Design, synthesis and biological evaluation of novel chalcone-like compounds as potent and reversible pancreatic lipase inhibitors

Article abstract of DOI:10.1016/j.bmc.2020.115853

Pancreatic lipase (PL), a crucial enzyme responsible for hydrolysis of dietary lipids, has been validated as a key therapeutic target to prevent and treat obesity-associated metabolic disorders. Herein, we report the design, synthesis and biological evaluation of a series of chalcone-like compounds as potent and reversible PL inhibitors. Following two rounds of structural modifications at both A and B rings of a chalcone-like skeleton, structure-PL inhibition relationships of the chalcone-like compounds were studied, while the key substituents that would be beneficial for PL inhibition were revealed. Among all tested chalcone-like compounds, compound B13 (a novel chalcone-like compound bearing two long carbon chains) displayed the most potent PL inhibition activity, with an IC₅₀ value of 0.33 μM. Inhibition kinetic analyses demonstrated that B13 could potently inhibit PL-mediated 4-MUO hydrolysis in a mixed inhibition manner, with the K_i value of 0.12 μM. Molecular docking simulations suggested that B13 could tightly bind on PL at both the catalytic site and a non-catalytic site that was located on the surface of PL, which was consistent with the mixed inhibition mode of this agent. In addition, B13 displayed excellent stability in artificial gastrointestinal fluids and good metabolic stability in human liver preparations. Collectively, our findings suggested that chalcone-like compounds were good choices for design and development of orally administrated PL inhibitors, while B13 could be served as a promising lead compound to develop novel anti-obesity agents via targeting on PL.

Full text of DOI:10.1016/j.bmc.2020.115853

Journal Pre-proofs
Design, synthesis and biological evaluation of novel chalcone-like compounds
as potent and reversible pancreatic lipase inhibitors
Peng-Chao Huo, Qing Hu, Sheng Shu, Qi-Hang Zhou, Rong-Jing He, Jie
Hou, Xiao-Qing Guan, Dong-Zhu Tu, Xu-Dong Hou, Peng Liu, Nan Zhang,
Zhi-Guo Liu, Guang-Bo Ge
PII:
S0968-0896(20)30683-0
https://doi.org/10.1016/j.bmc.2020.115853
BMC 115853
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
31 July 2020
15 October 2020
1 November 2020
Please cite this article as: P-C. Huo, Q. Hu, S. Shu, Q-H. Zhou, R-J. He, J. Hou, X-Q. Guan, D-Z. Tu, X-D. Hou,
P. Liu, N. Zhang, Z-G. Liu, G-B. Ge, Design, synthesis and biological evaluation of novel chalcone-like
compounds as potent and reversible pancreatic lipase inhibitors, Bioorganic & Medicinal Chemistry (2020), doi:
https://doi.org/10.1016/j.bmc.2020.115853
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Research paper
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Design, synthesis and biological evaluation of novel chalcone-like compounds as potent
and reversible pancreatic lipase inhibitors
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Peng-Chao Huo a,b,1, Qing Hu b,1, Sheng Shu c,1, Qi-Hang Zhou b, Rong-Jing He b, Jie Hou d,
Xiao-Qing Guan b, Dong-Zhu Tu b, Xu-Dong Hou d, Peng Liu c, Nan Zhang a,*, Zhi-Guo Liu
c,*, Guang-Bo Ge b,*
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a School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
b
Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of
Traditional Chinese Medicine, Shanghai, China
c
Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical
University, Wenzhou, Zhejiang, China
d Department of Biotechnology, College of Basic Medical Sciences, Dalian Medical University,
Dalian 116044, China.
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* Corresponding authors
E-mail address: zhangnan@zzu.edu.cn (Zhang N.)
lzgcnu@163.com (Liu Z.)
geguangbo@dicp.ac.cn (Ge G.)
1 contributed equally.

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Abstract
Pancreatic lipase (PL), a crucial enzyme responsible for hydrolysis of dietary lipids, has been
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validated as a key therapeutic target to prevent and treat obesity-associated metabolic disorders.
Herein, we report the design, synthesis and biological evaluation of a series of chalcone-like
compounds as potent and reversible PL inhibitors. Following two rounds of structural
modifications at both A and B rings of a chalcone-like skeleton, structure-PL inhibition
relationships of the chalcone-like compounds were studied, while the key substituents that
would be beneficial for PL inhibition were revealed. Among all tested chalcone-like
compounds, compound B13 (a novel chalcone-like compound bearing two long carbon chains)
displayed the most potent PL inhibition activity, with an IC50 value of 0.33 μM. Inhibition
kinetic analyses demonstrated that B13 could potently inhibit PL-mediated 4-MUO hydrolysis
in a mixed inhibition manner, with the Ki value of 0.12 μM. Molecular docking simulations
suggested that B13 could tightly bind on PL at both the catalytic site and a non-catalytic site
that was located on the surface of PL, which was consistent with the mixed inhibition mode of
this agent. In addition, B13 displayed excellent stability in artificial gastrointestinal fluids and
good metabolic stability in human liver preparations. Collectively, our findings suggested that
chalcone-like compounds were good choices for design and development of orally
administrated PL inhibitors, while B13 could be served as a promising lead compound to
develop novel anti-obesity agents via targeting on PL.
Keywords: Pancreatic lipase (PL); chalcone-like compounds; inhibition; anti-obesity agent

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1. Introduction
Obesity is a chronic, multifactorial problem which has become one of the biggest threats to
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global health in modern society [1, 2]. It has been reported that obesity is tightly linked to a
variety of metabolic disorders, such as insulin resistance, diabetes mellitus, cardiovascular
diseases and various types of cancer [3-6]. Especially, cardiovascular diseases caused by
obesity have become a serious public health problem in the worldwide [7]. Over the past few
decades, numerous therapeutic targets for the prevention and treatment of obesity have been
explored [8-15]. Among them, pancreatic lipase (PL), a crucial enzyme responsible for the
hydrolysis of dietary triglycerides (TAGs) into the fatty acids and glycerol in gastrointestinal
system [1, 16-19], has been validated as a key target to prevent and treat obesity or obesity-
related disorders. Orlistat, an FDA-approved orally administrated anti-obesity drug with potent
PL inhibition activity both in vitro and in vivo, has been widely used to reduce the absorption
of dietary lipids in clinical settings [20-22]. However, undesirable gastrointestinal (GI) adverse
effects (including flatulence with discharge and abdominal pain, oily spotting, bowel urgency,
oily stool leakage, steatorrhea, and intestinal borborygmi) strongly block its long-term use in
patients, especially the fragile populations (such as the elderly and young children) [3, 23].
Hence, it is highly desirable to develop more efficacious orally administrated PL inhibitors
with good safety profiles.
Over the past few decades, a wide range of natural products or herbal constituents have been
found with strong PL inhibition activities, such as pentacyclic triterpenoids, flavonoids and
chalcones [24-26]. In contrast to the synthetic PL inhibitors (such as orlistat), the natural PL
inhibitors possess relatively high safety profiles. But in most cases, the PL inhibition potency,
chemical stability, oral bioavailability and metabolic stability of naturally occurring PL
inhibitors are far away from that of synthetic PL inhibitors [27-29]. In these cases, it is
necessary to develop some semi-synthetic compounds or the derivatives of natural PL
inhibitors as novel PL inhibitors possessing high potency, low toxicity, and acceptable
metabolic stability. Notably, some natural chalcones isolated from herbs (such as morachalcone
A, licochalcone A, isobavachalcone and bavachalcone) have been found with relatively strong
PL inhibition activities (IC50 values of less than 10 μM) and good safety profiles [3, 23],
providing useful hits for the medicinal chemists to develop more efficacious PL inhibitors

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(Fig.1). However, natural chalcones are α,β-unsaturated ketones which display poor
photostability and can be transformed into various by-products following exposure to UV light
[30-34]. Meanwhile, natural chalcones can also be readily metabolized by a panel of human
drug-metabolizing enzymes, leading to short half-lives in vivo [35]. To overcome these
shortcomings, series of chemically stable chalcone-like compounds has been designed for
various purposes in recent years. For example, the synthetic indanone–chalcone hybrids have
been reported with MAO-B inhibition and CES2A inhibition activities [36, 37], while 2-
benzylidene-1-indanone derivatives have been found with AChE inhibitory activities [38].
Inspired by these studies, it is feasible to design and develop series of chemically stable
chalcone-like compounds as PL inhibitors via exploring their structure-PL inhibition activities
and improving their druglikeness properties.
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Herein, a total of 30 chalcone-like compounds were designed and synthesized as potential
PL inhibitors. Following two rounds of structural modifications and screening, as depicted in
Scheme 1, the structure-PL inhibition relationships of the chalcone-like compounds were well
investigated, while the key substituents that would be beneficial for PL inhibition were
revealed. Finally, B13, a novel chalcone-like compound bearing two long carbon chains, was
found with the most potent PL inhibition activity. After that, a set of inhibition kinetic analyses
and in silico analysis were performed to explore the inhibitory mechanism of B13 against PL,
while the stability of B13 in both artificial gastrointestinal fluids and human liver preparations
was also assayed. All these findings suggested that chalcone-like compounds were good
choices for design and development of orally administrated PL inhibitors, while B13 could be
acted as a promising candidate to develop novel anti-obesity agents via targeting on PL.
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2. Results and Discussion
2.1 Chemistry
In view of the fact that natural chalcones are chemically unstable at room temperature or
under physiological conditions, the α, β-unsaturated ketone moiety was slightly modified to
form a five-member carbon ring to improve the chemical stability of these compounds (Fig.1).
In this study, all chalcone-like compounds were synthesized according to a previously
described synthetic procedure [39]. As depicted in Scheme 2, A1, A2, A4-A7, A9-A16 were

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prepared via the Claisen Schmidt condensation reaction. The commercially available 6-
hydroxy-1-indanone was protected by tetrahydropyranyl (THP) to give 6-[(tetrahydro-2H-
pyran-2-yl)oxy]-1-indanone, and the chalcone-like compounds intermediates were obtained by
coupling THP protected 1-indanone with various benzaldehydes in the ethanolic sodium
hydroxide solution. In the last step, deprotection of the protective groups with hydrochloric
acid in ethanol used as an efficient reagent to give the desired chalcone-like compounds.
Alternatively, hydrochloric acid gas was employed to catalyze the aldol condensation to
provide the final products A3 and A8, with 6-hydroxy-1-indanone and hydroxyl-
benzaldehydes used as reaction materials. Similarly, the chalcone-like compounds derivatives
B1-B14 were synthesized via a condensation reaction, in which 4-hydroxy-3-
methoxybenzaldehyde and substituted 1-indanones were served as the start materials,
respectively. All of these synthetic compounds were fully characterized by 1H-NMR, 13C-NMR,
and ESI-MS, and their purities were over than 96% that were determined by high performance
liquid chromatography combined UV detection (LC-UV). In addition, the two-dimensional
nuclear magnetic resonance spectroscopy (2D NMR) clearly showed that no obvious coupling
effects between H3 and Hβ, suggesting that the aim compounds are in the trans conformation.
The details of spectral characterization of all final products and analytical data can be found in
the supplementary materials.
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Fig.1. A) Several reported chalcone derivatives with PL inhibition activity. B) Design of novel PL inhibitors
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using 1-indanone and chalcone as fragment hits.

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Scheme 1. The process of structural modifications and screening of chalcone-like derivatives as PL
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inhibitors.
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Scheme 2. A) General synthetic scheme for chalcone-like compounds A1-16. Reagents and conditions: (a)
3,4-dihydro-2H-pyran, PPTS, 4 h, CH2Cl2, 40°C; (b) various benzaldehydes, EtOH, 20% (w/v) NaOH, room
temperature, overnight; (c) 1.0 mol/L HCl, EtOH, room temperature, 5 h; (d) HCl (gas), EtOH, room
temperature, overnight. B) General synthetic scheme for chalcone-like compounds B1-14; 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/L HCl, EtOH, room temperature, 5 h.
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2.2 Inhibition of chalcone-like compounds against PL and structure–activity relationships
In this study, a total of 30 novel chalcone-like compounds were designed and synthesized
via Claisen Schmidt condensation reaction, and their PL inhibitory activities were routinely
assayed using 4-MUO as the fluorescent substrate. Firstly, the PL inhibitory activities of series
of A ring-modified chalcone-like compounds were assayed. As listed in Table 1 and Fig.S1,
several substituents on A ring strongly affected the PL inhibitory effects. Among them, the
compounds bearing a methoxyl group at the C3’ site and a hydroxyl at the C4’ site on A ring
were found with relatively strong PL inhibitory activities, such as A1 (6.94 ± 0.65 μM), A3
(10.42 ± 2.48 μM), and A4 (12.36 ± 2.04 μM). Especially, the methoxyl group at the C3’ site
was beneficial for PL inhibition, as indicated by IC50 values of A1 (6.94 ± 0.65 μM) vs. A2
(14.89 ± 3.96 μM), and A3 (10.42 ± 2.48 μM) vs. A8 (>100 μM). However, the polymethoxy
substituted derivatives of the A ring did not enhance PL inhibition activity significantly, as
evidenced by IC50 values of A5 (24.81 ± 3.29 μM), A6 (14.79 ± 2.07 μM), A7 (20.66 ± 2.09
μM) and A9 (35.01 ± 6.02 μM). Meanwhile, we also found that introduction of one or more
halogen substitutions on A ring will lead to dramatically decrease in PL inhibitory activity of
these chalcone-like compounds, e.g., A10-A16, all of these compounds showed moderate to
weak PL inhibitory activities (>30 μM). In addition, the results also suggested that the
compounds bearing an H-bonding donor (such as -NH2, -OH) at the C-6 site on B ring is good
for PL inhibition. With these findings in mind, A3 and A4 were selected as the optimal
compounds to further modify the chalcone-like skeleton on B ring.
Subsequently, series of B ring-modified chalcone-like compounds were synthesized and
their PL inhibitory activities were assayed under some conditions. As shown in Table 2 and
Fig.2, modification of the phenolic group at the C-6 site on B ring to ethers displayed
differential effects on PL inhibition. Among them, the methyl ether derivative leads to a
decrease in PL inhibition activities, evidence by IC50 values of B1 (37.15 ± 3.31 μM) vs. A3
(10.42 ± 2.48 μM). However, introduction of propyl ether and i-propyl ether slightly improved
PL inhibition activity, as indicated by IC50 values of B2 (2.54 ± 0.42 μM), B3 (8.61 ± 1.23 μM)
vs. A3 (10.42 ± 2.48 μM). By contrast, the inhibition potency of these compounds against PL
could be significantly enhanced when the amino group at the C-6 site on B ring was modified
by introduction N-alkyl substituents, such as B4 (3.32 ± 0.47 μM), B5 (3.50 ± 0.65 μM), B6

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(1.69 ± 0.23 μM) vs. A4 (12.36 ± 2.04 μM). It is evident that N-alkyl substituents at the C-6
site were beneficial for PL inhibition, especially the N, N-disubstituted derivatives. For
example, the IC50 value of compound B12 (84.09 ± 19.11 μM) is around 145-fold greater than
that of B11 (0.58 ± 0.12 μM), while the IC50 value of compound B8 (25.09 ± 1.93 μM) against
PL is 12.5-fold greater than that of B7 (2.09 ± 0.14 μM). And B9 (0.83 ± 0.13 μM) vs. B10
(2.52 ± 0.65 μM), B13 (0.33 ± 0.02 μM) vs. B14 (0.68 ± 0.09 μM) also displayed strong
inhibitory activity. Among all tested compounds, compound B13 (a novel chalcone-like
compound bearing two long carbon chains along with a polar tail at the end of each carbon
chain) displayed the most potent PL inhibitory activity, with a calculated IC50 value of 0.33 ±
0.02 μM (Table 2).
Collectively, the structure–activity relationships of these chalcone-like compounds are
summarized as follows, 1) the compounds bearing a methoxyl group at the C3’ site and a
hydroxyl at the C4’ site on A ring are beneficial for PL inhibition. 2) Introduction of one or
more halogen substitutions on A ring will lead to loss of PL inhibitory activity. 3) Introduction
of N-alkyl substituents at the C-6 site on B ring are beneficial for PL inhibition, while the PL
inhibition potency of N,N-disubstituted hydrocarbyl is better than that of monosubstituted
derivatives (Fig.3).
Table 1. IC50 values of A-ring modified chalcone-like derivatives against PL.
No.
Compound
Structure
IC50 (μΜ)
O
1
A1
6.94 ± 0.65
O
OH
O
2
A2
14.89 ± 3.96
OH

O
HO
3
4
5
6
7
8
9
A3
A4
A5
A6
A7
A8
A9
10.42 ± 2.48
12.36 ± 2.04
24.81 ± 3.29
14.79 ± 2.07
20.66 ± 2.09
>100
O
OH
O
H2N
O
OH
O
HO
O
O
O
HO
O
O
O
O
HO
O
O
O
O
HO
OH
O
HO
O
O
35.01 ± 6.02
O
O
HO
Br
10
11
A10
A11
28.93 ± 8.07
14.8 ± 1.95
O
HO
Br

O
HO
12
13
14
A12
A13
A14
75.58 ± 19.06
Br
Cl
O
HO
>100
O
HO
75.38 ± 15.96
Cl
Cl
O
HO
15
A15
32.67 ± 6.39
F
F
O
HO
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17
A16
>100
O
Chalcone
84.41 ± 13.32
O
O
O
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Orlistat
4.65±0.90 nM
O
O
HN
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Table 2. IC50 values of B-ring modified chalcone-like derivatives against PL.
No.
Compound
Structure
IC50 (μΜ)
O
O
1
B1
37.15 ± 3.31
O
OH

O
O
2
3
4
5
6
B2
B3
B4
B5
B6
2.54 ± 0.42
8.61 ± 1.23
3.32 ± 0.47
3.50 ± 0.65
1.69 ± 0.23
O
OH
O
O
O
OH
O
N
O
OH
O
N
O
OH
O
N
O
OH
O
N
7
8
B7
B8
2.09 ± 0.14
O
OH
O
NH
25.09 ± 1.93
O
OH
O
N
9
B9
0.83 ± 0.13
O
OH

O
H
N
10
B10
2.52 ± 0.65
O
OH
O
N
11
B11
0.58 ± 0.12
O
OH
O
H
N
O
12
B12
84.09 ± 19.11
OH
OH
O
13
B13
0.33 ± 0.02
N
O
OH
HO
O
H
N
O
14
B14
OH
0.68 ± 0.09
HO

O
15
16
Chalcone
Orlistat
84.41 ± 13.32
O
4.65±0.90
O
O
nM
O
O
HN
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Fig. 2. Dose-response inhibition curves of several chalcone-like compounds on PL-catalyzed 4-MUO
hydrolysis. All data were shown as mean ± SD of triplicate determinations.

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Fig.3. Structure-activity relationships of chalcone-like compounds as PL inhibitors.
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2.3 Inhibition kinetic analyses of B13 against PL
To further investigate the inhibitory mechanism of B13 against PL, a set of enzyme
inhibition kinetics analyzed were performed by using varying concentrations of both substrate
and the inhibitor. Prior to inhibition kinetic assays, time-dependent inhibition assays were
conducted to judge whether B13 was a time-dependent inhibitor/inactivator of PL. As shown
in Fig.S2, the inhibitory tendency and potency of B13 against PL didn’t change with different
pre-incubation times, indicating that B13 was a reversible inhibitor rather than a time-
dependent inactivator of PL. Subsequently, a set of inhibition kinetic assays of B13 against PL-
mediated 4-MUO hydrolysis were performed. As represented in Fig.4, the Lineweaver-burk
plots clearly demonstrated that compound B13 potently inhibited PL via a mixed inhibition
manner, with the Ki value as low as 0.12 μM. These findings suggested that B13 was a potent
and reversible inhibitor against PL, while this agent exhibits a mixed inhibition mode against
PL-mediated 4-MUO hydrolysis.
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Fig.4 A) The Lineweave-Burk plots of B13 against PL-catalyzed 4-MUO hydrolysis. B) The second plot of
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slopes from A. All data were shown as mean ± SD of triplicate assays.

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2.4 Molecular docking simulations
Next, molecular docking simulations were performed to decipher the key interactions
between B13 and PL. To identify the possible binding sites of compound B13, protein cavity
detection was executed on a web server, CavityPlus（http://repharma.pku.edu.cn/cavityplus）
[40]. As illustrated in Fig.5 and in Table S1, compound B13 could be well-docked into PPL at
both the catalytic pocket and another ligand-binding site that was located on the surface of PPL.
These two possible ligand-binding sites of B13 on PL were detected based on ligandability
scores, which were shown in Fig.S3. For further validation of the prediction results, a
molecular docking simulations of compound B13 binding on PPL (PDB code: 1ETH) at these
two binding sites was conducted. As shown in Table S1, the binding affinities of compound
B13 at two binding sites were -7.8 kcal/mol and -7.2 kcal/mol, for site I, and site II, respectively.
The favorable binding affinities indicated that the occurrence of an acceptable binding clefts in
the PPL cavities. These results elucidated that compound B13 could bind on PPL in multi-
modes, which well-explained the mixed inhibition mode of this agent against PL.
Furthermore, the key interactions between B13 and the residues of PPL in two identified
ligand-binding sites were performed by 2D interaction analysis. As shown in Fig.6, the
aromatic B ring could interact with Phe-78 in PPL via π-π T-shaped interaction. Meanwhile,
the 1-carbonyl group of B13 (on ring A) could create hydrogen-bond interactions with Arg-
257 of PPL, the ring B formulated an aromatic center, by interacting with His-264 through π-
cation interaction, which might keep the equilibrium of charge in catalytic pocket. In the middle
of the catalytic cavity, the aromatic ring of B13 could form van der waals interactions with
Gly-77 and Leu-265. All these findings suggested that B13 could create strong interactions
with PPL, through the forces mentioned above. In Fig.7, the hydroxyl of carbon chain produced
a hydrogen bond with Cys-238, the carbonyl on five-membered ring generated a hydrogen
bond with Gln-239 alongside the Cys-238. In addition, the hydroxyl on ring A could interact
with Asp-206, Ala-208, Ala-207 via hydrogen bonding. These observations suggested that both
of two poses could form stable interactions with PL.

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Fig.5. The predicted binding modes of compound B13 on pancreatic lipase (PDB code: 1ETH) within site I
(A, B) and site II (C, D). (A) The overview of B13 and 4-MU oleate docked into the catalytic cavity of PL.
(B) The detailed view of B13 docked into the catalytic cavity of PL (site I). (C) B13 could also be bound on
PL at a non-catalytic site that was located on the surface of PL. (D) The detail view of B13 bound on a non-
catalytic site of PL (site II). In these figures, red spheres represent the catalytic residue Ser153, while
compound B13 and 4-MU oleate are shown in yellow and red, respectively. Hydrogen bond, π-cation, and
π-π T shaped interactions were indicated as green, orange and yellow dashed lines, respectively.
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Fig.6. The key interactions between compound B13 and the residues surrounding in the catalytic cavity of
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PL.

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Fig.7. The key interactions between compound B13 and the residues surrounding in the site II of PL, another
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non-catalytic site that is located on the surface of PL.
2.5 Stability assays of B13 in gastrointestinal fluids and in human liver preparations
Finally, to decipher the potential of B13 as a good choice for design and development of
orally administrated PL inhibitors, the stability of B13 in artificial gastrointestinal fluids and
in human liver preparations were routinely assayed [41, 42]. As shown in Fig.S4, B13 is very
stable in artificial gastrointestinal fluids, with the half-life of 3.89 h in artificial gastric fluids
and >4 h in artificial intestine fluids, respectively. Meanwhile, as depicted in Table 3 and
Fig.S5, B13 also displayed good metabolic stability in human liver preparations, with the half-
lives of 221 min and 117.3 min in phase I and phase II metabolic systems, respectively.
Meanwhile, the in vitro half-lives of two positive reference drugs were also determined as 24
min and 15.5 min, for testosterone (phase I metabolism) and umbelliferone (phase II
metabolism), respectively. These results clearly demonstrated that B13 displayed good stability
in both artificial gastrointestinal fluids and in human liver preparations, suggesting that this

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compound could be served as a promising lead compound to develop novel orally administrated
anti-obesity agents via targeting on PL.
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Table 3. The half-life of B13 in phase I or phase II metabolic systems in human liver microsomes.
T1/2 (min)
Compound
Phase I
221
Phase II
117.3
--
B13
Testosterone
Umbelliferone
24
--
15.5
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3. Conclusion
In summary, two series of novel chalcone-like compounds were designed, synthesized, and
their PL inhibitory activities were assayed by a florescence-based biochemical assay.
Following two rounds of structural modifications at both A and B rings of a chalcone-like
skeleton, the structure-PL inhibition relationships of the chalcone-like compounds and the key
substituents affecting PL inhibition were revealed. Among all tested chalcone-like compounds,
B13 displayed the most potent PL inhibitory activity, with a calculated IC50 value of 0.33 μM.
Inhibition kinetic analyses showed that this agent could potently and dose-dependently inhibit
PL in a reversible and mixed inhibition manner, with the Ki value of 0.12 μM. Docking
simulations revealed that B13 could tightly bind on PL at both the catalytic site and a non-
catalytic site that was located on the surface of PL, which was highly consistent with the mixed
inhibition mode of this agent. Further investigations showed that B13 also displayed excellent
stability in gastrointestinal fluids and in human liver preparations, suggesting that this agent
hold great promise to develop novel orally administrated anti-obesity agents. Collectively, our
results demonstrated that chalcone-like compounds were good choices for the design and
development of PL inhibitors, while B13 could be used as a promising lead compound to
develop novel orally administrated anti-obesity agents.
4. Materials and experimental
4.1. Chemicals, reagents and instruments

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All starting materials for synthesis of chalcone-like compounds were obtained from
commercial suppliers and used without further purification. Thin layer chromatography (TLC)
was carried out on precoated silica gel plates (Merck 60 PF254). Column chromatography was
carried out on a column Merck silica gel 60 (200-400 mesh ASTM) (Merck KGaA, Darmstadt,
1
Germany). H and 13C NMR spectra were recorded in DMSO-d6 or CDCl3 on a Bruker
AVANCE 500/126 MHz instrument. Chemical shifts are reported in parts per million (ppm)
relative to tetramethylsilane (TMS). Electron-spray ionization mass spectra in positive mode
(ESI-MS) data were recorded on a Bruker Esquire 3000t spectrometer. The purities of all tested
compounds were higher than 96%, which was determined by high performance liquid
chromatography coupled to a UV detector.
Porcine pancreatic lipase (PL, type II, Lot.SLBN9099V), 4-methylumbelliferone (4-MU)
was purchased from Sigma-Aldrich (St. Louis, MO). 4-Methylumbelliferyl oleate (4-MUO)
was obtained from J&K chemical (Beijing, China). The positive PL inhibitor (orlistat),
pancreatin and pepsin were purchased from Dalian Meilun Biotech Co., Ltd (Dalian, China).
0.1 M citrate phosphate buffer (PH 7.4), 0.1 M phosphate buffer (pH 7.4) and 50 mM Tris-HCl
buffer were prepared by using Milli-Q Water (Millipore, Bedford, USA). The stock solutions
of 4-MUO (100 mM) and each tested compound were prepared using LC grade dimethyl
sulfoxide (DMSO, Tedia, USA) as the solvent, which was then kept at 4 °C until use.
4.2. PL inhibition assay
The procedure for PL inhibition was same as previous studies, in which 4-MUO was used
as the substrate to assess the residual activities of PL in the presence of each tested compound
[43-45]. Briefly, a mixture of 0.2 mL incubation system composed of PL solution (0.01 mg/mL,
final concentration), 0.1 M citrate phosphate buffer (pH 7.4), and each inhibitor (with different
concentrations) was vortexed for 10 sec and then pre-incubated at 37 °C for 10 min. After then,
4-MUO was added to initiate the hydrolytic reaction and the multi-mode microplate reader
(SpectraMax iD3, Molecular Devices, Austria) was used to analyze the florescence signals of
the hydrolytic product (4-MU). The detection conditions and the formula of residual activities
were consistent with the literature reports [43-45].
4.3. Inhibition kinetic analyses
Inhibition behavior of B13 against PL-mediated 4-MUO hydrolysis was carefully

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investigated by performing a set of inhibition kinetic analyses. The assays were conducted by
using different concentrations of substrates in the presence of increasing doses of B13. The
inhibition kinetic modes (competitive inhibition, noncompetitive inhibition, or mixed
inhibition) and the inhibition constant values (Ki) were determined by the inhibition kinetic
plots and the Lineweaver-Burk plots. The following equations for competitive inhibition Eq.
(a), noncompetitive inhibition Eq. (b), or mixed inhibition Eq. (c) were used to fit the inhibition
kinetic data of reversible PL inhibitors.
V=(VmaxS)/ [Km (1+I/Ki)+S]
V=(VmaxS)/ [(Km+S)×(1+I/Ki)]
V=(VmaxS)/ [(Km+S)×(1+I/αKi)]
(a)
(b)
(c)
in which V is the velocity of PL-mediated hydrolytic reaction, Vmax is the maximum velocity.
S and I are the substrate and inhibitor concentrations, respectively. Ki is the inhibition constant
of the inhibitor towards the target enzyme PL; Km is the Michaelis constant (the substrate
concentration at half of the Vmax) [43, 45-47].
4.4. Molecular docking simulations
Docking simulations were performed using the open source program AutoDock Vina
(version 1.1.2) [48], while the crystal structure of PPL (PDB code: 1ETH) was downloaded
from the Protein Data Bank (http://www.rcsb.org/pdb). MM2 force field in ChemBio 3D Ultra
14.0 was employed to obtain the energy minimized conformation of B13. The structure of PPL
occurred within a cubic box which was large enough to accommodate the inhibitor binding
pocket and space to search for possible binding poses. Then the position of compound B13 was
initialized from a random starting position. The docking poses with the lowest binding energy
were recorded for further analysis.
4.5. Stability assays of B13 in artificial gastrointestinal fluids
To investigate the potential for B13 as an orally administrated anti-obesity agent, the
stabilities of B13 in gastrointestinal systems were assayed in artificial gastric fluids and
artificial intestinal fluids, respectively according to the previously reported schemes [49, 50].
In brief, a total volume of 800 μL incubation mixture including artificial gastric fluids or
artificial intestine fluids was pre-incubated at 37 °C for 3 min, the incubation was subsequently
initiated by adding B13 (100 μM, final concentration). At the time points of 0-, 0.5-, 1-, 2- and

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4-hour, 100 μL of the reaction mixture was taken out and then mixed with equal volume of ice-
cold acetonitrile. The mixtures were then centrifuged at 20000 g for 20 min at 4°C, and the
supernatants (100 μL) were subsequently analyzed by LC-UV, as described below.
4.6. Metabolic stability assays of B13 in both phase I and phase II metabolic systems
The procedure for metabolic stability assays in both phase I and phase II metabolic systems
has been reported recently [37]. The final concentration of B13 or the positive control
testosterone was 10 μM. B13 was quantified by LC-UV, as described below.
4.7. Determination of B13 by LC-UV
B13 was analyzed by an ultra-fast liquid chromatography (UFLC) coupling with a UV
detector. The UFLC system (Shimadzu, Japan) equipped with a CBM-20A communication bus
module, two LC-20AD pumps, a SIL-20AC autosampler and a CTO-20AC column oven.
Chromatographic separation was performed on an ODS column (2.1 × 150 mm, 2.2 μm,
Shimadzu), using a flow rate of 0.4 mL/min. The column temperature was kept at 40 °C.
Acetonitrile (A) and water containing 0.2% formic acid (B) were used as the mobile phase,
with the following gradient: 0.01-13.00 min, 95% B-10% B; 13.00-13.50 min, 10% B; 13.50-
16.00 min, 10% B-95% B; 16.00-18.50 min, 95% B. The detection wavelength for B13 was
set to 370 nm.
4.8. Data analysis
The inhibition constants (including IC50 and Ki values) were calculated by using nonlinear
regression by GraphPad Prism 8.0 (GraphPad Software, Inc., La Jolla, USA), while all data
were expressed as mean ± SD of the triplicate assays.
Acknowledgments
This study was was financially supported by the Natural Science Foundation of China
(81922070, 81973286, 81773687), Program of Shanghai Academic/Technology Research
Leader (18XD1403600), Shanghai Talent Development Fund (2019093), the Natural Science
Foundation of Liaoning Province (20180530025), the Three-year Action Plan of Shanghai
TCM Development (ZY-(2018-2020)-CCCX-5001), the Wenzhou Public Welfare Science and
Technology Project (Y20180179) and Shuguang Program (18SG40) supported by Shanghai
Education Development Foundation and Shanghai Municipal Education Commission.

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Declaration of Interest
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The authors state that no conflicts of interest exist.
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Fig.1. A) Several reported chalcone derivatives with PL inhibition activity. B) Design of novel
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PL inhibitors using 1-indanone and chalcone as fragment hits.
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Scheme 1. The process of structural modifications and screening of chalcone-like derivatives
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as PL inhibitors.

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Scheme 2. A) General synthetic scheme for chalcone-like compounds A1-16. Reagents and
conditions: (a) 3,4-dihydro-2H-pyran, PPTS, 4 h, CH2Cl2, 40°C; (b) various benzaldehydes,
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 chalcone-like compounds B1-14; 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.