Design, synthesis and biological evaluation of indanone–chalcone hybrids as potent and selective hCES2A inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112856

Human carboxylesterase 2 (hCES2A), one of the major serine hydrolases distributed in the small intestine, plays a crucial role in hydrolysis of ester-bearing drugs. Accumulating evidence has indicated that hCES2A inhibitor therapy can modulate the pharmacokinetic and toxicological profiles of some important hCES2A-substrate drugs, such as the anticancer agent CPT-11. Herein, a series of indanone–chalcone hybrids are designed and synthesized to find potent and highly selective hCES2A inhibitors. Inhibition assays demonstrated that most indanone–chalcone hybrids displayed strong to moderate hCES2A inhibition activities. Structure-hCES2A inhibition activity relationship studies showed that introduction of a hydroxyl at the C4’ site and introduction of an N-alkyl group at the C6 site were beneficial for hCES2A inhibition. Particularly, B7 (an N-alkylated 1-indanone–chalcone hybrid) exhibited the most potent inhibition on hCES2A and excellent specificity (this agent could not inhibit other human esterases including hCES1A and butyrylcholinesterase). Inhibition kinetic analyses demonstrated that B7 potently inhibited hCES2A-mediated FD hydrolysis in a mixed inhibition manner, with a calculated K_i value of 0.068 μM. Furthermore, B7 was capable of inhibiting intracellular hCES2A in living cells and displayed good metabolic stability. Collectively, our findings show that indanone–chalcone hybrids are good choices for the development of hCES2A inhibitors, while B7 is a promising candidate for the development of novel anti-diarrhea agents to ameliorate irinotecan-induced intestinal toxicity.

Full text of DOI:10.1016/j.ejmech.2020.112856

European Journal of Medicinal Chemistry 209 (2021) 112856
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and biological evaluation of indanoneechalcone
hybrids as potent and selective hCES2A inhibitors
,
b
,
,
,
Peng-Chao Huo a 1, Xiao-Qing Guan a 1, Peng Liu c 1, Yun-Qing Song a, Meng-Ru Sun a,
Rong-Jing He a, Li-Wei Zou a, Li-Juan Xue a, Jin-Hui Shi a, Nan Zhang b **, Zhi-Guo Liu c
,
,
, ***
,
*
Guang-Bo Ge a
a Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, China
b School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, 450001, China
c Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Human carboxylesterase 2 (hCES2A), one of the major serine hydrolases distributed in the small intes-
tine, plays a crucial role in hydrolysis of ester-bearing drugs. Accumulating evidence has indicated that
hCES2A inhibitor therapy can modulate the pharmacokinetic and toxicological profiles of some impor-
tant hCES2A-substrate drugs, such as the anticancer agent CPT-11. Herein, a series of indanoneechalcone
hybrids are designed and synthesized to find potent and highly selective hCES2A inhibitors. Inhibition
assays demonstrated that most indanoneechalcone hybrids displayed strong to moderate hCES2A in-
hibition activities. Structure-hCES2A inhibition activity relationship studies showed that introduction of
a hydroxyl at the C4’ site and introduction of an N-alkyl group at the C6 site were beneficial for hCES2A
inhibition. Particularly, B7 (an N-alkylated 1-indanoneechalcone hybrid) exhibited the most potent in-
hibition on hCES2A and excellent specificity (this agent could not inhibit other human esterases
including hCES1A and butyrylcholinesterase). Inhibition kinetic analyses demonstrated that B7 potently
inhibited hCES2A-mediated FD hydrolysis in a mixed inhibition manner, with a calculated Ki value of
Received 21 June 2020
Received in revised form
9 September 2020
Accepted 17 September 2020
Available online 24 September 2020
Keywords:
Human carboxylesterase 2 (hCES2A)
Indanoneechalcone hybrids
Inhibition
Specificity
0.068 mM. Furthermore, B7 was capable of inhibiting intracellular hCES2A in living cells and displayed
good metabolic stability. Collectively, our findings show that indanoneechalcone hybrids are good
choices for the development of hCES2A inhibitors, while B7 is a promising candidate for the development
of novel anti-diarrhea agents to ameliorate irinotecan-induced intestinal toxicity.
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
extensively investigated. As one of the major hydrolases distributed
in the endoplasmic reticulum of intestinal epithelial cells, hCES2A
Human carboxylesterases (hCES, EC. 3.1.1.1) are important
members of the serine hydrolase superfamily, which catalyze the
hydrolytic metabolism of many endogenous substances and xeno-
biotics bearing ester- or amide-bond(s) to the corresponding free
acid and alcohol [1e4]. Over the past two decades, the substrate
spectra and biological functions of two predominant human car-
boxylesterases (including hCES1A and hCES2A) have been
plays a key role in hydrolytic metabolism of numerous therapeutic
agents bearing ester- or amide-bond(s), such as capecitabine, iri-
notecan and flutamide [5e11]. Increasing evidence has demon-
strated that hCES2A inhibitors can be used to increase the oral
bioavailabilities or to modulate therapeutic outcomes of hCES2A-
substrate drugs. Currently, loperamide (LPA,
a known anti-
diarrhea agent with hCES2A inhibition activity) has been used for
ameliorating the intestinal toxicity triggered by irinotecan in clin-
ical settings [12,13]. However, the moderate hCES2A inhibition
potency, as well as the side effects of LPA (such as drowsiness,
nausea, headache, cardiac dysrhythmia and constipation), strongly
block the clinical applications of this ester-bearing agent [14e16].
Therefore, it is urgent and necessary to find more efficacious
hCES2A inhibitors to ameliorate irinotecan-induced intestinal
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: zhangnan@zzu.edu.cn (N. Zhang), lzgcnu@163.com (Z.-G. Liu),
geguangbo@dicp.ac.cn (G.-B. Ge).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2020.112856
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

P.-C. Huo, X.-Q. Guan, P. Liu et al.
European Journal of Medicinal Chemistry 209 (2021) 112856
toxicity and intestinal dysfunction.
chemical structures were fully characterized by both NMR and
mass spectrometry. More details about the chemical synthesis and
structural characterization can be found in the Supplementary
materials.
In the past ten years, with the help of isoform-specific probe
substrates for two hCES isoenzymes [17e22], numerous com-
pounds with structurally diverse skeleton (such as flavonoids,
chalcones, triterpenoids and tanshinones) have been reported with
hCES inhibition activity [7,9,23e26], which provide useful hits for
the chemists to design and synthesize more efficacious hCES1A/
hCES2A inhibitors with high selectivity and good drug-likeness.
From the viewpoints of drug discovery and translational applica-
tions, ideal hCES2A inhibitors should have the following features:
highly specific, potent, low toxicity, cell-permeable (capable of
blocking intracellular hCES2A) and acceptable metabolic stability.
Unfortunately, the majority of reported hCES2A inhibitors (espe-
cially those natural compounds with hCES2A inhibition activity)
2.1. Design, synthesis and SAR of ring a modified indanone-chalcone
hybrids
Firstly, the possibility of two fragment hits (1-indanone and
chalcone) and their fusion type (indanone-chalcone) targeting on
hCES2A were investigated by docking simulations. As shown in
Table S1, both fragments (1-indanone and chalcone) and their
fusion type (indanone-chalcone) could be well-docked into the
catalytic cavity of hCES2A, while the fusion type (indanone-chal-
cone) gained relative high total score, implying that the fusion type
may possess relative high binding affinity toward hCES2A. Mean-
while, given that some natural chalcones bearing C40 phenolic
group (such as isobavachalcone, a bioactive compound in Fructus
Psoraleae) exhibited strong hCES2A inhibition activity [23], a
phenolic group was intentionally introduced on A ring at the C40
site. As shown in Table S1, the total score of the C40-hydroxyl
modified indanone-chalcone (fragment D in Table S1) is slightly
higher than that of the fusion skeleton (indanone-chalcone), sug-
gesting that the C40-hydroxyl modified indanone-chalcone may
also possess high binding affinity toward hCES2A. In this case, a
series of ring A modified indanone-chalcone hybrids (including A2,
A3, A4 and A5) were prepared via the Claisen Schmidt condensa-
tion reaction [27]. Alternatively, hydrochloric acid gas was
employed to catalyze the Aldol condensation between 6-hydroxy-
1-indanone and hydroxyl-benzaldehydes to give the products A1,
A6 and A7.
After that, the hCES2A inhibition activities of these ring A
modified indanone-chalcone hybrids were routinely assayed uti-
lizing FD as a fluorogenic substrate. The IC50 values of ring A
modified indanone-chalcone hybrids were listed in Table 1, while
the hCES2A inhibition activity of loperamide (LPA) was also assayed
under same conditions. As shown in Table 1, Fig. 2 and Fig. S1, most
of ring A modified indanone-chalcone hybrids exhibited moderate
to strong inhibition on hCES2A, while the phenolic group at the C40
site was beneficial for hCES2A inhibition when compared with the
C40-O-alkylated derivatives. Once the C40-hydroxyl was substituted
with O-alkylated groups (e.g., methoxyl and ethoxyl), the hCES2A
inhibition activity decreased dramatically, as indicated by IC50
display moderate inhibition potency (at the mM level), poor speci-
ficity, low cell permeability and unacceptable metabolic stability.
Therefore, it is necessary to develop more drug-like small mole-
cules possessing good hCES2A inhibition activity and improved
druglikeness properties, for ameliorating irinotecan-triggered in-
testinal toxicity.
This study aims to develop efficacious and highly selective
hCES2A inhibitor(s) with good drug-like properties as promising
drug candidate(s) for ameliorating irinotecan-induced intestinal
toxicity. Given that some chalcones and several compounds bearing
1-indanone have been reported with esterase inhibition activity
(Fig. 1), a series of novel indanone-chalcone hybrids are designed,
synthesized, while their hCES2A inhibition activities are routinely
assayed using an established biochemical assay. Meanwhile,
structureeactivity relationships (SAR) of the indanone-chalcone
hybrids as hCES2A inhibitors were studied to reveal the key sub-
stituents that would be beneficial for hCES2A inhibition. Following
two rounds optimization, B7, a novel indanone-chalcone hybrid,
was found with the most potent hCES2A inhibition activity.
Furthermore, the specificity, inhibitory mechanism and the drug-
likeness properties of B7 were carefully investigated utilizing a
set of in vitro assays and docking simulations. All these findings
demonstrate that indanone-chalcone hybrids are good choices to
develop efficacious hCES2A inhibitors.
2. Results and discussion
The synthetic schemes of indanone-chalcone hybrids are sum-
marized in Scheme 1. All of these synthetic compounds were ob-
tained in high purity via separation by flash column, while their
values of A1 (5.94
mM) vs. A2 (55.95 mM) and A3 (10.73 mM).
Fig. 1. A) Several reported 1-indanone or chalcones derivatives with esterase inhibition activity. B) The design strategy of hCES2A inhibitors using 1-indanone and chalcone as
fragment hits.
2

P.-C. Huo, X.-Q. Guan, P. Liu et al.
European Journal of Medicinal Chemistry 209 (2021) 112856
Scheme 1. A) General synthetic scheme for indanone-chalcone hybrids A1-A7. Reagents and conditions: (a) 3,4-dihydro-2H-pyran, PPTS, 4 h, CH2Cl2, 40 ꢀC; (b) various benzal-
dehydes, EtOH, 20% (w/v) NaOH, room temperature, overnight; (c) 1.0 mol/LHCl, EtOH, room temperature, 5 h; (d) HCl (gas), EtOH, room temperature, overnight. B) General
synthetic scheme for indanone-chalcone hybrids B1eB10; Reagents and conditions: (a) 3,4-dihydro-2H-pyran, PPTS, 4 h, CH2Cl2, 40 ꢀC; (b) various substituted 1-indanone, EtOH,
20% (w/v) NaOH, room temperature, overnight; (c) 1.0 mol/LHCl, EtOH, room temperature, 5 h.
Table 1
Structures, Log P and IC50 values of indanone-chalcone hybrids against hCES2A.
No.
Compound
R1
30
R2
MW
aLog P
IC50 (mМ)
20
40
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
A1
A2
A3
A4
A5
A6
A7
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
-H
-H
-H
-OMe
-OMe
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-OMe
-H
-OH
-OMe
-OEt
-OMe
-OMe
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-H
-NH2
252.08
266.30
280.32
326.35
296.32
268.27
282.30
266.30
281.31
296.32
324.38
324.38
349.43
335.40
363.46
337.42
323.12
477.05
2.64
3.05
3.37
3.00
3.03
2.27
2.67
3.09
2.53
3.05
3.78
3.7
3.78
3.50
4.05
3.72
2.64
5.358
5.94 1.26
55.95 9.10
10.73 1.13
5.23 0.23
18.21 1.46
2.81 0.17
1.09 0.17
1.31 0.13
1.48 0.16
2.91 0.17
1.62 0.12
2.86 0.16
0.61 0.09
0.052 0.004
0.81 0.13
0.38 0.06
0.63 0.09
6.80 0.79
-OH
-OMe
-OMe
-OMe
-OMe
-OMe
-OMe
-OMe
-OMe
-OMe
-OMe
-OMe
-OMe
-OPr
-O (i-Pr)
-N (eCH2e)5
-N (eCH2e)4
-N (eCH2e)6
-N (Et)2
-NH-COMe
Loperamide
a
Log P ¼ Predicted octanol/water partition coefficient. The *log P values were predicted using SwissADME.
Furthermore, addition of a hydroxyl or methoxyl group at the C-30
site slightly enhanced hCES2A inhibition activity, as shown by IC50
(18.21 mM). Among all tested ring A modified indanone-chalcone
hybrids, A7 (a derivative bearing a methoxyl and a hydroxyl at
the C-30, and C-40 site, respectively) displayed the most potent
hCES2A inhibition activity, suggesting that A7 could act as a leading
compound for the development of novel hCES2A inhibitors with
high potency and improved druglikeness properties.
values of A1 (5.94
polymethoxylated derivatives of ring
chalcone hybrids did not enhance hCES2A inhibition activity, as
evidenced by IC50 values of A1 (5.94 M) vs. A4 (5.23 M) or A5
mM) vs. A6 (2.81
mM) or A7 (1.09
mM). Meanwhile,
A
modified indanone-
m
m
3

P.-C. Huo, X.-Q. Guan, P. Liu et al.
European Journal of Medicinal Chemistry 209 (2021) 112856
Fig. 2. Dose-response inhibition curves of indanone-chalcone hybrids against hCES2A (AeI), loperamide (LPA, a known hCES2A inhibitor) was used as a positive inhibitor (J). All
data were expressed as mean SD of the triplicate assays.
4

P.-C. Huo, X.-Q. Guan, P. Liu et al.
European Journal of Medicinal Chemistry 209 (2021) 112856
Table 2
2.2. Design, synthesis and SAR of ring B modified indanone-
IC50 values of B7 against three predominant serine hydrolases (hCES2A, hCES1A and
BuChE) in the humans.
chalcone hybrids
Target enzyme
Enzyme source
Substrate
IC50 (mM)
To get more efficacious hCES2A inhibitors, a series of ring B
modified indanone-chalcone hybrids (B1eB10) were designed and
synthesized using 4-hydroxy-3-methoxybenzaldehyde and
substituted 1-indanones as the starting materials. Meanwhile, B1
and B2 were also synthesized to confirm whether introduction of
an H-Bond donor (-NH2, OH) at the C6 site was beneficial for
hCES2A inhibition.
After getting a series of ring B modified indanone-chalcone
hybrids, the hCES2A inhibition activities of these compounds
were routinely assayed as above mentioned. As listed in Table 1, as
well as depicted in Fig. 2 and Fig. S1, the results clearly showed that
the bearing of a polar group (such as phenolic or amino) at the C-6
site on ring B brought negligible effects on hCES2A inhibition, as
hCES2A
hCES1A
BuChE
HLM
HLM
Human plasma
FD
DME
BTch
0.052 0.004
>100
>100
highly overlapped, there is an imperative need to study the speci-
ficity of B7 on hCES2A over other human esterases, especially the
abundantly distributed esterases in the metabolic organ. In this
study, two esterases (hCES1A and BChE) were used to investigate
the specificity of B7 toward hCES2A. As depicted in Table 2, both
hCES1A and BChE were hardly inhibited by B7 (IC50 values > 100
m
М), suggesting that B7 was a highly selective inhibitor targeting
hCES2A over other human esterases.
evidenced by IC50 values of A7 (1.09
mM) vs. B1 (1.31 mM) and B2
(1.48 M). Considering that the amino and phenolic groups on ring
m
B are readily for chemical modifications, a series of O-alkylated and
N-alkylated derivatives are designed and synthesized. The results
showed that O-alkylation at the C-6 site (e.g., methoxyl and
ethoxyl) slightly decreased hCES2A inhibition, as evidenced by IC50
2.4. Inhibition kinetics of B7 against hCES2A
Next, the inhibitory mechanism of B7 against hCES2A was
investigated by performing a set of inhibition kinetic analyses and
in silico analysis. To judge whether the inhibition of B7 against
hCES2A was time-dependent, time-dependent inhibition assays of
B7 on hCES2A were performed at different pre-incubation times. As
shown in Fig. S2, both the inhibition-response curve and inhibition
potency (IC50 value) of B7 against hCES2A did not changed with
pre-incubation time, suggesting that B7 should be a reversible in-
hibitor rather than a time-dependent inactivator of hCES2A. After
then, a panel of inhibition kinetic assays of B7 against hCES2A-
catalyzed FD hydrolysis was performed. The results clearly
demonstrated that B7 potently inhibited hCES2A-mediated FD
values of A7 (1.09
(2.86 M). By contrast, introduction of a series of N-alkylated sub-
stitutes at the C6 site strongly enhanced the hCES2A inhibition
activity, as indicated by IC50 values of B2 (1.48 M) vs. B6eB9
(ranging from 0.81 to 0.052 M). Among all tested N-alkylated
mM) vs. B3 (2.91 mM), B4 (1.62 mM) and B5
m
m
m
derivatives, B7 (an N-tetrahydropyrrole substituted indanone-
chalcone hybrid) displayed the most potent hCES2A inhibition ac-
tivity (IC50 ¼ 0.052
fold when compared with the first designed indanone-chalcone
M). In addition, modification of the
mM), such activity had been enhanced by 114.2-
hybrid A1 (IC50 ¼ 5.94
m
amino group at the C-6 site to form a small amide also enhanced
the hCES2A inhibition activity, as evidenced by IC50 values of B2
hydrolysis in a mixed inhibition manner, with a Ki value of 0.068 mM
(Fig. 4). Notably, the inhibition potency of B7 was more much
potent than that of LPA, a currently used anti-diarrhea agent with
(1.48 mM) vs. B10 (0.65 mM).
In short summary, almost all synthesized indanone-chalcone
hybrids display strong to moderate hCES2A inhibition activities.
The structure-hCES2A inhibition relationships of these
indanoneechalcone hybrids are summarized as follows, (1) C-40
hydroxyl is beneficial for hCES2A inhibition, (2) C-3’ methoxyl
substitution can enhance hCES2A inhibition slightly, (3) Introduc-
tion of an amide or an N-alkyl groups at the C6 site strongly
enhance hCES2A inhibition activity (Fig. 3). These key findings are
very helpful for the development of novel anti-diarrhea agents
using indanoneechalcone hybrids as leading compounds.
moderate hCES2A inhibition activity (Ki ¼ 1.5
mM) [28].
2.5. Docking simulations
To further explore the interactions between compound B7 and
hCES2A at the molecular level, docking simulations were con-
ducted. As depicted in Fig. 5A, compound B7 could be well-docked
into the hydrophobic pocket in the catalytic cavity of hCES2A,
suggesting that hydrophobic interactions might be the major forces
for the binding of B7 on this enzyme. To validate this assumption,
the predicted octanol/water partition coefficients (log P) and cor-
responding docking score were calculated and listed in Table 1 and
Table S2. As listed in Table 1, the hCES2A inhibition activities of
most tested indanone-chalcone hybrids agreed well with their
2.3. Specificity of B7 on hCES2A
Given that the inhibitor spectra of mammalian esterases are
Fig. 3. The structure-activity relationship of indanone-chalcone hybrids as hCES2A inhibitors.
5

P.-C. Huo, X.-Q. Guan, P. Liu et al.
European Journal of Medicinal Chemistry 209 (2021) 112856
Fig. 4. A) The Lineweave-Burk plots of B7 against hCES2A-catalyzed FD hydrolysis. B) The second plot of slopes from A. All data were expressed as mean SD of the triplicate assays.
Fig. 5. A) The stereo view of hCES2A docked with compound B7. B) The detail view of the binding of compound B7 in the catalytic cavity of hCES2A.
Fig. 6. 2D interactions between compound B7 and the amino acid residues surrounding to the catalytic site of hCES2A.
6

P.-C. Huo, X.-Q. Guan, P. Liu et al.
European Journal of Medicinal Chemistry 209 (2021) 112856
hydrophobicity, except B10, which could be mainly attributed to
the hydrogen-bonding interactions between the amino group of
B10 and Lys-308 of hCES2A (Fig. S3). In these cases, 2D analysis of
the interactions between B7 and hCES2A were conducted. As
shown in Fig. 5B, the heterocyclic ring (ring B) of B7 could create
hydrophobic interactions with Phe-307, Lys-308, Met-309 of
hCES2A, suggesting that the heterocyclic ring (ring B) played a key
role for hCES2A inhibition. Meanwhile, we also found that the
hydrogen bonds could be formed between the C-40 hydroxyl of ring
A with the catalytic residue Ser-228 of hCES2A, suggesting that
introduction of the C-40 hydroxyl group on ring A was beneficial for
hCES2A inhibition (see Fig. 6). These observations were highly
consistent with the findings from structure-hCES2A inhibition
relationship studies of indanoneechalcone hybrids as hCES2A in-
hibitors, such as C-4’ hydroxyl on ring A is beneficial for hCES2A
inhibition.
inhibition potentials against hCES2A were assayed. The results
demonstrated that almost all tested indanone-chalcone hybrids
displayed strong to moderate inhibition against hCES2A. Following
two rounds of structural modifications on indanone-chalcone hy-
brids, we found that the C40-phenolic group and an amide or N-
alkyl groups at the C6 site were beneficial for hCES2A inhibition.
Among all tested indanone-chalcone hybrids, compound B7 (an N-
alkylated indanoneechalcone hybrid) exhibited the most potent
hCES2A inhibition activity and high specificity toward hCES2A over
other human esterases, including hCES1A and butyrylcholinester-
ase (BChE). Inhibition kinetic analyses demonstrated that B7
potently inhibited hCES2A in a mixed inhibition manner, with a Ki
value of 0.068
mM. Further investigations showed that B7 was
capable of inhibiting intracellular hCES2A in living cells and dis-
played good metabolic stability. Collectively, our findings suggest
that indanoneechalcone hybrids are good choices for design and
development of hCES2A inhibitors, while B7 hold great promise for
development of novel anti-diarrhea agents to ameliorate
irinotecan-induced intestinal toxicity.
2.6. Inhibition of intracellular hCES2A by B7 in living HepG-2 cells
Considering that hCES2A is an intracellular hydrolase that was
located in the lumen of the endoplasmic reticulum [29], it is very
necessary to test the inhibition potential of compound B7 on
intracellular hCES2A in living systems. To this end, NCEN (an
isoform-specific fluorogenic substrate for hCES2A) was employed
for sensing hCES2A activities in living HepG-2 cells [19]. As shown
in Fig. S4, B7 could dose-dependently inhibit hCES2A-catalyzed
NCEN hydrolysis in living HepG-2 cells, with the calculated IC50
4. Materials and experimental details
4.1. Chemicals and instruments
All starting materials for chemical synthesis of indanone-
chalcone hybrids were ordered from commercial suppliers (TCI,
Shanghai, China & Energy Chemical, Shanghai, China) and used
without further purification. The reaction was monitored by thin
layer chromatography on precoated silica gel plates (Merck 60
PF254). The final products were purified separately by using col-
umn chromatography, which was carried out on Merck silica gel 60
(200e400 mesh ASTM) packed columns (Merck KGaA, Darmstadt,
Germany). For structural characterization of indanone-chalcone
hybrids, both 1H and 13C NMR spectra were recorded on a Bruker
AVANCE 500/125 MHz instrument, using DMSO‑d6 or CDCl3 as the
solvent. Chemical shifts were reported in parts per million (ppm)
using tetramethylsilane (TMS) as the reference. A Bruker Esquire
3000t mass spectrometer equipped with an electron-spray ioni-
zation source (ESI-MS) was used to analyze the precursor ions of all
synthetic products under positive-ion mode. LC grade acetonitrile
and DMSO were obtained from Tedia Company (Fairfield, OH, USA).
D-luciferin methyl ester (DME) and D-luciferin were purchased
from AAT Bioquest (USA). Loperamide (LPA), fluorescein diacetate
(FD), butyrylthiocholineiodide (BTCh) and galanthamine hydro-
bromide were purchased from TCI (Shanghai, China). N-(2-butyl-
1,3-dioxo-2,3-dihydro-1H-phenalen-6-yl)-2-chloroacetamide
(NCEN) and its hydrolytic metabolite 4-amino-1,8-naphthalimide
(NAH) were synthesized according to the previously reported
scheme [19]. Human liver microsomes (HLMs) were ordered from
Bioreclamation IVT (Baltimore, MD, USA) and stored at ꢁ80 ꢀC until
use. Human plasma samples were obtained from healthy donors
and stored at ꢁ20 ꢀC until use. HepG2 cells were obtained from the
American Type Culture Collection (Teddington, Middlesex, UK). Cell
culture medium and fetal bovine serum were ordered from Hylcone
value as 4.45 mM. Such inhibition potency was even more potent
than that of LPA against hCES2A in human liver microsomes (HLM).
This finding demonstrated that B7 was a cell-permeable compound
and could strongly inhibit the hydrolytic activity of intracellular
hCES2A in living systems, which encouraged us to further investi-
gate the druglikeness properties of B7.
2.7. Metabolic stability assays
Next, the metabolic stability of B7 in human liver microsomes
were tested, while testosterone and umbelliferone were used as the
reference drugs for phase I and phase II metabolism, respectively.
As shown in Table 3 and Fig. S5, the metabolic half-lives of B7 in CYP
and UGT were 96 min, and 103.1 min, respectively, which were
much more stable than that of testosterone (24 min) and umbel-
liferone (15.5 min). This finding demonstrated that B7 exhibited
good metabolic stability, suggesting that this compound could
sever as a promising drug candidate for the development of novel
anti-diarrhea agent to ameliorate irinotecan-induced intestinal
toxicity.
3. Conclusion
In summary, to get an ideal hCES2A inhibitor, a series of
indanone-chalcone hybrids were designed, synthesized, and their
Table 3
(Logan, UK). Testosterone, MgCl2 and
obtained from Dalian Meilun Biotech Co., Ltd (Dalian, China).
Umbelliferone, glucose-6-phosphatedehydrogenase,
-NADPþ and
D-glucose-6-phosphate were
The half-life of B7 in phase
microsomes.
I
or phase II metabolic system in human liver
b
Compound
T1/2 (min)
uridine-50-diphosphoglucuronic acid (UDPGA) were ordered from
Sigma-Aldrich (St. Louis, MO, USA). The stock solutions of each
tested compound and substrate were prepared separately by using
LC grade DMSO (Tedia, USA), while 0.1 M Phosphate buffer (pH 7.4)
was prepared utilizing Millipore water (Millipore, Bedford, USA)
and then stored at 4 ꢀC until use.
Phase I
Phase II
B7
96
24
ND
103.1
NDa
15.5
Testosterone
Umbelliferone
a
ND ¼ Not detected.
7

P.-C. Huo, X.-Q. Guan, P. Liu et al.
European Journal of Medicinal Chemistry 209 (2021) 112856
4.2. Biological assays
V¼(VmaxS)/ [Km (1 þ I/Ki) þ S]
V ¼ (VmaxS)/ [(Km þ S) ꢂ (1 þ I/Ki)]
V¼(VmaxS)/ [(Km þ S) ꢂ (1 þ I/ Ki)]
(1)
(2)
(3)
4.2.1. hCES2A inhibition assay
To assess the inhibitory effects of each indanoneechalcone
hybrid against hCES2A, FD was used as the probe substrate, while
LPA was used as the positive inhibitor. Briefly, the incubation
mixture (0.2 mL) containing 0.1 M phosphate buffer (pH 7.4), HLM
a
where V is the hydrolytic velocity of FD, Vmax is the maximum
hydrolytic velocity. S and I are the substrate (FD) and inhibitor (B7)
concentration, respectively. Ki is the inhibition constant of B7 to-
ward target enzyme; Km is the Michaelis constant for this hydrolytic
reaction [31e33].
(2
varying concentration was incubated at 37 ꢀC for 10 min. After then,
FD (15 M, final concentration) was added to initiate the hydrolytic
mg/mL) and each inhibitor (indanone-chalcone hybrids or LPA) at
m
reaction. After 20 min incubation, ice-cold acetonitrile (0.2 mL) was
added into the incubation system to terminate the hydrolytic re-
action. Meanwhile, the negative control samples (DMSO only) were
also conducted to get the fluorescence signals of the hydrolytic
metabolite of FD without any inhibitor. The fluorescence intensity
of the hydrolytic metabolite of FD was recorded by a multi-Mode
microplate reader (SpectraMax® iD3, Molecular Devices, Austria),
with the excitation wavelength and the emission wavelength of
480 nm, and 525 nm, respectively. The formula for determining the
residual activities of hCES2A are identical to the previous report
[9,30].
4.4. Molecular docking simulations
Since the crystal structure of hCES2A has not been resolved,
homology modeling was used to construct the 3D structure of
hCES2A using a reported crystal structure of hCES1A (PDB ID:
5A7G). The amino acid sequence of hCES2A was retrieved from the
UniProt database (Entry code O00748). The homology modeling of
hCES2A was carried out in SWISS-MODEL. The NCBI Basic Local
Alignment Search Tool (BLAST) was used for searching the crystal
structure template in Protein Data Bank (PDB). The results revealed
hCES1A (PDB ID: 5A7G) with a resolution of 1.48 Å as a suitable
template and with an identity of 48.05% and Global Model Quality
Estimation (GMQE) value 0.74 [24]. Homology modeling was per-
formed by SWISS-MODEL Automated Mode. The validation for the
structure model was assessed using the QMEAN scoring function of
the value ꢁ2.30. Then molecular docking was carried out in Surflex-
Dock within Sybyl-X2.0 (Tripos, St. Louis, MO). Firstly, hydrogen
atoms were added and standard AMBER atomic partial charges
were assigned to hCES2A. The active site was defined by automatic
protomol generation mode in “Define SFXC File” module. After
then, the ligand structures were prepared to generate preferred
conformations at pH of 7.0 in “Ligand Structure Preparation”
module. Finally, Surflex-Dock was performed using default pa-
rameters. Pre/Post-Dock minimization was carried out to optimize
the results. Twenty poses per ligand were generated with a mini-
mum RMSD parameter of 0.50. The binding affinity was exhibited
by Surflex-Dock scores (total scores). The predicted octanol/water
partition coefficients (log P) of compounds were represented by the
value of Consensus Log Po/w was obtained using QikProp Swis-
4.2.2. hCES1A inhibition assay
The procedure and experimental details of hCES1A inhibition
assay have been reported previously, in which DME was used as a
probe substrate to investigate the inhibitory effects [10,30]. Briefly,
the inhibitor (indanone-chalcone hybrids or bis (4 nitrophenyl)
phosphate (BNPP)) mixed with HLM (1 mg/mL, final concentration),
phosphate buffer (0.1 M, pH 6.5) were dissolved in DMSO and
incubated at 37 ꢀC for 10 min. The hydrolytic reaction was initiated
by adding DME (3 mM, final concentration) into the reaction system,
with the final concentration of DMSO at 1% (v/v, without loss of the
catalytic activity). Following 10 min incubation at 37 ꢀC, equal
volume of LDR was added to stop reaction. The hydrolytic metab-
olite (D-luciferin) was determined by detecting the luminescence
signal. The detection conditions and the formula of residual activ-
ities were consistent with the literature reported.
4.2.3. BChE inhibition assay
The procedure and experimental details of BChE inhibition assay
have been reported previously [10,30], in which BTch was used as a
substrate while the diluted human plasma (1:9; V/V) was used as
enzyme source. Briefly, the inhibitor (indanone-chalcone hybrids or
ꢀ
sADME [34]. Discovery Studio Visualizer v19.1 (Dassault Systemes
Biovia Corp) was used to visualize the docking views.
galanthamine hydrobromide) mixed with human plasma (10 mL),
4.5. Inhibition of intracellular hCES2A in living cells
DTNB (2 mM, final concentration), phosphate buffer (0.1 M, pH 7.4)
were dissolved in DMSO and incubated at 37 ꢀC for 3 min. The
Here, NCEN was used as the isoform-specific fluorescent sub-
strate for sensing the residual hCES2A activities in living HepG-
2 cells, owing to its excellent isoform-specificity and outstanding
optical properties [19]. In brief, increasing concentrations of B7 was
hydrolytic reaction was initiated by adding BTch (15
mM, final
concentration) into the reaction system, and the hydrolytic
metabolite was analyzed by detecting the OD signals (optical
density at 412 nm). The detection conditions and the formula of
residual activities were consistent with the literature reported
[10,30].
added in 48-well plates with HepG-2 cells (40,000 cells/300
mL/
well) and incubated for 1 h at 37 ꢀC, then NCEN (10
m
M) was added
into 48-well plates and incubated at 37 ꢀC incubator for another
45 min. After that, equal volume of ice-cold acetonitrile was added
to terminate the hydrolytic reaction. The medium were then
transferred to Eppendorf Tubes and centrifuged at 20,000 g for
20 min at 4 ꢀC. Then the supernatant was taken for further LC-FD
analysis. The detection conditions for quantification of the hydro-
lytic metabolite of NCEN has been reported recently [35,36].
4.3. Inhibition kinetic analyses
The inhibition modes and inhibitory mechanism of B7 against
hCES2A-mediated FD hydrolysis were studied by performing a set
of inhibition kinetic analyses. The inhibition kinetic assays were
conducted utilizing various concentrations of substrates in the
presence of increasing concentrations of B7 (0
m
M, 0.025
mM,
4.6. Metabolic stability assays
0.05 M and 0.1 M). The inhibition mode and inhibition constant
m
m
(Ki) of B7 was determined by fitting the in vitro kinetic data to the
corresponding equations for competitive (1), noncompetitive (2),
or mixed (3) inhibition mode, as depicted follows,
For phase I metabolism, the incubation mixture (0.6 mL) was
consisted of human liver microsomes (0.1 mg/mL), MgCl2 (5 mM),
glucose-6-phosohate (1 mM), glucose-6-phosohate dehydrogenase
8

P.-C. Huo, X.-Q. Guan, P. Liu et al.
European Journal of Medicinal Chemistry 209 (2021) 112856
117 (2017) 84e95.
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3 min and then added with NADPþ (1 mM) to initiate the oxidative
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vortexed for 30
s at room temperature and centrifuged at
20,000 g at 4 ꢀC for 20 min. The supernatants were analyzed by
liquid chromatography coupled with an UV detector.
For phase II metabolism, the incubation mixture (0.6 mL) was
consisted of human liver microsomes (0.1 mg/mL), MgCl2 (5 mM),
B7 or reference drug (10
m
M), and 50 mM Tris-HCl buffer (pH ¼ 7.4).
The reaction mixture was incubated at 37 ꢀC for 3 min and then
added with UDPGA (2 mM) to initiate the reaction. At the different
time points (0-, 5-, 15-, 30-, 60-, 90-min), 100
were transferred to a tube containing 100 L ice-cold acetonitrile to
stop the reaction. The mixtures were then vortexed for 30 s at room
temperature and centrifuged at 20,000 g at 4 ꢀC for 20 min. The
supernatants were analyzed by liquid chromatography coupled
with an UV detector.
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Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
This work was finically supported by the National Key Research
and Development Program of China (2020YFC0845400,
2017YFC1700200, 2017YFC1702000), the NSF of China (81922070,
81973286, 81773687, 81973168), the Three-year Action Plan of
Shanghai TCM Development [ZY-(2018e2020)-CCCX-5001], Pro-
gram of Shanghai Academic/Technology Research Leader
(18XD1403600), Shanghai Talent Development Fund (2019093)
and Shuguang Program (18SG40) supported by Shanghai Education
Development Foundation and Shanghai Municipal Education
Commission.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112856.
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