Highly Potent and Selective Butyrylcholinesterase Inhibitors for Cognitive Improvement and Neuroprotection

Article abstract of DOI:10.1021/acs.jmedchem.1c00167

Butyrylcholinesterase (BChE) has been considered as a potential therapeutic target for Alzheimer's disease (AD) because of its compensation capacity to hydrolyze acetylcholine (ACh) and its close association with Aβ deposit. Here, we identified S06-1011 (hBChE IC50 = 16 nM) and S06-1031 (hBChE IC50 = 25 nM) as highly effective and selective BChE inhibitors, which were proved to be safe and long-acting. Candidate compounds exhibited neuroprotective effects and the ability to improve cognition in scopolamine- and Aβ1-42 peptide-induced cognitive deficit models. The best candidate S06-1011 increased the level of ghrelin, a substrate of BChE, which can function as improving the mental mood appetite. The weight gain of the S06-1011-treated group remarkably increased. Hence, BChE inhibition not only plays a protective role against dementia but also exerts a great effect on treating and nursing care.

Full text of DOI:10.1021/acs.jmedchem.1c00167

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Article
Highly Potent and Selective Butyrylcholinesterase Inhibitors for
Cognitive Improvement and Neuroprotection
Qi Li, Ying Chen, Shuaishuai Xing, Qinghong Liao, Baichen Xiong, Yuanyuan Wang, Weixuan Lu,
Siyu He, Feng Feng, Wenyuan Liu,* Yao Chen,* and Haopeng Sun*
Cite This: J. Med. Chem. 2021, 64, 6856−6876
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ABSTRACT: Butyrylcholinesterase (BChE) has been considered as a potential
therapeutic target for Alzheimer’s disease (AD) because of its compensation capacity
to hydrolyze acetylcholine (ACh) and its close association with Aβ deposit. Here, we
identified S06-1011 (hBChE IC50 = 16 nM) and S06-1031 (hBChE IC50 = 25 nM) as
highly effective and selective BChE inhibitors, which were proved to be safe and long-
acting. Candidate compounds exhibited neuroprotective effects and the ability to
improve cognition in scopolamine- and Aβ1−42 peptide-induced cognitive deficit
models. The best candidate S06-1011 increased the level of ghrelin, a substrate of
BChE, which can function as improving the mental mood appetite. The weight gain of
the S06-1011-treated group remarkably increased. Hence, BChE inhibition not only
plays a protective role against dementia but also exerts a great effect on treating and
nursing care.
plasma. Neural BChE is mostly of glial origin.18 However, the
compensatory character of BChE is greatly noticeable under
pathological conditions. In the AChE-knockout mouse model,
BChE was proven to compensate for hydrolyzing ACh due to
the lack of AChE, thereby maintaining normal cholinergic
pathways.18,19 In progressed AD, the level of AChE in the brain
sharply declines by 90%, while BChE increases to 165% of
normal levels, taking over the hydrolysis of ACh.20 Hence, BChE
is a potential target for the treatment of advanced AD. Generally,
central nervous system (CNS) drugs are required for good
blood−brain barrier (BBB) penetration capacity; however, most
drugs suffer from the restriction of tissue selectivity, which
commonly leads to peripheral side effects. Existing selective
AChE and nonselective ChE inhibitors exhibit typical
cholinergic toxicity, such as nausea and vomiting, due to
inhibition of peripheral AChE by residues.21 Furthermore, there
are no physiological deficiencies in BChE-knockout mice, and
people with silent BChE can live healthily to an old age.22,23
Based on these facts, suppression of BChE shows an advantage
in drug safety for AD treatment.
1. INTRODUCTION
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder, characterized by central cognitive and behavioral
dysfunction.1 As reported by the World Alzheimer Report 2018,
50 million people are affected by dementia, and this number will
more than triple to 152 million by 2050.2 Although the exact
etiology of AD has not been completely understood, several
common hallmarks, including cholinergic dysfunction, amyloid-
β (Aβ) deposits, τ-protein aggregation, neuroinflammation, and
mitochondrial dysfunction, are considered to be closely related
to the pathophysiology and progression of AD.3−7 Due to the
complexity of AD pathology, large investments result in low
returns, and few drugs have been clinically applied for AD
treatment.1,2 Except for memantine, an N-methyl-D-aspartic acid
receptor (NMDAR) blocker, all other approved drugs benefit
the dysfunctional neurocholinergic system.8−11 From this point
of view, designing cholinesterases (ChE) inhibitors remains a
reasonable and promising strategy for AD treatment.
According to the cholinergic hypothesis, damaged nerve cells
in the AD brain, especially in the cortex and hippocampus,
forebrain Meynert basal ganglia, and septal area, lead to an
abnormal decrease in acetylcholine (ACh).12−14 Low levels of
ACh are a symbolic pathological feature of AD, particularly for
its strong association with cognitive function.15 There are two
ChEs named acetylcholinesterase (AChE EC 3.1.1.7) and
butyrylcholinesterase (BChE, EC 3.1.1.8), but ACh is
predominantly decomposed by the former under physiological
conditions.16 Normally, AChE is predominant in muscles and
the nervous system accompanied by a lower level of BChE.17
BChE is notably synthesized in the liver and secreted into the
In addition to cholinergic benefits, BChE has been
demonstrated to be highly related to Aβ.24 Histochemical
localization has proven that BChE deposition is strongly
Received: January 29, 2021
Published: May 11, 2021
https://doi.org/10.1021/acs.jmedchem.1c00167
© 2021 American Chemical Society
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Figure 1. (A) Chemical structure of the lead compound S06-1001. (B) Substrate-binding pockets and superimposition of hAChE (PDB code: 4ey4)
and hBChE (PDB code: 1p0i). Residues (Phe297 and Tyr124) of hAChE are represented in red, and residues (Val288 and Gln119) of hBChE are in
blue. (C) Molecular dynamic result of S06-1001 and modification strategy purposed for further derivative design.
associated with progressive Aβ aggregation in senile plaques.25
Moreover, Aβ fibers in the brain were remarkably reduced in the
BChE-knockout mouse model, indicating that BChE may
promote Aβ deposition.26 The cognition of BChE-knockout
mice was found to not be influenced by injection of neurotoxic
Aβ25−35, whereas wild-type mice suffered from cognitive insults
after administration.27 From this point of view, BChE inhibition
can be an effective strategy for Aβ clearance.
1011 and S06-1031 containing a 3-(benzoimidazol-2-yl)-1,2,5-
oxadiazole core, were identified as drug candidates for a new
structural class of highly selective BChE inhibitors.
2. RESULTS AND DISCUSSION
2.1. Design, Synthesis, and Enzymic Inhibition Screen-
ing of Selective BChE Inhibitors. To better design the
derivatives, we first thoroughly explored the structure of BChE
and its structural differences from that of AChE. Generally, the
overall structures for the two ChEs are similar.21 Both enzymes,
which share more than 50% homology, contain a catalytic active
site (CAS), a deep and narrow gorge, and a peripheral anionic
site (PAS).40 Despite the high structural similarity, the subtle
but pivotal residue differences in substrate-binding pockets
provide an opportunity to exploit selective inhibitors. As shown
in Figure 1B, larger residues of hAChE, such as Phe297 and
Tyr124, protrude into the pocket, resulting in the formation of a
narrower site, while smaller residues of hBChE, such as Gln119
and Val286, create a wider cavity for ligands to bind to. This
structural difference dictates the substrate specificity of two
enzymes: AChE tends to bind small molecules such as ACh; as a
counterpart, BChE can hydrolyze various neuroactive peptides
due to its broader space for substrates.41
Based on the cocrystal structure of hBChE, we previously
discovered a lead compound S06-1001 with moderate BChE
inhibitory ability and high selectivity.39 According to the
molecular dynamics (MD) simulation result of S06-1001
(Supporting Information, Figure S1), an intramolecular hydro-
gen bond was formed between the N atom of benzimidazole and
the primary amino group on the oxadiazole, further constituting
a large polyaromatic-ring structure. This structure was involved
in a parallel π−π stacking interaction with Trp82 (Figure 1C),
which was a dominating residue for intermolecular interaction
between the compound and BChE. The subpocket at the
bottom of the gorge is pivotal to distinguish the two ChEs, and it
is also predominant in the design of highly selective inhibitors.
Based on the MD results obtained for S06-1001, we
hypothesized that the quinoline ring could be inserted into
this pocket (Figure 1C), contributing to the high selectivity of
S06-1001. Therefore, we proposed a modification strategy
(Moiety A, Linker B, and Moiety C) to support our speculation
and attempted to design and develop derivatives with higher
activity and selectivity.
AD is an age-related neurodisease; AD patients often have
metabolic dysfunction. Epidemiological studies have validated
that some diseases, such as metabolic syndrome, are also risk
factors for elderly AD patients.28,29 Weight loss has been
identified as a common syndrome in AD patients and appears
even earlier than damages in cognitive functions.30 It has been
demonstrated that weight gain may afford protection against
dementia, but this can be tough for AD patients owing to the
poor mental state.31 In 2004, Brimijoin et al. reported the
function of BChE as hydrolyzing ghrelin through the removal of
the octanoyl group.32 Ghrelin, a 28-amino acid peptide, plays an
essential role in energy homeostasis and metabolism.33,34 It is
secreted from the fundus of the human stomach and the
pancreas. Ghrelin is capable of penetrating the BBB and plays a
significant role in neurotrophy, particularly in the hippo-
campus.35 It is essential for improving cognitive adaptation to
the environment and the learning process and elevating mental
mood and appetite.36−38 Based on the hydrolysis influence of
BChE on ghrelin, BChE inhibition may help improve appetite
and food intake, which is a great benefit for AD patients.
Moreover, elevated appetite of patients is helpful in ameliorating
their nutrition state, which greatly reduces the pressure on
caregivers.
As concluded, selective BChE inhibitors are of vital
importance to prolonging the onset time, removing abnormal
brain Aβ deposits, protecting neural cells against oxidative stress,
evading adverse effects, and improving the mood and nutrition
state of patients, which are all beneficial for AD treatment.
Previously, a novel compound 8012-9656 (S06-1001, Figure
1A) was reported by our group to exhibit nanomolar-range
inhibitory activity (eeAChE IC50 > 100 μM, eqBChE IC50 = 0.18
0.03 μM, hBChE IC50 = 0.32 0.07 μM) and high selectivity
(AChE IC50/BChE IC50 ratio > 500).39 Herein, taking S06-
1001 as the lead, we report the medicinal chemistry
optimization of this compound, including both activity and
druglike properties. During this process, two compounds, S06-
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a
Scheme 1. General Procedure for the Preparation of Terminal Compounds S06-1002, S06-1007, and S06-1008
a
Reagents and conditions: (i) chloroacetyl chloride (or 3-chloropropionyl chloride or 5-chlorovaleryl chloride), NaHCO3, chloroform, r.t., 2 h; (ii)
NaNO2, AcOH, 0 °C, 0.5 h; (iii) NH2OH HCl, KOH, (MeOCH2CH2)2O, 110 °C, 6 h; (iv) Cs2CO3, dimethylformamide (DMF), 70 °C, 7 h.
a
Scheme 2. General Procedure for the Preparation of Terminal Compounds S06-1003 to S06-1006
a
Reagents and conditions: (v) NaHSO3, DMF, 80 °C, 5 h; (vi) BrCH2COOEt, MeCN, Cs2CO3, r.t., 30 min; (vii) 1 N LiOH H2O, tetrahydrofuran
(THF)/MeOH, r.t., 12 h; (viii) HATU, DIEA, DMF, r.t., 12 h; (ix) 10% Pd/C, N2H4 H2O, THF, reflux, 5 h.
To confirm the importance of the large polyaromatic Moiety
A and its nonpolar interaction with Trp82, we replaced the
oxadiazole ring with other aromatic rings, such as furan and
phenyl rings. Following Schemes 1 and 2, we synthesized
compounds S06-1002 to S06-1006. The synthetic route for
S06-1002 was initiated from commercially available 6-amino-
quinoline, which reacted with chloroacetyl chloride to afford 2a.
The commercially available reactant (2-benzimidazolyl)-
acetonitrile was mixed with NaNO2 in acetic acid as a solvent
to produce the transitional intermediate 1a, which was
subsequently reacted with hydroxylamine hydrochloride to
afford 1b. Terminal compounds S06-1002 were obtained with
1b and 2a as reactants at the temperature of 70 °C. For
compounds S06-1003 to S06-1005, the synthetic procedure
was initiated using o-phenylenediamine, which reacted with
aromatic aldehyde to afford intermediates 3a−5a. These
intermediates reacted with ethyl bromoacetate to produce
compounds 3b−5b, followed by hydrolysis to obtain 3c−5c.
Finally, terminal compounds were produced by amidation with
6-aminoquinoline, using HATU as a condensation agent and
N,N-diisopropylethylamine (DIEA) as the base.
From the results obtained from the target inhibition assay of
compounds S06-1002 to S06-1006 (Table 1), we found that the
activity of derivative S06-1002, although slightly lower than the
lead compound, was basically maintained. However, after
replacing the oxadiazole ring with other aromatic rings, such
as furan and benzene, the inhibitory ability of compounds S06-
1003 and S06-1004 obviously declined. Moreover, we
synthesized S06-1005 containing a 2-nitrobenzene ring and
S06-1006 containing a 2-aminobenzene ring to mimic the
primary amino group on oxadiazole and the large polyaromatic-
ring structure; however, these two compounds still presented
poor BChE inhibitory activity. We conducted a three-dimen-
sional configuration simulation to explain this phenomenon, and
the results are shown in Figure 2. The amino-oxadiazole moiety
is a prerequisite for the formation of the “tetraaromatic ring”
planar structure. Replacement with other ring systems was
unable to constitute the large planar system due to the deviation
of the dihedral angle, further leading to the complete loss of
inhibitory activity.
The tetracyclic system is a prerequisite for high enzyme
inhibitory activity, while the insertion of Moiety C into the
subpocket at the bottom of the gorge is presumed to be the key
to exerting high selectivity toward BChE against AChE.
Therefore, we fixed the oxadiazole ring at Moiety A and
synthesized S06-1007 and S06-1008 (Scheme 1) to explore the
effect of Linker B on inhibitory activity. The synthesis of S06-
1007 and S06-1008 was similar to that of S06-1002, using 3-
chloropropionyl chloride or 5-chlorovaleryl chloride to replace
chloroacetyl chloride to afford a longer linker. The other
reagents and conditions were conventional.
From the results given in Table 1, when the length of the
linker was slightly increased, the activity of the compounds was
enhanced accordingly. However, excessively long chains led to a
loss of inhibitory capacity. According to Figure 1C, we
speculated that a moderate length of the linker possibly
facilitated deep insertion of Moiety C into the subpocket;
however, excessive length resulted in a certain hindrance,
leading to the loss of activity (S06-1008). From this point of
view, we could infer that the pocket occupied by Moiety C also
needed to be carefully considered during the structural
modification process, as we found that the inhibitory ability of
compound S06-1002 was slightly lower than that of the lead
compound. Introducing proper substituents to Moiety C may
improve the activity.
To explore the structure−activity relationship (SAR) of
Moiety C and the volume of the pocket it occupies, we designed
and synthesized 34 derivatives, replacing the quinoline ring with
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a b c
, ,
Table 1. BChE Inhibition of Compounds S06-1002 to S06-1045
Cpd.
n
R1
R2
R3
eqBChE (IC50, μM)
hBChE (IC50, μM)
S06-1001
S06-1002
S06-1003
S06-1004
S06-1005
S06-1006
S06-1007
S06-1008
S06-1009
S06-1010
S06-1011
S06-1012
S06-1013
S06-1014
S06-1015
S06-1016
S06-1017
S06-1018
S06-1019
S06-1020
S06-1021
S06-1022
S06-1023
S06-1024
S06-1025
S06-1026
S06-1027
S06-1028
S06-1029
S06-1030
S06-1031
S06-1032
S06-1033
S06-1034
S06-1035
S06-1036
S06-1037
S06-1038
S06-1039
S06-1040
S06-1041
S06-1042
S06-1043
S06-1044
S06-1045
Tacrine
1
1
1
1
1
1
2
4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
4-amino-furazan-3-yl
4-amino-furazan-3-yl
furan-3-yl
phenyl
2-nitrophenyl
2-methylquinolin-6-yl
quinoline-6-yl
quinoline-6-yl
quinoline-6-yl
quinoline-6-yl
quinoline-6-yl
quinoline-6-yl
quinoline-6-yl
quinoline-5-yl
quinoline-4-yl
quinoline-3-yl
quinoline-2-yl
naphthalen-2-yl
naphthalen-1-yl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.177 0.029
0.705 0.213
n. a.
n. a.
n. a.
n. a.
0.253 0.105
n. a.
0.920 0.336
0.051 0.010
0.020 0.006
0.507 0.132
0.352 0.180
0.044 0.013
0.006 0.003
2.960 0.438
0.124 0.041
1.043 0.319
0.270 0.090
2.732 1.297
0.033 0.012
n. a.
0.322 0.073
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
2-aminophenyl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
4-amino-furazan-3-yl
0.212 0.059
0.016 0.004
n. d.
n. d.
0.022 0.011
0.032 0.016
n. d.
n. d.
n. d.
n. d.
n. d.
0.128 0.060
n. d.
0.212 0.086
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
0.025 0.009
0.080 0.031
n. d.
0.250 0.088
n. d.
n. d.
0.180 0.056
n. d.
n. d.
n. d.
n. d.
n. d.
4-bromonaphthalen-1-yl
phenyl
2-fluorophenyl
3-fluorophenyl
2,3-difluorophenyl
2-chlorophenyl
3-chlorophenyl
4-chlorophenyl
3-bromophenyl
4-bromophenyl
3-methylphenyl
4-methylphenyl
2,3-dimethylphenyl
3-methoxyphenyl
4-methoxyphenyl
2,3,4-trimethoxyphenyl
3-nitrophenyl
4-nitrophenyl
4-aminophenyl
3-cyanophenyl
3-acetylphenyl
4-acetylphenyl
3- methoxycarbonyl
4- methoxycarbonyl
3-acetamidophenyl
4-acetamidophenyl
biphenyl
benzo[1,3]dioxol-5-yl
quinoline-6-yl
quinoline-3-yl
0.083 0.022
n. a.
0.236 0.059
10.290 8.231
0.326 0.075
1.711 0.735
n. a.
6.643 2.276
0.031 0.003
0.030 0.007
n. a.
0.051 0.012
0.191 0.048
2.267 0.216
0.027 0.013
5.376 0.816
1.125 0.493
6.538 1.220
n. a.
2.206 1.133
11.25 10.03
28.53 56.50
0.108 0.037
0.005 0.001
H
H
H
H
H
H
H
H
H
H
5,6-dimethyl
5,6-dimethyl
5,6-dimethyl
n. d.
n. d.
n. d.
n. d.
naphthalen-1-yl
a
b
n. d., not determined. n. a., no activity for their inhibitory rates less than 50% at a concentration of 10 μM. Detailed inhibitory rates (IRs) are
c
summarized in the Supporting Information, Figure S2. All tests are expressed as the mean
standard error of the mean (SEM) of three
independent experiments.
diverse aromatic rings and fixing Linker B to n = 1, following
Scheme 3. The synthetic route was similar to that of S06-1002
and initiated using diverse commercially available amines, which
reacted with chloroacetyl chloride to afford 9a to 42a. The other
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Figure 2. Three-dimensional configuration simulation of S06-1002 and S06-1006.
a
Scheme 3. General Procedure for the Preparation of Terminal Compounds S06-1009 to S06-1042
a
Reagents and conditions: (i) chloroacetyl chloride, NaHCO3, chloroform, r.t., 2 h; (ii) NaNO2, AcOH, 0 °C, 0.5 h; (iii) NH2OH HCl, KOH,
(MeOCH2CH2)2O, 110 °C, 6 h; (iv) Cs2CO3, DMF, 70 °C, 7 h; (ix) 10% Pd/C, N2H4 H2O, THF, reflux, 5 h.
a
Scheme 4. General Procedure for the Preparation of Terminal Compounds S06-1043 to S06-1045
a
Reagents and conditions: (i) chloroacetyl chloride, NaHCO3, chloroform, r.t., 2 h; (ii) NaNO2, AcOH, 0 °C, 0.5 h; (iii) NH2OH HCl, KOH,
(MeOCH2CH2)2O, 110 °C, 6 h; (iv) Cs2CO3, DMF, 70 °C, 7 h; (x) ethyl cyanoacetate, 185 °C, under argon, 2 h.
reagents and conditions were the same as those used for S06-
1002.
BChE that was even higher than 3000-fold (Supporting
Information, Figure S2). However, compounds containing
non-, ortho-, and para-substituted benzenes showed reduced
inhibitory capacity. In particular, compounds containing p-
substituted benzene rings, except for S06-1032 (eqBChE IC50 =
0.030 0.007 μM), commonly lost their inhibitory activities
completely. Although several meta-substituted benzene-con-
taining inhibitors have comparable activity to compounds
containing bicyclic rings, they have much higher ligand
efficiency (LE), which is important for druggability. Due to
their excellent inhibitory activity against eqBChE, compounds
As shown in Table 1, derivatives containing a bicyclic
substitution at Moiety C commonly possessed good BChE
inhibitory capacities. In particular, compounds S06-1011, S06-
1014, and S06-1015 presented nanomolar inhibition against
eqBChE and hBChE. Derivatives S06-1016 to S06-1042
containing a benzene ring or a substituted benzene ring at
Moiety C displayed greatly varied capacities. Inhibitors with
substituents in the meta position showed preferable potency
with an IC50 value range of 27−1711 nM and a selectivity against
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Figure 3. MD results of compounds S06-1002 (A), S06-1011 (B), and S06-1014 (C). 5,6-Position of the benzimidazole has been marked by a blue
circle (PDB code: 5k5e).
a d
,
Table 2. In Vitro Potential Cytotoxicity on Neurocytes and Hepatocytes
SH-SY5Y
IC50, μM
PC12
IC50, μM
(SR, %)
BV2
HepG2
IC50, μM
(SR, %)
L02
Cpd.
IC50, μM
IC50, μM
(SR, %)
(SR, %)
(SR, %)
c
c
S06-1001
S06-1011
S06-1014
S06-1015
S06-1031
35.10 40.22
91.50 10.50
33.45 9.90
59.79%
69.72%
82.90%
55.66%
17.28 9.70
84.31 22.8
5.05 1.24
61.49%
70.64%
73.52%
86.55%
c
c
c
62.19%
40.84%
62.37%
b
b
c
c
44.44%
52.21%
c
c
c
c
c
73.24%
c
c
c
c
c
82.67%
58.34%
94.23%
75.46%
87.01%
a
b
c
d
SR, survival rate at 2.5 μM or 100 μM. All tests were expressed as the mean SEM of three independent experiments.
with IC50 values less than 100 nM against eqBChE were
subsequently evaluated for their inhibition against hBChE
(Table 1). As a result, four compounds, namely, S06-1011, S06-
1014, S06-1015, and S06-1031, exhibited target inhibition
values of less than 50 nM.
Finally, to explore the possibility of introducing substituents
on benzimidazole, we fixed Moiety C as bicyclic rings (quinoline
or naphthalene ring) and synthesized compounds S06-1043 to
S06-1045 following the procedure shown in Scheme 4. Due to
the commercial unavailability of substituted (2-benzimidazolyl)-
acetonitrile, we synthesized 43a with substituted 1,2-diamino-
benzene and ethyl cyanoacetate as reactants. 43a reacted with
sodium nitrite and then hydroxylamine hydrochloride to afford
43c. Terminal compounds S06-1043 to S06-1045 were finally
provided with 43c and 2a, 11a, or 14a.
From the results in Table 1, compounds containing
substituents on benzimidazole presented different inhibitory
activities due to the difference in Moiety C. The structures of
Moiety C could lead to variation in the orientation of the overall
configuration. We subsequently conducted molecular docking
(Supporting Information, Figure S3) and MD simulations
(Figure 3, Supporting Information, Figure S4) to explain this
phenomenon. Although all of the polyaromatic moieties
(Moiety A) formed π−π interactions with Trp82, the overall
compounds displayed diverse orientations induced by single-
bond rotation. When Moiety C was fixed as a naphthalene ring
(compound S06-1014), the 5,6-position of the benzimidazole
faced the solvent region, and its substitution exerted little effect
on the activity. However, other structures of Moiety C can cause
the 5,6-position of the benzimidazole to insert itself into a
subpocket (Figure 3A,B). Certain steric hindrances led to a great
decrease and loss of the BChE-inhibiting ability. Based on these
findings, structural modification of the benzimidazole is not
preferred.
After four rounds of structural modification and SAR
discussion, four compounds, namely, S06-1011, S06-1014,
S06-1015, and S06-1031 with high activity and supreme
selectivity regarding the target level, were concluded to be
representative BChE inhibitors for further evaluation of safety
and efficacy in vitro and in vivo.
2.2. Safety Investigations. In vitro potential cytotoxicity of
lead and active compounds was evaluated through the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay on
diverse types of neurocytes, PC12, SH-SY5Y, and BV2 cell
lines.42 Except for S06-1014, which was toxic to PC12 (40.84%
cell viability under 2.5 μM) and SH-SY5Y (44.44% cell viability
under 2.5 μM) cell lines, most of the active compounds were
proven to lead to weak or no toxicity (Table 2). These results
indicated that three active compounds (S06-1011, S06-1015,
and S06-1031) exhibited sufficient in vitro neurosafety and
could be further tested for their neuroprotective effects.
Subsequently, acute toxicity tests for active compounds S06-
1011, S06-1015, and S06-1031 were carried out in healthy ICR
mice at the single-dose level. No death occurred after intragastric
administration of compounds even at a relatively high dose of 1
g/kg. Mice in the drug administration groups (male and female)
grew normally. The body weights of the mice gradually
increased (Figure 4A), and no significant behavioral abnormal-
ities were observed over the 14 day period.
The toxicity to the liver was evaluated by monitoring in vitro
antiproliferative effects against hepatocytes (Table 2), the levels
of aspartate aminotransferase (AST) and alanine amino-
transferase (ALT) (Figure 4B), and hematoxylin−eosin (HE)
staining (Figure 4C). Except for S06-1014, compounds
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Figure 4. (A) Body weight of ICR mice (g)-time (day). (B) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities at time
points (8, 22, and 36 h) after the administration of vehicle only (blank control) or compounds S06-1011, S06-1015, and S06-1031. (C)
Histomorphological appearances of the liver of male mice after treatment with the vehicle only (blank control) or 22 h after administration of
compounds S06-1011, S06-1015, and S06-1031. Hematoxylin−eosin (HE) staining, original magnification ×100.
presented weak cytotoxicity against HepG2 or L02 cell lines.
Due to toxicity to some cell lines, S06-1014 was no longer used
as a representative for further studies. To ensure the hepatic
safety of S06-1011, S06-1015, and S06-1031, we next
investigated drug-induced hepatotoxicity. Heparinized serum
was collected at different time points (8, 22, and 36 h) after the
administration of normal saline (as a control) or compounds
S06-1011, S06-1015, and S06-1031. Compared to the blank-
control group, the three compounds did not show any
remarkable damage at any of the time points. Afterward,
morphological studies by immunohistochemical staining were
applied to assess the hepatotoxicities of S06-1011, S06-1015,
and S06-1031. Treatment with S06-1011, S06-1015, or S06-
1031 did not present obvious morphological changes in the liver
compared to the control group. Taken together, all three
compounds exhibited high safety for the center and periphery.
2.3. PK Evaluation and BBB Penetration. The metabolic
stabilities in the liver microsomes of S06-1011, S06-1015, and
S06-1031 were evaluated using the long-acting drug ketanserin
as a reference control. As expected, all three compounds were
stable in the presence of human liver microsomes (Figure 5A,B).
The intrinsic human microsome clearances for the active
compounds S06-1011, S06-1015, and S06-1031 were lower
than that for the positive control ketanserin, showing longer half-
lives. Furthermore, compound S06-1011 with the longest T1/2
in human liver microsomes was selected for in vitro metabolic
pathway analysis and in vivo pharmacokinetic (PK) evaluation.
After compound S06-1011 was incubated with liver microsomes
in vitro, two possible forms of metabolites were identified in the
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Figure 5. Human liver microsomal stability and metabolite analysis. (A) Percentage of remaining compounds at different time points. (B) Parameters
of liver microsomal stability. All data are the mean value of two independent experiments. (C) Possible biotransformation pathway of the compound
S06-1011 in the liver.
samples: mono-oxidation metabolites and double-oxidation
metabolites. The identification information is shown in Table
S1 (Supporting Information), and the metabolic pathway is
presented in Figure 5C. The metabolites all retain the pivotal
four-ring skeleton of benzimidazole-oxadiazole. Hence, we
considered that S06-1011 could exert its pharmacological
effects for an extended time.
Table 3. SD Rat PK Profiles of Compound S06-1011
a
parameter
10 mg/kg (p.o.)
10 mg/kg (i.v.)
AUC0‑t (ng h/mL)
AUC0‑inf (ng h/mL)
MRTinf (h)
627.52 45.35
661.74 65.66
11.56 1.89
5.47 1.59
859.64 81.54
1092.11 307.19
14.14 12.26
8.87 11.09
t
1/2 (h)
T
max (h)
9.50 2.07
0.017 0.00
The PK profiles (Table 3) in the male SD rat model showed
that compound S06-1011 was slowly absorbed with a Tmax value
of 9.5 h and a favorable half-life of 5.47 h following a single-dose
oral administration (10 mg/kg). Notably, compound S06-1011
enters the enterohepatic circulation (Supporting Information,
Figure S5), which means that it is excreted into the intestine via
bile and reabsorbed in the intestine. The overall PK property of
compound S06-1011 was desirable, as approved by its high
bioavailability (Foral = 73.37 0.06%). The results indicated that
compound S06-1011 was a potential drug candidate and
provided a solid basis for the further evaluation of in vitro and in
vivo biological activities.
As compounds were exploited for treating neurodegenerative
diseases, we performed a parallel artificial membrane permeation
assay of the blood−brain barrier (PAMPA−BBB) to assay the
BBB penetration potential of compounds S06-1011, S06-1015,
and S06-1031.43 According to the tested permeability (Figure 6,
C
F (%)
max (ng/mL)
44.73 3.40
488.33 32.32
73.37 0.06%
a
PK parameters in Sprague−Dawley rats (male). All data are the
mean SD of eight independent experiments. Abbreviations: AUC,
area under the concentration−time curve; MRT, mean residence
time; t1/2, time required for the concentration to diminish by a half;
T
max, time to reach Cmax; Cmax, peak plasma concentration of a drug
after administration.
Supporting Information, Figure S6), compounds S06-1015 and
S06-1031 presented a possibility for BBB penetration in vitro,
while it was uncertain whether or not compound S06-1011
penetrates the BBB and reached the biological targets. Hence,
we assayed the brain distribution of compound S06-1011 in
mice. The results (Figure 6) showed that although the brain
exposure was relatively low, it was stable, indicating that this
compound could be stabilized in the brain to exert its efficacy. As
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Figure 6. (A) PAMPA−BBB assay results and predicted penetration of active compounds S06-1011, S06-1015, and S06-1031 in the CNS. (B) BBB
exposure at several time points after oral administration of S06-1011. KD, ratio of plasma exposure to brain exposure. (C) Parameters of tissue
distribution. Abbreviations: Cmax, peak exposure of S06-1011 after administration; AUC, area under the concentration−time curve.
calculated by AUC(B) vs AUC(P), S06-1011 was able to partially
penetrate the BBB and enter the CNS.
neuronal death induced by Aβ1−42 in the absence or presence of
diverse concentrations of compounds S06-1011, S06-1015, and
S06-1031, an MTT assay was performed. As exhibited in Figure
7C, incubation of SH-SY5Y cells with 15 μM Aβ1−42 caused
pronounced cytotoxicity. To our delight, compounds S06-1011
and S06-1031 exhibited remarkable neuroprotective effects at a
relatively low concentration of 2.5 μM. All three representative
compounds presented concentration-dependent neuroprotec-
tive properties. Notably, at 10 μM, compound S06-1031 almost
protected SH-SY5Y cells from Aβ1−42 peptide toxicity.
ROS are a series of biological molecules produced by aerobic
organisms that maintain equilibrium and are important in
normal cell signaling under physiological conditions.44,53
However, it has been observed that the antioxidant mechanism
balance in the postmortem brains of AD patients could be
interrupted by aberrantly increased ROS levels. Hence, we
assessed the preliminary anti-inflammatory profiles of the best
molecules, namely, S06-1011, S06-1015, and S06-1031, by
examining the level of released ROS in the mouse neuroglial
BV2 cell line. A BV2 cell model with high ROS release
stimulated by lipopolysaccharides (LPS) (2.5 μg/mL) was
established.54 Compounds S06-1011, S06-1015, and S06-1031
were evaluated to pronouncedly hinder the increase of LPS-
induced ROS production without affecting the morphology of
BV2 cells, indicating their abilities to reduce oxidative stress
(Figure 7D, Supporting Information, Figure S7). The ROS
reduction capacities of S06-1011 and S06-1031 were better
than that of the lead compound S06-1001.
2.4. In Vitro Neuroprotection in SH-SY5Y Cells.
Neuroprotection by combating oxidative stress has long been
identified to be an important strategy for AD treatment.44
Hence, we next evaluated the cytoprotective effects of optimal
compounds against H2O2-, glutamate-, and Aβ1−42-induced cell
damage with the methyl thiazolyl tetrazolium (MTT) assay in
SH-SY5Y cells, three well-established cellular models of
neurodegenerative disorders.45−47 The addition of H2O2 (200
μM) to the growth media caused significant cell death by
reducing the cell viability (62.36%). To assess the neuro-
protective potential of the test compounds, cells were pretreated
for 2 h with compounds S06-1011, S06-1015, and S06-1031
(2.5 and 10 μM) prior to H2O2 insult for 24 h. As shown in
Figure 7A, the selected derivatives S06-1011 and S06-1031
exhibited pronounced neuroprotective effects at concentrations
of 2.5 and 10 μM in a dose-dependent manner.
Glutamate excitotoxicity is responsible for the pathology of
chronic disorders. Exposure to glutamate results in lowered
glutathione levels, leading to oxidative stress and cell death. As
shown in Figure 7B, the addition of glutamate (20 mM) elicited
a significant decline in cell viability (∼50%) compared to the
control. Preincubation with compounds S06-1011 and S06-
1031 moderately rescued SH-SY5Y cells insulted by glutamate
at a concentration of 10 μM.
The Aβ hypothesis has increasingly been proven to play a
pivotal role in AD pathology.48 Abnormal Aβ plaques can induce
a series of nerve injuries. It has been reported that Aβ
aggregation is related to the production of reactive oxygen
species (ROS), mitochondrial dysfunction, local microglial
activation, cytokine release, and multiprotein inflammation,
which are all harmful for central function.49−52 To compare
2.5. In Vivo Behavioral Assays. To determine whether
treatment with the representative compound S06-1011, S06-
1015, or S06-1031 could improve scopolamine-induced acute
memory impairment, we performed the Morris water maze test,
which has been extensively used to evaluate potential
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Figure 7. (A−C) Neuroprotective effects of compounds S06-1001, S06-1011, S06-1015, and S06-1031 against H2O2-, glutamate- and Aβ1−42
-
induced cell insults on the SH-SY5Y cell line. SH-SY5Y cells were preincubated with 2.5 and 10 μM of the test compounds (S06-1011, S06-1015, and
S06-1031) for 2 h and subsequently exposed to H2O2 (200 μM) (A), glutamate (20 mM) (B), or Aβ1−42 (15 μM) (C) for 24 h. The cell viability was
measured using an MTT assay. The data are expressed as mean SEM of three independent experiments (####p < 0.0001 vs the control group; *p <
0.05, ***p < 0.001, ****p < 0.0001 vs the model group). (D) Detection of reactive oxygen species (ROS) production in BV2 cells using fluorescence
microscopy analysis. The amount of intracellular ROS production was expressed by green fluorescence intensity (GFI). To avoid errors in the
fluorescence brightness, the exposure time was fixed at 10.2 ms.
Figure 8. (A, B) Effects of compounds on scopolamine-induced AD-like cognitive deficits in ICR mice. (A) Escape latency to the platform in Morris
water maze. (B) Average tracks of mice in Morris water maze. Data are presented as the mean SD (n = 8; ###p < 0.001 vs the control group; *p < 0.05,
**p < 0.01, ***p < 0.001, ****p < 0.0001 vs the scopolamine model group). (C−E) Effects of compounds on Aβ1−42 peptide-induced AD-like
cognitive deficits in ICR mice. (C) Escape latency to the platform in the Morris water maze. (D) Average tracks of mice in the Morris water maze. (E)
Spontaneous alternation performances in the Y maze test. Data are presented as the mean SD (n = 8; ##p < 0.01, ###p < 0.001 vs the sham-operation
group; **p < 0.01, ***p < 0.001, ****p < 0.0001 vs the model group).
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Figure 9. (A) Everyday body weight of ICR mice in 14 days. Data are presented as the mean (n = 8). (B) Body-weight changes in 14 days. Data are
presented as the mean SD (n = 8; ****p < 0.0001 vs the Aβ1−42 model group). (C) Ghrelin level in mice plasma. Data are presented as the mean
SD (n = 8; ****p < 0.0001 vs the control group).
therapeutic agents for treating AD. Memory training and
learning were conducted for 5 days, and a probe trial was
performed on the sixth day (Supporting Information, Figure
S8). The results are presented in Figure 8. Compared to the
control group, the spatial memory defects for mice treated with
scopolamine alone (3 mg/kg, model group) were significantly
shown to have a longer latency (37.45 vs 13.30 s) and a more
chaotic trajectory to the target. As a positive control group,
treatment with tacrine contributed to a simplified trajectory and
a shorter latency to the platform (6.48 vs 37.45 s). Furthermore,
compounds S06-1011, S06-1015, and S06-1031 significantly
attenuated scopolamine-induced cognitive impairment at a dose
of 30 mg/kg. Although the optimal compound S06-1011
presented a considerably short latency to the platform and a
simplified trajectory, its cognitive and memory improvement
effect was still inferior to that of tacrine at a low dose (15 mg/
kg).
compounds enhanced the body weight of mice, presenting
significant differences from the model group. Especially for the
mice in the S06-1011 and S06-1031 groups, there was an
obvious difference in weight gain compared with the donepezil
group (p < 0.01). To further corroborate this phenomenon, we
evaluated the plasma ghrelin levels of mice in the control and
S06-1011 groups. As a result, the ghrelin level of mice in the
S06-1011 group displayed a remarkable increase. Hence, we
concluded that inhibition of BChE led to an enhanced ghrelin
level, which helped improve mental mood and increase appetite.
This can not only play a protective role against dementia but also
exert a great influence on treatment and nursing care.
Overall, three optimal compounds showed good effects in
both investigated behavioral studies, successfully improving the
spatial exploration ability, cognition, and memory. Together
with in vitro neuroprotective effects, compounds S06-1011 and
S06-1031 were identified as effective candidates for BChE
inhibition and AD treatment.
To more closely investigate the anti-AD effects of compounds,
we carried out an accepted Aβ1−42-induced cognitive-impaired
model.55,56 The AD-like cognitive impairment mice model was
established by intracerebroventricular (icv) injection of the
oligomerized Aβ1−42 peptide (10 μg) on day 1 (Supporting
Information, Figure S8). Meanwhile, the same volume of saline
was icv-injected for the sham-operation group. Donepezil, which
could inhibit the formation of Aβ deposits and attenuate neural
toxicity of Aβ oligomers, contributing to cognition improve-
ment, was taken as a positive control. Donepezil, S06-1011,
S06-1015, and S06-1031 were orally administered from days 3
to 14. Behavioral studies were conducted using a Morris water
maze and a Y maze. As shown in Figure 8, no remarkable
differences in cognitive and learning capacities were observed
between the sham-operation group and the blank control group,
in terms of indistinctive latency to target, trajectory confusion,
and alternation performance. Treatment with donepezil
significantly shortened the latency, simplified the tracks to the
target, and improved alternation performance. Moreover,
intragastric administration of compounds S06-1011, S06-
1015, and S06-1031 at an equivalent (15 mg/kg) dose to
donepezil entirely prevented the impact of toxic Aβ1−42
oligomers. In particular, S06-1011 presented comparable
effectiveness to the positive control donepezil in the Morris
water maze test, reduced the target latency (51.98 to 13.83 s),
and simplified the trajectory. Compound S06-1031 showed the
most enhanced alternation performance in the Y maze test
(44.64% to 68.67%).
3. CONCLUSIONS
In our previous work, we disclosed a hierarchical structure-based
virtual screening workflow to identify a novel potent and
selective anti-BChE lead compound S06-1001 (eqBChE IC50
=
0.177 0.029 μM, hBChE IC50 = 0.322 0.073 μM). Based on
molecular binding and dynamics results, 44 derivatives were
synthesized and assayed for their BChE inhibitory efficacy,
among which four compounds (S06-1011, S06-1014, S06-
1015, and S06-1031) showed IC50 values lower than 50 nM
against human BChE. Furthermore, compounds S06-1011,
S06-1015, and S06-1031 were noncytotoxic in vitro and well-
tolerated in vivo at doses up to 1 g/kg, inducing no liver side
effects. Three compounds could penetrate the BBB and take
effect in the CNS. In addition, all three compounds presented
considerable pharmacokinetic properties, a long t1/2, and low
liver microsome clearances. Remarkably, the long-acting
compound S06-1011 showed a high oral bioavailability
(73.37%).
From the perspective of pharmacodynamics, the optimal
candidate compounds S06-1011 and S06-1031 exhibited
potent antioxidant activity and neuroprotective effects against
H2O2-, glutamate-, and Aβ1−42-mediated SH-SY5Y neuro-
toxicity. Moreover, three compounds reduced the production
of ROS induced by LPS at a concentration of 5 μM. Notably, the
BChE inhibitors S06-1011, S06-1015, and S06-1031 were
shown to improve cognitive and learning abilities in scopol-
amine- and Aβ1−42 peptide-induced cognitive deficit models.
This result demonstrated that the identified BChE inhibitors not
only improved memory impairments by symptomatically
Additionally, none of the treatments (icv for peptide or po for
compounds) resulted in a great loss of mouse body weight
during the therapy period, presenting good tolerability of the
test compounds (Figure 9). Instead, treatments with the
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vacuum to give the crude product, which was further purified by column
chromatography on silica gel (DCM/MeOH = 50:1, v/v) to obtain the
products S06-1003 to S06-1005.
Compound S06-1006 was obtained by reduction of S06-1005.
Compound S06-1005 was dissolved in 10 mL of THF, followed by
addition of palladium 10% on carbon and N2H4·H2O at 0 °C. The
reaction was refluxed and completed by a TLC monitor. The reaction
was filtered, and the filtrate was evaporated under a vacuum and purified
by column chromatography on silica gel (DCM/MeOH = 30:1, v/v) to
obtain the desired product S06-1006.
rescuing the cholinergic function but also protected the neural
environment from Aβ oligomers and oxidative insults, which
might be a potential strategy of BChE inhibition for AD
treatment. Moreover, BChE inhibition by compound admin-
istration can increase the level of blood ghrelin, contributing to a
significant increase in body weight by improving appetite. This
important finding also indicates that inhibiting BChE can not
only improve the neurological environment and cognitive
impairment but also provide great convenience for caregivers.
4.1.2.1. 2-(2-(Furan-2-yl)-1H-benzo[d]imidazol-1-yl)-N-(quinolin-
6-yl) Acetamide (S06-1003). According to the general procedure
described above, compound S06-1003 was synthesized with 6-
aminoquinoline and 3c. The product S06-1003 was further purified
by column chromatography (DCM/MeOH = 50:1, v/v), with a yield of
4. EXPERIMENTAL SECTION
4.1. Chemistry. 4.1.1. General Chemistry. Common reagents and
solvents were purchased from commercial suppliers and used without
further purification. Melting points were determined on a Mel-TEMP II
melting point apparatus. 1H and 13C NMR spectra were recorded with a
Bruker Avance 300 and a 100 MHz spectrometer at 300 K, using TMS
as an internal standard in dimethyl sulfoxide (DMSO)-d6. Chemical
shifts (δ) are reported in parts per million (ppm), and coupling
constants (J) are reported in hertz (Hz). MS spectra were recorded on
an Agilent LC-MS 6120 instrument with an electrospray ionization
(ESI) mass selective detector in the positive ion mode. All reaction
progresses were monitored by analytical thin-layer chromatography
(TLC) on precoated silica gel GF254 plates under UV light at 254 and
365 nm or through staining with iodine. Compounds were purified with
flash column chromatography using silica gel (100−300 mesh)
purchased from Qingdao Haiyang Chemical Co. Ltd. (China) as the
stationary phase and petroleum ether/ethyl acetate or dichloromethane
(DCM)/methanol mixtures as eluent systems. Purity for final
compounds (higher than 95%) was measured by high-performance
liquid chromatography (HPLC) with an Agilent Technologies 1260
infinity C18 4.60 mm × 150 mm × 5 μm column, eluted with a mixture
of solvent methanol/distilled water [0.1% formic acid] at the flow rate
of 1.0 mL/min.
4.1.2. General Synthetic Procedure for Compounds S06-1002 to
S06-1006. o-Phenylenediamine (1 mmoL) and benzaldehyde or furan-
3-carbaldehyde or 2-nitrobenzaldehyde (1 mmoL) were dissolved in 10
mL of DMF, followed by addition of sodium bisulfite (0.3 mmoL). The
reaction was stirred at 80 °C for 5 h, and its completion was monitored
by TLC. Thereafter, the solvent was dripped into stirred water (15 mL)
to precipitate a large amount of solid. The crude products 3a−5a were
obtained by filtration and used for subsequent reaction without
purification.
The products 3a−5a (1 mmoL) obtained in the last step were
dissolved in 50 mL of acetonitrile, followed by addition of cesium
carbonate (2 mmoL). The mixture was stirred for 15 min, and then
ethyl carbonobromidate (2.5 mmoL) was added dropwise. The
reaction was stirred at room temperature for another 30 min and
completed by thin-layer chromatography (TLC). The solvent was
evaporated under a vacuum to give the crude solid product.
Subsequently, the solid was again dissolved in 50 mL of DCM and
then washed with saturated sodium bicarbonate (aq, 3 × 50 mL). The
organic layer was then dried over sodium sulfate and filtered. The
solvent was evaporated under a vacuum to give the crude product,
which was further purified by column chromatography on silica gel
(DCM/MeOH = 100:1, v/v) to obtain the desired products 3b−5b.
3b−5b (1 mmoL) were dissolved in a 12 mL mixed solvent of THF/
MeOH (3:1, v/v). Later, 12 mL of 1 N LiOH·H2O (aq) was dropped
into the reaction solvent at 0 °C. The reaction was stirred at room
temperature overnight and evaporated under a vacuum to remove the
organic layer. The solvent was adjusted to pH = 2 with 9 M
hydrochloric acid and filtered to give the crude products 3c−5c.
6-Aminoquinoline (1 mmoL) was dissolved in 10 mL of DMF,
followed by addition of N,N-diisopropylethylamine (DIEA, 1 mmoL).
The solvent was stirred at room temperature for 15 min before addition
of 3c−5c (1 mmoL) and HATU (1 mmoL). The reaction was then
stirred overnight at room temperature. Thereafter, 25 mL of water and
25 mL of ethyl acetate were added to the solution and the water phase
was washed by 25 mL of ethyl acetate. The organic layer was collected
and dried over sodium sulfate. The solvent was evaporated under a
1
80%, mp 238−241 °C, 95.4% HPLC purity. H NMR (300 MHz,
DMSO-d6): δ = 10.93 (s, 1H), 8.82 (dd, J = 4.2, 1.7 Hz, 1H), 8.36 (d, J =
2.3 Hz, 1H), 8.28 (m, 1H), 8.04 (d, J = 9.1 Hz, 1H), 7.96 (d, J = 1.7 Hz,
1H), 7.86 (dd, J = 9.1, 2.4 Hz, 1H), 7.71 (qd, J = 4.1, 1.3 Hz, 2H), 7.50
(dd, J = 8.3, 4.2 Hz, 1H), 7.32 (m, 2H), 7.25 (d, J = 3.5 Hz, 1H), 6.76
(dd, J = 3.5, 1.8 Hz, 1H), 5.53 (s, 2H) ppm. 13C NMR (125 MHz,
DMSO-d6): δ = 166.6, 149.7, 145.7, 145.4, 145.3, 144.7, 142.9, 137.0,
136.8, 136.0, 130.2, 128.8, 123.7, 123.3, 122.9, 122.3, 119.4, 115.7,
112.9, 112.6, 110.9, 48.1 ppm. HRMS (m/z): calcd for C22H16N4O2 [M
+ H]+ 369.1346; found, 369.1354.
4.1.2.2. 2-(2-Phenyl-1H-benzo[d]imidazol-1-yl)-N-(quinolin-6-yl)
Acetamide (S06-1004). According to the general procedure described
above, compound S06-1004 was synthesized with 6-aminoquinoline
and 4c. The product S06-1004 was further purified by column
chromatography (DCM/MeOH = 50:1, v/v), with a yield of 87%, mp
256−259.5 °C, 95.0% HPLC purity. 1H NMR (300 MHz, DMSO-d6):
δ = 10.89 (s, 1H), 8.82 (dd, J = 4.2, 1.7 Hz, 1H), 8.38 (d, J = 2.3 Hz,
1H), 8.30 (m, 1H), 8.03 (d, J = 9.0 Hz, 1H), 7.84 (td, J = 6.7, 5.9, 3.7
Hz, 3H), 7.76 (m, 1H), 7.61 (m, 4H), 7.50 (dd, J = 8.3, 4.2 Hz, 1H),
7.35 (m, 2H), 5.26 (s, 2H) ppm. 13C NMR (125 MHz, DMSO-d6): δ =
166.6, 154.1, 149.8, 145.3, 143.0, 137.0, 136.8, 136.0, 130.6, 130.3,
130.2, 129.6, 129.3, 128.7, 123.7, 123.2, 122.7, 122.4, 119.6, 115.9,
111.1, 48.2 ppm. HRMS (m/z): calcd for C24H18N4O [M + H]+
379.1551; found, 379.1559.
4.1.2.3. 2-(2-(2-Nitrophenyl)-1H-benzo[d]imidazol-1-yl)-N-(qui-
nolin-6-yl) Acetamide (S06-1005). According to the general
procedure described above, compound S06-1005 was synthesized
with 6-aminoquinoline and 5c. The product S06-1005 was further
purified by column chromatography (DCM/MeOH = 45:1, v/v), with
a yield of 84%, mp 246−249.5 °C, 98.8% HPLC purity. 1H NMR (300
MHz, DMSO-d6): δ = 10.72 (s, 1H), 8.80 (dd, J = 4.3, 1.7 Hz, 1H), 8.32
(d, J = 2.4 Hz, 1H), 8.28 (ddd, J = 7.8, 6.0, 1.5 Hz, 2H), 8.00 (d, J = 9.0
Hz, 1H), 7.92 (td, J = 7.5, 1.3 Hz, 1H), 7.85 (m, 2H), 7.75 (dd, J = 9.1,
2.4 Hz, 1H), 7.71 (d, J = 7.9 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.49 (dd,
J = 8.3, 4.2 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H),
5.13 (s, 2H) ppm. 13C NMR (125 MHz, DMSO-d6): δ = 166.2, 149.9,
149.5, 149.3, 144.4, 142.7, 137.0, 136.9, 136.0, 134.2, 132.9, 132.2,
129.5, 128.8, 125.3, 125.0, 124.1, 123.6, 122.8, 122.4, 119.7, 115.9,
111.3, 47.7 ppm. HRMS (m/z): calcd for C24H17N5O3 [M + H]+
424.1404; found, 424.1421.
4.1.2.4. 2-(2-(2-Aminophenyl)-1H-benzo[d]imidazol-1-yl)-N-(qui-
nolin-6-yl) Acetamide (S06-1006). According to the general
procedure described above, compound S06-1006 was synthesized by
reduction of S06-1005. The product S06-1006 was further purified by
column chromatography (DCM/MeOH = 30:1, v/v), with a yield of
1
60%, mp 240−242.5 °C, 95.9% HPLC purity. H NMR (300 MHz,
DMSO-d6): δ = 10.91 (d, J = 6.4 Hz, 1H), 8.82 (dd, J = 4.2, 1.7 Hz, 1H),
8.38 (d, J = 2.4 Hz, 1H), 8.30 (dd, J = 8.5, 1.8 Hz, 1H), 8.03 (d, J = 9.1
Hz, 1H), 7.85 (m, 1H), 7.75 (dd, J = 6.2, 2.9 Hz, 1H), 7.61 (m, 1H),
7.50 (dd, J = 8.4, 4.2 Hz, 1H), 7.33 (m, 3H), 7.23 (m, 1H), 6.90 (d, J =
8.2 Hz, 1H), 6.70 (m, 1H), 5.84 (s, 2H), 5.17 (s, 2H) ppm. 13C NMR
(125 MHz, DMSO-d6): δ = 166.7, 153.1, 149.8, 148.4, 145.3, 142.8,
136.8, 136.3, 136.0, 131.1, 130.4, 130.2, 128.8, 123.7, 122.9, 122.5,
122.3, 119.2, 116.3, 116.2, 115.9, 112.7, 111.1, 48.2 ppm. HRMS (m/
z): calcd for C24H19N5O [M + H]+ 394.1662; found, 394.1675.
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Article
4.1.3. General Synthetic Procedure for Compounds S06-1002,
S06-1007, and S06-1008. 6-Aminoquinoline (1 mmoL) was diluted
in chloroform (4 mL), and sodium bicarbonate (1.2 mmoL) was added.
The mixture was stirred at room temperature for 15 min before
chloroacetyl chloride (1.5 mmoL) was dropped in. The reaction was
stirred at room temperature for 4 h and then monitored by TLC. The
solution was diluted with chloroform (10 mL) and washed with water
(2 × 15 mL), dried over sodium sulfate, and filtered. The solvent was
evaporated under a vacuum to give the crude product 2a, which was
used for subsequent reaction without purification. 7a and 8a were
synthesized with 3-chloropropionyl chloride and 5-chlorovaleryl
chloride replacing chloroacetyl chloride.
4.1.4. General Synthetic Procedure for Compounds S06-1009 to
S06-1042. The synthesis approaches of compounds S06-1009 to S06-
1042 were similar to that of S06-1002, replacing the initial reactant 6-
aminoquinoline with diverse commercially available aromatic amines.
Similar to the compound S06-1006, S06-1033 was obtained by
reduction of S06-1032 under the same reaction condition.
4.1.4.1. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(quinolin-5-yl)acetamide (S06-1009). According
to the general procedure described above, compound S06-1009 was
synthesized with 9a and 1b. The product S06-1009 was further purified
by column chromatography (DCM/MeOH = 70:1, v/v) with a yield of
1
48%, mp 295−298.5 °C, 95.1% HPLC purity. H NMR (300 MHz,
2-Benzimidazolylacetonitrile (1 mmoL) was dissolved with acetic
acid (3 mL), followed by dropping sodium nitrite (1 mmoL) in water
under an ice bath. The mixture was stirred under an ice bath for 40 min,
filtered, and washed with ice-water once and ether twice. In another
reactive flask, to an ice-cooled stirred solution of hydroxylamine
hydrochloride (1.2 mmoL) in 1.5 mL of water, potassium hydroxide
(1.5 mmoL) was slowly added. Then, 1 mL of diglyme and the above
residue were added. The ice bath was removed, and the reaction
mixture was heated to 110 °C for 6 h. After cooling to room
temperature, the reaction mixture was filtered and the residue was
washed with water to give the product 1b as a pale-yellow solid.
Compound 2a, 7a, or 8a (1 mmoL) was dissolved in DMF (12 mL).
Cesium carbonate (1 mmoL) and intermediate compound 1b (0.9
mmoL) were successively added. The solution was stirred at 70 °C for 9
h, and the completion of the reaction was monitored by TLC. Then, 80
mL of water was added into the reaction to produce a large amount of
precipitation. The mixture was filtered and the residue was washed with
water and methanol to produce the desired products S06-1002, S06-
1007, and S06-1008 as a yellow solid.
DMSO-d6): δ = 10.86 (s, 1H), 8.78 (d, J = 5.01 Hz, 1H), 8.52 (d, J =
8.25 Hz, 1H), 8.04 (d, J = 7.77 Hz, 1H), 7.97 (d, J = 5.01 Hz, 1H), 7.90
(d, J = 8.61 Hz, 2H), 7.85−7.80 (m, 1H), 7.76−7.71 (m, 1H), 7.49−
7.38 (m, 2H), 7.03 (s, 2H), 5.85 (s, 2H) ppm. 13C NMR (125 MHz,
DMSO-d6): δ = 166.0, 156.5, 149.7, 145.2, 142.0, 141.9, 138.8, 136.9,
136.6, 136.0, 130.2, 128.8, 125.2, 123.7, 123.5, 122.3, 120.4, 155.6,
111.7, 48.7 ppm. HRMS (m/z): calcd for C20H15N7O2 [M + H]+
386.1360; found, 386.1369.
4.1.4.2. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(quinolin-4-yl)acetamide (S06-1010). According
to the general procedure described above, compound S06-1010 was
synthesized with 10a and 1b. The product S06-1010 was further
purified by column chromatography (DCM/MeOH = 50:1, v/v) with a
1
yield of 32%, mp 289−291 °C, 95.6% HPLC purity. H NMR (300
MHz, DMSO-d6): δ = 10.86 (s, 1H), 8.77 (d, J = 4.98 Hz, 1H), 8.51 (d,
J = 8.19 Hz, 1H), 8.04 (d, J = 8.40 Hz, 1H), 7.96 (d, J = 5.01 Hz, 1H),
7.89 (d, J = 8.49 Hz, 2H), 7.85−7.80 (m, 1H), 7.65 (t, J = 7.25 Hz, 1H,
ArH), 7.47−7.40 (m, 2H), 7.01 (s, 2H), 5.85 (s, 2H) ppm. 13C NMR
(125 MHz, DMSO-d6): δ = 167.3, 156.5, 155.9, 151.3, 149.1, 142.0,
141.8, 141.5, 138.8, 136.5, 130.1, 130.0, 126.6, 125.2, 123.8, 122.8,
121.2, 120.5, 111.6, 49.0 ppm. HRMS (m/z): calcd for C20H15N7O2 [M
+ H]+ 386.1360; found, 386.1362.
4.1.3.1. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(quinolin-6-yl)acetamide (S06-1002). According
to the general procedure described above, compound S06-1002 was
synthesized with 2a and 1b. The product S06-1002 was further purified
by column chromatography (DCM/MeOH = 70:1, v/v) with a yield of
4.1.4.3. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(quinolin-3-yl)acetamide (S06-1011). According
to the general procedure described above, compound S06-1011 was
synthesized with 11a and 1b, with a yield of 82%, mp 293−294 °C,
1
70%, mp 282−284 °C, 96.2% HPLC purity. H NMR (300 MHz,
DMSO-d6): δ = 10.95 (s, 1H), 8.80 (s, 1H), 8.33 (s, 1H), 8.24 (d, J =
8.34 Hz, 1H), 8.02 (d, J = 9.39 Hz, 1H), 7.91−7.81 (m, 3H) 7.49−7.38
(m, 3H), 7.05 (s, 2H), 5.69 (s, 2H) ppm. 13C NMR (125 MHz, DMSO-
d6): δ = 166.0, 156.5, 149.7, 145.2, 142.0, 141.9, 138.8, 136.9, 136.6,
136.0, 130.2, 128.8, 125.2, 123.7, 123.5, 122.3, 120.4, 115.6, 111.5, 48.7
ppm. HRMS (m/z): calcd for C20H15N7O2 [M + H]+ 386.1365; found,
386.1359.
1
96.7% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 11.12 (s,
1H), 8.97 (d, J = 2.43 Hz, 1H), 8.64 (d, J = 2.19 Hz, 1H), 7.93 (d, J =
8.28 Hz, 1H), 7.91−7.86 (m, 3H), 7.68−7.62 (m, 1H), 7.58−7.53 (m,
1H), 7.49−7.38 (m, 2H), 7.04 (s, 2H), 5.71 (s, 2H) ppm. 13C NMR
(125 MHz, DMSO-d6): δ = 166.6, 156.5, 144.8, 144.6, 142.0, 141.8,
138.8, 136.6, 132.8, 129.0, 128.5, 128.2, 128.2, 127.6, 125.2, 123.7,
122.7, 120.4, 111.7, 48.7 ppm. HRMS (m/z): calcd for C20H15N7O2 [M
+ H]+ 386.1360; found, 386.1365.
4.1.3.2. 3-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(quinolin-6-yl)propenamide (S06-1007). Accord-
ing to the general procedure described above, compound S06-1007 was
synthesized with 7a and 1b, with a yield of 39%, mp 235−237 °C, 98.7%
HPLC purity. 1H NMR (300 MHz, DMSO-d6): δ = 10.30 (s, 1H), 8.76
(dd, J = 1.56, 4.14 Hz, 1H), 8.25−8.21 (m, 2H), 7.91 (d, J = 9.06 Hz,
1H), 7.83 (dd, J = 8.13, 13.59 Hz, 2H), 7.65 (dd, J = 2.28, 9.06 Hz, 1H),
7.47 (q, J = 3.54 Hz, 1H), 7.40 (d, J = 7.26 Hz, 1H) 7.32 (t, J = 7.38 Hz,
1H), 7.01 (s, 2H), 5.02 (t, J = 6.66 Hz, 2H), 3.03 (t, J = 6.54 Hz, 2H).
13C NMR (125 MHz, DMSO-d6): δ = 169.4, 156.6, 149.5, 145.1, 142.1,
4.1.4.4. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(quinolin-2-yl)acetamide (S06-1012). According
to the general procedure described above, compound S06-1012 was
synthesized with 12a and 1b, with a yield of 52%, mp 249−250 °C,
1
98.2% HPLC purity. H NMR (300 MHz, DMSO-d6): δ =11.46 (s,
1H), 8.33 (d, J = 9.03 Hz, 1H), 8.10 (d, J = 8.76 Hz, 1H), 7.88 (dd, J =
9.21, 16.31 Hz, 3H), 7.74 (t, J = 8.28 Hz, 1H), 7.54−7.37 (m, 3H), 7.01
(s, 2H), 6.84 (s, 1H), 5.75 (s, 2H) ppm. 13C NMR (125 MHz, DMSO-
d6): δ = 167.2, 156.5, 151.6, 146.8, 142.0, 141.9, 139.1, 138.8, 136.5,
130.6, 128.3, 127.5, 126.1, 125.6, 125.2, 123.7, 120.4, 114.5, 111.6, 48.8
ppm. HRMS (m/z): calcd for C20H15N7O2 [M + H]+ 386.1360; found,
386.1361.
141.1, 138.7, 137.1, 135.9, 135.6, 129.9, 128.7, 125.0, 123.7, 123.6,
122.2, 120.3, 115.5, 112.0, 42.0, 36.9 ppm. HRMS (m/z): calcd for
C21H17N7O2 [M + H]+ 400.1522; found, 400.1523.
4.1.3.3. 5-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(quinolin-6-yl)pentanamide (S06-1008). Accord-
ing to the general procedure described above, compound S06-1008 was
synthesized with 8a and 1b, with a yield of 45%, mp 219.5−223 °C,
4.1.4.5. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(naphthalen-2-yl)acetamide (S06-1013). Accord-
ing to the general procedure described above, compound S06-1013 was
synthesized with 13a and 1b, with a yield of 78%, mp 300−303 °C,
1
98.6% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.25 (s,
1H), 8.78 (d, J = 3.60 Hz, 1H), 8.37 (s, 1H), 8.26 (d, J = 8.19 Hz, 1H),
7.95 (d, J = 8.91 Hz, 1H), 7.85 (d, J = 7.92 Hz, 2H), 7.76 (d, J = 9.12 Hz,
1H), 7.49−7.35 (m, 3H), 7.04 (s, 2H), 4.74 (t, J = 6.81 Hz, 2H), 2.44 (t,
J = 7.35 Hz, 2H), 1.96−1.86 (m, 2H), 1.76−1.66 (m, 2H) ppm. 13C
NMR (125 MHz, DMSO-d6): δ = 171.8, 156.6, 149.3, 145.1, 142.1,
140.9, 138.6, 137.5, 135.8, 135.7, 129.9, 128.8, 125.0, 123.7, 123.6,
122.1, 120.5, 115.2, 111.7, 45.3, 36.4, 29.5, 22.7 ppm. HRMS (m/z):
calcd for C23H21N7O2 [M + H]+ 428.1835; found, 428.1836.
1
97.0% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.81 (s,
1H), 8.25 (d, J = 1.65 Hz, 1H), 7.91−7.83 (m, 4H), 7.77 (d, J = 7.83 Hz,
1H), 7.61 (dd, J = 2.07, 8.85 Hz, 1H), 7.48−7.38 (m, 4H), 7.05 (s, 2H),
5.67 (s, 2H) ppm. 13C NMR (125 MHz, DMSO-d6): δ = 165.8, 156.5,
142.0, 141.9, 138.8, 136.7, 136.6, 133.8, 130.3, 129.0, 127.9, 127.8,
127.0, 125.2, 125.2, 123.7, 120.4, 120.2, 115.8, 111.7, 48.7 ppm. HRMS
(m/z): calcd for C21H16N6O2 [M + H]+ 385.1408; found, 385.1411.
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Article
4.1.4.6. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(naphthalen-1-yl)acetamide (S06-1014). Accord-
ing to the general procedure described above, compound S06-1014 was
synthesized with 14a and 1b, with a yield of 72%, mp 273−274 °C,
4.1.4.12. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(2-chlorophenyl)acetamide (S06-1020). According
to the general procedure described above, compound S06-1020 was
synthesized with 20a and 1b, with a yield of 66%, mp 284−288 °C,
1
1
95.1% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.09 (s,
95.3% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.47 (s,
1H), 7.84 (q, J = 7.53 Hz, 2H), 7.67 (dd, J = 1.59, 8.03 Hz, 1H), 7.53−
7.36 (m, 3H), 7.34−7.28 (m, 1H), 7.23−7.17 (m, 1H), 6.96 (s, 2H),
5.68 (s, H) ppm. 13C NMR (125 MHz, DMSO-d6): δ = 166.2, 156.5,
155.9, 142.0, 141.9, 140.7, 139.0, 138.7, 136.4, 134.9, 130.1, 127.9,
125.2, 123.7, 120.4, 111.5, 48.5 ppm. HRMS (m/z): calcd for
C17H13ClN6O2 [M + H]+ 369.0867; found, 369.0862.
1H), 8.23 (d, J = 7.59 Hz, 1H), 7.97−7.87 (m, 3H), 7.79 (d, J = 7.92 Hz,
1H), 7.54−7.65 (m, 3H), 7.48 (t, J = 7.68 Hz, 2H), 7.40 (t, J = 7.92 Hz,
2H) 6.99 (s, 2H), 5.78 (s, 2H) ppm. 13C NMR (125 MHz, DMSO-d6):
δ = 166.6, 156.6, 142.1, 142.0, 138.9, 136.6, 134.2, 133.5, 128.6, 128.4,
126.7, 126.4, 126.2, 126.0, 125.2, 123.7, 123.3, 122.3, 120.5, 111.6, 48.6
ppm. HRMS (m/z): calcd for C21H16N6O2 [M + H]+ 385.1413; found,
385.1411.
4.1.4.13. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(3-chlorophenyl)acetamide (S06-1021). According
to the general procedure described above, compound S06-1021 was
synthesized with 21a and 1b, with a yield of 72%, mp 283−285 °C,
4.1.4.7. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(4-bromonaphthalen-1-yl)acetamide (S06-1015).
According to the general procedure described above, compound S06-
1015 was synthesized with 15a and 1b, with a yield of 72%, mp 293−
1
98.9% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.80 (s,
1
1H), 7.88 (q, J = 7.89 Hz, 2H), 7.79 (s, 1H), 7.49−7.39 (m, 4H), 7.17
(d, J = 7.80 Hz, 1H), 7. 05 (s, 2H), 5.63 (s, 2H) ppm. 13C NMR (125
MHz, DMSO-d6): δ = 166.0, 156.5, 141.9, 141.8, 140.5, 138.7, 136.5,
133.7, 131.1, 125.2, 123.8, 123.7, 120.4, 119.0, 118.0, 111.6, 48.6 ppm.
HRMS (m/z): calcd for C17H13ClN6O2 [M + H]+ 369.0861; found,
369.0863.
296 °C, 98.0% HPLC purity. H NMR (300 MHz, DMSO-d6): δ =
10.65 (s, 1H), 8.35−8.31 (m, 1H), 8.19−8.16 (m, 1H), 7.92 (d, J = 8.16
Hz, 1H), 7.87 (dd, J = 3.09, 8.08 Hz, 2H), 7.76−7.72 (m, 2H), 7.58 (d, J
= 8.10 Hz, 1H), 7.50−7.39 (m, 2H), 7.04 (s, 2H), 5.79 (s, 2H) ppm.
13C NMR (125 MHz, DMSO-d6): δ = 166.8, 156.5, 141.9, 141.8, 138.8,
136.5, 133.7, 132.0, 130.1, 129.6, 128.6, 127.5, 127.2, 125.3, 124.0,
123.8, 123.1, 120.4, 119.1, 111.5, 48.5 ppm. HRMS (m/z): calcd for
C21H15BrN6O2 [M + H]+ 463.0513; found, 463.0513.
4.1.4.14. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(4-chlorophenyl)acetamide (S06-1022). According
to the general procedure described above, compound S06-1022 was
synthesized with 22a and 1b, with a yield of 54%, mp 288.5−290 °C,
4.1.4.8. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-phenylacetamide (S06-1016). According to the
general procedure described above, compound S06-1016 was
synthesized with 16a and 1b, with a yield of 68%, mp 286.5−288 °C,
1
98.2% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.71 (s,
1H), 7.86 (q, J = 8.13 Hz, 2H), 7.53 (dd, J = 8.85, 15.09 Hz, 4H), 7.47−
7.36 (m, 2H), 7.02 (s, 2H), 5. 59 (s, 2H). 13C NMR (125 MHz, DMSO-
d6): δ = 165.7, 156.5, 141.9, 141.8, 138.7, 138.5, 136.5, 132.2 (2C),
125.2, 123.7, 121.5 (2C), 120.4, 115.6, 111.6, 48.7 ppm. HRMS (m/z):
calcd for C17H13ClN6O2 [M + H]+ 369.0867; found, 369.0866.
4.1.4.15. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(3-bromophenyl)acetamide (S06-1023). Accord-
ing to the general procedure described above, compound S06-1023 was
synthesized with 23a and 1b, with a yield of 58%, mp 278−281 °C,
1
96.5% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.51 (s,
1H), 7.88 (d, J = 7.83 Hz, 1H), 7.81 (d, J = 7.89 Hz, 1H), 7.57 (d, J =
14.10 Hz, 2H), 7.47−7.39 (m, 2H), 7.36−7.29 (m, 2H), 7.07 (t, J =
7.29 Hz, 1H), 6.98 (s, 2H), 5.59 (s, 2H) ppm. 13C NMR (125 MHz,
DMSO-d6): δ = 165.5, 156.5, 142.0, 141.8, 139.1, 138.7, 136.6, 129.3
(2C), 125.2, 124.0, 123.7, 120.4, 119.5 (2C), 111.6, 48.6 ppm. HRMS
(m/z): calcd for C17H14N6O2 [M + H]+ 335.1521; found, 334.1245.
4.1.4.9. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(2-fluorophenyl)acetamide (S06-1017). According
to the general procedure described above, compound S06-1017 was
synthesized with 17a and 1b, with a yield of 73%, mp 289−291.5 °C,
1
96.1% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.81 (s,
1H), 7.94−7.84 (m, 3H), 7.53−7.41 (m, 3H), 7.32 (d, J = 7.47 Hz,
2H), 7.06 (s, 2H), 5.63 (s, 2H) ppm. 13C NMR (125 MHz, DMSO-d6):
δ = 166.0, 156.5, 141.9, 141.8, 140.6, 138.7, 136.5, 131.4, 126.7, 125.2,
123.7, 122.1, 121.9, 120.4, 118.3, 111.6, 48.6 ppm. HRMS (m/z): calcd
for C17H13BrN6O2 [M + H]+ 415.0356; found, 415.1521.
1
97.2% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.42 (s,
1H), 7.87 (dd, J = 7.92, 12.66 Hz, 3H), 7.38−7.49 (m, 2H), 7.32 (t, J =
7.68 Hz, 1H), 7.17 (s, 2H), 7.03 (s, 2H), 5.69 (s, 2H) ppm. 13C NMR
(125 MHz, DMSO-d6): δ = 166.2, 156.5, 142.0, 141.8, 138.7, 136.5,
126.2−126.1, 125.9−125.9, 125.2, 124.9, 124.1, 123.7, 120.4, 116.1,
116.0, 111.6, 48.5 ppm. HRMS (m/z): calcd for C17H13FN6O2 [M +
H]+ 353.1157; found, 353.1164.
4.1.4.16. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(4-bromophenyl)acetamide (S06-1024). Accord-
ing to the general procedure described above, compound S06-1024 was
synthesized with 24a and 1b, with a yield of 45%, mp 279.5−284 °C,
1
98.6% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.70 (s,
4.1.4.10. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(3-fluorophenyl)acetamide (S06-1018). According
to the general procedure described above, compound S06-1018 was
synthesized with 18a and 1b, with a yield of 62%, mp 259.5−262 °C,
1H), 7.85 (q, J = 7.59 Hz, 2H), 7.60 (d, J = 8.85 Hz, 2H), 7.45−7.36 (m,
4H), 7.01 (s, 2H), 5.59 (s, 2H) ppm. 13C NMR (125 MHz, DMSO-d6):
δ = 165.7, 156.5, 142.0, 141.8, 138.7, 138.0, 136.5, 129.3 (2C), 127.6,
125.2, 123.7, 121.1 (2C), 120.4, 111.6, 48.6 ppm. HRMS (m/z): calcd
for C17H13BrN6O2 [M + H]+ 413.0356; found, 413.0356.
1
96.8% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.99 (s,
1H), 7.86 (dd, J = 7.29, 12.51 Hz, 2H), 7.54 (d, J = 11.40 Hz, 1H),
7.34−7.47 (m, 4H), 7.02 (s, 2H), 6.87−6.94 (m, 1H), 5. 62 (s, 2H)
ppm. 13C NMR (125 MHz, DMSO-d6): δ = 165.9, 163.6, 161.7, 156.5,
142.0−141.9, 140.9−140.8, 138.7, 136.5, 131.0-130.9, 125.1, 123.7,
120.4, 115.4−115.4, 111.5, 110.5−110.4, 106.6−106.4, 48.7 ppm.
HRMS (m/z): calcd for C17H13FN6O2 [M + H]+ 353.1157; found,
353.1166.
4.1.4.17. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(m-tolyl)acetamide (S06-1025). According to the
general procedure described above, compound S06-1025 was
synthesized with 25a and 1b, with a yield of 55%, mp 282−286.5 °C,
1
97.0% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.49 (s,
1H), 7.85 (q, J = 7.23 Hz, 2H), 7.47−7.33 (m, 4H), 7.19 (t, J = 7.68 Hz,
1H), 7.02 (s, 2H), 6.89 (d, J = 7.53 Hz, 1H), 5. 58 (s, 2H), 2.26 (s, 3H)
ppm. 13C NMR (125 MHz, DMSO-d6): δ = 165.4, 156.5, 142.0, 141.9,
139.0, 138.7, 138.6, 136.6, 128.1, 125.1, 124.7, 123.7, 120.4, 120.1,
116.7, 111.6, 48.6, 21.6 ppm. HRMS (m/z): calcd for C18H16N6O2 [M
+ H]+ 349.1408; found, 349.1413.
4.1.4.11. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(2,3-difluorophenyl)acetamide (S06-1019). Ac-
cording to the general procedure described above, compound S06-
1019 was synthesized with 19a and 1b, with a yield of 78%, mp 283−
1
286.5 °C, 95.3% HPLC purity. H NMR (300 MHz, DMSO-d6): δ
4.1.4.18. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(p-tolyl)acetamide (S06-1026). According to the
general procedure described above, compound S06-1026 was
synthesized with 26a and 1b, with a yield of 62%, mp 276−277 °C,
=10.66 (s, 1H), 7.90 (t, J = 8.34 Hz, 2H), 7.69 (t, J = 7.65 Hz, 1H),
7.52−7.40 (m, 2H), 7.25−7.17 (m, 2H), 7.05 (s, 2H), 5.73 (s, 2H)
ppm. 13C NMR (125 MHz, DMSO-d6): δ = 166.4, 156.5, 151.5−151.4,
149.6−149.5, 142.0, 141.8, 138.7, 136.5, 128.2-128.1, 125.2, 124.8−
124.7, 123.7, 120.4, 119.4, 113.2−113.0, 111.5, 48.6 ppm. HRMS (m/
z): calcd for C17H12F2N6O2 [M + H]+ 371.1096; found, 371.1067.
1
96.3% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.46 (s,
1H), 7.85 (q, J = 7.44 Hz, 2H), 7.47−7.36 (m, 4H), 7.12 (d, J = 8.34 Hz,
2H), 7.02 (s, 2H), 5. 57 (s, 2H), 2.25 (s, 3H) ppm. 13C NMR (125
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MHz, DMSO-d6): δ = 165.2, 156.5, 142.0, 141.8, 138.7, 136.6, 136.6,
133.0, 127.0 (2C), 125.1, 123.6, 120.4, 119.5 (2C),111.6, 48.6, 20.9
ppm. HRMS (m/z): calcd for C18H16N6O2 [M + H]+ 349.1408; found,
349.1408.
4.1.4.25. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(4-aminophenyl)acetamide (S06-1033). Accord-
ing to the general procedure described above, compound S06-1033
was synthesized by reduction of S06-1032, with a yield of 24%, mp
231−239 °C, 95.2% HPLC purity. 1H NMR (300 MHz, DMSO-d6): δ
= 10.08 (s, 1H), 7.85 (d, J = 7.05 Hz, 1H), 7.76 (d, J = 7.77 Hz, 1H),
7.40 (t, J = 8.82 Hz, 3H), 7.18 (d, J = 5.79 Hz, 2H), 6.94 (s, 2H), 6.50
(d, J = 5.82 Hz, 1H), 5.51 (s, 2H), 4.81 (s, 2H) ppm. 13C NMR (125
MHz, DMSO-d6): δ = 164.3, 156.5, 145.5, 142.0, 141.9, 138.7, 136.6,
128.2, 125.1, 123.6, 121.3 (2C), 120.4, 114.3 (2C), 111.6, 48.4 ppm.
HRMS (m/z): calcd for C17H15N7O2 [M + H]+ 350.1360; found,
350.1349.
4.1.4.19. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(2,3-dimethylphenyl)acetamide (S06-1027). Ac-
cording to the general procedure described above, compound S06-
1027 was synthesized with 27a and 1b, with a yield of 60%, mp 289−
290.5 °C, 98.7% HPLC purity. 1H NMR (300 MHz, DMSO-d6): δ =
9.94 (s, 1H), 7.86 (q, J = 4.74 Hz, 2H), 7.48 (t, J = 6.90 Hz, 1H), 7.36 (t,
J = 6.87 Hz, 1H), 7.08 (t, J = 3.18 Hz, 1H), 7.01−7.04 (m, 4H), 5.62 (s,
2H), 2.24 (s, 3H), 2.13 (s, 3H) ppm. 13C NMR (125 MHz, DMSO-d6):
δ = 165.8, 156.5, 142.0, 141.9, 138.7, 137.6, 136.6, 135.9, 131.8, 127.6,
125.7, 125.1, 124.0, 123.6, 120.4, 111.6, 48.3, 20.6, 14.4 ppm. HRMS
(m/z): calcd for C19H18N6O2 [M + H]+ 363.1564; found, 363.1574.
4.1.4.20. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(3-methoxyphenyl)acetamide (S06-1028). Ac-
cording to the general procedure described above, compound S06-
1028 was synthesized with 28a and 1b, with a yield of 52%, mp 284−
4.1.4.26. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(3-cyanophenyl)acetamide (S06-1034). According
to the general procedure described above, compound S06-1034 was
synthesized with 34a and 1b, with a yield of 56%, mp 255−258 °C,
1
95.9% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 11.20 (s,
1H), 8.08 (s, 1H), 7.92−7.83 (m, 3H), 7.61−7.54 (m, 2H), 7.50−7.38
(m, 2H), 7.08 (s, 2H), 5.66 (s, 2H) ppm. 13C NMR (125 MHz, DMSO-
d6): δ = 166.3, 156.5, 142.0, 141.8, 139.9, 138.7, 136.5, 130.8, 127.6,
125.2, 124.1, 123.7, 122.2, 120.4, 119.0, 112.2, 111.5, 48.7 ppm. HRMS
(m/z): calcd for C18H13N7O4 [M + H]+ 360.1203; found, 360.1209.
4.1.4.27. N-(3-Acetylphenyl)-2-(2-(4-amino-1,2,5-oxadiazol-3-yl)-
1H-benzo[d]imidazol-1-yl)acetamide (S06-1035). According to the
general procedure described above, compound S06-1035 was
synthesized with 35a and 1b, with a yield of 58%, mp 285−286.5 °C,
1
288 °C, 98.7% HPLC purity. H NMR (300 MHz, DMSO-d6): δ =
10.57 (s, 1H), 7.85 (q, J = 7.41 Hz, 2H), 7.48−7.36 (m, 2H), 7.29 (t, J =
2.13 Hz, 1H), 7.23 (t, J = 8.13 Hz, 1H), 7.11 (d, J = 8.07 Hz, 1H), 7.03
(s, 2H), 6.65 (dd, J = 2.34, 7.92 Hz, 1H), 5. 59 (s, 2H), 3.70 (s, 3H)
ppm. 13C NMR (125 MHz, DMSO-d6): δ = 165.6, 160.1, 156.5, 142.0,
141.8, 140.3, 138.7, 136.6, 130.2, 125.2, 123.7, 120.4, 111.8, 111.6,
109.6, 105.2, 55.4, 48.7 ppm. HRMS (m/z): calcd for C18H16N6O3 [M
+ H]+ 365.1357; found, 365.1361.
4.1.4.21. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(4-methoxyphenyl)acetamide (S06-1029). Ac-
cording to the general procedure described above, compound S06-
1029 was synthesized with 29a and 1b, with a yield of 52%, mp 281−
285.5 °C, 98.4% HPLC purity. 1H NMR (300 MHz, DMSO-d6): δ =
10.41 (s, 1H), 7.84 (q, J = 7.26 Hz, 2H), 7.48 (d, J = 9.09 Hz, 2H),
7.45−7.38 (m, 2H), 7.03 (s, 2H), 6.89 (d, J = 9.09 Hz, 2H), 5.56 (s,
2H), 3.72 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ = 164.9, 156.5,
155.8, 142.0, 141.9, 138.7, 136.6, 132.2, 125.1, 123.6, 121.1 (2C),
120.4, 114.4 (2C), 111.6, 55.6, 48.5 ppm. HRMS (m/z): calcd for
C18H16N6O3 [M + H]+ 365.1362; found, 365.1357.
4.1.4.22. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (S06-1030).
According to the general procedure described above, compound S06-
1030 was synthesized with S06-1030 and 1b, with a yield of 56%, mp
255−258 °C, 97.9% HPLC purity. 1H NMR (300 MHz, DMSO-d6): δ
= 10.43 (s, 1H), 7.87 (d, J = 7.62 Hz, 1H), 7.79 (d, J = 7.47 Hz, 1H),
7.48−7.36 (m, 2H), 6.96 (s, 4H), 5. 57 (s, 2H), 3.72 (s, 6H), 3.63 (s,
3H). 13C NMR (125 MHz, DMSO-d6): δ = 165.3, 156.5, 153.3 (2C),
142.0, 141.8, 138.7, 136.5, 135.3, 134.0, 125.2, 123.7, 120.4, 111.6, 97.0
(2C), 60.6, 56.09 (2C), 48.6 ppm. HRMS (m/z): calcd for
C20H20N6O5 [M + H]+ 425.1573; found, 425.1558.
1
97.1% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.85 (s,
1H), 8.21 (s, 1H), 7.87 (dd, J = 8.58, 20.55 Hz, 3H), 7.77 (d, J = 7.62
Hz, 1H), 7.53−7.41 (m, 3H), 7.07 (s, 2H), 5.64 (s, 2H), 2.56 (s, 3H)
ppm. 13C NMR (125 MHz, DMSO-d6): δ = 198.0, 165.9, 156.5, 142.0,
141.8, 139.5, 138.7, 137.9, 136.6, 129.8, 125.2, 124.1, 124.0, 123.7,
120.4, 118.7, 111.6, 48.7, 27.2 ppm. HRMS (m/z): calcd for
C19H16N6O3 [M + H]+ 377.1357; found, 377.1362.
4.1.4.28. N-(4-Acetylphenyl)-2-(2-(4-amino-1,2,5-oxadiazol-3-yl)-
1H-benzo[d]imidazol-1-yl)acetamide (S06-1036). According to the
general procedure described above, compound S06-1036 was
synthesized with 36a and 1b, with a yield of 52%, mp 292−297 °C,
1
97.4% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.87 (s,
1H), 7.94 (d, J = 8.67 Hz, 2H), 7.86 (dd, J = 7.44, 14.80 Hz, 2H), 7.71
(d, J = 8.79 Hz, 2H), 7.48−7.36 (m, 2H), 6.98 (s, 2H), 5.63 (s, 2H),
2.53 (s, 3H) ppm. 13C NMR (125 MHz, DMSO-d6): δ = 197.0, 166.2,
156.5, 143.4, 141.9, 141.8, 138.7, 136.5, 132.5, 130.1 (2C), 125.2,
123.7, 120.4, 118.9 (2C), 111.6, 48.8, 26.9 ppm. HRMS (m/z): calcd
for C19H16N6O3 [M + H]+ 377.1362; found, 377.1356.
4.1.4.29. Methyl 3-(2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-
benzo[d]imidazol-1-yl)acetamido)benzoate (S06-1037). According
to the general procedure described above, compound S06-1037 was
synthesized with 37a and 1b, with a yield of 52%, mp 286.5−289 °C,
1
95.3% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.84 (s,
1H), 8.29 (s, 1H), 7.90−7.79 (m, 3H), 7.67 (d, J = 7.83 Hz, 1H), 7.51−
7.37 (m, 3H), 7.04 (s, 2H), 5.62 (s, 2H), 3.83 (s, 3H) ppm. 13C NMR
(125 MHz, DMSO-d6): δ = 166.5, 165.9, 156.4, 141.9, 141.8, 139.3,
138.7, 136.4, 130.7, 129.9, 125.3, 124.7, 124.1, 123.8, 120.4, 120.0,
111.4, 52.7, 48.5 ppm. HRMS (m/z): calcd for C19H16N6O4 [M + H]+
393.1306; found, 393.1314.
4.1.4.23. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(3-nitrophenyl)acetamide (S06-1031). According
to the general procedure described above, compound S06-1031 was
synthesized with 31a and 1b, with a yield of 64%, mp 249−253 °C,
98.1% HPLC purity. 1H NMR (300 MHz, DMSO-d6): δ = 11.12. (s,
1H), 8.61 (s, 1H), 7.98−7.86 (m, 4H), 7.68 (t, J = 7.89 Hz, 1H), 7.51−
7.40 (m, 2H), 7.06 (s, 2H), 5.67 (s, 2H) ppm. 13C NMR (125 MHz,
DMSO-d6): δ = 166.5, 156.5, 148.5, 141.9, 141.8, 140.1, 138.7, 136.5,
130.9, 125.5, 125.2, 123.7, 120.4, 118.6, 113.7, 111.6, 48.7 ppm. HRMS
(m/z): calcd for C17H13N7O4 [M + H]+ 380.1102; found, 380.1106.
4.1.4.24. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(4-nitrophenyl)acetamide (S06-1032). According
to the general procedure described above, compound S06-1032 was
synthesized with 32a and 1b, with a yield of 64%, mp 249−253 °C,
4.1.4.30. Methyl 4-(2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-
benzo[d]imidazol-1-yl)acetamido)benzoate (S06-1038). According
to the general procedure described above, compound S06-1038 was
synthesized with 38a and 1b, with a yield of 58%, mp 285.5−288 °C,
1
96.7% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.99 (s,
1H), 7.96 (d, J = 8.70 Hz, 2H), 7.89 (t, J = 8.88 Hz, 2H), 7.74 (d, J =
8.64 Hz, 2H), 7.50−7.39 (m, 2H), 7.06 (s, 2H), 5. 65 (s, 2H), 3.84 (s,
3H). 13C NMR (125 MHz, DMSO-d6): δ = 166.2 (2C), 156.5, 143.4,
141.9, 141.8, 138.7, 136.5, 130.9 (2C), 125.2, 124.8, 123.7, 120.4, 119.0
(2C), 111.6, 52.4, 48.7 ppm. HRMS (m/z): calcd for C19H16N6O4 [M
+ H]+ 393.1311; found, 393.1307.
4.1.4.31. N-(3-Acetamidophenyl)-2-(2-(4-amino-1,2,5-oxadiazol-
3-yl)-1H-benzo[d]imidazol-1-yl)acetamide (S06-1039). According
to the general procedure described above, compound S06-1039 was
synthesized with 39a and 1b, with a yield of 48%, mp 292.5−295.5 °C,
1
97.6% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 11.20 (s,
1H), 8.24 (d, J = 9.27 Hz, 2H), 7.90−7.80 (m, 4H), 7.48−7.37 (m,
2H), 7.01 (s, 2H), 5. 66 (s, 2H). 13C NMR (125 MHz, DMSO-d6): δ =
166.7, 156.5, 145.1, 142.9, 141.9, 141.7, 138.7, 136.5, 125.6 (2C),
125.2, 123.7, 120.4, 119.4 (2C), 111.6, 48.9 ppm. HRMS (m/z): calcd
for C17H13N7O4 [M + H]+ 380.1107; found, 380.1104.
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97.5% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.61 (s,
cording to the general procedure described above, compound S06-
1044 was synthesized with 11a and 43c, with a yield of 42%, mp 271−
1H), 9.97 (s, 1H), 7.95 (s, 1H), 7.88 (dd, J = 7.50, 12.81 Hz, 2H),
7.50−7.38 (m, 2H), 7.33−7.19 (m, 3H), 7.05 (s, 2H), 5.61 (s, 2H),
2.05 (s, 3H) ppm. 13C NMR (125 MHz, DMSO-d6): δ = 168.8, 165.5,
156.5, 141.9, 141.8, 140.1, 139.3, 138.7, 136.6, 129.4, 125.1, 123.6,
120.4, 114.6, 114.3, 111.6, 110.3, 48.6, 24.5 ppm. HRMS (m/z): calcd
for C19H17N7O3 [M + H]+ 392.1466; found, 392.1476.
1
274 °C, 97.9% HPLC purity. H NMR (300 MHz, DMSO-d6): δ =
11.06 (s, 1H), 8.96 (d, J = 2.52 Hz, 1H), 8.63 (d, J = 2.31 Hz, 1H), 7.97
(d, J = 8.28 Hz, 1H), 7.87 (d, J = 7.32 Hz, 1H), 7.68−7.62 (m, 3H),
7.58−7.53 (m, 1H), 7.00 (s, 2H), 5.63 (s, 2H), 2.38 (s, 3H), 2.38 (s,
3H) ppm. 13C NMR (125 MHz, DMSO-d6): δ = 166.6, 156.4, 155.9,
144.8, 140.8, 140.6, 139.6, 138.8, 135.1, 134.5, 132.8, 132.5, 129.0,
128.2, 127.6, 122.7, 120.1, 111.3, 48.6, 20.7, 20.3 ppm. HRMS (m/z):
calcd for C22H19N7O2 [M + H]+ 414.1673; found, 414.1684.
4.1.5.3. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-5,6-dimethyl-1H-
benzo[d]imidazol-1-yl)-N-(naphthalen-1-yl)acetamide (S06-1045).
According to the general procedure described above, compound S06-
1045 was synthesized with 14a and 43c, with a yield of 38%, mp 279−
282.5 °C, 98.4% HPLC purity. 1H NMR (300 MHz, DMSO-d6): δ =
10.51 (s, 1H), 8.24 (s, 1H), 7.96 (s, 1H), 7.78 (s, 1H), 7.61 (s, 5H),
7.48 (s, 1H), 7.00 (s, 2H), 5.70 (s, 2H), 2.39 (s, 3H), 2.36 (s, 3H) ppm.
13C NMR (125 MHz, DMSO-d6): δ = 166.6, 156.4, 140.9, 140.7, 138.8,
4.1.4.32. N-(4-Acetamidophenyl)-2-(2-(4-amino-1,2,5-oxadiazol-
3-yl)-1H-benzo[d]imidazol-1-yl)acetamide (S06-1040). According
to the general procedure described above, compound S06-1040 was
synthesized with 40a and 1b, with a yield of 42%, mp 296.5−299 °C,
1
96.4% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.48 (s,
1H), 9.89 (s, 1H), 7.84 (dd, J = 5.16, 15.22 Hz, 2H), 7.49−7.39 (m,
6H), 7.02 (s, 2H), 5. 57 (s, 2H), 2.02 (s, 3H) ppm. 13C NMR (125
MHz, DMSO-d6): δ = 168.5, 165.1, 156.5, 142.0, 141.8, 138.7, 136.6,
135.6, 134.3, 125.1, 123.6, 120.4, 120.0 (2C), 119.9 (2C), 111.6, 48.5,
24.4 ppm. HRMS (m/z): calcd for C19H17N7O3 [M + H]+ 392.1466;
found, 392.1453.
135.2, 134.5, 134.2, 133.5, 132.4, 128.6, 128.3, 126.6, 126.4, 126.1,
126.0, 123.2, 122.2, 120.1, 111.3, 48.5, 20.8, 20.3 ppm. HRMS (m/z):
calcd for C23H20N6O2 [M + H]+ 413.1721; found, 413.1739.
4.1.4.33. N-([1,1′-Biphenyl]-4-yl)-2-(2-(4-amino-1,2,5-oxadiazol-
3-yl)-1H-benzo[d]imidazol-1-yl)acetamide (S06-1041). According
to the general procedure described above, compound S06-1041 was
synthesized with 41a and 1b, with a yield of 65%, mp 299.5−303 °C,
4.2. Biological Evaluation. 4.2.1. In Vitro AChE/BChE Inhibitory
Evaluation. The inhibitory activities of test compounds against AChE
(EC 3.1.1.7) and BChE (EC 3.1.1.8) were carried out following the
method of Ellman et al., using a Shimadzu 160 spectrophotometer.
AChE (EC 3.1.1.7, Type VI-S, from electric eel, C3389), BChE (EC
3.1.1.8, from equine serum, C0663; from human, B4186), 5,5′-dithiobis
(2-nitrobenzoic acid) (DTNB, D218200), acetylthiocholine iodide
(ATC, A5751), and butyrylthiocholine iodide (BTC, B3253) were
purchased from Sigma-Aldrich (St. Louis, MO). AChE/BChE stock
solution was prepared by adjusting 500 units of the enzyme in gelatin
solution (1% in deionized water) and further diluted with water to give
the final concentrations of 2.5 units/mL (for eeAChE and eqBChE) or
0.015 units/mL (for hBChE). ATC/BTC iodide solutions were
prepared with deionized water before using to give the concentration
of 0.075 M. DTNB solution (0.01 M) was prepared in water containing
0.15% (w/v) sodium bicarbonate. The buffer solution was prepared
with potassium dihydrogen phosphate (1.36 g, 10 mmol) in 100 mL of
water. The pH of the solution was adjusted to 8.0 0.1 with KOH. The
test compounds were dissolved in DMSO to give a final concentration
of 10−1 M to produce stock solutions. Subsequently, at least six different
concentrations of each compound (normally 10−4−10−9, diluted with
ethanol) were used to determine the ChE inhibitory activities.
The measurement was performed using a 96-well plate. Then, 40 μL
of buffer, 10 μL of test compounds with different concentrations, 10 μL
of AChE or BChE, and 20 μL of DTNB were added and mixed in a well.
The solution in a 96-well plate was preincubated at room temperature
for 5 min (for eeAChE and eqBChE) or 20 min (for hBChE). After the
addition of 20 μL of ATC or BTC, the reaction was initiated. After 2
min of incubation at room temperature, the absorption was determined
at 25 °C at 412 nm. The sample replacing the compound solution with
10 μL of ethanol was used to determine 100% of the enzyme activity.
For determining the blank value, 10 μL of water replaced the enzyme
solution. All of the measurements for each concentration were
performed in triplicate and at least in three independent runs. The
inhibition curve was fitted by plotting the percentage enzyme activity
(100% for the reference) vs the logarithm of the test compound
concentration. The IC50 values were calculated by GraphPad Prism 6.0
software, and the data were expressed as the mean SD.
1
96.1% HPLC purity. H NMR (300 MHz, DMSO-d6): δ = 10.70 (s,
1H), 7.87 (t, J = 8.64 Hz, 2H), 7.70−7.63 (m, 6H), 7.49−7.33 (m, 5H),
7.04 (s, 2H), 5.62 (s, 2H) ppm. 13C NMR (125 MHz, DMSO-d6): δ =
165.6, 156.5, 142.0, 141.9, 140.0, 138.7, 138.6, 136.6, 135.7, 129.4
(2C), 127.5 (2C), 126.7 (2C), 125.2, 123.7, 120.4, 119.9 (2C), 111.6,
48.7 ppm. HRMS (m/z): calcd for C23H18N6O2 [M + H]+ 411.1564;
found, 411.1564.
4.1.4.34. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1H-benzo[d]-
imidazol-1-yl)-N-(benzo[d][1,3]dioxol-5-yl)acetamide (S06-1042).
According to the general procedure described above, compound S06-
1042 was synthesized with 42a and 1b, with a yield of 56%, mp 280−
1
286 °C, 97.7% HPLC purity. H NMR (300 MHz, DMSO-d6): δ =
10.49 (s, 1H), 7.84 (dd, J = 7.62, 15.69 Hz, 2H), 7.47−7.36 (m, 2H),
7.24 (d, J = 1.95 Hz, 1H), 7.02 (s, 2H), 6.97 (dd, J = 2.07, 8.40 Hz, 1H),
6.87 (d, J = 8.40 Hz, 1H), 5.98 (s, 2H) 5. 55 (s, 2H). 13C NMR (125
MHz, DMSO-d6): δ = 165.1, 156.5, 147.6, 143.6, 142.0, 141.8, 138.7,
136.5, 133.5, 125.1, 123.6, 120.4, 112.4, 111.6, 108.6, 101.7, 101.5, 48.5
ppm. HRMS (m/z): calcd for C18H14N6O4 [M + H]+ 379.1149; found,
379.1158.
4.1.5. General Synthetic Procedure for Compounds S06-1043 to
S06-1046. The synthesis approaches of compounds S06-1043 to S06-
1046 were similar to that of S06-1002. However, as compounds 43a
and 44a could not be purchased, they were synthesized from 4,5-
dimethyl-1,2-phenylenediamine. 4,5-Dimethyl-1,2-phenylenediamine
(1 mmoL) was mixed with ethyl cyanoacetate (1.5 mmoL) and reacted
under argon at 185 °C for 2 h. Completion of the reaction was
monitored by TLC. Afterward, 5 mL of water was added into the
reaction system and washed by ether (3 × 30 mL). The ether phase was
collected, dried over sodium sulfate, and evaporated under a vacuum to
give a crude product. It was subsequently purified by column
chromatography on silica gel (DCM/methanol (MeOH) = 20:1, v/
v) to obtain 43a. 43c was synthesized similar to 1c and then reacted
with 2a, 11a, or 14a to produce S06-1043 to S06-1046.
4.1.5.1. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-5,6-dimethyl-1H-
benzo[d]imidazol-1-yl)-N-(quinolin-6-yl)acetamide (S06-1043). Ac-
cording to the general procedure described above, compound S06-
1043 was synthesized with 2a and 43c, with a yield of 48%, mp 279.5−
1
4.2.2. Molecular Modeling and Molecular Dynamics Studies. The
docking study was performed by the CDOCKER module implemented
in Discovery Studio 2019 (BIOVIA). CDOCKER is a grid-based
molecular docking method that employs CHARMm. Briefly, random
ligand conformations were generated by high-temperature molecular
dynamics using a modified CHARMm forcefield (Prepare Ligands).
The acquired structures were subsequently minimized under the same
forcefield (Minimize Ligands). The solutions were then clustered based
on the positions and conformations and ranked by energy. The
cocrystal structure of hBChE bound with the reported screening-
template compound was selected for molecular docking. The structure
282 °C, 96.7% HPLC purity. H NMR (300 MHz, DMSO-d6): δ =
10.90 (s, 1H), 8.78 (d, J = 2.61 Hz, 1H), 8.31 (s, 1H), 8.22 (d, J = 7.92
Hz, 1H), 8.00 (d, J = 9.39 Hz, 1H), 7.82 (dd, J = 3.96, 9.08 Hz, 1H),
7.62 (d, J = 4.41 Hz, 2H), 7.46 (dd, J = 4.14, 8.28 Hz, 1H), 7.01 (s, 2H),
5.60 (s, 2H), 2.37 (s, 3H), 2.37 (s, 3H) ppm. 13C NMR (125 MHz,
DMSO-d6): δ = 166.1, 156.4, 149.6, 145.2, 140.9, 140.7, 138.8, 137.0,
135.9, 135.2, 134.5, 132.5, 130.2, 128.8, 123.5, 122.3, 120.1, 115.6,
111.4, 48.6, 20.7, 20.4 ppm. MS (ESI): calcd for C22H19N7O2 [M + H]+
414.1673; found, 414.1685.
4.1.5.2. 2-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-5,6-dimethyl-1H-
benzo[d]imidazol-1-yl)-N-(quinolin-3-yl)acetamide (S06-1044). Ac-
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was downloaded from Protein Data Bank (PDB id: 5k5e). The receptor
was prepared by the “Prepare Protein” module in DS for further
docking. A sphere (in 10 Å radius) around the original ligand was
defined as the binding site. All other parameters were kept as default.
After the docking procedure, 10 top-ranked conformations were
obtained.
4.2.6. ROS Measurement. The intracellular ROS levels were
determined with an oxidation-sensitive fluorescent probe, dichloro-
dihydro-fluorescein diacetate (DCFH-DA). It passively diffuses into
cells and is subsequently deacetylated by esterases to produce
nonfluorescent DCFH. In the presence of ROS, DCFH reacts with
ROS to form a fluorescent product dichlorofluorescein (DCF), which is
trapped inside the cells. For this measurement, BV2 cells were seeded in
96-well plates at a density of 1 × 104 per well and incubated for 24 h at
37 °C. The cells were exposed to the test compounds at diverse
concentrations and preincubated for 1 h and then treated with LPS (2.5
μg/mL) for an additional 24 h. Afterward, the harvested cells were
Molecular dynamics (MD) simulations were conducted after
molecular docking to explore the dynamic binding process using the
PMEMD module in AMBER 16. To start with, the previously obtained
docking modes were imported and prepared with the AMBER ff99SB
forcefield (for proteins), the ANTECHAMBER module, and the
general AMBER forcefield (for ligands). Thereafter, hydrogen atoms of
both the receptor and ligand were added using the “Reduce” module to
ensure the integrity of simulations. The simulation systems were
solvated in a TIP3P water box in a 9 Å hexahedron, and sodium ions
were added to neutralize the systems. To reduce possible steric stresses,
the minimum steps of the systems were set as 1000 in the way of the
steepest descent method. Additionally, the conjugate gradient method
was applied in another 1000 steps. The systems were then heated from 0
to 300 K in a linear way using a Langevin thermostat; the protein
backbone atoms were exerted with a weak restraint of 10 kcal/mol over
1 ns. Finally, MD simulation of 100 ns in the NPT ensemble was set at 1
atm and 300 K. The MMGBSA method in AMBER 16 software was
performed to calculate the binding free energies and energy
decomposition.
4.2.3. Cell Culture and Treatments. All cells were purchased from
the Cell Culture Center at the Institute of Basic Medical Sciences,
Chinese Academy of Medical Sciences. Dulbecco’s modified Eagle’s
medium (DMEM) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium (MTT) were purchased from Sigma (M2128, St. Louis, MO).
Recombinant human 1,1,1,3,3,3-hexafluoro-2-propanol-pretreated
Aβ1−42 peptide was obtained from Merck Millipore (Darmastadt,
Germany). H2O2 and β-glutamate were purchased from Aladdin.
Cells were cultured in DMEM supplemented with 10% fetal bovine
serum, 100 U/mL penicillin, and 100 μg/mL streptomycin in a
humidified incubator at 37 °C with 5% CO2. The test compounds were
prepared as stock solutions of 10−1 M in DMSO and were subsequently
diluted to the test concentrations with DMEM. MTT was dissolved in
phosphate-buffered saline (PBS) to a stock concentration of 5 mg/mL
and stored at −20 °C. For the cytotoxic stimuli, the Aβ1−42 peptide was
dissolved in DMSO as a 5 mM stock solution and incubated for 24 h at
37 °C in PBS at a final concentration of 2 mg/mL to induce Aβ1−42
aggregation.
washed with PBS (pH 7.2
0.1) and incubated with DCFH-DA
(diluted to give a final concentration of 5 μM with DMEM) for 30 min.
Then, the DCFH-DA medium was removed; the cells were washed
three times with DMEM before being observed under an inverted
microscope. Tacrine and test compounds were correspondingly orally
administered to mice in groups iii−ix.
4.2.7. In Vitro Blood−Brain Barrier (BBB) Permeation Assay. The
ability of compounds to permeate into the brain was evaluated using a
parallel artificial membrane permeation assay (PAMPA) for BBB
according to the method described by Di et al. Commercial drugs,
DMSO, and dodecane were obtained from Aladdin and Energy
Chemical. Porcine brain lipid (PBL) was purchased from Avanti Polar
Lipids. The donor microplate (96-well filter plate, poly(vinylidene
difluoride) (PVDF) membrane, pore size 0.45 μM) and the acceptor
microplate (indented 96-well plate) were both obtained from Millipore.
The 96-well UV plate (COSTAR) was purchased from Corning Inc.
The test compounds were initially dissolved in DMSO at a
concentration of 5 mg/mL. Then, it was diluted 200-fold with a
solution of PBS (pH 7.4 0.1)/EtOH (70:30, V/V) to give a final
concentration of 100 μg/mL. The filter membrane in the donor
microplate was impregnated with 4 μL of PBL in dodecane (20 mg/
mL). Thereafter, 200 μL of the diluted compound solution and 300 μL
of PBS/EtOH (70:30, V/V) were added into the donor wells and the
acceptor wells, respectively. The donor filter plate was carefully placed
on the acceptor plate to form a “sandwich,” making the filter membrane
contact the buffer solution. The sandwich assembly was left
undisturbed for 18 h at 25 °C. After that, the donor plate was carefully
removed; the concentrations of test compounds in the donor and
acceptor wells were measured with a UV plate spectroscopy reader
(SpectraMax Plus 384, Molecular Devices, Sunnyvale, CA). Each
sample was analyzed at five wavelengths, in at least three independent
runs, in four wells, and the results are given as the means SD. In each
experiment, nine quality control standards of known BBB permeability
were included to validate the analysis set.
4.2.8. Liver Microsome Stability Assay and Metabolites Analysis.
All liver microsome assays were performed by Shanghai ChemPartner
Co., Ltd. Metabolic stability of testing compounds was evaluated using
human liver microsomes to predict intrinsic clearance. The assay was
conducted in 96-well plates at 37 °C. The solution (45 μL) contained a
final concentration of 1 μM test compound, 0.5 mg/mL liver
microsome protein, and 2 mM NADPH. At each of the time points
(0-, 5-, 15-, 30-, and 45-min), 135 μL of acetonitrile containing an
internal standard (imipramine) was transferred to corresponding wells
to stop the reaction. Ketanserin was included as the positive control to
validate the assay performance.57 Plates were sealed, shaken (600 rpm
for 10 min), and then centrifuged at 4 °C at 5500g for 15 min. Then, 50
μL of the supernatant was transferred into a fresh 96-well sample plate
containing 50 μL of ultrapure water (Millipore, ZMQS50F01) for
liquid chromatography with tandem mass spectrometry (LC/MS/MS)
analysis. All samples were analyzed on LC/MS/MS using an API 4000
instrument. Analytical samples were separated using an Ultimate-XB-
C18 (5 μm, 2.1 × 50 mm2) at a flow rate of 0.6 mL/min. The mobile
phase consists of 0.025% formic acid in water (as solvent A) and
methanol (as solvent B). The metabolism extent was calculated as the
clearance of compounds, compared to the 0 min time incubation. Initial
rates were calculated for the compound concentration and used to
determine t1/2 values and the intrinsic clearance.
4.2.4. Cell Viability Assay. Cells were seeded in 96-well plates (1 ×
104 per well) and incubated overnight. After attachment, the cells were
treated with DMEM or compounds at various concentrations for 24 h at
37 °C. Then, 15 μL of MTT solution was added into each well and
incubated for 4 h. Then, the solution was removed and 100 μL of
DMSO was added to dissolve the MTT formazan crystals. Thereafter,
the absorbance values (OD values) of cell samples were read at 492 nm
by a spectrophotometer. The percentage of cell viability was calculated
using the following formula: cell viability (%) = OD(compound‑treated)
/
OD(DMEM‑treated) × 100%. All data were calculated from three
independent experiments. The IC50 values were calculated by
Graphpad Prism 6.0, and the data were expressed as mean SD.
4.2.5. Neuroprotective Effect. Generally, 80% confluent SH-SY5Y
cells were seeded with 5000 cells per well into sterile 96-well plates and
incubated for 24 h. The previous medium was removed; the cells were
exposed to DMEM or the test compounds at different concentrations
and preincubated for 2 h. Subsequently, H2O2 (200 μM, diluted with
DMEM), glutamate (20 mM, dissolved in DMEM), or Aβ1−42 (15 μM,
diluted with DMEM) was added sequentially to induce cell insults.
After 24 h, 15 μL of MTT was added to each well; the 96-well plates
were incubated for another 4 h at 37 °C. The blue-purple crystal MTT
formazan was dissolved in DMSO after discarding the supernatant. The
absorbance was determined at 492 nm using a microplate reader.
Percentages of cell protection against H2O2, glutamate, and Aβ1−42 were
calculated by considering the absorbance of the control cells (100% cell
viability).
Metabolite analysis was evaluated using human liver microsomes.
The assay was conducted in 96-well plates at 37 °C. The solution
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contained a final concentration of 20 μM test compound, 1 mg/mL
liver microsome protein, 10 mM MgCl2, 1 mM NADPH, 10 mM G-6-P,
and 1.0 unit/mL PDH. After 120 min, a triple amount of acetonitrile
was added to stop the reaction. The plate was sealed, shaken, and then
centrifuged at 4 °C at 5500g for 15 min. Then, 50 μL of the supernatant
was transferred into a fresh 96-well sample plate containing 50 μL of
ultrapure water (Millipore, ZMQS50F01) for LC/MS/MS analysis.
The analysis approach was the same as that of the stability assay.
4.2.9. In Vivo PK Study. The study was conducted in the
Pharmaceutical Laboratory Animal Center of China Pharmaceutical
University (IACUC). The experiment meets the ethical considerations.
Healthy male Sprague−Dawley rats (180−220 g, Nanjing Qinglong-
shan Animal Breeding Factory, China) were randomly divided into two
groups (n = 8) to analyze PK properties of the test compound. Eight rats
were used for oral administration (10 mg/kg), while the other eight rats
were used for intravenous administration (10 mg/kg). The compound
sample was dissolved in a mixture of DMSO, poly(ethylene glycol)
(PEG) 400, and normal saline (10:80:10, V/V/V) before the
experiments. The animals were fasted overnight before initiating the
experiment but had free access to water. After administration, the blood
samples were collected from the retro-orbital plexus at 1, 5, 10, 20, 30,
60, 120, 240, 360, 480, 720, and 1440 min into heparinized tubes. Blood
samples were immediately centrifuged at 8000 rpm for 10 min to
separate the plasma. Then, 100 μL of plasma was precipitated by
addition of 300 μL of chromatographic methanol containing an internal
standard (theophylline), vortexed for 10 min, and centrifuged at 15 000
rpm for 10 min. The supernatant (100 μL) was collected and
transferred into HPLC vials for LC/MS/MS to determine the
concentrations in plasma, using the mobile phase consisting of an
aqueous solution containing 0.1% formic acid (as solvent A, 30%) and
methanol (as solvent B, 70%) at a flow rate of 0.6 mL/min using a
Hedera ODS-2 column (5 μm, 2.1 × 50 mm2). The standard curve was
generated by the addition of serial concentrations of compound with an
internal standard to blank plasma. The PK parameters were calculated
using the plasma concentration data of the test compound at different
time points using extravascular analysis of the noncompartmental
(NCA) model with PK Solver software.
4.2.10. In Vivo BBB Penetration Determination. The study was
conducted in the Pharmaceutical Laboratory Animal Center of China
Pharmaceutical University (IACUC). The experiment meets the ethical
considerations. Compound S06-1011 was dissolved in 5% DMSO, 40%
PEG 400, and 55% saline to yield a nominal concentration of 1 mg/mL
and was dosed at 10 mg/kg in ICR mice by oral administration. Samples
were collected at 30, 60, 120, and 240 min postdose. Mice were
anesthetized and sacrificed, blood was collected by retro-orbital bleeds,
and brains were collected. Blood samples were processed and analyzed
as described above. Brain samples were mixed with a double volume of
PBS and fully homogenized. Subsequently, a triple volume of ethyl
acetate was added and vortexed for 2 min. The mixture was centrifuged
at 15 000 rpm for 10 min, and the supernatant (1000 μL) was collected.
The solvent was allowed to evaporate in the fume hood, and the residue
was reconstituted with the mobile phase (250 μL). The samples were
analyzed as described above.
meets the ethical considerations. The experiment was performed using
ICR male mice (8−10 weeks old, weight 18−20 g), which were
purchased from Nanjing Qinglongshan Animal Breeding Factory
(Nanjing, China). The modeling stimulus, scopolamine, was obtained
from Aladdin. Tacrine, used as the positive control, was purchased from
Sigma. The cognitive function of mice was evaluated by the Morris
water maze analysis-management system (Viewer2 Tracking Software,
Ji Liang Instruments, Shanghai, China) to record the escape latency and
the trajectory to the platform.58 The test compounds were suspended in
a mixture of DMSO and normal saline (10:90, V/V) before the
experiments.
The mice were separated into nine groups as follows (n = 8): (i)
vehicle as the blank control group, (ii) scopolamine (3 mg/kg,
intraperitoneal injection) as the model group, (iii) tacrine (15 mg/kg,
intragastric administration) + scopolamine (3 mg/kg, intraperitoneal
injection) as the positive control group, (iv) test compound S06-1011
(15 mg/kg, intragastric administration) + scopolamine (3 mg/kg,
intraperitoneal injection) group, (v) test compound S06-1011 (30 mg/
kg, intragastric administration) + scopolamine (3 mg/kg, intra-
peritoneal injection) group, (vi) test compound S06-1015 (15 mg/
kg, intragastric administration) + scopolamine (3 mg/kg, intra-
peritoneal injection) group, (vii) test compound S06-1015 (30 mg/
kg, intragastric administration) + scopolamine (3 mg/kg, intra-
peritoneal injection) group, (viii) test compound S06-1031 (15 mg/
kg, intragastric administration) + scopolamine (3 mg/kg, intra-
peritoneal injection) group, and (ix) test compound S06-1031 (30
mg/kg, intragastric administration) + scopolamine (3 mg/kg, intra-
peritoneal injection) group. Tacrine and test compounds were
correspondingly orally administered to mice in groups iii−ix, 30 min
before the intraperitoneal injection of scopolamine for 10 consecutive
days.
During the last 5 days, spatial learning and memory were evaluated
by the Morris water maze. The maze was placed in a lit room with visual
cues at 25 °C. A platform (6 cm radius, 1.0 cm submerged under the
water level) was put in the center of one quadrant of the circular pool
(60 cm radius, 45 cm height) with a depth of 30 cm water. The
behavioral study of each mouse consisted of 5 days of learning and
memory training and a probe trail on day 6. The mice started facing the
pool wall and were pseudorandomized for each trial. For cognitive
evaluation, each mouse was separately evaluated by a visible platform
(days 1−2) and a hidden platform (days 3−5) of the water maze. Mice
received a nonspatial pretraining during the training days 1−2, prepared
for the subsequent spatial learning test. During these 2 days, mice were
trained to find the platform that was labeled by a small flag. On days 3−
5, the hidden-platform version was used to explore the retention of
memory to find the platform. The hidden platform was placed 1 cm
below the water level. All mice were subjected to two trials, each of
which was lasting 90 s. The mouse successfully reaching the platform
was called “a successful escape”, and its escape latency was recorded. If a
mouse failed to arrive at the platform within 90 s, the test was
terminated. The mouse was navigated to the platform by hand and got
used to the platform for 1 min. On the last day (day 6), the platform was
removed from its location and the probe trial was performed. The
escape latency and the traveled trajectory of all mice were recorded by
Panlab SMART 3.0 and processed by Graphpad Prism 6.0 software.
4.2.13. Behavioral Study II (Oligomerized Aβ1−42 as the Modeling
Stimuli). The study was conducted in the Pharmaceutical Laboratory
Animal Center of China Pharmaceutical University (IACUC). The
experiment meets the ethical considerations. ICR mice (6−8 weeks old,
18−22 g, Nanjing Qinglongshan Animal Breeding Factory, China)
were separated into seven groups (n = 8) for this experiment.
Recombinant human 1,1,1,3,3,3-hexafluoro-2-propanol-pretreated
Aβ1−42 peptide was obtained from Merck Millipore (Darmastadt,
Germany). Aβ1−42 peptide was dissolved in DMSO as a 5 mM stock
solution and incubated for 24 h at 37 °C in normal saline at a final
concentration of 2 mg/mL to induce Aβ1−42 aggregation.59 Donepezil,
used as the positive control, was purchased from Sigma. The cognitive
function was evaluated by the Morris water maze and the Y maze, using
a computer equipped with an analysis-management system (Viewer2
Tracking Software, Ji Liang Instruments, Shanghai, China). The test
4.2.11. Acute Toxicity Evaluation. The study was conducted in the
Pharmaceutical Laboratory Animal Center of China Pharmaceutical
University (IACUC). The experiment meets the ethical considerations.
Three test compounds were serially suspended in 5% DMSO, 40% PEG
400, and 55% saline. Forty male and forty female mice (15−18 g) were
divided into eight groups (n = 9): male control group, female control
group, and male test groups for three compounds, and female test
groups for three compounds. All mice had been fasted overnight, and
then the mice in test groups were intragastrically administered with a
dosage of 1 g/kg of test compounds while the mice in control groups
were administered with the same volume of vehicle solution on the first
day. The death, daily behavior, and body mass were monitored during
the subsequent 2 weeks. All mice were dissected on day 14, and the liver
was extracted and then examined by HE to see if any damages occurred.
4.2.12. Behavioral Study I (Scopolamine as the Modeling Stimuli).
The study was conducted in the Pharmaceutical Laboratory Animal
Center of China Pharmaceutical University (IACUC). The experiment
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compounds were suspended in a mixture of DMSO and normal saline
(10:90, V/V) before the experiments.
Haopeng Sun − School of Pharmacy, China Pharmaceutical
University, Nanjing 211198, People’s Republic of China;
orcid.org/0000-0002-5109-0304; Phone: +86-
The oligomerized Aβ1−42 peptide was injected (intracerebroven-
tricular injection, icv) on the first day of the experiment, and test
compounds were intragastrically injected during days 2−14. The mice
were grouped as follows (n = 8): (i) vehicle (intragastric
administration) as the blank control group; (ii) oligomerized Aβ1−42
peptide (10 μg per mouse, icv) as the model group; (iii) normal saline
(icv) as the sham-operation group; (iv) oligomerized Aβ1−42 peptide
(10 μg per mouse, icv) + donepezil (15 mg/kg, intragastric
administration) as the positive control group; (v) oligomerized
Aβ1−42 peptide (10 μg per mouse, icv) + test compound S06-1011
(15 mg/kg, intragastric administration) group; (vi) oligomerized
Aβ1−42 peptide (10 μg per mouse, icv) + test compound S06-1015 (15
mg/kg, intragastric administration) group; and (vii) oligomerized
Aβ1−42 peptide (10 μg per mouse, icv) + test compound S06-1031 (15
mg/kg, intragastric administration) group. The behavioral studies
included the Morris water maze on days 9−14 and the Y maze on day
15.
13951934235; Email: sunhaopeng@163.com
Authors
Qi Li − School of Pharmacy, China Pharmaceutical University,
Nanjing 211198, People’s Republic of China; School of Basic
Medicine, Qingdao University, Qingdao 266071, People’s
Republic of China
Ying Chen − Department of Natural Medicinal Chemistry,
China Pharmaceutical University, Nanjing 211198, People’s
Republic of China
Shuaishuai Xing − School of Pharmacy, China Pharmaceutical
University, Nanjing 211198, People’s Republic of China
Qinghong Liao − Department of Natural Medicinal Chemistry,
China Pharmaceutical University, Nanjing 211198, People’s
Republic of China
The Morris water maze was performed like described before (in
section Section 4.2.12). The spatial learning and memory were
conducted on days 9−13, and the probe trail was performed on day 14.
On day 15, all mice were tested for spontaneous alternation
performance in the Y maze to define the spatial working memory.27,60
The Y maze assembly was made of gray poly(vinyl chloride), with three
arms (40 cm long, 13 cm high, 3 cm wide at the bottom, 10 cm wide at
the top, converging at an angle of 120°). Each mouse was placed at the
end of one arm and explored freely in the maze for 5 min. The sequence
of arm entries of the Y maze was checked visually and recorded. The
mouse entering the three arms on consecutive occasions was defined as
an alternation. The total number of arm entries minus two was the
maximum alternation. The percentage of alternation performance was
calculated as (actual alternations/maximum alternations) × 100%. If an
extreme behavior (alternation percentage <20 or >90%) occurs, this
alternation was not involved. All values were expressed as means SD
by Graphpad Prism 6.0 software.
Baichen Xiong − School of Pharmacy, China Pharmaceutical
University, Nanjing 211198, People’s Republic of China
Yuanyuan Wang − School of Pharmacy, China Pharmaceutical
University, Nanjing 211198, People’s Republic of China
Weixuan Lu − School of Pharmacy, China Pharmaceutical
University, Nanjing 211198, People’s Republic of China
Siyu He − State Key Laboratory of Natural Medicines, Jiangsu
Key Laboratory of Carcinogenesis and Intervention, School of
Basic Medicine and Clinical Pharmacy, China Pharmaceutical
University, Nanjing 210009, People’s Republic of China
Feng Feng − Department of Natural Medicinal Chemistry,
China Pharmaceutical University, Nanjing 211198, People’s
Republic of China; Jiangsu Food and Pharmaceutical Science
College, Huai’an 223003, People’s Republic of China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00167
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00167.
■
sı
Notes
The authors declare no competing financial interest.
Molecular dynamics8012-9656 (PDB)
ACKNOWLEDGMENTS
■
Molecular dynamicsS06-1002 (PDB)
We gratefully thank the support from the grants (Nos.
81872728, 81573281, and 81973207) of the National Natural
Science Foundation of China (Nos. BK20191411) of the
Natural Science Foundation of Jiangsu Province. We thank the
support from the “Double First-Class” initiative Innovation
team project of China Pharmaceutical University (Nos.
CPU2018GF11 and CPU2018GY34). We thank the ACS
Authoring Services for English editing.
Molecular dynamicsS06-1011 (PDB)
Molecular dynamicsS06-1014 (PDB)
Molecular string formulas (CSV)
Compound characterization; inhibitory rates on eeAChE
and on eqBChE of partial compounds; molecular docking
modes of S06-1002, S06-1011, and S06-1014; molecular
dynamic simulation results of S06-1001, S06-1002, S06-
1011, and S06-1014; chromatographic and mass
spectrometric identification information for S06-1011;
standard curves for pharmacokinetics and PAMPA study;
quantization of ROS production; protocol for the in vivo
experiments (PDF)
ABBREVIATIONS
■
AD, Alzheimer’s disease; β-amyloid, Aβ; NMDAR, N-methyl-D-
aspartic acid receptor; ChE, cholinesterases; ACh, acetylcholine;
AChE, acetylcholinesterase; BChE, butyrylcholinesterase; CNS,
central nervous system; BBB, blood−brain barrier; eeAChE,
electrophorus electricus AChE; eqBChE, equine serum BChE;
CAS, catalytic active site; PAS, peripheral anionic site; hAChE,
human AChE; hBChE, human BChE; MD, molecular dynamics;
IR, inhibitory rates; LE, ligand efficiency; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; AST, aspartate
aminotransferase; ALT, alanine aminotransferase; HE, hema-
toxylin−eosin; PK, pharmacokinetics; PAMPA−BBB, parallel
artificial membrane permeation assay of the blood−brain
barrier; BBB, blood−brain barrier; Cmax, peak exposure; AUC,
area under the concentration−time curve; ROS, reactive oxygen
AUTHOR INFORMATION
Corresponding Authors
■
Wenyuan Liu − School of Pharmacy, China Pharmaceutical
University, Nanjing 211198, People’s Republic of China;
Phone: +86-18012955795; Email: liuwenyuan@
cpu.edu.cn
Yao Chen − School of Pharmacy, Nanjing University of Chinese
Medicine, Nanjing 210023, People’s Republic of China;
Phone: +86-13921442470; Email: clarissa0710@163.com,
300630@njucm.edu.cn
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