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
15.5
Testosterone
Umbelliferone
a
ND ¼ Not detected.
7