Discovery of pyrazolones as novel carboxylesterase 2 inhibitors that potently inhibit the adipogenesis in cells

Article abstract of DOI:10.1016/j.bmc.2021.116187

Carboxylesterase 2 (CES2) is one of the most important Phase I drug metabolizing enzymes in the carboxylesterase family. It plays crucial roles in the bioavailability of oral ester prodrugs and the therapeutic effect of some anticancer drugs such as irino

Full text of DOI:10.1016/j.bmc.2021.116187

Bioorg. Med. Chem. 40 (2021) 116187
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of pyrazolones as novel carboxylesterase 2 inhibitors that
potently inhibit the adipogenesis in cells
,
,
Xing-Kai Qiana 1, Jing Zhanga 1, Pei-Fang Songa, Yi-Su Zhaoa, Hong-Ying Maa, Qiang Jina,
Dan-Dan Wanga, Xiao-Qing Guana, Shi-Yang Lic, XiaoZe Baob *, Li-Wei Zoua
,
,*
a Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
b College of Pharmaceutical Science & Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, Zhejiang University of Technology,
Hangzhou 310014, China
c Analytical Central Laboratory, Shengyang Harmony Health Medical Laboratory Co Ltd, 19 Wen Hui Road Shenyang 210112, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Carboxylesterase 2 (CES2) is one of the most important Phase I drug metabolizing enzymes in the carbox-
ylesterase family. It plays crucial roles in the bioavailability of oral ester prodrugs and the therapeutic effect of
some anticancer drugs such as irinotecan (CPT11) and capecitabine. In addition to the well-known roles of CES2
in xenobiotic metabolism, the enzyme also participates in endogenous metabolism and the production of lipids.
In this study, we synthesized a series of pyrazolones and assayed their inhibitory effects against CES2 in vitro.
Structure-activity relationship analysis of these pyrazolones reveals that the introduction of 4-methylphenyl unit
(R1), 4-methylbenzyl (R2) and cyclohexyl (R3) moieties are beneficial for CES2 inhibition. Guided by these SARs
results, 1-cyclohexyl-4-(4-methylbenzyl)-3-p-tolyl-1H- pyrazol-5(4H)-one (27) was designed and synthesized.
Further investigations demonstrated that the compound 27 exhibited stronger CES2 inhibition activity with a
Carboxylesterase 2
Inhibitor
Pyrazolones
Structure-activity relationship
Adipogenesis
lower IC50 value (0.13 μM). The inhibition kinetic study demonstrated that compound 27 inhibited the hydro-
lysis of CES2-fluorescein diacetate (FD) through non-competitive inhibition. In addition, the molecular docking
showed that the core of pyrazolone, the cyclohexane moiety, 4-methylbenzyl and 4-methylphenyl groups in
compound 27 all played important roles with the amino acid residues of CSE2. Also, compound 27 could inhibit
adipocyte adipogenesis induced by mouse preadipocytes. In brief, we designed and synthesized a novel pyr-
azolone compound with a strong inhibitory ability on CES2 and could inhibit the adipogenesis induced by mouse
preadipocytes, which can be served as a promising lead compound for the development of more potent
pyrazolone-type CES2 inhibitors, and also used as a potential tool for exploring the biological functions of CES2
in human being.
1. Introduction
Hydroxy-camptothecin, the hydrolytic metabolite of irinotecan) could
cause severe delayed diarrhea. Increasing evidences have indicated that
Carboxylesterase (CES, E.C. 3.1.1.1) is a super family with 93 sub-
families.1 There are two main types of aryl esterases in the human body,
namely human carboxylesterase 1 (CES1) and human carboxylesterase 2
(CES2). The CES2 is an esterase mainly distributed in human intestine
and colon, which could hydrolyze exogenous compounds containing
ester- and amide,2,3 and metabolize the ester-containing drugs and
environmental toxicants. For example, when the prodrug irinotecan
(CPT-11) featuring a carbamate was used to treat patients with colo-
rectal cancer, the excessive accumulation of SN-38 (7-Ethyl-10-
potent inhibition on intestinal CES2 may reduce the excessive accu-
mulation of SN-38 in the intestinal tract and thereby alleviate the in-
testinal toxicity triggered by irinotecan.4 Therefore, the combination of
CES2 inhibitors and CPT-11 was considered to be an effective way to
relieve the persistent diarrhea.5–7
In addition, CES2 also participates in the metabolism of endogenous
substances in vivo, such as triacylglycerol and diacylglycerol.8 CES2
expression has a certain correlation in non-alcoholic steatohepatitis and
obesity.9 Despite it was reported that CES2 was a triacylglycerol
* Corresponding authors.
E-mail addresses: baoxiaoze@zjut.edu.cn (X. Bao), chemzlw@163.com (L.-W. Zou).
1
These authors contribute equally to this work.
https://doi.org/10.1016/j.bmc.2021.116187
Received 1 March 2021; Received in revised form 9 April 2021; Accepted 26 April 2021
Available online 30 April 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

X.-K. Qian et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116187
hydrolase that assumed as a fundamental part in regulating the hepatic
triglyceride homeostasis by managing endoplasmic reticulum stress,
lipolysis, fatty acid oxidation, and lipogenesis,10 there were articles
showing that knocking out mice’s carboxylesterase 3 (Ces3, homologous
to human CES1) would cause the decrease of its blood lipids, the
improvement of its glucose tolerance and the increase of its energy
consumption.11 Meanwhile, mice’s Ces3 activation contributed to the
browning of white adipocytes in vitro, resulting in the reduction of the
fat formation of 3 T3-L1 adipocytes.12 However, the adipogenic effect of
CES2 in 3 T3-L1 adipocytes has not been reported yet. The relationship
between CES2 and obesity, diabetes and other diseases are hot research
topics. Therefore, the development of CES2 inhibitors are particularly
important for the research of metabolic function and the state and
development of diseases.9,13,14
ester 1S (10 mmol) in 5 mL of anhydrous THF was added dropwise. The
resulting mixture was then warmed to rt for 1 h. After cooled to 0 ◦C, the
corresponding alkyl bromide (10 mmol) or alkyl chloride (10 mmol) was
added dropwise to the mixture. The mixture was then stirred at rt (for
alkyl bromide) or at 70 ◦C (for alkyl chloride). The reaction process was
detected by TLC analysis. After completion, the reaction mixture was
cooled to 0 ◦C, and then quenched by 10 mL of saturated NH4Cl aqueous
carefully. The reaction mixture was extracted with ethyl acetate (3 × 10
mL). The combined organic phase was then washed with brine and dried
over Na2SO4, concentrated. The crude mono-substituted β-ketone ester
2S was used without further purification for next step with quantitive
yield.
To a 50 mL round-bottom flask was added mono-substituted β-ke-
tone ester 2S (10 mmol) and mono-substituted hydrazine (11 mmol),
under argon. The neat mixture was then stirred at 120 ◦C for 4–6 h. The
reaction was detected with TLC analysis. After cooling to rt, the resulting
solid was recrystallized with EtOH to yield the final pyrazolone. If no
product was precipitated, the mixture was then purified by column
chromatography on silica gel with petroleum ether/ethyl acetate as
eluent.
Pyrazolone is a nitrogen-containing five-membered heterocyclic
compound, which is the core structure of many analgesic drugs. For
example, antipyrine, propyphenazone and famprofazone.15 In addition,
pyrazolones have a wide range of biological activities in anti-tubercu-
losis,16 anti-viral,17 anti-hypertension,18 anti-oxidation,19 neuro-
protection,20 anti-diabetic,21 anti-inflammatory22 and anti-cancer.23 In
our previous report, we have developed several highly specific optical
substrates that produced fluorescence after hydrolysis of probes. These
substrates have been successfully used for high-throughput screening of
natural CES2 inhibitors.24–26 Herein, we designed and synthesized a
series of pyrazolone derivatives featuring easily accessible scaffold and a
simple two-step protocol. Through the transformation of the substituent-
properties of these pyrazolones, we established the structure–activity
relationship between CES2 and pyrazolones for the further design and
synthesis of more potent CES2 inhibitor. Finally, the use of the CES2
inhibitor to inhibit the formation of fat granules in induced mouse
preadipocytes was verified in cells.
The pyrazolones exist in tautomeric forms (ketone form and enol
form).30 So the NMR data was usually showed as a mixture of ketone
form and enol form.
4-(4-methylbenzyl)-1,3-diphenyl-1H-pyrazol-5(4H)-one (2) white
solid, yield 40%; 1H NMR for the enol form (400 MHz, DMSO‑d6) δ 11.14
(s, 1H), 7.95 (d, J = 8.0 Hz, 2H), 7.65 (d, J = 7.2 Hz, 2H), 7.55–7.26 (m,
6H), 7.18–7.04 (m, 4H), 3.97 (s, 1H), 2.25 (s, 3H); 13C NMR (101 MHz,
DMSO‑d6) δ 149.6, 138.3, 135.2, 129.4, 128.9, 128.2, 127.5, 126.1,
121.8, 27.9, 21.1; HRMS (EI) m/z Calcd. for C23H20N2O ([M]+)
340.1570, Found 340.1577.
4-(2-methylbenzyl)-1,3-diphenyl-1H-pyrazol-5(4H)-one (4) yellow
solid, yield 48%; 1H NMR for a mixture of ketone form and enol form
(400 MHz, CDCl3) δ 10.07 (s, 1H), 7.94–7.82 (m, 1.6 Hketone), 7.52 (d, J
= 7.8 Hz, 2H), 7.49–7.44 (m, 1.6 Hketone), 7.40–7.34 (m, 2.4H),
7.33–7.28 (m, 1.8H), 7.25–7.17 (m, 8H), 7.09 (t, J = 7.3 Hz, 1H),
7.04–6.92 (m, 6H), 6.89 (d, J = 7.1 Hz, 1H), 3.93–3.84 (m, 0.8H), 3.39
(s, 2H), 3.27 (dd, J = 14.4, 5.9 Hz, 0.8 Hketone), 3.20 (dd, J = 14.4, 7.0
Hz, 0.8 Hketone)., 2.06 (s, 5.4H); 13C NMR (101 MHz, CDCl3) δ 173.2,
159.2, 149.9, 138.1, 138.0, 136.5, 135.9, 134.5, 130.8, 130.5, 130.4,
129.8, 129.7, 129.2, 128.9, 128.7, 128.6, 127.7, 127.2, 126.8, 126.0,
125.9, 125.5, 125.4, 120.6, 119.3, 50.4, 32.7, 25.7, 19.6, 19.50; HRMS
(EI) m/z Calcd. for C23H20N2O ([M]+) 340.1570, Found 340.1566.
4-(4-bromobenzyl)-1,3-diphenyl-1H-pyrazol-5(4H)-one (7) White
solid, yield 50%; 1H NMR (400 MHz, DMSO‑d6) δ 11.07 (br. s, 1H), 7.85
(d, J = 8.0 Hz, 2H), 7.56 (d, J = 6.4 Hz, 2H), 7.52–7.42 (m, 4H),
7.42–7.21 (m, 4H), 7.13 (d, J = 7.2 Hz, 2H), 3.92 (s, 2H). 13C NMR (101
MHz, CDCl3) δ 172.8, 157.8, 137.8, 134.4, 131.5, 131.0, 130.8, 130.1,
129.3, 129.0, 127.9, 126.6, 125.7, 121.4, 119.6, 50.8, 34.8. HRMS (ESI):
calcd. for C22H17BrN2ONa (M+Na)+ 427.0422 found: 427.0414.
1,3-diphenyl-4-(4-(trifluoromethyl)benzyl)-1H-pyrazol-5(4H)-one
(9) white solid, yield 70%; 1H NMR for a mixture of ketone form and
enol form (400 MHz, CDCl3) δ 7.78–7.72 (m, 2H), 7.72–7.65 (m, 2H),
7.64–7.55 (m, 1.3Henol), 7.54–7.46 (m, 3H), 7.45–7.31 (m, 8.3H),
7.30–7.23 (m, 1.7H), 7.23–7.11 (m, 3H), 7.01 (d, J = 8.1 Hz, 2H),
4.11–4.02 (m, 1H), 3.62 (s, 1.2Henol), 3.48 (dd, J = 13.8, 4.5 Hz, 1H),
3.37 (dd, J = 13.8, 5.5 Hz, 1H); 19F NMR (470 MHz, CDCl3) δ ꢀ 62.35,
ꢀ 62.62; 13C NMR (101 MHz, CDCl3) δ 172.6, 157.7, 150.0, 144.6, 139.4,
137.6, 130.7, 129.7, 129.5 (q, J = 32.3 Hz), 129.2, 128.9, 128.8, 128.4,
127.7, 126.5, 125.7, 125.2 (q, J = 4.0 Hz), 124.0 (q, J = 272.7 Hz),
120.4, 119.5, 50.6, 35.0, 28.1; HRMS (EI) m/z Calcd. for C23H17F3N2O
([M]+) 394.1287, Found 394.1283.
2. Methods and materials
2.1. Reagents and standards
Fluorescein diacetate (FD), loperamide (LPA) and bis-p-nitro-phenyl
phosphate (BNPP) were purchased from TCI (Tokyo, Japan). Human
liver microsomes (HLM) were obtained from Research Institute for Liver
Diseases (RILD, Shanghai, China). Insulin, 3-isobutyl1-methylxanthine
were obtained from Sigma (St. Louis, MO, US). Dexamethasone was
obtained from ICN BIOMEDICALS INC. (CA, US). Oil red O staining kit
was purchased from Solarbio (Beijing, China). All other reagents and
fine chemicals with the highest grade were from Sigma, Tedia (Fairfield,
OH, USA), and J&K Chemical Ltd. (Shang-hai, China). Substrate and
compounds were dissolved in DMSO (Tedia, USA) and stored in re-
frigerators at 4 ℃ until use. All 1H NMR and 19F NMR spectra were
recorded on a Bruker Avance II 400 MHz and Bruker Avance III 471 MHz
respectively, 13C NMR spectra were recorded on a Bruker Avance II 101
MHz or Bruker Avance III 126 MHz with chemical shifts reported as ppm
(in CDCl3, TMS as internal standard). Data for 1H NMR are recorded as
follows: chemical shift (δ, ppm), multiplicity (s = singlet, d = doublet, t
= triplet, m = multiplet, br = broad singlet, dd = doublet doublet,
coupling constants in Hz, integration). HRMS (ESI) was obtained with a
HRMS/MS instrument (LTQ Orbitrap XL TM).
2.2. The synthesis of pyrazolones derivatives
Pyrazolones 1, 3, 5, 6, 8, 10, 11, 13, 15, 17, 21–24 were prepared
according to the literature.27–29 Pyrazolones 2, 4, 7, 9, 12, 14, 16,
18–20, 25–27 were prepared from the corresponding β-keto esters 1S
according to the slightly modified procedure from the general
procedure.
4-(naphthalen-1-ylmethyl)-1,3-diphenyl-1H-pyrazol-5(4H)-one (12)
light yellow solid, yield 46%; 1H NMR for a mixture of ketone form and
enol form(400 MHz, CDCl3) δ 10.35 (s, 1H), 8.01–7.94 (m, 0.6Hketone),
7.88–7.69 (m, 3.8H), 7.64 (d, J = 8.3 Hz, 0.6Hketone), 7.57 (d, J = 8.2 Hz,
1H), 7.45–7.26 (m, 7.4H), 7.23–6.87 (m, 13.9H), 4.07–4.00 (m,
General procedure: To a 50 mL round-bottom flask was added 10 mL
of anhydrous THF and NaH (60% in mineral oil, 0.48 g, 12 mmol) under
argon. After cooled to 0 ◦C, a solution of the corresponding β-ketone
2

X.-K. Qian et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116187
0.6Hketone), 3.83 (dd, J = 14.4, 5.3 Hz, 0.6Hketone), 3.72 (s, 2H), 3.46
(dd, J = 14.5, 7.6 Hz, 0.6Hketone); 13C NMR (101 MHz, CDCl3) δ 173.2,
159.6, 149.8, 137.9, 135.5, 133.8, 133.7, 132.2, 131.8, 131.5, 130.9,
130.3, 129.1, 128.9, 128.7, 128.6, 128.5, 128.2, 128.1, 127.6, 126.9,
126.7, 126.2, 125.7, 125.6, 125.5, 125.4, 125.0, 124.9, 123.5, 120.8,
119.3, 50.3, 33.2, 25.2; HRMS (EI) m/z Calcd. for C26H20N2O+ ([M]+)
376.1570, Found 376.1581.
4.20–4.09 (m, 1H), 3.82 (s, 2H), 2.27 (s, 3H), 2.23 (s, 3H), 1.90–1.77 (m,
6H), 1.67 (d, J = 12.6 Hz, 1H), 1.42–1.33 (m, 2H), 1.27–1.18 (m, 1H);
13C NMR (400 MHz, DMSO‑d6) δ 150.10, 146.8, 138.93, 136.5, 134.8,
129.8, 129.2, 128.7, 128.1, 126.9, 126.4, 96.8, 55.1, 32.5, 27.9, 25.7,
25.5, 21.2, 21.0; HRMS (ESI): calcd. for
361.2274, found 361.2288.
C
24H29N2O ([M+H]+)
4-allyl-1,3-diphenyl-1H-pyrazol-5(4H)-one (14) light yellow solid,
yield 43%; 1H NMR (400 MHz, DMSO‑d6) δ 10.82 (br. s, 1H), 7.82 (d, J
= 8.0 Hz, 2H), 7.68 (d, J = 7.2 Hz, 2H), 7.58–7.32 (m, 5H), 7.27 (t, J =
2.3. CES2 inhibitor screening method
The CES 2 inhibitor screening method refers to the previously re-
ported FD as substrate to produce fluorescence product after being hy-
drolyzed. The reaction consists of the following materials: phosphate
7.2 Hz, 1H), 6.06–5.84 (m, 1H), 5.10–4.91 (m, 2H), 3.30 (m, 2H). 13
C
NMR (101 MHz, DMSO‑d6) δ 149.1, 138.6, 137.0, 134.2, 128.9, 128.5,
127.1, 125.7, 121.3, 114.9, 97.7, 26.3. HRMS (ESI): calcd. for
buffer (194
centration 20
dissolved in DMSO) were suspended in an EP tube, and then pre-
incubated at 37℃ for 3 mins. The substrate FD (2 L, dissolved in
DMSO final concentration 50 M) was added to initiate the reaction.
L) was added to
μ
L 50 mM, pH 7.4), CES2 enzyme source (2
μL, final con-
C
18H16N2ONa (M+Na)+ 299.1160 found: 299.1168.
μ
g/mL HLM) and the compound to be screened (2 μL,
4-benzyl-3-(4-bromophenyl)-1-phenyl-1H-pyrazol-5(4H)-one (16)
white solid, yield 65%; 1H NMR for the enol form(400 MHz, DMSO‑d6) δ
11.16 (s, 1H), 7.87 (d, J = 7.9 Hz, 2H), 7.67–7.44 (m, 6H), 7.36–7.09 (m,
6H), 3.99 (s, 2H); 13C NMR (400 MHz, DMSO‑d6) δ 151.0, 148.3, 141.2,
131.8, 129.4, 128.8, 128.3, 126.3, 121.9, 28.1; HRMS (ESI): calcd. for
μ
μ
After 20 mins, the same volume of acetonitrile (200
μ
terminate the reaction. Finally, 200 mL of the mixture was added to the
microplate. Put it into the microplate reader for detection at excitation
wavelength/emission wavelength = 495/520 nm.
C
22H17BrN2NaO+ ([M+Na]+) 427.0416, found 427.0409.
4-benzyl-1-phenyl-3-p-tolyl-1H-pyrazol-5(4H)-one (18) white solid,
yield 55%; 1H NMR for the enol form(400 MHz, DMSO‑d6) δ 11.01 (s,
1H), 7.88 (d, J = 7.1 Hz, 2H), 7.50 (t, J = 7.7 Hz, 4H), 7.30–7.11 (m,
8H), 3.99 (s, 2H), 2.30 (s, 3H); 13C NMR (400 MHz, DMSO‑d6) δ 151.5,
149.5, 141.4, 139.3, 129.4, 128.8, 128.3, 127.3, 126.2, 121.8, 98.8,
28.2, 21.3; HRMS (ESI): calcd. for C23H21N2O ([M+H]+) 341.1648,
found 341.1636.
2.4. Statistical analysis
The calculation methods of half-maximal inhibitory concentration
(IC50) and inhibition kinetics are as previously reported.31 The residual
activities of CES2 were calculated using the following formula: the re-
sidual activity (%) = (the florescence intensity in the presence of in-
hibitor)/the florescence intensity in negative control (DMSO only)
100%. Different inhibitor concentrations were used to determine IC50 of
each compound by Inhibition log(inhibitor) vs. normalized response
using GraphPad Prism 6.0 software. In order to determine the type of
inhibition kinetics (mixed inhibition, non-competitive inhibition or
competitive inhibition), multiple substrate concentrations and various
inhibitor concentrations can be used to determine the corresponding
reaction rate. The second plot from the slope of the Lineweaver-Burk
plot is used as a function of [inhibitor] to calculate the corresponding
inhibition constant (Ki) value. Data are expressed as mean ± SD.
341.1648
4-benzyl-1-phenyl-3-m-tolyl-1H-pyrazol-5(4H)-one (19) white solid,
yield 48%; 1H NMR for the enol form(400 MHz, DMSO‑d6) δ 11.05 (s,
1H), 7.88 (d, J = 7.3 Hz, 2H), 7.50 (t, J = 7.8 Hz, 2H), 7.44–7.07 (m,
10H), 3.99 (s, 2H), 2.27 (s, 3H); 13C NMR (400 MHz, DMSO‑d6) δ 151.5,
149.6, 141.6, 137.8, 134.5, 129.4, 128.8, 128.3, 128.1, 126.3, 124.5,
121.9, 119.1, 99.1, 28.3, 21.5; HRMS (ESI): calcd. for C23H21N2O
([M+H]+) 341.1648, found 341.1641.
4-benzyl-1-phenyl-3-(thiophen-2-yl)-1H-pyrazol-5(4H)-one
(20)
white solid, yield 56%; 1H NMR for the enol form(400 MHz, DMSO‑d6) δ
11.18 (s, 1H), 7.87 (d, J = 8.0 Hz, 2H), 7.56–7.35 (m, 4H), 7.32–7.16 (m,
4H), 7.13–6.99 (m, 3H), 3.85 (s, 2H); 13C NMR (101 MHz, DMSO‑d6) δ
144.7, 140.9, 139.0, 129.4, 128.8, 128.1, 126.3, 121.8, 28.1; HRMS (EI)
m/z Calcd. for C20H14N2OS ([Mꢀ H2]+) 330.0821, Found 330.0832.
4-benzyl-1-methyl-3-phenyl-1H-pyrazol-5(4H)-one (25) light yellow
solid, yield 54%; 1H NMR for a mixture of ketone form and enol form
(400 MHz, CDCl3) δ 8.03–7.98 (m, 1.7H), 7.55 (dd, J = 6.6, 3.1 Hz,
1.3H), 7.44–7.38 (m, 4.5H), 7.33 (dd, J = 7.5, 1.8 Hz, 2.1H), 7.25–7.20
(m, 2.7H), 7.14–7.03 (m, 9.6H), 6.85–6.78 (m, 2.8H), 3.79 (t, J = 5.1 Hz,
0.6Hketone), 3.64 (s, 2H), 3.32 (d, J = 1.1 Hz, 1.8Hketone and enol), 3.28 (d,
J = 4.8 Hz, 0.6Hketone), 3.21 (s, 3H), 3.15 (s, 1.8Hketone), 2.99 (s,
2.4Henol); 13C NMR (101 MHz, CDCl3) δ 175.3, 174.5, 160.9, 157.9,
157.8, 146.6, 140.8, 135.4, 132.5, 131.1, 130.5, 130.4, 130.2, 129.7,
129.1, 129.0, 128.8, 128.6, 128.3, 128.1, 128.0, 127.9, 127.7, 127.3,
127.1, 126.7, 126.3, 125.8, 102.5, 81.0, 49.6, 44.2, 35.1, 31.2, 31.1,
30.9, 28.2; HRMS (EI) m/z Calcd. for C17H16N2O+ ([M]+) 264.1257,
Found 264.1268.
2.5. Molecular docking
The 3D structure of CES2 was downloaded from the SWISS-MODEL
library (a database of annotated 3D protein structure models gener-
ated by homology modeling pipeline), and the accession number is
O00748.32 The docking protocol of CES2 and ligand was as reported in
the previous article.33 Molecular docking was performed using Auto-
Dock Vina version 1.1.2.34 The crystallized waters were removed, and
the protein was assigned polar hydrogens and united atom Kollman
charges. The docking grid was generated with a volume of 80 × 80 × 80
and centered at the catalytic triad (Ser228, Glu345, and His457).2 The
docking simulation was carried out based on the Lamarckian genetic
algorithm. The protein–ligand pose with the lowest binding energy was
displayed in full text.
4-benzyl-1-cyclohexyl-3-phenyl-1H-pyrazol-5(4H)-one (26) white
solid, yield 44%; 1H NMR for a mixture of ketone form and enol form
(400 MHz, DMSO‑d6) δ10.26 (s, 1H), 7.48 (d, J = 6.7 Hz, 2H), 7.33–7.07
(m, 8H), 4.17 (brs, 1H), 3.91 (s, 2H), 1.95–1.80 (m, 6H), 1.67 (d, J =
11.1 Hz, 1H), 1.45–1.34 (m, 2H), 1.25–1.15 (dd, J = 18.5, 10.8 Hz, 1H);
13C NMR (101 MHz, DMSO‑d6) δ 150.1, 146.8, 142.0, 135.5, 128.7,
128.6, 128.2, 127.2, 127.0, 126.1, 96.8, 55.0, 32.6, 28.3, 25.6, 25.5;
2.6. Cell culture
Caco-2 (ATCC HTB-37, Manassas, VA, USA) and HepG2 (ATCC HB-
8065, Manassas, VA, USA) cells were cultured in GibcoDulbecco’s
Modified Eagle medium (DMEM), supplemented with 10% fetal bovine
serum (FBS, Gibco, US), and 1% penicillin–streptomycin (Dalian Meilun
Biotechnology Co., Ltd., Dalian, China) at 37℃ with 5% CO2.35 3 T3-L1
cells (ATCC CL-173, Manassas, VA, USA) were cultured in Gibco-
Dulbecco’s Modified Eagle Medium/F:12 (DMEM/F:12), supplemented
with 10% FBS, and 1% penicillin–streptomycin at 37℃ with 5% CO2.
The method of 3 T3-L1 cells differentiation into adipocytes was
improved on the basis of previous reports.36 The cells were planted in a
HRMS (EI) m/z Calcd. for
C
22H24N2O ([M]+) 332.1883, Found
332.1896.
1-cyclohexyl-4-(4-methylbenzyl)-3-p-tolyl-1H-pyrazol-5(4H)-one
(27) white foam, yield 45%; 1H NMR for the enol form(400 MHz,
DMSO‑d6) δ10.16 (s, 1H), 7.37 (d, J = 8.1 Hz, 2H), 7.10–6.98 (m, 6H),
3

X.-K. Qian et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116187
well plate and cultured for 48 h after the cells were 100% confluent. The
Table 1
The IC50 values of pyrazolones derivatives toward CES2.
CES2 inhibitors were mixed with solution I (10 μg/mL insulin, 0.5 mM 3-
Compound
R1
R2
R3
IC50
isobutyl ꢀ 1- methylxanthine, and 1 mM dexamethasone dissolved in
DMEM/F:12 medium) to different concentrations, then added to
different wells and incubated for 48 h Subsequently, the CES2 inhibitors
(µM)
1
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Bn
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
12.19 ±
1.29
were mixed with solution II (10 μg/mL insulin dissolved in DMEM/F:12
2
4-methylbenzyl
3-methylbenzyl
2-methylbenzyl
4-methoxylbenzyl,
4-fluorobenzyl
4-bromobenzyl
2-chlorobenzyl
5.16 ±
0.43
medium) at different concentrations, and then added to the wells of the
same concentration and incubated for 48 h. Afterwards, the medium in
the wells were replaced with DMEM/F:12 medium with different con-
centrations of CES2 inhibitors and continued to be cultured for 48 h.
3
7.11 ±
1.75
4
23.25 ±
3.92
5
6.68 ±
1.10
2.7. Cytotoxicity assays
6
19.40 ±
3.64
Cell viability was investigated by CCK-8 Kit ((Dalian Meilun
Biotechnology Co., Ltd., Dalian, China)). 3 T3-L1, Caco-2,HepG2 cells (5
7
8.24 ±
0.68
× 104/mL, 200
μL) were seeded in the 96-well plate and maintained at
8
23.70 ±
4.15
37℃ in a 5% CO2 incubator for 24 h. Then the cells were incubated with
different concentrations of compound 27 (0–100 μM) for another 48 h.
9
4-trifluoromethyl-
benzyl
17.01 ±
4.12
CCK-8 (10%, w/v, 100 μL) was added into the adherent cells and fixed in
37℃ for 2 h. The absorbance was measured at 450 nm.
10
3,5-
14.29 ±
3.97
ditrifluoromethyl-
benzyl
2.8. Oil red O staining
11
Ph
Ph
Ph
Ph
2- nitrobenzyl
Ph
10.90 ±
1.40
After the above-mentioned adipocytes were cultured for 48 h. 3 T3-
L1 cells were washed with PBS and fixed for 20–30 mins at room tem-
perature, and then washed again with deionized water for 3 times.
Added 60% isopropanol and washed for 5 mins. The oil red O solution
was soaked in the wells for 20 mins, then washed with deionized water 3
times. Added hematoxylin staining solution to stain the nucleus for 2
mins, and finally added deionized water to observe the stained lipid
droplets under an inverted microscope (Echo Laboratories RevolveFL).
12
1-naphthyl methyl
Ph
22.35 ±
9.48
13
ethyl
Ph
9.04 ±
2.23
14
allyl
Ph
7.76 ±
1.91
15
4-
Bn
Ph
15.06 ±
2.81
methoxylphenyl
4-bromophenyl
16
Bn
Ph
6.19 ±
0.66
17
2-fluorobenzyl
4-methylphenyl
3-methylphenyl
2-thienyl
methyl
Bn
Ph
13.00 ±
3.05
3. Results
18
Bn
Ph
3.82 ±
0.84
3.1. Synthesis of pyrazolones derivatives
19
Bn
Ph
11.17 ±
2.10
A series of pyrazolone compounds were synthesized by a simple two-
step process, as shown in Table 1. Derivatives with different R1, R2 and
R3 were labeled as compounds 1–27. The synthetic route was shown in
Fig. 1. See materials and methods for specific synthetic methods.
20
Bn
Ph
14.09 ±
3.41
21
Bn
Ph
38.13 ±
9.40
22
methyl
2-methylbenzyl
Ph
46.46 ±
12.10
78.20 ±
13.69
24.87 ±
7.43
3.2. CES2 inhibition of pyrazolone derivatives
23
methyl
2-chlorobenzyl
Ph
24
ethyl
Bn
Ph
In the previous works, we screened a series of polycyclic compounds
for CES2 inhibitory activity, such as triterpenes and bis(indolyl)methane
derivatives.24–26,33,37 And tried to explain the relationship between the
modification of different sites on the original nucleus and the inhibition
of CES2. In this study, we performed CES2 activity inhibition screening
on twenty-seven pyrazolone derivatives and measured all the half
inhibitory concentration (IC50), which were listed in Table 1.
25
Ph
Bn
methyl
cyclohexyl
cyclohexyl
50.90 ±
15.27
9.12 ±
2.81
26
Ph
Bn
27
4-methylphenyl
4-methylbenzyl
0.13 ±
0.01
BNPPa
LPAa
6.40 ±
1.20
1.57 ±
0.22
3.3. SAR analysis of pyrazolones derivatives
a
We modified the pyrazolones at positions C3, C4 and N1, which were
showed as R1, R2, and R3, respectively. Combining the results of their
inhibitions to CES2, the structure–activity relationship could be derived.
Compounds 1–14 had the same R1 and R3 (phenyl group). When R2 were
4-methylbenzyl, 3-methylbenzyl, 4-methoxylbenzyl, 4-bromobenzyl,
ethyl, or allyl groups, these pyrazolones showed IC50 values against
Positive inhibitor against CES.
variation of the phenyl group (R1) to methyl or ethyl (compounds
22–24) group finally resulted in a loss of potency (Table 1). Compound
25 with methyl substitute (R3) displayed poor inhibition toward CES2
than compound 1 (R3, Ph), however, the variation of the methyl unit to
cyclohexyl unit (compound 26) finally resulted in a significant increase
of potency (IC50, 9.12 µM). To summarize the above SAR of these
compounds, 4-methylphenyl unit (R1), 4-methylbenzyl (R2) and cyclo-
hexyl (R3) moiety were beneficial for pyrazolone’s inhibitory property
toward CES2, resulting in a dramatic increase of the inhibitory effect on
CES2. Guided by these SARs results, we synthesized a new compound 27
CES2 less than 10 μM (Table 1). Among them, compound 2 with 4-meth-
ylbenzyl unit showed the best inhibitory effect against CES2 with IC50
value of 5.16 µM. Compounds 15–21 were substituted with benzyl and
phenyl at the C4 and N1 positions, respectively. Further characteriza-
tions of compounds 15–21 with the variation of R1 unit demonstrated
that compound 18 with 4-methylphenyl substitute was more beneficial
for inhibitory property towards CES2 with IC50 value of 3.82 µM. The
4

X.-K. Qian et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116187
Fig. 1. Chemical structures and synthetic route of pyrazolone.
with 4-methylphenyl (R1), 4-methylbenzyl (R2), and cyclohexyl (R3)
unit. This novel compound 27 showed potent inhibitory activity against
CES2 with the IC50 value of 0.13 µM (Fig. 2a). Compared with the IC50
values of CES2′s positive inhibitors BNPP and LAP, which were 6.40
μM
and 1.57 μM, respectively, the IC50 value of compound 27 were about 10
to 60 times lower than the aforementioned two positive inhibitors.
3.4. Inhibition kinetic analyses
The strong inhibitory ability of compound 27 on CES2 prompted us
to further study the inhibitory mechanism of this pyrazolone derivative
on CES2. As shown in Fig. 2b-c, the enzymatic kinetics of FD, Michaelis-
Menten equations, and linewever-burk diagram showed that compound
27 inhibited CES2 activity with Ki value of 0.09 μM. The results showed
that the type of inhibition of compound 27 was non-competitive inhi-
bition, which had a quite prominent ability to inhibit CES2 activity.
3.5. Docking simulation of compound 27 into human CES2
In order to explore the interaction mode between compound 27 and
CES2, molecular docking was simulated. The pyrazolones exist in
tautomeric forms (keto form and enol form) (Chauhan et al., 2015). So
the ligand 27 position of the enol form and keto form with the lowest
binding energy in the CES2 complex were shown in Fig. 3 and Fig. S2,
respectively. When compound 27 core existed in enol form, the binding
energy of compound 27 on CES2′s catalytic cavity was lower than that of
FD on CES2 (-9.1 kcal/mol VS ꢀ 8.9 kcal/mol), indicating that the
binding energy of compound 27 on CES2′s catalytic cavity was more
stable than FD. Compound 27 could interact well with the active cavity
of CES2, and the main interaction was hydrophobic. Residue Ala150
Fig 3. (a) CES2 structure and stereogram of compound 27 (enol form); (b)
Detailed view of compound 27 (enol form) in the active site of CES2. (c) CES2
structure and stereogram of compound 27 (keto form); (d) Detailed view of
compound 27 (keto form) in the active site of CES2.
His457, Phe106 and Ile350 residues). Meanwhile, the Ala150 residue
could simultaneously form two alkyl-π interaction with the cyclohexane
located at the bottom of the gorge could form an σ-π interaction with the
group (R3) and the 4-methylbenzyl group (R2). Both Met309 and Leu262
core of pyrazolone and form alkyl-π interaction with 4-methylphenyl
residues formed alkyl-
π
interaction with the 4-methylbenzyl group (R2).
group (R1), 4-methylbenzyl group (R2) and the cyclohexane group
(R3). Also, Met354 near the entrance of the gorge could form a sulfur-
π
Similarly, the Met354 formed alkyl-π, σ-π, sulfur-π interaction with the
4-methylphenyl group (R1). The molecular docking results indicated
that two tautomeric forms of compound 27 had a stronger binding
ability with the active cavity of CES2 than that of FD, the pyrazolone
interaction with the 4-methylphenyl group (R1). The His457 could form
cation-
π
with 4-methylbenzyl group (R2). When compound 27 core was
in the form of keto, its binding energy with CES2 (ꢀ 8.6 kcal/mol) was
slightly larger than that of FD, as shown in Fig. 3 and Fig. S2. The
cyclohexane group (R3) of compound 27 penetrates into the active
core of enol form 27 could form an σ-π interaction with the residue
Ala150 of CES2 located at the bottom of the gorge, and the cyclohexane
group (R3) of keto form 27 penetrates into the active cavity to form
cavity to form alkyl-π conjugates with multiple amino acids (Val353,
Fig. 2. (a) The log(inhibitor) vs. normalized response of 27; (b) Enzymatic kinetics of FD and Michaelis-Menten equation; (c) The Lineweave-Burk plot of 27 against
CES2-mediated FD.
5

X.-K. Qian et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116187
multiple amino acids alkyl-π conjugates.
3.6. Pyrazolones inhibit the formation of lipid droplets in 3 T3-L1 cells
In order to study the effect of CES2 inhibitors on the formation of
lipid droplets during the induction of mouse preadipocytes 3 T3-L1 into
adipocytes, compound 27 was added for pretreatment before cell in-
duction. The results of the cell viability test showed that compound 27
had almost no toxicity to 3 T3-L1, Caco-2 and HepG2 below 40 μM, as
shown in Fig. S3. We formulated compound 27, which was the strongest
inhibitor of CES2, into different concentrations and mixed with an
inducing agent that induces adipocyte differentiation, and incubated
with the cells for 72 h. The cells were stained with oil red O dye after
culturing with compound 27 in normal medium for 48 h. The results
showed that the lipid droplets in the induced 3 T3-L1 cells (Fig. 4a) were
significantly higher than those in the uninduced control group (Fig. 4b).
However, there was almost no formation of lipid droplets in the cells
treated with compound 27 of high concentration (40
μ
M). The forma-
tion of lipid droplets in cells treated with low concentration (10
μ
M) was
similar to that of the uninduced control. It could be seen from the Fig. 4c-
f that the content of lipid droplets of compound 27 gradually increased
from high concentration to low concentration. The results indicated that
CES2 inhibitors may inhibit the formation of lipid droplets in adipocytes
induced by preadipocytes.
Fig. 4. Oil red O staining results of 3 T3-L1 cells (a) normally cultured and (b-f)
differentiation culture treated with compound 27 (0–40
tion, 20×).
μM) (magnifica-
will increase the differentiation of adipocytes and reduce the fat pro-
duction. Inhibition of CES1 increased fat production.12 Different from
CES2, the above research results showed that CES2 inhibitors could also
inhibit adipogenesis and lipogenesis. This might be related to the fact
that CES1 was more involved in the metabolism of endogenous fatty
acids, while CES2 was less involved in endogenous metabolism. This
also indicated that compound 27 may inhibit adipogenesis and lipo-
genesis through the inhibition of CES2 or other metabolic mechanisms.
In summary, through the SAR analysis of the inhibitory effects of
synthesized pyrazolones against CES2, a novel pyrazolone 27 featuring
4-Methylphenyl (R1), 4-methylbenzyl (R2), and cyclohexyl (R3) moieties
was designed and synthesized. This new molecule had the best inhibi-
tory effect on CES2 via a non-competitive manner with IC50 value of
4. Discussion
The results indicated that pyrazolone compounds exhibited an
inhibitory effect on CES2. By comparing the inhibitory activity of a se-
ries of compounds on CES2, we found that compound 27 has a strong
inhibitory effect on CES2. The discovery and use of pyrazolone com-
pounds provides a new way of thinking, as well as the treatment of
metabolic toxicity caused by CES2 provide a more effective drug can-
didates or lead compounds.
A large number of drugs were purposely or accidentally designed as
prodrugs, which could be hydrolyzed by CES2.38 For example, although
the metabolism of CPT-11 in the human body might be mainly partici-
pated by P450 enzymes, the metabolite of CES2 was the main structure
that exerts its efficacy.39 Especially in the intestine, the activity of CES2
was the highest relative to other organs. Mass production of SN-38 could
cause delayed diarrhea. Therefore, the most effective way was to alle-
viate the hydrolysis rate of CPT-11 by inhibiting the activity of CES2 in
the intestine. According to our results, compound 27 might be a po-
tential treatment drug for persistent diarrhea caused by CPT-11. The
results showed that compound 27 inhibited the hydrolysis of FD through
non-competitive inhibition. The hydrolysis of CPT-11 might be similar
to FD, and it also inhibit the production of SN-38 through non-
competitive inhibition. The advantage of this type of inhibition was
that it could prevent compound 27 from inhibiting product production
by competing with the substrate. Vmax becomes smaller but km remains
unchanged, which only affects the amount of product produced without
affecting the structure of the enzyme.40 The docking results also verified
the intermolecular interaction between compound 27 and amino acid
residues of CES2. The results showed that compound 27 might have
tautomerism, and the two forms of structure have strong interactions
with CES2. Although compound 27 could dock in the active cavity, they
do not interact with Ser228 and Glu345 in the active cavity triplet. This
was also consistent with the non-competitive inhibition of compound 27
and the substrate. There is also a possibility that CES2 does not have a
crystal structure and can only be docked by homology modeling, which
means that there may be other potential sites that interact better with
compound 27 and affect the conformation and activity of the active
cavity.41
0.13 μM. In addition, the molecular docking showed that the core of
pyrazolone, the cyclohexane group (R3), 4-methylbenzyl group (R2),
and the 4-methylphenyl group (R1) in compound 27 all played impor-
tant roles with the amino acid residues of CSE2. The property of 27 to
inhibit the formation of intracellular lipid droplets was also confirmed
by the mouse preadipocyte induction experiments, which may explained
by the obstruction of intracellular adipogenesis after inhibiting CES2.
Thus, this new molecule can be served as a promising lead compound for
the medicinal chemists to design and develop more efficacious CES2
inhibitors and a potential tool for the biologists to explore the biological
functions of CES2 in human being.
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.
Acknowledgements
This work was supported by the NSF of China (82073813, 82003847,
81803489, 81703604, 81903576 and 81973393), the National Science
and Technology Major Project of China (2018ZX09731016) and the
National Key Research and Development Program of China
(2017YFC1702000).
Compounds may have multiple effects in the human body.42 The
highly homologous enzyme CES1 has been reported to increase adipo-
genesis and weaken lipogenesis after been activated. Inhibition of CES1
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
6

X.-K. Qian et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116187
aminosulfonyl pharmacophore as inhibitors of COX-2/5-LOX enzymes:
org/10.1016/j.bmc.2021.116187.
Histopathological and docking studies. Bioorg Chem. 2018;78:103–114.
23 Sun X, Zhang L, Gao M, et al. Nanoformulation of a novel pyrano[2,3-c] pyrazole
heterocyclic compound AMDPC exhibits anti-cancer activity via blocking the cell
cycle through a P53-independent pathway. Molecules. 2019;24.
24 Bao X, Wei S, Qian X, et al. Asymmetric construction of a multi-pharmacophore-
containing dispirotriheterocyclic scaffold and identification of a human
carboxylesterase 1 inhibitor. Org Lett. 2018;20:3394–3398.
References
1 Long JZ, Cravatt BF. The metabolic serine hydrolases and their functions in
mammalian physiology and disease. Chem Rev. 2011;111:6022–6063.
2 Wang D, Zou L, Jin Q, Hou J, Ge G, Yang L. Human carboxylesterases: a
comprehensive review. Acta pharmaceutica Sinica. B. 2018;8:699–712.
3 Laizure SC, Herring V, Hu Z, Witbrodt K, Parker RB. The role of human
carboxylesterases in drug metabolism: have we overlooked their importance?
Pharmacotherapy. 2013;33:210–222.
25 Zou LW, Li YG, Wang P, et al. Design, synthesis, and structure-activity relationship
study of glycyrrhetinic acid derivatives as potent and selective inhibitors against
human carboxylesterase 2. Eur J Med Chem. 2016;112:280–288.
26 Zou LW, Dou TY, Wang P, et al. Structure-activity relationships of pentacyclic
triterpenoids as potent and selective inhibitors against human carboxylesterase 1.
Front Pharmacol. 2017;8:435.
4 Xu G, Zhang W, Ma MK, McLeod HL. Human carboxylesterase 2 is commonly
expressed in tumor tissue and is correlated with activation of irinotecan. Clin Cancer
Res: Off J Am Assoc Cancer Res. 2002;8:2605–2611.
27 Gagnon PE, Boivin JL, Paquin RJ. Synthesis, potentiometric titrations, and spectra of
pyrazolones. Can J Chem. 1953;31:1025–1039.
5 Wang DD, Jin Q, Hou J, et al. Highly sensitive and selective detection of human
carboxylesterase 1 activity by liquid chromatography with fluorescence detection.
J Chromatogr B, Anal Technol Biomed Life Sci. 2016;1008:212–218.
6 Wang DD, Zou LW, Jin Q, Hou J, Ge GB, Yang L. Recent progress in the discovery of
natural inhibitors against human carboxylesterases. Fitoterapia. 2017;117:84–95.
7 Zou LW, Jin Q, Wang DD, et al. Carboxylesterase inhibitors: an update. Curr Med
Chem. 2018;25:1627–1649.
28 Liao YH, Chen WB, Wu ZJ, et al. Organocatalytic asymmetric michael addition of
pyrazolin-5-ones to nitroolefins with bifunctional thiourea: stereocontrolled
construction of contiguous quaternary and tertiary stereocenters. Adv Synth Catal.
2010;352:827–832.
29 Wang Z, Yang ZG, Chen DH, Liu XH, Lin LL, Feng XM. Highly enantioselective
michael addition of pyrazolin-5-ones catalyzed by chiral metal/N, N ’-dioxide
complexes: metal-directed switch in enantioselectivity. Angew Chem-Int Ed. 2011;50:
4928–4932.
8 Ross MK, Crow JA. Human carboxylesterases and their role in xenobiotic and
endobiotic metabolism. J Biochem Mol Toxicol. 2007;21:187–196.
9 Ruby MA, Massart J, Hunerdosse DM, et al. Human carboxylesterase 2 reverses
obesity-induced diacylglycerol accumulation and glucose intolerance. Cell reports.
2017;18:636–646.
30 Chauhan P, Mahajan S, Enders D. Asymmetric synthesis of pyrazoles and pyrazolones
employing the reactivity of pyrazolin-5-one derivatives. Chem Commun. 2015;51:
12890–12907.
31 Weng ZM, Ge GB, Dou TY, et al. Characterization and structure-activity relationship
studies of flavonoids as inhibitors against human carboxylesterase 2. Bioorg Chem.
2018;77:320–329.
10 Li Y, Zalzala M, Jadhav K, et al. Carboxylesterase 2 prevents liver steatosis by
modulating lipolysis, endoplasmic reticulum stress, and lipogenesis and is regulated
by hepatocyte nuclear factor 4 alpha in mice. Hepatology. 2016;63:1860–1874.
11 Wei E, Ben Ali Y, Lyon J, et al. Loss of TGH/Ces3 in mice decreases blood lipids,
improves glucose tolerance, and increases energy expenditure. Cell Metab. 2010;11:
183–193.
32 Waterhouse A, Bertoni M, Bienert S, et al. SWISS-MODEL: homology modelling of
protein structures and complexes. Nucleic Acids Res. 2018;46:W296–W303.
33 Zhao Y-S, Ruan H-L, Wang X-Y, et al. Catalyst-free visible-light-induced condensation
to synthesize bis(indolyl)methanes and biological activity evaluation of them as
potent human carboxylesterase 2 inhibitors. RSC Adv. 2019;9:40168–40175.
34 Wang Z, Sun HY, Yao XJ, et al. Comprehensive evaluation of ten docking programs
on a diverse set of protein-ligand complexes: the prediction accuracy of sampling
power and scoring power. PCCP. 2016;18:12964–12975.
12 Mukherjee S, Choi M, Yun JW. Novel regulatory roles of carboxylesterase 3 in lipid
metabolism and browning in 3T3-L1 white adipocytes. Appl Physiol Nutrit Metab =
Physiologie appliquee, nutrition et metabolisme. 2019;44:1089–1098.
13 Li B, Cheng Y, Yu S, et al. Human umbilical cord-derived mesenchymal stem cell
therapy ameliorates nonalcoholic fatty liver disease in obese type 2 diabetic mice.
Stem Cells Int. 2019;2019:8628027.
35 Xue L, Qian X, Jin Q, et al. Construction and application of a high-content analysis
for identifying human carboxylesterase 2 inhibitors in living cell system. Anal Bioanal
Chem. 2020;412:2645–2654.
14 Tian X, Yan F, Zheng J, et al. Endoplasmic reticulum targeting ratiometric
fluorescent probe for carboxylesterase 2 detection in drug-induced acute liver injury.
Anal Chem. 2019;91:15840–15845.
36 Zebisch K, Voigt V, Wabitsch M, Brandsch M. Protocol for effective differentiation of
3T3-L1 cells to adipocytes. Anal Biochem. 2012;425:88–90.
15 Levy M. Hypersensitivity to pyrazolones. Thorax. 2000;55(Suppl 2):S72–74.
16 Sweeney NL, Lipker L, Hanson AM, et al. Docking into mycobacterium tuberculosis
thioredoxin reductase protein yields pyrazolone lead molecules for methicillin-
resistant Staphylococcus aureus. Antibiotics. 2017;6.
37 Song P-F, Zhu Y-D, Ma H-Y, et al. Discovery of natural pentacyclic triterpenoids as
potent and selective inhibitors against human carboxylesterase 1. Fitoterapia. 2019;
137.
38 Yao M, Ma X, Zhang X, et al. Lectin-mediated pH-sensitive doxorubicin prodrug for
pre-targeted chemotherapy of colorectal cancer with enhanced efficacy and reduced
side effects. Theranostics. 2019;9:747–760.
17 Ramajayam R, Tan KP, Liu HG, Liang PH. Synthesis and evaluation of pyrazolone
compounds as SARS-coronavirus 3C-like protease inhibitors. Bioorg Med Chem. 2010;
18:7849–7854.
39 Rivory LP, Riou JF, Haaz MC, et al. Identification and properties of a major plasma
metabolite of irinotecan (CPT-11) isolated from the plasma of patients. Cancer Res.
1996;56:3689–3694.
18 Hassan MQ, Akhtar MS, Afzal O, et al. Edaravone and benidipine protect myocardial
damage by regulating mitochondrial stress, apoptosis signalling and cardiac
biomarkers against doxorubicin-induced cardiotoxicity. Clin Exp Hypertens. 2020;42:
381–392.
40 Rafiq M, Saleem M, Hanif M, Kang SK, Seo SY, Lee KH. Synthesis, structural
elucidation and bioevaluation of 4-amino-1,2,4-triazole-3-thione’s Schiff base
derivatives. Arch Pharm Res. 2016;39:161–171.
19 Bailly C. Potential use of edaravone to reduce specific side effects of chemo-, radio-
and immuno-therapy of cancers. Int Immunopharmacol. 2019;77, 105967.
20 Bhandari R, Kuhad A, Kuhad A. Edaravone: a new hope for deadly amyotrophic
lateral sclerosis. Drugs Today. 2018;54:349–360.
41 Chae J, Choi I, Kim C. Homology modeling and molecular docking study of
translationally controlled tumor protein and artemisinin. Arch Pharm Res. 2006;29:
50–58.
21 Haroun M. Novel hybrids of pyrazolidinedione and benzothiazole as TZD analogues.
Rationale design, synthesis and in vivo anti-diabetic evaluation. Med Chem. 2019;15:
624–633.
42 Kong TY, Kim JH, Kim DK, Lee HS. Synthetic cannabinoids are substrates and
inhibitors of multiple drug-metabolizing enzymes. Arch Pharm Res. 2018;41:
691–710.
22 Abdelgawad MA, Labib MB, Ali WAM, Kamel G, Azouz AA, El-Nahass ES. Design,
synthesis, analgesic, anti-inflammatory activity of novel pyrazolones possessing
7