Synthesis, biological evaluation and SAR studies of ursolic acid 3β-ester derivatives as novel CETP inhibitors

Article abstract of DOI:10.1016/j.bmcl.2019.126824

Cholesteryl ester transfer protein (CETP) is an attractive therapeutic target for the prevention and treatment of cardiovascular diseases by lowering low-density lipoprotein cholesterol levels as well as raising high-density lipoprotein cholesterol levels in human plasma. Herein, a series of ursolic acid 3β-ester derivatives were designed, synthesized and evaluated for the CETP inhibiting activities. Among these compounds, the most active compound is U12 with an IC₅₀ value of 2.4 μM in enzymatic assay. The docking studies showed that the possible hydrogen bond interactions between the carboxyl groups at both ends of the molecule skeleton and several polar residues (such as Ser191, Cys13 and Ser230) in the active site region of CETP could significantly enhance the inhibition activity. This study provides structural insight of the interactions between these pentacyclic triterpenoid 3β-ester derivatives and CETP protein for the further modification and optimization.

Full text of DOI:10.1016/j.bmcl.2019.126824

Journal Pre-proofs
Synthesis, biological evaluation and SAR studies of ursolic acid 3β-ester de-
rivatives as novel CETP inhibitors
Chao Chen, Renhua Sun, Yan Sun, Xuan Chen, Fei Li, Xiaoan Wen, Haoliang
Yuan, Dongyin Chen
PII:
S0960-894X(19)30793-0
DOI:
https://doi.org/10.1016/j.bmcl.2019.126824
BMCL 126824
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
24 August 2019
12 October 2019
12 November 2019
Please cite this article as: Chen, C., Sun, R., Sun, Y., Chen, X., Li, F., Wen, X., Yuan, H., Chen, D., Synthesis,
biological evaluation and SAR studies of ursolic acid 3β-ester derivatives as novel CETP inhibitors, Bioorganic &
Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126824
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Chao Chen, Renhua Sun, Yan Sun, Xuan Chen, Fei Li, Xiaoan Wen, Haoliang Yuan* and Dongyin Chen*

Bioorganic & Medicinal Chemistry Letters
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Synthesis, biological evaluation and SAR studies of ursolic acid 3β-ester
derivatives as novel CETP inhibitors
Chao Chenc,‡, Renhua Sund,‡, Yan Suna, Xuan Chena, Fei Lia, Xiaoan Wenb, Haoliang Yuanb,* and Dongyin
Chena,
aDepartment of Medicinal Chemistry, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
b State Key Laboratory of Natural Medicines and Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, China
cKey Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 211166, China
dDepartment of Cardiology, First People’s Hospital of Yancheng, Fourth Affiliated Hospital of Nantong University, Yancheng 224005, China
———
ARTICLE INFO
ABSTRACT
 Corresponding author. E-mail: chendongyin@njmu.edu.cn (D. Chen), yhl@cpu.edu.cn (H. Yuan).
‡ These authors contributed equally to this work.
Article history:
Received
Cholesteryl ester transfer protein (CETP) is an attractive therapeutic target for the prevention
and treatment of cardiovascular diseases by lowering low-density lipoprotein cholesterol levels
as well as raising high-density lipoprotein cholesterol levels in human plasma. Herein, a series of
ursolic acid 3β-ester derivatives were designed, synthesized and evaluated for the CETP
inhibiting activities. Among these compounds, the most active compound is U12 with an IC50
value of 2.4 μM in enzymatic assay. The docking studies showed that the possible hydrogen
bond interactions between the carboxyl groups at both ends of the molecule skeleton and several
polar residues (such as Ser191, Cys13 and Ser230) in the active site region of CETP could
significantly enhance the inhibition activity. This study provides structural insight of the
interactions between these pentacyclic triterpenoid 3β-ester derivatives and CETP protein for the
further modification and optimization.
Revised
Accepted
Available online
Keywords:
Cardiovascular diseases
CETP inhibitor
Pentacyclic triterpenes
Usolic acid
2009 Elsevier Ltd. All rights reserved.
Molecular docking
Cardiovascular diseases (CVDs) are identified as the leading
cause of death in low-income and middle-income countries.1
Epidemiological studies confirmed that lowering low-density
lipoprotein cholesterol (LDL-C) levels as well as raising high-
density lipoprotein cholesterol (HDL-C) levels in human plasma
appeared as a promising treatment strategy for cardiovascular
events.2 Cholesteryl ester transfer protein (CETP) is a crucial
modulator of plasma lipoproteins that promotes the migration of
cholesteryl esters from high density lipoprotein (HDL) to low-
density lipoprotein (LDL) and very-low-density lipoproteins
(VLDL).3 The growing experimental and clinical studies have
suggested that a high CETP concentration is associated with an
increased risk of CVDs by raising proatherogenic LDL-C and
lowering atheroprotective HDL-C.4 This suggests that CETP
inhibition could be a promising approach for the prevention and
treatment of CVDs. In the past two decades, several CETP
inhibitors with different structural scaffolds have been identified,
and four of them have been evaluated in large scale, randomized
cardiovascular clinical outcome trials.5 While the trials with
torcetrapib, dalcetrapib and evacetrapib with the purpose of
reducing CVDs risk have been unsuccessful, treatment with
anacetrapib shows significant benefit for major coronary events.6
However, as the manufacturers of anacetrapib recently decided to
suspend development of the drug,6d the future of CETP inhibition
discovery of effective and safe CETP inhibitors with different
structural scaffolds has been an important area of pharmaceutical
research due to the association of CETP activity with the risk of
CVDs.
Pentacyclic triterpenoids (PTs), a group of widespread natural
products that exhibiting similar structures to cholesterol and
cholesteryl esters (CE), were observed to possess hypolipidemic
activity more than thirty years ago.8 In China, ursolic acid (2) as
the main triterpene ingredients from Fructus Ligustri Lucidi has
been marketed as an OTC drug (trade name Taizhi'an capsule)
for the treatment of hyperlipidemia.9 Many studies have indicated
that sustained treatment with the plant extracts containing
pentacyclic triterpene compounds could cause a significant
decrease in TC, TG and LDL-C levels, and increase in HDL-C
levels.10 However, the biological mechanism of hypolipidemic
activity of PTs is still under investigation.10 As part of our
ongoing research to find novel CETP inhibitors, we have
designed and synthesized a series of oleanolic acid (1) 3β-ester
derivatives, some of them displayed moderate inhibiting human
CETP activity with IC50s less than 10 μM.11 The most potent
compound 20 with an IC50 value of 2.3 μM (Figure 1) showed
robust potential to regulate the lipid profile both in ap2-CETPTg
mice and high-fat fed guinea pig models, meanwhile it exhibited
an excellent pharmacokinetic profile without changes on the
systolic blood pressure in guinea pigs.11 Additionally, Chang and
as
a
potential therapeutic option for reducing major
cardiovascular events is currently uncertain.7 Therefore, the

co-workers have reported a new class of CETP inhibitors by
linking the PTs scaffolds and the active fragment from
Torcetrapib through fragment-based drug design approaches.12 In
this manuscript, we have designed and synthesized a series of
ursolic acid 3β-ester derivatives U1-U15 and explored them as
novel CETP inhibitors. In vitro screening assay showed that the
most active compound was U12 with an IC50 value of 2.4 μM
(Figure 1). The molecular docking studies were carried out to
investigate the mode of interaction of these compounds and their
structure-activity relationship (SAR), which revealed that these
pentacyclic triterpene 3β-ester derivatives might bind at the
active site and interact with Cys13, Ser191, Gln199, Ser230 and
His232 present in the binding site. The molecular docking
demonstrated excellent co-relations with the experimental
findings.
monoester U6 and glutaric acid monoester U7 exhibited
moderate inhibitory effect toward CETP with an IC50 of 10.9 μΜ
and 3.2 μΜ, respectively, while adipi acid monoester U8 and
heptane diacid monoester U9 have hardly any inhibitory potency.
Compared with U7, due to the steric hindrance of methyl group,
U10 and U11 with glutaric acid monoester moiety showed a
complete loss in inhibitory potency against CETP. Quite
unexpectedly, 1,2-cyclopropanedicarboxylic acid monoester U12
showed the most potent CETP inhibitory activity; its IC50 reached
2.4 μΜ. Similarly, the existence of branched methyl group of
U13 caused a complete loss in inhibitory potency. It is regrettable
that 1,2-cyclopentanedicarboxylic acid monoester U14 and 1,2-
cyclohexanedicarboxylic acid monoester U15 also have no CETP
inhibitory activities. Through a series of further comparisons (U2
vs U3, U7 vs U8, U7 vs U11, and U12 vs U13), we concluded
that the carboxyl group at the end of skeleton chain and the
length of skeleton chain are both key factors to maintain the
inhibitory potency against CETP. Overall, U12 emerged as the
most potent compound in this series, with an IC50 value of 2.4
μM in the in vitro enzymatic assay.
Previous work
H
H
CO2H
CO2H
O
H
H
O
OH
H
HO
H
oleanolic acid (1)
20
CETP IC50 = 2.3 M
O
H
no CETP inhibitory activity
Eur. J. Med. Chem. 139 (2017) 201-213
OH
ii, iv
O
O
H
This work
R
O
H
H
U1, U6-U7, U10-U15
OBn
H
i
H
Ursolic acid (2)
O
CO2H
H
CO2H
3
HO
O
H
H
H
H
3
OH
HO
O
H
H
iii, iv
O
O
H
ursolic acid (2)
CETP IC50 > 50 M
U12
OH
O
R
O
H
CETP IC50 = 2.4 M
U2-U5, U8-U9
Figure 1. Discovery of pentacyclic triterpene 3β-ester derivatives as novel
Scheme 1. Synthesis of the target compounds U1-U15. Reaction conditions:
(i) BnBr, K2CO3, DMF, r.t.; (ii) anhydrides, DMAP, pyridine, reflux; (iii)
carboxylic acids, DCC, DMAP, DCM, reflux; (iv) H2, 10% Pd/C, THF, r.t.
CETP inhibitors.
The general synthetic route for the target compounds U1-U15
is shown in Scheme 1. A typical condensation method was
embraced by treating 2 with benzyl chloride and K2CO3 in DMF
to provide benzyl ester 3. Then, esterification of the 3β-hydroxyl
group of 3 with corresponding anhydrides in anhydrous pyridine,
followed by debenzylation with H2 over Pd/C in THF to give the
desired compounds U1, U6-U7 and U10-U15 in 41-93% yields
for two steps. Similarly, the product 3 was reacted with various
carboxylic acid compounds in the present of DCC and DMAP,
followed by debenzylation with H2 over Pd/C in THF to afford
the target products U2-U5 and U8-U9 in 35-85% yields for two
steps. The structures of all synthesized compounds were
Table 1. Screening assay of ursolic acid 3β-ester derivatives as
novel CETP inhibitors in vitro. a
In vitro CETP inhibitory activity
Compd.
% Inhibition at 10 μΜ
IC50 (μΜ)
H
CO2H
O
H
H
3
R1
O
OH
47.9
61.8
24.5
5.5 ± 1.0 d
3.3 ± 0.5 d
established by spectroscopic data i.e. H NMR, 13C NMR and
1
U1 (R1 =
U2 (R1 =
)
O
ESI Mass spectrometry.
All the target compounds were evaluated for their lipid-
regulating effect by in vitro CETP enzymatic inhibition assay,
and the results are summarized in Table 1. The initial screening
was carried out at a concentration of 10 μМ for each compound,
and compounds that displayed >30% inhibition at 10 μМ were
further evaluated for their IC50s. We found that compounds U1
and U2 with the phthalic acid monoester or isophthalic acid
monoester moiety displayed moderate inhibition of CETP with
IC50 values 5.5 μM and 3.3 μM, respectively. However, a
significant loss of inhibitory potency was observed for compound
U3, which having terephthalic acid monoester moiety. Further
comparison of U2 with U4 (% inhibition at 10 μΜ = 22.7%) and
U5 (% inhibition at 10 μΜ = 11.2%) revealed that carboxyl
group on the skeleton chain was essential to enhance the
inhibitory activity. Moreover, the compounds U6-U11 with
carboxyl group at the end of the alkyl chain contributed
significantly to our understanding of the linker region on the
potency of inhibitory activity. Among them, succinic acid
O
OH
)
e
HO
/
U3 (R1 =
U4 (R1 =
)
)
O
22.7
11.2
/
/
)
)
U5 (R1 =
U6 (R1 =
U7 (R1 =
U8 (R1 =
U9 (R1 =
N
HO
42.8
55.1
23.3
11.1
1.1
10.9 ± 1.3 d
O
O
)
3.2 ± 0.8 d
HO
HO
/
/
/
O
O
)
HO
)
O
U10 (R1 =
)
HO

O
O
According to our docking analysis, compound U12 displayed a
similar binding mode to CETP protein. The docked view of U12
showed that it almost occupied the binding position, and got the
key hydrogen bond interactions with Ser191 and Ser230 (Figure
0
/
U11 (R1 =
U12 (R1 =
)
HO
HO
53.2
2.4 ± 0.37 d
)
)
O
3a).
Moreover,
the
carboxyl
group
from
1,2-
HO
9.0
0
/
/
cyclopropanedicarboxylic acid moiety of U12 was also observed
to stretch itself into the hydrophilic pocket, therefore the
compounds U12 and 20 showed similar activities of IC50 values
of 2.4 and 2.3 μM, respectively. We reasoned that the hydrogen
bond interactions involving Ser191 and Ser230 might explain the
SAR observed in other ursolic acid 3β-ester derivatives as CETP
inhibitors. For example, docking of glutaric acid monoester U7
into the active site of CETP showed the similar combination, and
two hydrogen bond interactions between U7 and Ser191, Cys13
were observed (Figure 3b). In contrast, the dimethy moiety of
U11 (Figure 3c) and the cyclohexyl moiety of U15 (Figure 3d)
created steric hindrance, which losing the interactions with
Ser191, Cys13 and Ser230, and resulted in a complete loss in
activity.
U13 (R1 =
U14 (R1 =
O
O
HO
HO
)
)
1.5
/
U15 (R1 =
Anacetrapib f
88.4
0.036 ± 0.004 c
a
The initial screening was carried out at a concentration of 10 μΜ for
each compound and IC50s were measured for compounds that displayed
>30% inhibition of CETP. The value of % inhibition at 10 μΜ is the
mean of more than two independent assays. The IC50 value was
b
c
d
expressed as the mean ± SEM of two separate tests. The IC50 value
e
was expressed as the mean ± SEM of three separate tests. “/” means
that no experiment was conducted. f The reference drug.
Molecular docking studies were carried out to explore the
mode of interaction of these novel CETP inhibitors with PTs
scaffolds and their SAR. Here we applied Glide-docking module
of Schrödinger software (Maestro v9.0, Schrödinger 2009, LLC,
NEW YORK, NY) for docking studies. The crystal structure of
CE in complex with CETP (PDB ID: 2OBD) was retrieved from
the protein data bank.3 In our previous research,11 we
hypothesized that PTs might be substrate analogue inhibitors of
CETP, and designed a series of oleanolic acid (1) 3β-ester
derivatives, which mimicking the protein-ligand interactions
between the active site and CE scaffold. Among them, compound
20 (IC50 = 2.3 μM) was identified as a potent CETP inhibitor,
which could regulate the lipid profile both in ap2-CETPTg mice
and high-fat fed guinea pig models. Therefore, compound 20 was
first selected for docking studies, and the binding mode was
depicted in Figure 2. We found that 20 almost occupied the
binding position of the endogenous ligand CE in the active site.
Besides, the hydrogen bond interaction between 20 and Ser191
was observed (Figure 2a). Interestingly, the carboxyl group on
the benzene ring of 20 was observed to stretch itself into a
hydrophilic pocket, which was consisted by four polar residues
Cys13, Gln199, Ser230 and His232 (Figure 2b). Recent studies
also showed that it might be possible to incorporate some
hydrophilic groups that interact with Ser230, His232, or Gln199,
which located at the interior of the inhibitor binding pocket, to
improve compound solubility while maintaining binding
affinities.13 Therefore, we concluded that the carboxyl groups at
the end of molecular scaffold of 20 could form crucial hydrogen
bond interactions with several polar residues in the active site
region of CETP, which might contribute significantly to its
inhibitory activity.
(c)
(d)
Figure 3. The binding modes of compounds U12 (pink), U7 (bright green),
U11 (blue) and U15 (gray) are presented. The active site residues involved in
interactions are depicted in blackish green sticks. The blue dash line indicates
hydrogen bond interaction. Light pink shadow region indicates a hydrophilic
pocket.
To further validate our docking model, a series of pentacyclic
triterpenoid derivatives including oleanolic acid 3β-ester
derivatives11 and ursolic acid 3β-ester derivatives were docked
into the active site of CETP protein, and the relationship between
the docking scores produced by several scoring functions and the
known inhibition ratios of CETP at 10 μΜ of these docked
compounds was computed. The docking scores are depicted in
Supporting Information (SI) Table 1, and the relationship is
shown in SI Figure 1. LigScore2, XP-Score, and PMF-Score
provided better correlations than the other scoring functions (SI
Figure 1a), however, which were not able to satisfactorily predict
the binding affinities for ligands bound to the target protein.
From their docking results in the active site of CETP, we found
that the carboxyl groups of some docked compounds could not
form the hydrogen bond interactions with the hydrophilic pocket.
Thus, we reasoned that the conformation of polar residues in the
hydrophilic pocket changed in the ligand-binding process, and
the computation directly on unaltered crystal structure was not
suitable for the investigation of above hydrogen bond interactions.
In order to introduce some flexibility into the docking process,
the induced-fit docking protocol of Schrödinger was used.
Induce-fit docking aims to improve the docking of ligands in
which it is believed that the receptor adjusts significantly to the
(a)
(b)
Figure 2. (a) Alignment of docking conformation of compound 20 (orange)
and the crystal conformation of CE (pink). (b) Proposed binding mode of
compound 20 (orange) with CETP. Residues involved in interactions are
shown as blackish green sticks. The blue dash line indicates hydrogen bond
interaction. Light pink shadow region indicates a hydrophilic pocket, which
was consisted by Cys13, Gln199, Ser230 and His232.
presence of the ligand.14 By performing
a constrained
minimization of the receptor following by initial Glide docking
and redocking the ligands, better receptor structures for each
ligand can be obtained. With this method, molecular docking of
compound U12 into the active site of CETP showed that the
polar residues (Cys13, Ser230 and His232) in the hydrophilic
Next, several ursolic acid 3β-ester derivatives were selected to
further investigate the mode of interaction with CETP protein.

(b)
pocket would turn toward the ligand molecule to form the
hydrogen bond interactions after the ligand binding to the
receptor (Figure 4). With this new conformation, the relationship
between the binding scores produced by XP-Score, PMF-Score
and LigScore2 and the known inhibition ratios of these docked
compounds was recomputed. The docking results showed that the
relationship between inhibition ratios and binding scores was
significantly increased (SI Figure 1b), which indicated that the
new docking model might be more suitable to predict the
inhibitory activity of these triterpenoid derivatives against CETP.
Furthermore, the hydrogen bond interactions between carboxyl
groups at both ends of the molecule skeleton and several polar
residues (such as Ser191, Cys13 and Ser230) in the active site
region of CETP are the key factor to maintain the inhibitory
activity of these triterpenoid derivatives.
Figure 5. (a) SAR for 3β-ester derivatives of PTs as CETP inhibitors. (b) The
superimposed binding modes of active compounds 20 (orange), U1 (dark
blue), U2 (purple), U7 (bright green) and U12 (pink) are presented. The
active site residues involved in interactions are depicted in blackish green
sticks. The blue dash line indicates hydrogen bond interaction. Light pink
shadow region indicates a hydrophilic pocket.
In summary, a series of ursolic acid 3β-ester derivatives were
designed, synthesized and evaluated for the CETP inhibiting
activities. In vitro screening assay showed that 5 out of 15
compounds displayed moderate inhibiting human CETP activity;
the most active compound was U12 with an IC50 value of 2.4 μM.
The docking studies showed that the possible hydrogen bond
interactions between the carboxyl groups at both ends of these
triterpenoid 3β-ester derivatives and several polar residues (such
as Ser191, Cys13 and Ser230) in the active site region of CETP
could significantly enhance the inhibition activity, which gave us
a direction for further lead optimization. We believe that ursolic
acid 3β-ester derivative U12 may serve as a lead compound for
the design of more effective and safe CETP inhibitors.
Figure 4. The conformation transitions in the induced-fit docking study of
compound 12 to the active site of CETP protein. The polar residues in the
hydrophilic pocket (residues displayed in blackish green stick style) would
turn toward the ligand molecule to form the hydrogen bond interactions
(residues displayed in yellow stick style).
Acknowledgments
Combined with our previous research results,11 the whole
SAR of these 3β-ester derivatives of PTs has been summarized in
Figure 5a. A good CETP inhibiting activity may be attributed to
the presence of carboxyl groups at opposite ends on the
molecular scaffold, which could form the hydrogen bond
interactions with the polar residues (such as Ser191, Cys13 and
Ser230) in the active site of CETP protein. The hydrophobic
linking chains at C-3 position of molecular skeleton could be aryl
and alkyl groups; the length of carbon chains prefers 2~3 carbon
atoms. In addition, oleanane-type and ursane-type scaffolds
might be more suitable to fit the active pocket of CETP than
other pentacyclic scaffolds. As outlined above, through the
superimposed binding modes of five active compounds (Figure
5b), we can clearly observed that all these conformations have
very similar binding interactions in the binding pocket except for
minor differences.
We gratefully acknowledge financial support from the
National Natural Science Foundation of China (No. 81602957,
81703367), and the Natural Science Foundation of Jiangsu
Province, China (No. BK20161035).
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R2
Hydrophobic linker: phenyl, cyclopropyl, alkyl groups
R1
The length of linker: 2~3 carbon atoms
H
17
OH
Gln199
O
O
Cys13
His232
H
A
HO
O
3
Ser191
Linking chain
O
H
Ser230
Hydrophilic pocket
Oleanane-type scaffold: R1 = H, R2 = CH3
Ursane-type scaffold: R1 = CH3, R2 = H
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Declaration of interests
☒ 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.
☐The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests:

Highlights
A series of ursolic acid 3β-ester derivatives were
synthesized and evaluated for the CETP inhibiting
activities.
In vitro screening assay showed that the most active
compound was U12 with an IC50 value of 2.4 μM.
The molecular docking studies were carried out to
investigate the structure-activity relationships of
these pentacyclic triterpene 3β-ester derivatives.