Synthesis and evaluation of novel oleanolic acid derivatives as potential antidiabetic agents

Article abstract of DOI:10.1111/cbdd.12241

Antidiabetic agents simultaneously inhibiting hepatic glucose production and stimulating hepatic glucose consumption could apply a better control over hyperglycemia. A series of oleanolic acid derivatives with bulky substituents at C-3 position were designed and synthesized in order to search for this kind of agents. All of the compounds were evaluated biologically in vitro using glycogen phosphorylase and HepG2 cells. The results indicated that several derivatives exhibited moderate-to-good inhibitory activities against glycogen phosphorylase. Compound 8g showed the best inhibition with an IC₅₀ value of 5.4 μm. Moreover, most of the derivatives were found to increase the glucose consumption in HepG2 cells in a dose-dependent manner. The possible binding mode of compound 8g with glycogen phosphorylase was also explored by docking study. 8g was found to have hydrogen bonding interactions with Arg193, Arg310, and Arg60 of the allosteric site.

Full text of DOI:10.1111/cbdd.12241

Chem Biol Drug Des 2014; 83: 297–305
Research Article
Synthesis and Evaluation of Novel Oleanolic Acid
Derivatives as Potential Antidiabetic Agents
Liying Zhang1, Xiaojian Jia2, Jizhe Dong3,
inhibitory concentration; SD, standard deviation; RMGPa,
rabbit muscle glycogen phosphorylase a; SAR, structure–
activity relationship.
Dongyin Chen3, Jun Liu4,*, Luyong Zhang4 and
Xiaoan Wen3,*
Received 21 June 2013, revised 28 August 2013 and
1Key Laboratory of Traditional Chinese Medicine Research
and Development of Hebei Province, Institute of Traditional
accepted for publication 24 September 2013
Chinese Medicine, Chengde Medical University, Chengde
067000, China
2School of Life Science & Technology, China
The liver plays a dominant role in the regulation of glucose
homeostasis because it is an organ of glucose production
as well as glucose consumption. When blood glucose
declines, the liver breaks down glycogen and releases
glucose into the bloodstream. When plasma glucose levels
are high, the liver takes up glucose, replenishes depleted
glycogen stores, thus helping to dampen variations in
blood glucose (1).
Pharmaceutical University, Nanjing 21009, China
3Center for Drug Discovery, China Pharmaceutical
University, Nanjing 21009, China
4Jiangsu Center for Drug Screening, China Pharmaceutical
University, Nanjing 21009, China
*Corresponding authors: Jun Liu, junliu@cpu. edu.cn;
Xiaoan Wen, wxagj@126.com
Antidiabetic agents simultaneously inhibiting hepatic
glucose production and stimulating hepatic glucose
Type 2 diabetes is associated with the liver’s inability to con-
trol glucose homeostasis (2). The defects of insulin to trigger
downstream actions in liver are defined as hepatic insulin
resistance, including increased hepatic glucose production
and decreased hepatic glucose consumption (3). Therefore,
in principle, any effect that decreases glucose production or
increases glucose consumption, or both, would lead to a
reduction in plasma glucose levels. From the perspective of
enzymes involved in glucose metabolism, glycogen pho-
sphorylase (GP), phosphoenolpyruvate carboxykinase
(PEPCK), fructose-1,6-bisphosphatase (FBPase), and glu-
cose-6-phosphatase (G6Pase) work together to generate
glucose, whereas glycogen synthase (GS), glucokinase
(GK), 6-phosphofructo-1-kinase (6PFK1), and pyruvate
kinase (PK) work together for glucose disposal in the liver (4).
consumption could apply
a
better control over
hyperglycemia. A series of oleanolic acid derivatives
with bulky substituents at C-3 position were designed
and synthesized in order to search for this kind of
agents. All of the compounds were evaluated biologi-
cally in vitro using glycogen phosphorylase and HepG2
cells. The results indicated that several derivatives
exhibited moderate-to-good inhibitory activities against
glycogen phosphorylase. Compound 8g showed the
best inhibition with an IC50 value of 5.4 lM. Moreover,
most of the derivatives were found to increase the glu-
cose consumption in HepG2 cells in a dose-dependent
manner. The possible binding mode of compound 8g
with glycogen phosphorylase was also explored by
docking study. 8g was found to have hydrogen bond-
ing interactions with Arg193, Arg310, and Arg60 of the
allosteric site.
Oleanolic acid (OA, 1), a pentacyclic triterpenoid compound
isolated from the traditional Chinese herb Ligustrum lucidum
Ait, exhibits a significant activity in lowering fasting blood
glucose level (5). Unlike the commonly used antidiabetic
therapeutics such as insulin or thiazolidinediones that
up-regulate glucose transport in periphery and often lead to
weight gain, OA possesses glucose-lowering activity, but
lacks the adipogenic activity (6). Studies indicated that the
liver may be the major site for the sustained improvement of
glycemia after the treatment with OA (7). Previously, we
have found that OA and its derivatives represented a novel
class of inhibitors of GP (8), and their anti-hyperglycemia
activity could, at least in part, be due to the modulation of
hepatic glucose production. Recently, we first screened
Key words: binding mode, glycogen phosphorylase inhibitors,
hepatic glucose consumption, hepatic glucose production,
oleanolic acid
Abbreviations: GP, glycogen phosphorylase; PEPCK, pho-s-
phoenolpyruvate carboxykinase; FBPase, fructose-1,6-bis-
phosphatase; G6Pase, glucose-6-phosphatase; GS, glycogen
synthase; GK, glucokinase; 6PFK1, 6-phosphofructo-1-
kinase; PK, pyruvate kinase; OA, oleanolic acid; HepG2,
human hepatocellular carcinoma cell line; NMR, nuclear mag-
netic resonance; IR, infrared spectrometry; DMF, N, N-dim-
ethylformamide; THF, tetrahydrofuran; Pd-C, palladium on
carbon; DMSO, dimethyl sulfoxide; IC50
, half maximal
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12241
297

Zhang et al.
the effect of OA on glucose consumption in human
hepatocellular carcinoma cell line (HepG2) cells. OA signifi-
cantly promoted the glucose consumption in HepG2 cells.
Considering the hypoglycemic phenomenon, we postulated
that the antidiabetic pharmacological effects of OA and its
derivatives were not only due to the suppression of hepatic
glucose production but also maybe partly due to the
enhancement of hepatic glucose consumption.
obtained on a MEL-TEMP II melting-point apparatus and
are uncorrected. NMR spectra were recorded on a BRU-
KER-ACF-300 instrument (300 MHz for 1H NMR and
75 MHz for 13C NMR spectra, chemical shifts are
expressed as values relative to TMS as internal standard).
Mass spectral data were obtained on Agilent 1100
LC/DAD/MSD or Q-Tof Micro MS/MS. IR spectra were
recorded on Shimadzu FTIR-8400S spectrometer. Com-
pounds 4–6 were prepared according to the procedures
as previously reported in the literature (9).
Our previously reported data suggested that the proper
substitutions at C-3 position conferred enhanced hypo-
glycemic activities (9). However, the C-3 ester groups of
prior OA analogs have mainly contained freely rotating
short alkyl chains. It is still unclear whether the increas-
ing alkyl chain length or introducing aromatic substitu-
tions is associated with increased hypoglycemic
activities. Therefore, in the present study, a series of OA
derivatives with long alkyl chains or aromatic rings at C-
3 position were designed in order to further explore the
conformational space of the C-3 pharmacophore. Fur-
thermore, an attractive strategy for the linking conjuga-
tion involves the click reaction of azide-bearing OA with
alkyne-bearing alkyl or aromatic groups to join them via
a triazole linkage. The triazole linkage is more than just a
passive linker because it readily associates with biological
targets, through hydrogen bonding and dipole interac-
tions (10).
General procedure for the synthesis of 3a–3g
To a solution of carboxylic acid (2a–2g, 3.20 mmol) in
DMF (10 mL) were added propargyl bromide (3.58 mmol)
and K2CO3 (6.40 mmol). The reaction mixture was stirred
at room temperature for 6–24 h and filtered. The filtrate
was concentrated under vacuum, and the resultant resi-
due was redissolved in ethyl acetate (50 mL). The organic
layer was washed with 1 N HCl, saturated NaHCO3, and
brine and then dried (Na2SO4) and filtered. The filtrate was
evaporated under vacuum to give a crude product alkyne-
functionalized intermediate (3a–3g), which was used for
the next reaction without further purification.
General procedure for the synthesis of 7a–7g
To a mixture of alkyne-functionalized intermediate (3a–3g,
In this article, we report our preliminary attempt to discover
a novel of antidiabetic agents that inhibit hepatic glucose
production and promote hepatic glucose consumption
based on a simple and economical synthetic procedure
(Figure 1). Biological evaluation of the derivatives as new
GP inhibitors is also presented. Moreover, the glucose
consumption was examined by measuring the glucose
concentration in the medium after treatment with the
synthesized compounds. To the best of our knowledge,
no other dual-action compounds targeting both hepatic
glucose production and hepatic glucose consumption
have been reported.
0.48 mmol) and azide 6 (9) (0.48 mmol) in CH2Cl2
(1.5 mL) and H2O (1.5 mL) were added CuSO4Á5H2O
(0.095 mmol) and sodium ascorbate (0.19 mmol). The
resulting solution was stirred at room temperature for
2–8 h and filtered. The filtrate was concentrated under vac-
uum, and the resultant residue was redissolved in CH2Cl2
(5 mL). The reaction mixture was extracted with CH2Cl2
(3 9 5 mL). The combined organic layers were washed
with brine, dried (Na2SO4), filtered, and concentrated. The
residue was purified by flash column chromatography.
Benzyl 3b-[2-(4-nonanoyloxymethyl-1H-1,2,3-
triazol-1-yl)acetoxy]olean-12-en-28-oate (7a)
Materials and Methods
White solid; yield: 91%; mp 63–65 °C. IR (KBr,per cm) m
2948, 2856, 2106, 1732, 1464. 1H-NMR (CDCl3, 300
MHz) d 0.63, 0.77, 0.86, 0.88, 0.91, 0.93, 0.95, 1.15 (s,
each 3H), 2.34 (t, J = 7.5 Hz, 2H), 2.93 (dd, J = 3.9,
13.8 Hz, 1H), 4.58–4.63 (m, 1H), 5.07 (d, J = 12.6 Hz,
Chemistry
Purifications by column chromatography were carried out
over silica gel (200–400 mesh). Melting points were
H
H
COOH
O
R
COOH
N
O
N
N
O
H
H
O
H
HO
H
1
8a-8g
Figure 1: Structure of oleanolic acid (OA, 1) and OA derivatives (8a–8g).
298
Chem Biol Drug Des 2014; 83: 297–305

Oleanolic Acid Derivatives as Antidiabetic Agents
1H), 5.10 (d, J = 12.6 Hz, 1H), 5.18 (s, 2H), 5.27 (s, 2H),
5.31(br s, 1H), 7.29–7.36 (m, 5H), 7.77 (s, 1H). 13C-NMR
(CDCl3, 75 MHz) d 177.4, 173.7, 165.7, 143.8, 143.5,
136.4, 128.4, 127.9, 127.8, 125.1, 122.3, 83.9, 65.9,
57.3, 55.2, 51.1, 47.5, 46.7, 45.8, 41.6, 41.4, 39.2, 38.0,
34.1, 33.8, 33.1, 32.6, 32.3, 31.7, 30.6, 29.2, 29.1, 28.1,
27.5, 25.8, 24.8, 23.6, 23.4, 23.0, 22.6, 18.1, 16.8, 16.5,
15.3, 14.0. MS (ESI, m/z): 848.6 [M+Na]+.
37.7, 36.9, 34.1, 33.9, 33.0, 32.6, 32.4, 31.9, 30.7, 29.7,
29.6, 29.4, 29.3, 29.2, 29.1, 28.1, 27.6, 25.8, 24.8, 23.6,
23.4, 23.0, 22.6, 18.1, 16.9, 16.5, 15.3, 14.0. MS (ESI, m/
z): 924.9 [M+H]+.
Benzyl 3b-[2-(4-stearoyloxymethyl-1H-1,2,3-
triazol-1-yl)acetoxy]olean-12-en-28-oate (7e)
White solid; yield: 89%; mp 50–52 °C. IR (KBr, per cm)
m 2924, 2852, 1737, 1464. 1H-NMR (CDCl3, 300 MHz)
d 0.60, 0.74, 0.83, 0.88, 0.89, 0.89, 0.91, 1.11 (s, each
3H), 2.31 (t, J = 7.7 Hz, 2H), 2.90 (dd, J = 4.2,
13.7 Hz, 1H), 4.54–4.60 (m, 1H), 5.02 (d, J = 12.6 Hz,
1H), 5.06 (d, J = 12.6 Hz, 1H), 5.14 (s, 2H), 5.23 (s,
2H), 5.27 (t, J = 3.6 Hz, 1H), 7.27–7.35 (m, 5H), 7.73
Benzyl 3b-[2-(4-decanoyloxymethyl-1H-1,2,3-
triazol-1-yl)acetoxy]olean-12-en-28-oate (7b)
White solid; yield: 87%; mp 65–67 °C. IR (KBr, per cm) m
3447, 2948, 2853, 1735, 1464. 1H-NMR (CDCl3,
300 MHz) d 0.60, 0.73, 0.83, 0.89, 0.89, 0.91, 1.11,
1.25 (s, each 3H), 2.31 (t, J = 7.3 Hz, 2H), 2.89 (d,
J = 9.7 Hz, 1H), 4.54–4.60 (m, 1H), 5.03 (d,
J = 12.4 Hz, 1H), 5.09 (d, J = 12.4 Hz, 1H), 5.15 (s, 1H),
5.24 (s, 2H), 5.28 (br s, 1H), 7.32-7.35 (m, 5H), 7.73 (s,
1H). 13C-NMR (CDCl3, 75 MHz) d 177.4, 173.7, 165.8,
143.8, 143.5, 136.4, 128.4, 128.0, 127.9, 125.1, 122.3,
83.9, 65.9, 57.4, 55.2, 51.1, 47.5, 46.7, 45.8, 41.7,
41.4, 39.3, 38.0, 37.7, 36.8, 34.1, 33.8, 32.6, 32.3,
31.9, 30.7, 29.4, 29.3, 29.1, 28.1, 27.6, 25.8, 24.8,
23.6, 23.4, 23.0, 22.7, 18.1, 16.8, 16.5, 15.3, 14.1. MS
(ESI, m/z): 840.8 [M+H]+.
(s, 1H). 13C-NMR (CDCl3, 75 MHz)
d 177.4, 173.7,
165.7, 143.8, 143.5, 136.5, 128.4, 128.0, 127.9, 125.1,
122.3, 84.0, 65.9, 57.4, 55.3, 51.2, 47.6, 45.9, 46.7,
41.7, 41.4, 39.3, 38.0, 37.8, 36.9, 34.2, 33.9, 33.1,
32.6, 32.4, 31.9, 30.7, 29.7, 29.7, 29.6, 29.5, 29.3,
29.2, 29.1, 28.1, 27.6, 25.8, 24.8, 23.6, 23.4, 23.0,
22.6, 18.1, 16.9, 16.5, 15.3, 14.0. MS (ESI, m/z): 953.0
[M+H]+.
Benzyl 3b-{2-[4-(4-methoxybenzoyloxy)methyl-1H-
1,2,3-triazol-1-yl]acetoxy}olean-12-en-28-oate (7f)
White solid; yield: 91%; mp 89–91 °C. IR (KBr, per cm) m
2948, 1720, 1606, 1511. 1H-NMR (CDCl3, 300 MHz) d
0.58, 0.65, 0.78, 0.83, 0.89, 0.91, 1.20 (s, each 3H), 2.90
(dd, J = 3.5, 13.7 Hz, 1H), 3.86 (s, 3H), 4.52–4.57 (m,
1H), 5.03 (d, J = 12.6 Hz, 1H), 5.09 (d, J = 12.6 Hz, 1H),
5.16 (s, 2H), 5.28 (t, J = 3.8 Hz, 1H), 5.47 (br s, 2H), 6.89
(d, J = 8.8 Hz, 2H), 7.28–7.33 (m, 5H), 7.82 (br s, 1H),
7.98 (d, J = 8.8 Hz, 2H). 13C-NMR (CDCl3, 75 MHz) d
177.4, 166.1, 165.7, 163.6, 143.7, 136.8, 131.8, 128.4,
128.0, 127.9, 122.3, 122.2, 113.6, 83.9, 65.9, 57.7, 55.4,
55.2, 51.2, 47.5, 46.7, 45.9, 41.6, 41.4, 39.2, 37.9, 37.7,
36.8, 33.8, 33.1, 32.6, 32.4, 30.7, 28.0, 27.6, 25.8, 23.6,
23.4, 23.0, 18.1, 16.8, 16.5, 15.2. MS (ESI, m/z): 842.5
[M+Na]+.
Benzyl 3b-[2-(4-dodecanoyloxymethyl-1H-1,2,3-
triazol-1-yl)acetoxy]olean-12-en-28-oate (7c)
White solid; yield: 81%; mp 66–68 °C. IR (KBr, per cm) m
2926, 2853, 1735, 1465. 1H-NMR (CDCl3, 300 MHz) d
0.60, 0.74, 0.83, 0.89, 0.91, 1.16, 1.24 (s, each 3H),
2.31 (t, 2H), 2.88 (dd, J = 4.1, 13.8 Hz, 1H), 4.54–4.60
(m, 1H), 5.03 (d, J = 12.5 Hz, 1H), 5.09 (d, J = 12.5 Hz,
1H), 5.14 (s, 2H), 5.23 (s, 2H), 5.27 (t, 1H, J = 3.0 Hz),
7.29–7.36 (m, 5H), 7.73 (s, 1H). 13C-NMR (CDCl3,
75 MHz) d 177.3, 173.6, 165.7, 143.8, 143.5, 136.4,
128.4, 127.9, 127.8, 125.0, 122.3, 83.9, 65.9, 57.3,
55.2, 51.1, 47.5, 46.7, 45.9, 41.7, 41.4, 39.3, 38.0,
37.7, 36.9, 34.1, 33.9, 33.0, 32.6, 32.4, 31.9, 30.7,
29.6, 29.4, 29.3, 29.2, 29.1, 28.1, 27.6, 25.8, 24.8,
23.6, 23.4, 23.0, 22.6, 18.1, 16.9, 16.5, 15.3, 14.0.
ESI-MS (ESI, m/z): 868.8 [M+H]+.
Benzyl 3b-{2-[4-(2-naphthalen-2-yl)acetoxymethyl-
1H-1,2,3-triazol-1-yl]acetoxy}olean-12-en-28-oate
(7 g)
Benzyl 3b-[2-(4-palmitoyloxymethyl-1H-1,2,3-
triazol-1-yl)acetoxy]olean-12-en-28-oate (7d)
White solid; yield: 86%; mp 91–93 °C. IR (KBr, per cm) m
2947, 2854, 1734, 1700. 1H-NMR (CDCl3, 300 MHz) d
0.60, 0.72, 0.81, 0.88, 0.89, 0.92, 1.12 (s, each 3H), 2.89
(d, J = 10.0 Hz, 1H), 4.01 (s, 2H), 4.53–4.56 (m, 1H),
5.02–5.12 (m, 4H), 5.27 (br s, 3H), 7.33–7.53 (m, 9H),
7.78–7.94 (m, 3H). 13C-NMR (CDCl3, 75 MHz) d 177.4,
165.7, 143.8, 143.2, 136.5, 128.7, 128.4, 128.2, 128.1,
128.0, 127.9, 126.4, 125.8, 125.5, 125.1, 123.8, 122.3,
83.9, 65.9, 58.1, 55.3, 51.1, 47.6, 46.8, 45.9, 41.7, 41.4,
39.3, 38.9, 38.0, 37.8, 36.9, 33.9, 33.1, 32.6, 32.4, 30.7,
28.1, 27.6, 25.8, 23.6, 23.4, 23.1, 18.2, 16.9, 16.5, 15.3.
MS (ESI, m/z): 854.6 [M+H]+.
White solid; yield: 83%; mp 56–58 °C. IR (KBr, per cm) m
2924, 2852, 1735, 1465. 1H-NMR (CDCl3, 300 MHz) d
0.60, 0.73, 0.83, 0.85, 0.88, 0.89, 0.91, 1.11 (s, each
3H), 2.31 (t, J = 7.4 Hz, 2H), 2.91 (dd, J = 4.2, 13.8 Hz,
1H), 4.54–4.59 (m, 1H), 5.03 (d, J = 12.5 Hz, 1H), 5.09
(d, J = 12.5 Hz, 1H), 5.14 (s, 2H), 5.23 (s, 2H), 5.27(t,
J = 3.4 Hz, 1H), 7.29–7.36 (m, 5H), 7.73 (s, 1H). 13C-
NMR (CDCl3, 75 MHz) d 177.3, 173.6, 165.7, 143.7,
143.5, 136.4, 128.4, 127.9, 125.0, 122.3, 83.9, 65.9,
57.3, 55.2, 51.1, 47.5, 46.7, 45.9, 41.7, 41.4, 39.3, 38.0,
Chem Biol Drug Des 2014; 83: 297–305
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Zhang et al.
General procedure for the synthesis of 8a-8 g
To a solution of benzyl ester (7a–7g, 0.35 mmol) in THF
(5 mL) was added 10% Pd-C (0.3 g), with stirring under
hydrogen atmosphere at room temperature for 12 h. The
catalyst was filtered through Celite, and the filtrate was
concentrated under vacuum to give a crude product that
was purified by flash column chromatography.
3b-[2-(4-palmitoyloxymethyl-1H-1,2,3-triazol-1-yl)
acetoxy]olean-12-en-28-oic acid (8d)
White solid; yield: 93%; mp 129–131 °C. IR(KBr, per cm) m
2924, 2852, 1740, 1693, 1465. 1H-NMR (CDCl3,
300 MHz) d 0.75, 0.75, 0.84, 0.86, 0.88, 0.90, 0.92, 1.13
(s, each 3H), 2.31 (t, J = 7.5 Hz, 2H), 2.82 (dd, J = 3.5,
13.5 Hz, 1H), 4.56–4.61 (m, 1H), 5.15 (s, 2H), 5.24 (s,
2H), 5.28 (br s, 1H), 7.74 (s, 1H). 13C-NMR (CDCl3,
75 MHz) d 173.7, 165.8, 143.6, 143.5, 125.1, 122.4,
83.9, 57.3, 55.2, 51.1, 47.5, 46.5, 45.8, 41.6, 41.0, 39.3,
37.9, 37.7, 36.9, 34.1, 33.8, 33.0, 32.4, 31.9, 30.7, 29.7,
29.6, 29.4, 29.3, 29.2, 29.1, 28.1, 27.6, 25.9, 24.8, 23.6,
23.4, 22.9, 22.7, 18.1, 17.1, 16.5, 15.3, 14.1. MS (ESI, m/
z): 832.5[MÀH]À. HR-MS (ESI, M-H) m/z: calcd for
C51H82N3O6 832.6204, found 832.6202.
3b-[2-(4-nonanoyloxymethyl-1H-1,2,3-triazol-1-yl)
acetoxy]olean-12-en-28-oic acid (8a)
White solid; yield: 87%; mp 132–134 °C. IR (KBr, per
cm)
m
2928, 1743, 1692, 1465. 1H-NMR (CDCl3,
300 MHz) d 0.74 (s, 6H), 0.81, 0.84, 0.87, 0.90, 0.92,
1.12 (s, each 3H), 2.31 (t, J = 7.5 Hz, 2H), 2.82
(d, J = 9.9 Hz, 1H), 4.55–4.60 (m, 1H), 5.15 (s, 2H), 5.24
(s, 2H), 5.31 (br s, 1H), 7.74 (s, 1H). 13C-NMR (CDCl3,
75 MHz) d 183.6, 165.8, 143.6, 143.4, 125.1, 122.4,
83.9, 57.3, 55.2, 51.1, 47.5, 46.5, 45.8, 41.6, 40.9,
39.2, 37.9, 37.7, 36.9, 34.1, 33.8, 33.0, 32.5, 31.8,
30.6, 29.2, 29.1, 28.1, 27.6, 25.9, 24.8, 23.5, 23.4,
22.9, 22.6, 18.1, 17.1, 16.5, 15.3, 14.1. MS (ESI, m/z):
734.7[MÀH]À. HR-MS (ESI, M+H) m/z: calcd for C44
H70N3O6 736.52591, found 736.52825.
3b-[2-(4-stearoyloxymethyl-1H-1,2,3-triazol-1-yl)
acetoxy]olean-12-en-28-oic acid (8e)
White solid; yield: 90%; mp 149–150 °C. IR(KBr, per cm) m
2923, 2850, 1741, 1693, 1466. 1H-NMR (CDCl3,
300 MHz) d 0.74 (s, 6H), 0.81, 0.84, 0.88, 0.90, 0.92,
1.12 (s, each 3H), 2.31 (t, J = 7.4 Hz, 2H), 2.80 (dd,
J = 2.4, 12.9 Hz, 1H), 4.55–4.60 (m, 1H), 5.15 (s, 2H),
5.24 (s, 2H), 5.27 (br s, 1H), 7.74 (s, 1H). 13C-NMR
(CDCl3, 75 MHz) d 182.8, 173.7, 165.8, 143.7, 143.5,
125.1, 122.4, 83.9, 57.4, 55.3, 51.2, 47.6, 46.5, 45.9,
41.7, 41.1, 39.3, 38.0, 37.8, 37.0, 34.2, 33.8, 33.0, 32.6,
32.5, 31.9, 30.7, 29.5, 29.3, 29.3, 29.1, 28.1, 27.7, 25.9,
24.9, 23.6, 23.4, 23.0, 22.7, 18.2, 17.1, 16.5, 15.3, 14.1.
MS (ESI, m/z): 862.6[M+H]+. HR-MS (ESI, M+Na) m/z:
calcd for C53H87N3Na1O6 884.64871, found 884.64672.
3b-[2-(4-decanoyloxymethyl-1H-1,2,3-triazol-1-yl)
acetoxy]olean-12-en-28-oic acid (8b)
White solid; yield: 88%; mp 160–162 °C. IR (KBr, per cm)
m 3382, 2925, 1741, 1691. 1H-NMR (CDCl3, 300 MHz) d
0.75, 0.75, 0.84, 0.88, 0.90, 0.92, 0.92, 1.13 (s, each
3H), 2.31 (t, J = 7.4 Hz, 2H), 2.81 (d, J = 9.5 Hz, 1H),
4.56–4.61 (m, 1H), 5.16 (s, 2H), 5.24 (s, 2H), 5.28 (br s,
1H), 7.74 (s, 1H). 13C-NMR (CDCl3, 75 MHz) d 183.7,
173.6, 165.7, 143.6, 143.4, 125.1, 122.3, 83.8, 57.3,
55.1, 51.0, 47.4, 46.4, 45.8, 41.5, 40.9, 39.2, 37.9, 37.7,
36.9, 34.1, 33.7, 33.0, 32.3, 31.8, 30.6, 29.3, 29.2, 29.0,
28.0, 27.6, 25.8, 24.8, 23.5, 23.3, 22.8, 22.6, 18.1, 17.0,
16.4, 15.3, 14.0. MS (ESI, m/z): 788.0 [M+K]+. HR-MS
(ESI, M-H) m/z: calcd for C45H70N3O6 748.5265, found
748.5263.
3b-{2-[4-(4-methoxybenzoyloxy)methyl-1H-1,2,3-
triazol-1-yl]acetoxy}olean-12-en-28-oic acid (8f)
White solid; yield: 75%; mp 174–175 °C. IR(KBr, per cm) m
2946, 1745, 1712, 1694. 1H-NMR (CDCl3, 300 MHz) d
0.67, 0.73, 0.80, 0.87, 0.90, 0.92, 1.12 (s, each 3H), 2.81
(dd, J = 3.8, 13.6 Hz, 1H), 3.84 (s, 3H), 4.53–4.58 (m,
1H), 5.16 (s, 2H), 5.26 (br s, 1H), 5.47 (s, 2H), 6.89 (d,
J = 9.7 Hz, 2H), 7.83 (s, 1H), 7.99 (d, J = 9.7 Hz, 2H).
13C-NMR (CDCl3, 75 MHz) d 165.7, 143.6, 131.8, 125.3,
122.4, 113.6, 83.8, 57.7, 55.4, 55.2, 51.2, 47.5, 46.5,
45.8, 41.6, 41.0, 39.3, 37.9, 37.7, 36.9, 33.8, 33.0, 32.5,
32.4, 30.6, 28.0, 27.6, 25.9, 23.6, 23.4, 22.9, 18.1, 17.0,
16.4, 15.3. MS (ESI, m/z): 728.4[MÀH]À. HR-MS (ESI,
M+H) m/z: calcd for C43H60N3O7 730.44258, found
730.44429.
3b-[2-(4-dodecanoyloxymethyl-1H-1,2,3-triazol-1-
yl)acetoxy]olean-12-en-28-oic acid (8c)
White solid; yield: 79%; mp 163–164 °C. IR(KBr, per cm) m
2928, 2852, 1740, 1693. 1H-NMR (CDCl3, 300 MHz) d
0.75, 0.75, 0.84, 0.86, 0.88, 0.90, 0.92, 1.13 (s, each
3H), 2.31 (t, J = 7.4 Hz, 2H), 2.81 (d, J = 13.8 Hz, 1H),
4.55-4.61 (m, 1H), 5.16 (s, 2H), 5.24 (s, 2H), 5.28 (br s,
1H), 7.73 (s, 1H). 13C-NMR (CDCl3, 75 MHz) d 183.6,
173.7, 165.8, 143.6, 143.4, 125.1, 122.4, 83.9, 57.3,
55.2, 51.1, 47.5, 46.5, 45.8, 41.6, 40.9, 39.2, 37.9, 37.7,
36.9, 34.1, 33.8, 33.0, 32.4, 31.9, 30.6, 29.6, 29.4, 29.3,
29.2, 29.1, 28.1, 27.6, 25.9, 24.8, 23.5, 23.4, 22.9, 22.7,
18.1, 17.1, 16.5, 15.3, 14.1. MS (ESI, m/z): 776.6 [M-H]À.
HR-MS (ESI, M-H) m/z: calcd for C47H74N3O6 776.5578,
found 776.5581.
3b-{2-[4-(2-naphthalen-1-yl)acetoxymethyl-1H-
1,2,3-triazol-1-yl]acetoxy}olean-12-en-28-oic acid
(8 g)
White solid; yield: 84%; mp 100–102 °C. IR(KBr, per cm) m
2945, 2873, 1740, 1691. 1H-NMR (CDCl3, 300 MHz) d
0.65, 0.69, 0.75, 0.84, 0.85, 0.86, 1.06 (s, each 3H), 2.75
(d, J = 10.2 Hz, 1H), 4.01 (s, 2H), 4.47–4.52 (m, 1H), 4.99
300
Chem Biol Drug Des 2014; 83: 297–305

Oleanolic Acid Derivatives as Antidiabetic Agents
(s, 2H), 5.20 (s, 3H), 7.32–7.45 (m, 4H), 7.70–7.87 (m,
3H). 13C-NMR (CDCl3, 75 MHz): d 183.0, 171.4, 165.7,
143.7, 143.2, 133.9, 132.1, 130.2, 128.7, 128.2, 128.1,
126.4, 125.8, 125.5, 125.1, 123.7, 122.4, 83.9, 58.1,
55.2, 51.1, 47.6, 46.5, 45.9, 41.6, 41.0, 39.3, 38.9, 38.0,
37.8, 37.0, 33.8, 33.0, 32.5, 32.5, 30.7, 28.1, 27.7, 25.9,
23.6, 23.4, 22.9, 18.2, 17.1, 16.5, 15.3. MS (ESI, m/z):
762.4 [MÀH]À. HR-MS (ESI, M-H) m/z: calcd for
followed by Dunnett’s multiple comparison test was per-
formed for multiple comparisons between the groups. Sta-
tistical significance was accepted at p < 0.05, indicated in
Figure 3 as follows: *p < 0.05; **p < 0.01; ***p < 0.001.
Docking study
The docking analysis was performed using the sketch
molecule module of Sybyl 6.9 package (Tripos Inc., St.
Louis, MO, USA). The crystal structure of GP complexed
with maslinic acid at the allosteric site was received from the
Protein Data Bank (PDB code: 2QN2) (8). All the hydrogen
atoms were added to specify the correct configuration and
tautomeric states. Then, the modeled structure was energy-
minimized using AMBER 7 F99 force field with distance-
dependent dielectric function and current charges. The
Powell method was used for energy minimization. All the co-
crystallized ligands and waters were removed, and the ran-
dom hydrogen atoms were added. The 3D structures of all
compounds were constructed by using the sketch molecu-
C
47H60N3O6 762.4482, found 762.4484.
Biological studies
Assay of glycogen phosphorylase inhibitory
activity
The inhibitory activity of the synthesized compounds against
rabbit muscle glycogen phosphorylase a (RMGPa) was
monitored using microplate reader (BIO-RAD, Hercules, CA,
USA) based on the published method (11). In brief, RMGPa
activity was measured in the direction of glycogen synthesis
by the release of phosphate from glucose-1-phosphate.
Each test compound was dissolved in DMSO and diluted at
different concentrations for IC50 determination. The enzyme
was added into 100 lL of buffer containing 50 mM Hepes
(pH = 7.2), 100 mM KCl, 2.5 mM MgCl2, 0.5 mM glucose-
1-phosphate, 1 mg/mL glycogen, and the test compound
in 96-well microplates (Costar, Cambridge, MA, USA). After
the addition of 150 lL of 1 M HCl containing 10 mg/mL
ammonium molybdate and 0.38 mg/mL malachite green,
reactions were run at 22 °C for 25 min, and then, the phos-
phate absorbance was measured at 655 nm. The IC50 val-
ues were estimated by fitting the inhibition data to a dose-
dependent curve using a logistic derivative equation.
€
lar modules. Hydrogen and Gasteiger–Huckel charges were
assigned to each compound. Then, their geometries were
optimized by the conjugate gradient method in TRIPOS
force field. The energy convergence criterion is 0.001 kcal/
mol. Default values were chosen to finish this work when the
protomol was generated. The analysis of intermolecular
interactions was performed using Accelrys Discovery Studio
2.5 (Accelrys Software Inc., San Diego, CA, USA).
Results and Discussion
Chemistry
Preparation of the target compounds is shown in
Scheme 1. According to the procedures reported previ-
ously (9), esterification of acids 2a–2g with propargyl bro-
mide afforded alkyne-functionalized intermediates 3a–3g
for further click coupling. Reaction of OA with BnCl in the
presence of K2CO3 in DMF gave benzyl ester 4 in high
yield. Then, 4 reacted with 2-chloroethyl chloroformate to
afford the chloroacetyl ester 5. The ester 5 was treated
with NaN3 in the presence of K2CO3 in anhydrous DMF at
75 °C for 2 h to give azide 6. Click chemistry cycloaddition
between the azide 6 and corresponding alkyne intermedi-
ates 3a–3g was carried out at room temperature in aque-
ous condition in the presence of catalytic sodium
Measurement of the glucose consumption in
HepG2 cells
HepG2 cells (ATCC, Manassas, VA, USA) were cultured in
Gibco RPMI-1640 media (Invitrogen, Carlsbad, CA, USA)
supplemented with 10% fetal bovine serum (Invitrogen),
and maintained at 37 °C with 5% CO2. The glucose con-
centration of RPMI-1640 is 11.11 mM. For screening, cells
were plated into 96-well plates (Corning Incorporated, NY,
USA) at a density of 1 9 105 cells/well in serum-contain-
ing medium. After 20 h, cells (80–90% confluence) were
washed once and incubated in serum-free RPMI-1640
containing DMSO (0.1%), insulin (0.1 lM), and different
doses of target compounds (1 and 10 lM). Changes in the
glucose concentrations in aliquots from the culture media
after HepG2 incubation for 24 h were determined by
glucose oxidase–peroxidase method with a commercial
diagnostic kit (Jiancheng Bioengineering Institute, Nanjing,
China) (12). Results reported in all analyses were derived
from at least three independent experiments. All values
were presented as mean Æ SD. Data are presented as
mean values, and error bars indicate SD in chart. Stu-
dent’s t-test was used to determine the difference
between control and treatment groups. One-way ANOVA
.
ascorbate and CuSO4 5H2O to produce triazoles 7a–7g in
83–91% yields. Hydrogenolysis of the triazoles 7a–7g over
Pd-C (10%) in THF-MeOH at room temperature gave
triterpene acids 8a–8g. The synthesis of proposed
structures was found in conformity with spectral analysis.
Biological studies
Glycogen phosphorylase inhibitory activity
The novel compounds described above were evaluated in
an enzyme inhibition assay against rabbit muscle glycogen
Chem Biol Drug Des 2014; 83: 297–305
301

Zhang et al.
O
O
a
R
OH
R
O
2a-2g
3a-3g
H
H
H
COOH
COOBn
COOBn
b
c
O
H
H
H
Cl
HO
HO
O
H
H
H
4
5
1
H
H
COOBn
COOBn
d
e
O
R
N
O
O
N
N
O
H
H
N3
O
O
H
H
6
7a-7g
Scheme 1: The synthesis of OA
derivatives (8a–8g). (a) Propargyl
bromide, K2CO3, DMF, r.t.
H
COOH
(b) K2CO3, BnCl, DMF, 100 ꢀC.
(c) ClCH2COCl, K2CO3, DMAP,
CH2Cl2, 0 ꢀC to r.t. (d) NaN3, DMF,
75 ꢀC. (e) 3a-3g, CuSO4Á5H2O,
sodium ascorbate, CH2Cl2-H2O, r.t.
(f) H2, Pd/C (10%), THF, r.t.
O
f
N
O
N
N
O
H
H
a
b
R = C8H17
R = C9H19
e
R = C17H35
R
O
f
R =
O
c
d
R = C11H21
R = C15H31
8a-8g
g
R =
phosphorylase a (RMGPa), an enzyme that shares consid-
erable sequence similarity with human liver GPa. As
described previously, the activity of RMGPa was measured
through detecting the release of phosphate from glucose-
1-phosphate in the direction of glycogen synthesis (11).
Caffeine, a known allosteric GP inhibitor that shares the
same binding site with OA, was used as a positive control
(13).
Among all these compounds, 8g is the most potent GP
inhibitor with an IC50 value of 5.4 lM, stronger than the
parent OA (IC50 = 14 lM).
Glucose consumption in HepG2 cells
HepG2 cell line is a common vitro model to study anti-
hyperglycemic agents. It maintains the morphology and
function of live cell and is valuable for investigating the
liver glucose metabolism (14). To evaluate the effects of
synthesized compounds on hepatic glucose consumption,
we treated HepG2 cells with different doses of syn-
The assay results showed that most of the synthesized
compounds exhibited moderate-to-good inhibitory activi-
ties against RMGPa (Table 1). The SAR analysis showed
that the introduction of an aromatic group led to an
increased inhibitory activity. Among the compounds with
fatty moieties (7a–7e, 8a–8e), optimal GPa inhibition
activity was observed with the derivative 7c composed of
12-carbon fatty acid. Predictive SAR was difficult to
discern in this case, such as the good potency observed
with 7c, but the lower potency observed with closely
related compounds (e.g., 7a, 7b, 7d, 7e, 8a, 8b, 8d, and
8e). Among the compounds with aromatic moieties (9),
introduction of benzene or substituted benzene ring (e.g.,
7f, and 8f) resulted in a great loss of activity, while the
introduction of naphthalene moiety (8g) enhanced the GP
inhibitory activity. However, a clear preference could not
be established due to the insufficient number of com-
pounds. In this study, SAR analysis of 28-carboxyl group
showed that the 28-O-benzyl esters are less potent than
the corresponding carboxylic acids (e.g., 7a versus 8a; 7b
versus 8b; 7d versus 8d; 7e versus 8e; 7f versus 8f; 7g
versus 8g) with the exception of derivative 7c.
Table 1: IC50 values (lM) for RMGPa inhibition assay results
a
Compound RMGPa IC50 (lM) Compound RMGPa IC50 (lM)
1(OA)
4
14
7g
8a
23.9
77.1
25.6
8.4
221.3
11.2
34.6
5.4
461.0
12.6
12.7
80.1
143.9
5
8b
8c
6
7a
7b
7c
7d
7e
7f
8d
8e
7.76
8f
8g
Caffeinec
NIb
NI
144
38.9
aEach value represents the mean of three determinations.
bNI means no inhibition.
cCaffeine was used as positive control.
The bold values indicates the best inhibition value with an IC50
value of 5.4 μM.
302
Chem Biol Drug Des 2014; 83: 297–305

Oleanolic Acid Derivatives as Antidiabetic Agents
Table 2: Effects of OA, OA derivatives on glucose consumption
in HepG2 cells
untreated cells. As shown in Figure 2, compounds 8a, 8c,
8d, 8e, 8g (each at 1 and 10 lM) and positive reference
insulin (at 0.1 lM) induced a significant increase in the
consumption of extracellular glucose compared to the
blank vehicle 0.1% DMSO (p < 0.01). However, the former
analysis on glycogen phosphorylase shows that com-
pound 8g displayed the best inhibition against RMGPa.
Taken together, compound 8g exerted both good GP
inhibitory activity (IC50 = 5.4 lM) and good activity of
glucose consumption enhancement. These results should
be helpful to the further modification of OA and thus
benefit the potential use of OA in the treatment for hyper-
glycemia. To further identify the proteins responsible for
the glucose consumption activity of these compounds, we
have carried out a search. The findings will be reported in
due course.
Compounds
1 lM (%)
10 lM (%)
Negative control, NC, 0.1%
DMSO
1.68 Æ 0.70
OA
8a
9.96 Æ 1.31** 17.02 Æ 1.56***
27.11 Æ 4.34*** 35.23 Æ 2.94***
13.88 Æ 2.89** 19.77 Æ 1.15***
16.05 Æ 4.21*** 25.12 Æ 2.24***
16.27 Æ 4.74*** 24.28 Æ 1.54***
20.23 Æ 5.66*** 31.74 Æ 3.23***
9.37 Æ 2.73** 17.91 Æ 0.71***
23.96 Æ 5.20*** 33.21 Æ 1.95***
9.45 Æ 1.72**
8b
8c
8d
8e
8f
8g
Positive control, PC,
0.1 lM Insulin
Values are mean Æ SD; n = 6 in each condition. Incubation time:
24 h. The glucose concentration of medium: 11.11 mM.
**p < 0.01 for comparisons against the negative control group
(0.1% DMSO).
Docking study
***p < 0.001 for comparisons against the negative control group
(0.1% DMSO).
The bold values indicates the best inhibition value with an IC50
value of 5.4 μM.
To explore the binding mode of the synthesized com-
pounds with GP, the most potent compound 8g was
selected for subsequent molecular docking simulation.
The crystal structure of GP complexed with maslinic acid
(PDB code: 2QN2) was used in the molecular docking
simulation. Compound 8g was predicted to bind at the
allosteric site exclusively as shown in Figure 3. This com-
plex was structurally refined by energy minimization in
Sybyl X 2.0. This model showed that the OA moiety of
compound 8g occupied the location same as malinic acid
at the allosteric site in the crystal structure. The triazole
linker and naphthalene ring of compound 8g extended out
into a deep hydrophobic pocket. This might explain why
compound 8g (IC50 = 5.4 lM) exhibited more potency of
GP inhibition than compound 8f (IC50 = 34.6 lM). The
hydrogen bonding interactions formed between the com-
pound 8g and the protein are illustrated in Figure 3A.
Specifically, the hydroxyl oxygen at C-3 position formed
hydrogen bonding interactions with Arg193, while the
carboxylate oxygen exploited the allosteric effector
thesized compounds and measured the glucose con-
sumption by glucose oxidase method. The assay results
(Table 2) show that most of the newly synthesized
compounds exhibited strong activities in the stimulation of
glucose consumption in vitro. The glucose consumption
enhancement was notably in a dose-dependent manner.
Moreover, SAR analysis suggests that the introduction of
bulky groups at C-3 position increased the glucose
consumption activity except for compound 8f (e.g., 8a,
8b, 8c, 8d, 8e, 8g). Furthermore, it is implied that the
substituents surrounding C-3 might prefer to fatty moieties
as evidenced by that good potency was observed with
compounds 8a–8e. Within these compounds, compound
8a exhibited the best activity,
a 20.9-fold increase
observed at the concentration of 10 lM compared with
Figure 2: Effects of OA, OA derivatives (8a–8g, 1 or 10 lM), and insulin (PC, positive control, 0.1 lM) on glucose consumption of HepG2 cells.
Mean Æ SD; n = 6; **p < 0.01 and ***p < 0.001, for comparisons against the negative control group (0.1% DMSO).
Chem Biol Drug Des 2014; 83: 297–305
303

Zhang et al.
A
B
Figure 3: Binding details of glycogen phosphorylase complexed (A) with compound 8g and (B) with reference compound malinic acid.
Residues at the allosteric site are displayed in line style and colored differently. Compounds are represented with ball and stick
representation. Hydrogen bonding interactions are shown as red dashed lines. This figure was generated by DS Visualizer 2.5.
phosphate-recognition subsite by forming hydrogen bond-
ing interactions with Arg310. There were additional hydro-
gen bond formations between the naphthalene ester
carboxylate oxygens and Arg60. These two hydrogen
bonds may account for the significant increase in GP
inhibitory activity of 8g compared with reference com-
pound malinic acid (IC50 = 28 lM) (8).
Youth Scholars of Education Department of Hebei Province,
China (No. Y2012026).
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