seed was then subjected to high temperature molecular dynamics (MD) using a
23

modified version of CHARMm force field. Each of the structures from the MD was
run then located and fully minimized. The solutions were then clustered according to
position and conformation and ranked by energy. The co-crystal structures of
N-((1-(2,3-dihydro-1H-inden-2-yl)piperidin-3-yl)methyl)-N-(2-methoxyethyl)-2-napt
hamide-BuChE (PDB id: 4TPK) and NLG919 analogue-IDO1 (PDB id: 5EK3) were
used for molecular docking. The binding sites were defined by residues around the
BuChE (in 10 Å radius). Other parameters were kept as default.
Supplementary data
1
The hepatotoxicity test of miconazole, kinetic study, H-NMR and 13C-NMR spectra
of the target compounds are supplied in the Supplementary data.
Additional information
Competing financial interests: The authors declare no competing financial interests.
Acknowledgment
We gratefully thank the support from the grants 81402851, 81573281 and 81603529
of National Natural Science Foundation of China, BK20140957 of Natural Science
Foundation of Jiangsu Province. We also thank the support from Fundamental
Research Funds for the Central Universities (2015ZD009), Jiangsu Qing Lan Project,
Top-notch Academic Programs Project of Jiangsu Higher Education Institutions
(TAPP-PPZY2015A070) and Priority Academic Program Development of Jiangsu
Higher Education Institutions (PAPD).
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28

Highlight:
Attractive dual BuChE/IDO1inhibitory activity was determined in target compounds.
Significantly reversed scopolamine-induced memory deficit in mice.
Not trigger acute hepatotoxicity on mice.
Highlight a new multi-target-directed ligands (MTDLs) design strategy against AD.
29