Synthesis and evaluation of novel triterpene analogues of ursolic acid as potential antidiabetic agent

Article abstract of DOI:10.1371/journal.pone.0138767

Ursolic acid (UA) is a naturally bioactive compound that possesses potential anti-diabetic activity. The relatively safe and effective molecule intrigued us to further explore and to improve its anti-diabetic activity. In the present study, a series of novel UA analogues was synthesized and their structures were characterized. Their bioactivities against the α-glucosidase from baker's yeast were determined in vitro. The results suggested that most of the analogues exhibited significant inhibitory activity, especially analogues 8b and 9b with the IC₅₀ values of 1.27 ± 0.27 μM (8b) and 1.28 ± 0.27 μM (9b), which were lower than the other analogues and the positive control. The molecular docking and 2D-QSAR studies were carried out to prove that the C-3 hydroxyl could interact with the hydrophobic region of the active pocket and form hydrogen bonds to increase the binding affinity of ligand and the homology modelling protein. Thus, these results will be helpful for understanding the relationship between binding mode and bioactivity and for designing better inhibitors from UA analogues.

Full text of DOI:10.1371/journal.pone.0138767

RESEARCH ARTICLE
Synthesis and Evaluation of Novel Triterpene
Analogues of Ursolic Acid as Potential
Antidiabetic Agent
Panpan Wu1, Jie Zheng1, Tianming Huang1, Dianmeng Li2, Qingqing Hu1, Anming Cheng1,
Zhengyun Jiang1, Luoying Jiao1, Suqing Zhao1*, Kun Zhang1*
1 Department of Pharmaceutical Engineering, Faculty of Chemical Engineering and Light Industry,
Guangdong University of Technology, Guangzhou, China, 2 Guangxi Institute of Botany, Guangxi Zhuang
Autonomous Region and Chinese Academy of Sciences, Guilin, China
* sqzhao@gdut.edu.cn (SQZ), kzhang@gdut.edu.cn (KZ)
Abstract
Ursolic acid (UA) is a naturally bioactive compound that possesses potential anti-diabetic
activity. The relatively safe and effective molecule intrigued us to further explore and to
improve its anti-diabetic activity. In the present study, a series of novel UA analogues was
synthesized and their structures were characterized. Their bioactivities against the α-gluco-
sidase from baker's yeast were determined in vitro. The results suggested that most of the
analogues exhibited significant inhibitory activity, especially analogues 8b and 9b with the
IC50 values of 1.27 0.27 μM (8b) and 1.28 0.27 μM (9b), which were lower than the other
analogues and the positive control. The molecular docking and 2D-QSAR studies were car-
ried out to prove that the C-3 hydroxyl could interact with the hydrophobic region of the
active pocket and form hydrogen bonds to increase the binding affinity of ligand and the
homology modelling protein. Thus, these results will be helpful for understanding the rela-
tionship between binding mode and bioactivity and for designing better inhibitors from UA
analogues.
OPEN ACCESS
Citation: Wu P, Zheng J, Huang T, Li D, Hu Q,
Cheng A, et al. (2015) Synthesis and Evaluation of
Novel Triterpene Analogues of Ursolic Acid as
Potential Antidiabetic Agent. PLoS ONE 10(9):
e0138767. doi:10.1371/journal.pone.0138767
Editor: Jie Zheng, University of Akron, UNITED
STATES
Received: July 19, 2015
Accepted: September 3, 2015
Published: September 25, 2015
Copyright: © 2015 Wu et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Introduction
November 14th in every year, launched by the World Health Organization (WHO) and the
International Diabetes Federation (IDF) in 1991, is the United Nations Diabetes Day, which is
commonly known as the World Diabetes Day and aims to arouse the global awareness and dis-
illusion of diabetes mellitus (DM) [1]. DM is a complex metabolic disorder characterized by
persistent hyperglycaemia, which may cause the patient to acute or chronic complications,
including blindness, heart disease, stroke, kidney failure and amputations [2]. WHO projects
that DM will be the 7th leading cause of death in 2030 [3]. More than 80% of DM deaths occur
in middle- and low-income countries [4]. Type 2 diabetes (T2D) is a chief form of DM which
results from the body’s ineffective use of insulin, it comprises 90% of people with diabetes
around the world [5,6]. There are an enormous number of therapies available for the treatment
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This study was supported by National
Natural Science Foundation of China (Grant
No.21172046). The authors are also grateful to the
combination research projects of Guangdong
Province and Ministry of Education for financial
support (Grant No. 2011B090600033), Guangdong
Province Higher Education “Qianbaishi Engineering”,
and Guangzhou Science and Technology Plan (Grant
No.2013Y2-00081).
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Novel Ursolic Acid Analogues as Antidiabetic Agent
Competing Interests: The authors have declared
that no competing interests exist.
and the prevention of DM and its complications, such as insulin therapy, α-glucosidase inhibi-
tion, protein tyrosine phosphatase 1B inhibition, along with metabolism adjustment [7–10].
Ursolic acid (UA, 3β-hydroxy-urs-12-en-28-oic acid, 1) is a well-known natural pentacyclic
triterpenoid carboxylic acid which is ubiquitous in some traditional medicinal herbs. UA and
its analogues exhibit a wide range of biological activities, including antibacterial [11], antitu-
mor [12,13], antiviral [14], anti-HIV [15], antioxidative [16] and antimalarial activities [17].
Among them, the anti-diabetic activity is one of the most prominent in both in vitro and in
vivo according to our previous studies [18–20]. In recent years, more and more studies indi-
cates that UA and its analogues are potential therapeutic agents for the treatment of DM and
its complications [21–24]. In order to find new potential UA analogues with higher activities,
considerable attempts on structural modification of UA have been made, especially at the
3-OH and/or 17-COOH positions [25,26]. However, few studies of UA analogues focus on the
anti-diabetic.
According to our previous work, a series of halogen-containing UA analogues has been syn-
thesized [18,20]. However, their efficacy on α-glucosidase inhibition was decreased while com-
pared with the parent compound UA. Therefore, a series of new hydrolyzation analogues has
been synthesized in our study. In an attempt to explore the activity and mechanisms of these
new analogues, and to study their structure-activity relationships, the bioactivities of these new
analogues against α-glucosidase were evaluated in vitro. Furthermore, molecular docking stud-
ies were also carried out with binding of UA analogues in the active site of α-glucosidase, to
demonstrate that the hydrophilic moieties can interact with the hydrophobic group of the cata-
lytic pocket and form hydrogen bonds. In addition, this study was also supported by 2D-QSAR
model which was set up by partial least squares modelling with R software, in order to explore
the structural requirements controlling the observed activities. This is the first study focusing
on the anti-diabetic properties of these new hydrolysed analogues.
Results and Discussion
Chemistry
Based on the previous study, with a slight modification using UA as the lead compound, struc-
tural modifications were made at the 3-OH and/or 17-COOH positions to get a series of new
UA analogues. The synthetic routes are presented in Fig 1 and Fig 2.
UA (1) was first esterified with acetic anhydride in anhydrous pyridine to produce its 3-O-
acetate (2), which was then treated with oxalyl chloride to give the intermediate 28-acyl chlo-
ride [18]. This intermediate was dissolved in dichloromethane and then condensed with the
appropriate amino compounds (aminobenzene, o-fluoroaniline, o-chloroaniline, o-bromoben-
zenamine, p-fluoroaniline, p-chloroaniline, p-bromobenzenamine, p-methoxylaniline) in the
presence of triethylamine to produce analogues 3a-10a, then the saponification analogues of
3a-10a were hydrolyzed to give the corresponding analogues 3b-10b (Fig 1) [27]. UA was oxi-
dized with pyridinium chlorochromate (PCC) to give the 3-oxo analogue (11) [28]. All the tar-
get analogues were purified by column chromatography with petroleum ether/ethyl acetate
and/or chloroform/methanol as the eluent. Their structures were confirmed by the application
of 1H NMR (S1 File), 13C NMR (S1 File), mp, electrospray ionization mass spectrometry
(ESI-MS) (S2 File), high resolution mass spectrometry (HRMS) (S2 File).
In vitro α-glucosidase inhibition assay of the UA analogues
In this experiment, α-glucosidase from baker’s yeast was the model which has been widely cho-
sen to determine the anti-diabetic activity of all tested analogues with a slight modification
[29,30]. Acarbose was chosen as the positive control, it act by competitively inhibiting the
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Novel Ursolic Acid Analogues as Antidiabetic Agent
Fig 1. Synthesis of analogues 10a, 3b-10b from UA. Reagents and conditions: (a) acetic anhydride/Pyr/DMAP, r.t.; (b) (COCl)2, CH2Cl2, r.t.; (c) CH2Cl2,
Et3N, R-NH2, r.t.; (d) NaOH, THF/CH3OH, r.t.
doi:10.1371/journal.pone.0138767.g001
α-glucosidase, a group of key intestinal enzymes involved in the digestion of carbohydrates. A
stock solution of each sample, which has been dissolved in dimethylsulfoxide (DMSO) at the
concentrations of 0.05 μM to 500 μM, was diluted with 0.1 M phosphate buffer solution
(pH = 6.8) containing an appropriate concentration of enzyme solution (0.1 U/mL). After a 10
min pre-incubation at 37°C of the reactions, the substrate (1mM p-nitrophenyl-α-D-glucopyr-
anoside) was added to initiate them. Then the reactions were incubated for 30 min at 37°C
before they were terminated by adding 1 M Na2CO3, and their optical density values were mea-
sured at 405 nm by using a Multimodel Plate Reader (Infinite 200). Each experiment was
repeated at least four times.
To determine the inhibition activity of each sample, the enzyme activity was measured at a
fixed substrate concentration, in which a sequence of sample concentrations were tested. The
IC50 value was calculated according to the curve fit to the sequence of concentrations versus
the corresponding inhibition abilities. The results were illustrated in Table 1 and Fig 3. The
IC50 values of tested samples against α-glucosidase from baker’s yeast ranged from 1.27 μM to
2.56 μM, and it could be concluded that all of them had lower IC50 than both positive control
and UA, indicating that all the synthesized UA analogues had significant effect on α-glucosi-
dase inhibition.
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Novel Ursolic Acid Analogues as Antidiabetic Agent
Fig 2. Synthesis of analogue 11 from UA. Reagent and condition: (e) PCC, r.t.
doi:10.1371/journal.pone.0138767.g002
Structure activity relationship
A total of eleven analogues of UA (10a, 3b-10b and 11) were synthesized and their structure
activity relationship (SAR) against α-glucosidase was derived. The UA (IC50 5.04 0.80 μM)
into its ester 3-O-acetate (2, IC50 5.27 0.35 μM) at the position of C-3 slightly decreased the
activity. Similarly, the analogue 2 into unsubstituted aryl amide analogue, 3a (IC50 5.64
1.12 μM), showed a slight reduction in activity, and its halogen-containing amide analogues
from 4a to 9a (IC50 ꢀ 6.53 1.33 μM) drastically reduced the activity except 8a (IC50 3.24
0.21 μM) [18]. However, the activities of C-3 hydroxyl analogues 3b-10b were more active
than those of the corresponding ester analogues. Furthermore, electronegative (-F,-Cl,-Br) sub-
stitutions at the para position in the aryl amide analogues were more potential than the ortho
position ones. It is worth noting that electronegative (-OCH3) substitution at the para position
of the aryl amide analogue has a positive effect on α-glucosidase inhibition. As is indicated in
Table 1 and Fig 3, among the C-3 hydroxyl analogues, analogues 8b and 9b were better than
the others in this enzyme inhibition model, the IC50 values of the two analogues were 1.27
0.27 μM (8b) and 1.28 0.27 μM (9b), which were about quadruple the starting material UA.
According to the above results, it might be concluded that 8b and 9b possessed potential activ-
ity against α-glucosidase.
Table 1. Observed and predicted inhibitory effects of UA analogues against α-glucosidase from baker’s yeast.
Analogue
Polar moiety
IC50a (μM)
Observed pIC50
Predicted pIC50
Binding free energy (kcal/mol)
1
1×OH, 1×COOH
1×OH
5.08 0.70
1.69 0.08
1.51 0.10
1.31 0.19
2.56 0.06
1.67 0.09
1.27 0.27
1.28 0.27
2.01 0.12
2.55 1.32
2.47 0.14
573.50 15.17
5.294
5.772
5.821
5.883
5.592
5.777
5.896
5.893
5.697
5.593
5.607
—
5.282
5.765
5.821
5.875
5.591
5.782
5.903
5.909
5.718
5.599
5.597
—
-3.694
-4.092
-3.309
-3.157
-2.879
-3.375
-3.891
-3.488
-4.482
-4.738
-4.332
-9.134
3b
4b
5b
6b
7b
8b
9b
10a
10b
11
1×OH
1×OH
1×OH
1×OH
1×OH
1×OH
—
1×OH
1×COOH
13×OH
12b
Each experiment was performed in quadruplicate. The data presented represent the means (n = 4) SD.
aIC50 value representing the concentration that caused a 50% loss of activity.
bAcarbose, positive control.
doi:10.1371/journal.pone.0138767.t001
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Novel Ursolic Acid Analogues as Antidiabetic Agent
Fig 3. Inhibitory activities of UA analogues against α-glucosidase from baker’s yeast. (a) 10a, (b-i) 3b-10b, (j) 11. The data reported represent the
mean (n = 4) SD.
doi:10.1371/journal.pone.0138767.g003
Molecular docking mode
SYBYL 2.0, a molecular docking software was introduced to predict the enzyme inhibition of
these UA analogues. It can not only expound how these UA analogues conjugate with the
enzyme, but also can provide a guidance for the design of α-glucosidase inhibitors in the future.
The molecular docking studies was carried out to survey the binging model of UA analogues
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Novel Ursolic Acid Analogues as Antidiabetic Agent
Fig 4. (a) The binding mode of acarbose docked with α-glucosidase. (b) Acarbose with the active site MOLCAD surface representation. (c) The
binding mode of UA docked with α-glucosidase. (d) UA with the active site MOLCAD surface representation.
doi:10.1371/journal.pone.0138767.g004
within the binding pocket of α-glucosidase and to understand their structure-activity
relationship.
According to the previous study, homology modelled structure of α-glucosidase has been
used for molecular modelling study to identify the reasonable binding mode [20,31,32]. The
structure of oligo-1,6-glucosidase from Saccharomyces cerevisiae (PDB: 1UOK) was selected as
the template because the sequence similarity and identity between α-glucosidase and the tem-
plate were around 62.0% and 38.0%, respectively [33].
As is indicated in Fig 4, the positive control, acarbose showed higher binding affinity with
the homology protein than the parent compound UA, and the binding free energy of the both
analogues were -9.134 kcal/mol and -3.694 kcal/mol, respectively. From Fig 4A and 4C, acar-
bose could be formed into hydrogen bonds with ASP60, ASP199, GLU255, GLY258, ASP285,
SER288, ASP329 and ARG415 residues in the active site. UA which could be interacted with
SER222, ASP329 and ARG415 residues possessed lower binding affinity while compared with
the positive control. It could be concluded that this binding mode might owning to the large
number of hydroxyl groups and the hydrophobic interaction. Above all, as is depicted in Fig
4B and 4D, the analysis of interaction between UA and the catalytic pocket is similar with that
of acarbose.
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Novel Ursolic Acid Analogues as Antidiabetic Agent
Fig 5. (a) The binding mode of analogue 8b docked with α-glucosidase. (b) Active site MOLCAD surface representation lipophilic potential. (c)
Active site MOLCAD surface representation hydrogen bonding.
doi:10.1371/journal.pone.0138767.g005
All of our synthesized UA analogues were docked with the developed homology model of
α-glucosidase (PDB: 1UOK). The docking studies of two potential analogues (8b and 9b)
against α-glucosidase were presented in Figs 5 and 6. The binding free energy of analogues 8b
and 9b was calculated as -3.891 kcal/mol and -3.488 kcal/mol, which were similar with that of
UA itself. The two analogues were mainly surrounded by the residues of ASP329, ARG415 and
GLU255 in the catalytic pocket. As is shown in Fig 5, analogue 8b was formed into hydrogen
bonds with the residues of ASP329 and ARG415 through the C-3 free hydroxyl group with the
inside catalytic pocket. As is depicted in Fig 6, analogue 9b was formed into hydrogen bonds
with the residue of GLU255 through the C-3 free hydroxyl group with the inside catalytic
pocket. The MOLCAD lipophilic potential study revealed that the free hydroxyl group at C-3
position of analogues 8b and 9b were closed to the hydrophobic region of the active pocket,
and it also indicated that more hydrophilic group could improve the inhibitory activity.
Besides, the MOLCAD hydrogen bonding study of the binding surface exhibited that several
hydrogen bond donors were presented in the hydrophobic pocket while analogues 8b and 9b
were served as an acceptor by forming two and one hydrogen bonds, respectively. Analogues
8b and 9b have significant inhibitory activity through the interaction with the α-glucosidase,
which presumably via competitively binding active site [20]. Thus, the release of the C-3 free
hydroxyl group and the modification on UA with more hydrophilic moieties could be of great
importance for improving the inhibitory activity of the new UA analogues.
In order to have a deep insight into the relationship between the in vitro inhibitory activity
and the docking study, the predicted binding free energies of all tested analogues were calcu-
lated by the docking procedures [20,34], presented in Table 1. According to the correlations of
predicted binding free energies and the inhibitory activities (see Fig 7), analogues 3b, 8b, 10a,
10b and 11 exhibited lower predicted binding free energy which were lower than -3.891 kcal/
mol, while the other analogues showed a slight higher binding free energy than that of the
Fig 6. (a) The binding mode of analogue 9b docked with α-glucosidase. (b) Active site MOLCAD surface representation lipophilic potential. (c)
Active site MOLCAD surface representation hydrogen bonding.
doi:10.1371/journal.pone.0138767.g006
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Novel Ursolic Acid Analogues as Antidiabetic Agent
Fig 7. Correlation of binding free energies with inhibitory activities for UA and its analogues.
doi:10.1371/journal.pone.0138767.g007
parent compound UA. However, there is no significant difference between the result of pre-
dicted binding free energy and in vitro enzyme inhibition. It also indicated that choosing the
homology protein of α-glucosidase as the docking model could afford some guidance for the
selection of α-glucosidase inhibitor.
2D-QSAR study
About 30 UA analogues (S3 File) were studied in the QSAR model, their structures (S3 File)
and bioactivities could be obtained in our present and previous studies [18,20]. QSAR analyses
of these analogues for anti-diabetic activities were performed to correlate the bioactivities with
the synthesized analogues, and to identify positive and negative structural features within the
four series. The analysis was run by means of Sybyl molecular modelling package, version 2.0
(Tripos, shanghai, China).
The 2D-QSAR model was constructed by partial least squares modelling with R software
and then evaluated using a training set of 25 analogues and a test set of 5 analogues. The
observed pIC50 values were validated by measuring the residuals between the observed and the
predicted pIC50 values of the training set. As shown interestingly in Table 1 and S4 File, the
predicted pIC50 values which were measured by the QSAR model were very close to those
observed with very low error (S4 File). In addition, from the QSAR model results of predicted
pIC50 values versus Observed pIC50 values (see Fig 8), the root mean square errors (RMSE) of
training and test sets were 0.0158 and 0.0113, respectively. The R-squared of this two sets were
0.9986 and 0.9996, respectively. It was indicated that this model could be applied for prediction
of more effective hits having the same skeletal framework. Furthermore, from the calculation
of the other 2D Descriptors (S4 File), each analogue in series b has one more 'hydrogen bond
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Novel Ursolic Acid Analogues as Antidiabetic Agent
Fig 8. Observed pIC50 values versus predicted pIC50 values of UA and its analogues against α-glucosidase by 2D-QSAR model. (a) the integration of
training and test set, (b) training set, (c) test set.
doi:10.1371/journal.pone.0138767.g008
donor' than the others, but one less 'hydrogen bond donor acceptor' than the others (except
UA), together with their bioactivities, indicating that the C-3 hydroxyl was necessary for their
activities against α-glucosidase.
Conclusions
In summary, we have reported a series of UA analogues with a free hydroxyl group at the C-3
position, and their potential in vitro inhibitory activity against α-glucosidase has been investi-
gated. All tested UA analogues exhibited greater potency than the acarbose and the parent
compound UA in this α-glucosidase inhibition assay, the bioactivity of C-3 hydroxyl analogues
were more potential than their corresponding ester analogues. Among them, electronegative
(-F,-Cl,-Br) substitutions at the para position analogues were more active than the ortho ones,
especially analogues 8b and 9b, whose IC50 were 1.27 0.27 μM and 1.28 0.27 μM, respec-
tively. Molecular docking was also studied to identify the binding mode and to afford some
guidance for α-glucosidase inhibitor development. The results indicated that the hydrogen
bonds formation with the residues of SER222, GLU415, ASP329 and ARG415 between ligands
and protein might play an important role in enhancing their inhibitory activity by improving
the binding affinity. In addition, validation was employed by measuring the residuals between
the observed and the predicted pIC50 values by the 2D-QSAR model, indicated that UA new
analogues which were retained the C-3 hydroxyl and modified with more hydrophilic groups
in other positions could be a class of promising analogues as potential α-glucosidase inhibitor.
Materials and Methods
General Remarks
UA was supplied by Nanjing Zelang Medical Technology Co., Ltd., with a purity of over 98%.
Silica gel (100–200 or 200–300 mesh) used in column chromatography was bought from Tsing-
tao Marine Chemistry Co., Ltd. Other reagents were purchased from commercial suppliers in
their chemically or analytically pure grade without further purification unless otherwise noted.
1H NMR and 13C NMR spectra were recorded on a Bruker AVANCE 400 or Mercury-Plus
300 NMR spectrometers under a standard condition, chemical shifts were measured in ppm
downfield from TMS as internal standard (S1 File). The melting points were determined on a
Fischer-Johns apparatus and are uncorrected. Electrospray ionization (ESI) mass spectra were
measured on an LC-MS-2010A and reported as m/z (S2 File). High resolution mass spectra of
analogues 10a, 3b-10b, and 11 were measured on a Bruker maXis impact (S2 File). The
enzyme inhibition activity was measured using a Multimodel Plate Reader (Infinite 200).
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Novel Ursolic Acid Analogues as Antidiabetic Agent
Synthesis
General procedure for the preparation of analogues (2, 3a-10a). Through the procedure
of our previous study, analogue 2 could be obtained after UA was treated with acetic anhydride
and purified on a silica gel column with petroleum ether/ethyl acetate (v/v 10:1). Then ana-
logue 2 was treated with oxalyl chloride to get the intermediate of 3-O-acetylursolyl chloride,
analogues 3a-10a could be obtained after an equal amount of appropriate amino was added
and purified on a silica gel column with petroleum ether/ethyl acetate (v/v 10:1) as the eluent.
Eight analogues of UA were synthesized according to the procedure reported in our earlier
publications [18].
N-[3β-acetoxy-urs-12-en-28-oyl]-p-methoxyamiline (10a)
Yield 95%; amorphous white powder; 1H NMR (300 MHz, DMSO) δ 7.39 (d, J = 9.0 Hz,
2H), 6.81 (d, J = 9.1 Hz, 2H), 5.26 (s, 1H), 4.38 (dd, J = 11.0, 4.7 Hz, 1H), 3.69 (s, 3H), 3.34 (s,
1H), 2.35 (d, J = 10.7 Hz, 1H), 1.99 (s, 3H), 1.93–1.73 (m, 4H), 1.73–1.38 (m, 10H), 1.30 (ddd,
J = 29.2, 12.8, 6.0 Hz, 3H), 1.08 (s, 3H), 1.00 (d, J = 1.5 Hz, 2H), 0.94 (d, J = 6.0 Hz, 4H), 0.87
(d, J = 6.5 Hz, 7H), 0.80 (s, 3H), 0.79 (s, 3H), 0.65 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
176.1, 171.1, 156.3, 140.3, 131.5, 126.1, 121.5, 114.2, 81.0, 55.6, 55.3, 54.4, 48.5, 47.6, 42.8, 40.0,
39.7, 39.3, 38.5, 37.8, 37.2, 36.9, 32.8, 31.1, 28.5, 28.0, 25.2, 23.68, 23.66, 23.4, 21.4, 21.3, 18.2,
17.4, 17.1, 16.8, 15.7; ESI-MS m/z 602.2 [M-H]-; HRMS (ESI) calculated for C39H58NO4
[M+H]+ = 604.4360, found: 604.4383.
General procedure for the preparation of analogues (3b-10b). Analogue 3a or (4a-10a),
which was obtained from our laboratory, was dissolved in CH3OH/THF (1:1.5, 10 mL) and
treated with aqueous NaOH (4N), the reaction was stirred for 4h at room temperature and
concentrated in vacuo. The residue was suspended in distilled water and neutralized with 2 N
HCl to pH 3, filtered. The filter was washed with distilled water to pH 6, and dried to get an
amorphous white powder.
N-[3β-Hydroxy-urs-12-en-28-oyl]-aminobenzene (3b)
Yield 98%; amorphous white powder; 1H NMR (300 MHz, DMSO) δ 7.51 (d, J = 7.5 Hz,
2H), 7.23 (t, J = 7.9 Hz, 2H), 6.99 (t, J = 7.3 Hz, 1H), 5.27 (d, J = 3.4 Hz, 1H), 4.28 (d, J = 5.2 Hz,
1H), 3.04–2.92 (m, 1H), 2.37 (d, J = 10.8 Hz, 1H), 2.02 (dt, J = 14.3, 7.1 Hz, 1H), 1.95–1.64 (m,
5H), 1.60–1.32 (m, 9H), 1.25 (dd, J = 21.8, 11.1 Hz, 3H), 1.06 (s, 3H), 0.96 (t, J = 12.0 Hz, 5H),
0.87 (d, J = 6.5 Hz, 6H), 0.81 (s, 3H), 0.65 (t, J = 5.4 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ
176.4, 140.2, 138.3, 129.0, 126.3, 124.1, 119.8, 79.0, 55.2, 54.4, 48.7, 47.7, 42.8, 40.0, 39.7, 39.2,
38.9, 38.8, 37.2, 37.0, 32.9, 31.0, 28.2, 28.1, 27.3, 25.2, 23.7, 23.4, 21.3, 18.3, 17.4, 17.0, 15.7, 15.6;
ESI-MS m/z 530.3 [M-H]-; HRMS (ESI) calculated for C36H54NO2 [M+H]+ = 532.4149, found:
532.4163.
N-[3β-Hydroxy-urs-12-en-28-oyl]-o-fluoroaniline (4b)
Yield 96%; amorphous white powder; 1H NMR (300 MHz, DMSO) δ 7.55 (ddd, J = 7.9, 4.2,
2.9 Hz, 1H), 7.25–7.06 (m, 3H), 5.29 (s, 1H), 4.29 (d, J = 5.1 Hz, 1H), 3.05–2.94 (m, 1H), 2.30
(d, J = 10.9 Hz, 1H), 2.11–1.96 (m, 1H), 1.91–1.67 (m, 5H), 1.65–1.32 (m, 9H), 1.26 (t, J = 11.8
Hz, 3H), 1.08 (s, 3H), 1.05–0.91 (m, 5H), 0.88 (d, J = 5.7 Hz, 4H), 0.84 (d, J = 4.4 Hz, 5H), 0.68
(s, 3H), 0.66 (s, 4H); 13C NMR (100 MHz, CDCl3) δ 176.8, 153.6, 139.0, 127.2, 126.9, 124.7,
123.8, 121.5, 114.7, 79.1, 55.3, 54.3, 49.4, 47.7, 42.6, 40.0, 39.6, 39.2, 38.9, 38.8, 37.3, 37.0, 32.9,
31.1, 28.3, 28.1, 27.4, 25.3, 23.6, 23.5, 21.3, 18.4, 17.4, 16.6, 15.7, 15.6; ESI-MS m/z548.2
[M-H]-; HRMS (ESI) calculated for C36H53FNO2 [M+H]+ = 550.4055, found: 550.4067.
N-[3β-Hydroxy-urs-12-en-28-oyl]-o-chloroaniline (5b)
Yield 96%; amorphous white powder; 1H NMR (300 MHz, DMSO) δ 7.72 (d, J = 8.0 Hz,
1H), 7.45 (d, J = 8.0 Hz, 1H), 7.28 (t, J = 7.7 Hz, 1H), 7.13 (t, J = 7.7 Hz, 1H), 5.31 (s, 1H), 4.27
(d, J = 5.1 Hz, 1H), 2.98 (dd, J = 14.8, 6.2 Hz, 1H), 2.25 (d, J = 10.8 Hz, 1H), 2.15–1.97 (m, 1H),
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Novel Ursolic Acid Analogues as Antidiabetic Agent
1.97–1.69 (m, 5H), 1.68–1.33 (m, 9H), 1.33–1.16 (m, 3H), 1.08 (s, 3H), 1.05–0.91 (m, 5H), 0.87
(d, J = 8.2 Hz, 5H), 0.82 (s, 4H), 0.67 (s, 3H), 0.66 (s, 4H); 13C NMR (100 MHz, CDCl3) δ
176.6, 138.3, 135.2, 129.1, 127.8, 127.4, 124.2, 122.8, 121.5, 79.1, 55.3, 54.1, 49.6, 47.7, 42.4,
40.0, 39.7, 39.2, 38.9, 38.8, 37.6, 37.0, 32.8, 31.0, 28.2, 28.0, 27.3, 25.3, 23.8, 23.5, 21.3, 18.4, 17.4,
16.8, 15.7, 15.6; ESI-MS m/z 564.8 [M-H]-; HRMS (ESI) calculated for C36H53ClNO2 [M+H]+ =
566.3759, found: 566.3768.
N-[3β-Hydroxy-urs-12-en-28-oyl]-o-bromoaniline (6b)
Yield 95%; amorphous white powder; 1H NMR (300 MHz, DMSO) δ 7.69 (d, J = 8.0 Hz,
1H), 7.61 (d, J = 8.0 Hz, 1H), 7.33 (t, J = 7.2 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 5.32 (s, 1H), 4.28
(d, J = 4.7 Hz, 1H), 3.06–2.92 (m, 1H), 2.24 (d, J = 10.9 Hz, 1H), 2.17–1.99 (m, 1H), 1.81 (dd,
J = 24.4, 13.1 Hz, 5H), 1.70–1.34 (m, 9H), 1.24 (dd, J = 20.7, 12.6 Hz, 3H), 1.17–1.06 (m, 3H),
0.98 (dd, J = 23.8, 8.1 Hz, 5H), 0.88 (d, J = 8.3 Hz, 6H), 0.83 (s, 3H), 0.80–0.74 (m, 1H), 0.68 (s,
3H), 0.66 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 176.4, 138.2, 136.3, 132.4, 128.5, 127.4, 124.8,
122.0, 113.5, 79.1, 55.3, 54.0, 49.6, 47.7, 42.4, 40.0, 39.7, 39.2, 38.9, 38.7, 37.6, 37.1, 32.8, 31.0,
28.2, 28.0, 27.3, 25.3, 23.8, 23.4, 21.3, 18.4, 17.4, 16.9, 15.7, 15.6; ESI-MS m/z 608.1 [M-H]-;
HRMS (ESI) calculated for C36H53BrNO2 [M+H]+ = 610.3254, found: 610.3262.
N-[3β-Hydroxy-urs-12-en-28-oyl]-p-fluoroaniline (7b)
Yield 97%; amorphous white powder; 1H NMR (300 MHz, DMSO) δ 7.51 (dd, J = 9.0, 5.1
Hz, 2H), 7.08 (t, J = 8.9 Hz, 2H), 5.26 (s, 1H), 4.28 (d, J = 5.1 Hz, 1H), 3.06–2.91 (m, 1H), 2.35
(d, J = 10.7 Hz, 1H), 2.12–1.61 (m, 7H), 1.61–1.32 (m, 9H), 1.32–1.16 (m, 3H), 1.06 (s, 3H),
0.96 (t, J = 11.9 Hz, 5H), 0.87 (d, J = 7.9 Hz, 6H), 0.81 (s, 3H), 0.65 (t, J = 6.0 Hz, 6H); 13C NMR
(100 MHz, CDCl3) δ 176.4, 159.3, 140.3, 134.3, 126.3, 121.5, 121.4, 115.7, 79.1, 55.3, 54.4, 48.7,
47.7, 42.8, 40.0, 39.7, 39.3, 38.9, 38.8, 37.2, 37.0, 32.9, 31.0, 28.3, 28.0, 27.3, 25.3, 23.7, 23.4, 21.3,
18.3, 17.4, 17.0, 15.71, 15.65; ESI-MS m/z 548.3 [M-H]-; HRMS (ESI) calculated for
C36H53FNO2 [M+H]+ = 550.4055, found: 550.4065.
N-[3β-Hydroxy-urs-12-en-28-oyl]-p-chloroaniline (8b)
Yield 97%; amorphous white powder; 1H NMR (300 MHz, DMSO) δ 7.56 (d, J = 8.9 Hz,
2H), 7.29 (d, J = 8.8 Hz, 2H), 5.26 (s, 1H), 4.27 (s, 1H), 3.05–2.91 (m, 1H), 2.36 (d, J = 10.7 Hz,
1H), 2.03 (td, J = 14.2, 3.8 Hz, 1H), 1.94–1.62 (m, 5H), 1.59–1.32 (m, 9H), 1.24 (dd, J = 23.0,
11.4 Hz, 3H), 1.06 (s, 3H), 0.96 (t, J = 11.8 Hz, 5H), 0.87 (d, J = 7.4 Hz, 6H), 0.81 (s, 3H), 0.66
(d, J = 5.9 Hz, 4H), 0.61 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 176.6, 140.4, 137.4, 132.0,
126.4, 121.2, 116.6, 79.1, 55.3, 54.5, 48.8, 47.7, 42.8, 40.0, 39.7, 39.2, 38.9, 38.8, 37.1, 37.0, 32.8,
31.0, 28.3, 28.0, 27.3, 25.3, 23.7, 23.4, 21.3, 18.3, 17.4, 17.0, 15.71, 15.65; ESI-MS m/z 564.2
[M-H]-; HRMS (ESI) calculated for C36H53ClNO2 [M+H]+ = 566.3759, found: 566.3767.
N-[3β-Hydroxy-urs-12-en-28-oyl]-p-bromoaniline (9b)
Yield 94%; amorphous white powder; 1H NMR(300 MHz, DMSO) δ 7.52 (d, J = 8.8 Hz,
2H), 7.42 (d, J = 8.8 Hz, 2H), 5.26 (s, 1H), 4.28 (d, J = 5.1 Hz, 1H), 2.98 (dd, J = 13.1, 7.5 Hz,
1H), 2.35 (d, J = 10.5 Hz, 1H), 2.11–1.95 (m, 1H), 1.95–1.61 (m, 5H), 1.61–1.33 (m, 9H), 1.24
(dd, J = 23.5, 11.0 Hz, 3H), 1.06 (s, 3H), 0.96 (t, J = 11.6 Hz, 5H), 0.87 (d, J = 7.6 Hz, 6H), 0.80
(s, 3H), 0.65 (s, 4H), 0.60 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 176.5, 140.3, 137.4, 132.0,
126.3, 121.2, 116.6, 79.0, 55.2, 54.4, 48.8, 47.6, 42.8, 40.0, 39.7, 39.2, 38.9, 38.8, 37.1, 37.0, 32.8,
31.0, 28.2, 28.0, 27.3, 25.3, 23.7, 23.4, 21.3, 18.3, 17.4, 16.9, 15.7, 15.6. ESI-MS m/z 610.1
[M-H]-; HRMS (ESI) calculated for C36H53BrNO2 [M+H]+ = 610.3254, found: 610.3260.
N-[3β-Hydroxy-urs-12-en-28-oyl]-p-methoxylaniline (10b)
Yield 96%; amorphous white powder; 1H NMR (300 MHz, DMSO) δ 7.38 (d, J = 8.9 Hz,
2H), 6.81 (d, J = 8.9 Hz, 2H), 5.75 (s, 1H), 5.26 (s, 1H), 4.29 (d, J = 5.0 Hz, 1H), 3.69 (s, 3H),
2.99 (dd, J = 13.2, 7.8 Hz, 1H), 2.35 (d, J = 11.3 Hz, 1H), 2.01 (t, J = 13.0 Hz, 1H), 1.89–1.64 (m,
5H), 1.62–1.34 (m, 10H), 1.26 (dd, J = 26.7, 13.0 Hz, 3H), 1.06 (s, 3H), 0.94 (d, J = 5.7 Hz, 4H),
0.87 (d, J = 9.2 Hz, 7H), 0.81 (s, 3H), 0.65 (s, 3H), 0.64 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
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Novel Ursolic Acid Analogues as Antidiabetic Agent
176.1, 156.3, 140.3, 131.5, 126.2, 121.5, 114.2, 79.1, 55.6, 55.3, 54.4, 48.5, 47.7, 42.8, 40.0, 39.7,
39.2, 38.9, 38.8, 37.2, 37.0, 32.9, 31.1, 28.2, 28.1, 27.3, 25.2, 23.7, 23.4, 21.3, 18.3, 17.4, 17.1, 15.7,
15.6; ESI-MS m/z 560.3 [M-H]-; HRMS (ESI) calculated for C37H56NO3 [M+H]+ = 562.4255,
found: 562.4274.
Preparation of 3-Oxo-urs-12-en-28-oic acid (11). PCC (1.42 g, 6.6 mmol) was added to a
solution of UA (1.0 g, 2.2 mmol) in acetone-CH2Cl2 (15 mL), after stirred at room temperature
for 8 h, the reaction was concentrated and partitioned with distilled water and CHCl3. The
organic layer was concentrated and purified by silica gel column chromatography using n-hex-
ane-acetone (v/v 95:5) as the eluent to give analogue 11.
Yield 55%; amorphous white powder; 1H NMR (300 MHz, DMSO) δ 5.14 (t, J = 3.5 Hz,
1H), 2.36–2.24 (m, 1H), 2.12 (d, J = 10.8 Hz, 1H), 2.04–1.70 (m, 4H), 1.68–1.15 (m, 13H), 1.06
(s, 3H), 1.02–0.93 (m, 10H), 0.92 (s, 5H), 0.82 (d, J = 8.5 Hz, 7H); 13C NMR (100 MHz, CDCl3)
δ 217.9, 184.0, 138.2, 125.7, 55.4, 52.7, 48.2, 47.5, 46.9, 42.2, 39.6, 39.4, 39.2, 39.0, 36.9, 36.8,
34.3, 32.6, 30.7, 28.1, 26.7, 24.2, 23.7, 23.6, 21.6, 21.3, 19.7, 17.2, 17.1, 15.4; ESI-MS m/z 453.2
[M-H]-.
α-glucosidase inhibitory activity
The α-glucosidase inhibition assay was performed according to the method of Worawalai et al
[30]. with a slight modification. The α-glucosidase enzyme (0.1 U/mL) and substrate (1 mM p-
nitrophenyl-α-D-glucopyranoside) were dissolved in 0.1 M phosphate buffer, pH 6.8. 10 μL of
each synthesized analogue (1 mg/mL in DMSO) was pre-incubated with 8 μL of α-glucosidase
at 37°C for 10 min. A 100 μL of substrate solution was then added to the reaction mixture,
which was further incubated at 37°C for 30 min. Then, the reaction was terminated by adding
100 μL of 1 M Na2CO3 solution. Enzymatic activity was quantified by measuring the absor-
bance at 405 nm with a use of a Multimodel Plate Reader (Infinite 200). The percentage of inhi-
bition was calculated by using [(A0-A1)/A0]×100%, where A0 was the absorbance without the
sample, and A1 was the absorbance with the sample. The IC50 value was determined from a
plot of the percentage of inhibition versus the sample concentration. Acarbose was used as the
standard control and the experiment was performed in duplicate.
Molecular modelling
The molecular minimizing of UA analogues were built by use of the Sybyl molecular modelling
package, version 2.0 (Tripos, shanghai, China). All structures of test analogues were minimized
with the Tripos force field, and the hydrogen atoms were added. Powel optimize the energy
gradient, the maximum times to 1000 times the energy convergence criterion reached 0.005
kcal/mol, using Gasteiger–Hückle charges. Ligand-protein docking was performed by the Sur-
flex-Dock in Sybyl 2.0. The crystal structure of α-glucosidase was retrieved from RCSB Protein
Data Bank (PDB: 1UOK). Biopolymer module was then used to repair the crystal structure of
the protein termini treatment, fix side chain amides, residues and add charges. The potent UA
analogues docking with the α-glucosidase selected catalytic pocket of acarbose as active site.
The active pocket form by computing, the others are the default settings. The binding free ener-
gies (ΔGbinding) were estimated by an extended model of the unbound states of the receptor and
the ligands (1):
DGbinding ¼ RTlnKd
ð1Þ
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Novel Ursolic Acid Analogues as Antidiabetic Agent
Supporting Information
S1 File. Characterization of UA analogues 10a, 3b-10b, and 11 by application of 1H NMR
and13C NMR.
(PDF)
S2 File. Characterization of UA analogues 10a, 3b-10b, and 11 by application of ESI-MS
and HRMS.
(PDF)
S3 File. The structure of UA and its analogues which were studied in QSAR model.
(DOCX)
S4 File. Computer descriptors and pIC50 of UA analogues.
(XLSX)
Acknowledgments
Sincere and heartfelt thanks must go to Miss Sulian Liang who has given generous suggestions
on the language modification of the thesis.
Author Contributions
Conceived and designed the experiments: SZ KZ JZ DL. Performed the experiments: PW ZJ JZ.
Analyzed the data: PW TH AC LJ. Contributed reagents/materials/analysis tools: SZ KZ.
Wrote the paper: PW QH JZ.
References
1. W.H.O. (Diabetes), fact sheet N°312, updated January/2015, available from,http://www.who.int/
mediacentre/factsheets/fs312/en, accessed: June/2015.
2. Amos AF, McCarty DJ, Zimmet P. The rising global burden of diabetes and its complications: estimates
and projections to the year 2010. Diabetic Med. 1997; 14(S5): S7–S85.
3. Mathers CD, Loncar D. Projections of global mortality and burden of disease from 2002 to 2030. Plos
Med. 2006; 3(11): e442. PMID: 17132052
4. Abegunde DO, Mathers CD, Adam T, Ortegon M, Strong K. The burden and costs of chronic diseases
in low-income and middle-income countries. The Lancet. 2007; 370(9603): 1929–1938.
5. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care.
2010; 33(Supplement 1): S62–S69.
6. Smyth S, Heron A. Diabetes and obesity: the twin epidemics. Nat Med. 2006; 12(1): 75–80. PMID:
16397575
7. Tuomilehto J, Lindström J, Eriksson JG, Valle TT, Hämäläinen H, Ilanne-Parikka P, et al. Prevention of
type 2 diabetes mellitus by changes in lifestyle among subjects with impaired glucose tolerance. New
Engl J Med. 2001; 344(18): 1343–1350. PMID: 11333990
8. DeFronzo RA. Pharmacologic therapy for type 2 diabetes mellitus. Ann. Intern Med. 1999; 131(4):
281–303. PMID: 10454950
9. Diabetes Prevention Program Research Group. Reduction in the incidence of type 2 diabetes with life-
style intervention or metformin. New Engl J Med. 2002; 346(6): 393. PMID: 11832527
10. Buckley DA, Cheng A, Kiely PA, Tremblay ML, O'Connor R. Regulation of insulin-like growth factor
type I (IGF-I) receptor kinase activity by protein tyrosine phosphatase 1B (PTP-1B) and enhanced IGF-
I-mediated suppression of apoptosis and motility in PTP-1B-deficient fibroblasts. Mol Cell Biol. 2002;
22(7): 1998–2010. PMID: 11884589
11. Gu W, Hao Y, Zhang G, Wang SF, Miao TT, Zhang KP. Synthesis, in vitro antimicrobial and cytotoxic
activities of new carbazole derivatives of ursolic acid. Bioorg Med Chem Lett.2015; 25(3): 554–557.
doi: 10.1016/j.bmcl.2014.12.021 PMID: 25537271
PLOS ONE | DOI:10.1371/journal.pone.0138767 September 25, 2015
13 / 15

Novel Ursolic Acid Analogues as Antidiabetic Agent
12. Hua SX, Huang RZ, Ye MY, Pan YM, Yao GY, Zhang Y, et al. Design, synthesis and in vitro evaluation
of novel ursolic acid derivatives as potential anticancer agents. Eur J Med Chem. 2015; 95: 435–452.
doi: 10.1016/j.ejmech.2015.03.051 PMID: 25841199
13. Wang JC, Jiang Z, Xiang LP, Li YF, Ou MR, Yang X, et al. Synergism of ursolic acid derivative US597
with 2-deoxy-D-glucose to preferentially induce tumor cell death by dual-targeting of apoptosis and gly-
colysis. Sci Rep. 2014; 4: 5006. doi: 10.1038/srep05006 PMID: 25833312
14. Song GP, Shen XT, Li SM, Li YB, Liu YP, Zheng YS, et al. Structure–activity relationships of 3-O-β-cha-
cotriosyl ursolic acid derivatives as novel H5N1 entry inhibitors. Eur J Med Chem.2015; 93: 431–442.
doi: 10.1016/j.ejmech.2015.02.029 PMID: 25728024
15. Kashiwada Y, Nagao T, Hashimoto A,Ikeshiro Y, Okabe H, Cosentino LM, et al. Anti-AIDS Agents 38.
Anti-HIV Activity of 3-O-Acyl Ursolic Acid Derivatives 1. J Nat Prod. 2000; 63(12): 1619–1622. PMID:
11141100
16. Saravanan R, Pugalendi V. Impact of ursolic acid on chronic ethanol-induced oxidative stress in the rat
heart. Pharmacol. Rep. 2006; 58(1): 41–47. PMID: 16531629
17. Kalani K, Cheema HS, Tripathi H, Khan F, Daroker MP, Srivastava SK. QSAR-guided semi-synthesis
and in vitro validation of antiplasmodial activity in ursolic acid derivatives. RSC Adv. 2015; 5(41):
32133–32143.
18. Wu PP, Zhang K, Lu YJ, He P, Zhao SQ. In vitro and in vivo evaluation of the antidiabetic activity of
ursolic acid derivatives. Eur J Med Chem. 2014; 80: 502–508. doi: 10.1016/j.ejmech.2014.04.073
PMID: 24813878
19. Wu PP, He P, Zhao SQ, Huang TM, Lu YJ, Zhang K. Effects of ursolic acid derivatives on Caco-2 cells
and their alleviating role in streptozocin-induced type 2 diabetic rats. Molecules, 2014; 19(8): 12559–
12576. doi: 10.3390/molecules190812559 PMID: 25153871
20. Huang TM, Wu PP, Cheng AM, Jing Qin, Zhang K, Zhao SQ. A hydrophilic conjugate approach toward
the design and synthesis of ursolic acid derivatives as potential antidiabetic agent. RSC Adv. 2015; 5
(55): 44234–44246.
21. Jayaprakasam B, Olson LK, Schutzki RE, Tai MH, Nair MG. Amelioration of obesity and glucose intoler-
ance in high-fat-fed C57BL/6 mice by anthocyanins and ursolic acid in Cornelian cherry (Cornus mas).
J Agr Food Chem. 2006; 54(1): 243–248.
22. Ali H, Houghton PJ, Soumyanath A. α-Amylase inhibitory activity of some Malaysian plants used to
treat diabetes; with particular reference to Phyllanthus amarus. J Ethnopharmacol. 2006; 107(3): 449–
455. PMID: 16678367
23. Ramírez-Espinosa JJ, Rios MY, López-Martínez S, López-Vallejo F, Medina-Franco JL, Paoli P, et al.
Antidiabetic activity of some pentacyclic acid triterpenoids, role of PTP–1B: In vitro, in silico, and in vivo
approaches. Eur J Med Chem. 2011; 46(6): 2243–2251. doi: 10.1016/j.ejmech.2011.03.005 PMID:
21453996
24. Castro AJG, Frederico MJS, Cazarolli LH, Mendes CP, Bretanha LC, Schmidt ÉC, et al. The mecha-
nism of action of ursolic acid as insulin secretagogue and insulinomimetic is mediated by cross-talk
between calcium and kinases to regulate glucose balance. BBA-Gen Subjects. 2015; 1850(1): 51–61.
25. Dong HY, Yang X, Xie JJ, Xiang LP, Li YF, Ou MR, et al. UP12, a novel ursolic acid derivative with
potential for targeting multiple signaling pathways in hepatocellular carcinoma. Biochem Pharmacol.
2015; 93(2): 151–162. doi: 10.1016/j.bcp.2014.11.014 PMID: 25522955
26. Meng YQ, Liu D, Cai LL, Chen H, Cao B, Wang YZ. The synthesis of ursolic acid derivatives with cyto-
toxic activity and the investigation of their preliminary mechanism of action. Bioorg Med Chem. 2009;
17(2): 848–854. doi: 10.1016/j.bmc.2008.11.036 PMID: 19091579
27. Bai KK, Yu Z, Chen FL, Li F, Li WY, Guo YH. Synthesis and evaluation of ursolic acid derivatives as
potent cytotoxic agents. Bioorg Med Chem Lett. 2012; 22(7): 2488–2493. doi: 10.1016/j.bmcl.2012.02.
009 PMID: 22370266
28. Ma CM, Cai SQ, Cui JR, Wang RQ, Tu PF, Hattori M, et al. The cytotoxic activity of ursolic acid deriva-
tives. Eur J Med Chem. 2005; 40(6): 582–589. PMID: 15922841
29. Schäfer A, Högger P. Oligomeric procyanidins of French maritime pine bark extract (Pycnogenol1
effectively inhibit α-glucosidase. Diabetes Res Clin Pr. 2007; 77(1): 41–46.
)
30. Worawalai W, Wacharasindhu S, Phuwapraisirisan P. Synthesis of new N-substituted aminoquercitols
from naturally available (+)-proto-quercitol and their α-glucosidase inhibitory activity. MedChemComm.
2012; 3(11): 1466–1470.
31. Watanabe K, Hata Y, Kizaki H, Katsube Y, Suzuki Y. The refined crystal structure of Bacillus cereus
oligo-1, 6-glucosidase at 2.0 Å resolution: structural characterization of proline-substitution sites for pro-
tein thermostabilization. J Mol Biol. 1997; 269(1): 142–153. PMID: 9193006
PLOS ONE | DOI:10.1371/journal.pone.0138767 September 25, 2015
14 / 15

Novel Ursolic Acid Analogues as Antidiabetic Agent
32. Lee Y, Kim S, Kim JY, Arooj M, Kim S, Hwang S, et al. Binding Mode Analyses and Pharmacophore
Model Development for Stilbene Derivatives as a Noveland Competitive Class of a-Glucosidase Inhibi-
tors. PLoS One. 2014; 9(1): e85827. doi: 10.1371/journal.pone.0085827 PMID: 24465730
33. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and
Clustal X version 2.0. Bioinformatics, 2007; 23(21): 2947–2948. PMID: 17846036
34. Liu Y, Ke ZF, Wu KY, Liu SW, Chen WH, Jiang SB, et al. An amphiphilic conjugate approach toward
the design and synthesis of betulinic acid–polyphenol conjugates as inhibitors of the HIV‐1 gp41 fusion
core formation. ChemMedChem. 2011; 6(9): 1654–1664. doi: 10.1002/cmdc.201100149 PMID:
21688394
PLOS ONE | DOI:10.1371/journal.pone.0138767 September 25, 2015
15 / 15