In vitro and in vivo evaluation of the antidiabetic activity of ursolic acid derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.04.073

In this study, a series of ursolic acid derivatives were synthesized, and their structures were confirmed. The activity of the synthesized compounds against α-glucosidase was determined in vitro. The results suggested that all compounds have significant inhibitory activity, especially compounds 3-5 and 8, the IC₅₀ values of which were 2.66 ± 0.84, 1.01 ± 0.44, 3.26 ± 0.22, and 3.24 ± 0.21 μM. These compounds were more potent than acarbose (positive control) against α-glucosidase. Kinetic studies were performed to determine the mechanism of inhibition by compounds 3-5 and 8. The kinetic inhibition studies indicated that compound 3 was a non-competitive inhibitor, and the inhibition constant K_i was calculated to be 2.67 ± 0.19 μM. Moreover, the kinetic inhibition studies of compounds 4, 5 and 8 demonstrated that they were mixed-type inhibitors. Furthermore, the actual pharmacological potentials of synthesized compounds 3 and 4 were demonstrated by the reduction of postprandial blood glucose levels in normal Kunming mice. The hypoglycemic effects of these compounds were more evident 30 and 60 min after maltose ingestion (P < 0.05), which was similar to the effect displayed by the positive control, acarbose.

Full text of DOI:10.1016/j.ejmech.2014.04.073

Accepted Manuscript
In vitro and in vivo evaluation of the antidiabetic activity of ursolic acid derivatives
Pan-Pan Wu, Kun Zhang, Yu-Jing Lu, Ping He, Su-Qing Zhao
PII:
S0223-5234(14)00402-4
10.1016/j.ejmech.2014.04.073
EJMECH 6944
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 September 2013
Revised Date: 5 March 2014
Accepted Date: 25 April 2014
Please cite this article as: P.-P. Wu, K. Zhang, Y.-J. Lu, P. He, S.-Q. Zhao, In vitro and in vivo evaluation
of the antidiabetic activity of ursolic acid derivatives, European Journal of Medicinal Chemistry (2014),
doi: 10.1016/j.ejmech.2014.04.073.
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*Highlights (for review)
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Highlights
● A series of ursolic acid derivatives were synthesized and spectrally
characterized.
● Compounds 3-5 and 8 are more potent in vitro than acarbose and UA.
● The kinetic study of compounds 3-5 and 8 was carried out.
● Both compounds 3 and 4 have hypoglycemic effect in vivo.

*Revised Manuscript
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1 In vitro and in vivo evaluation of the antidiabetic activity of
2 ursolic acid derivatives
3
4
Pan-Pan Wu, Kun Zhang*, Yu-Jing Lu, Ping He, Su-Qing Zhao*
Department of Pharmaceutical Engineering, Faculty of Chemical Engineering and
Light Industry, Guangdong University of Technology Guangzhou, 510006 P.R. China
*Corresponding Authors:
5
6
7
Prof. Kun Zhang, Prof. Su-Qing Zhao
8
Tel.: +86 15820258676 Fax: +86 020-61017546.
9
E-mail address: sqzhao@gdut.edu.cn (S.-Q. Zhao), kzhang@gdut.edu.cn (K. Zhang).
Email addresses for other authors:
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12
E-mail address: wyuwpp@126.com (P.-P. Wu), luyj@gdut.edu.cn (Y.-J. Lu),
hepingjyjy@sina.com (P. He).
13 Authors’ contributions
14 Pan-Pan Wu performed the experimental work and drafted the manuscript.
1
15 Kun Zhang was the project coordinator. Yu-Jing Lu performed the H
13
16 NMR and C NMR analysis. Ping He participated in the synthesis and
17 testing work. Su-Qing Zhao was the thesis director and designer. All
18 authors read and approved the final manuscript.
19 Abstract
20 In this study, a series of ursolic acid derivatives were synthesized, and
21 their structures were confirmed. The activity of the synthesized
22 compounds against α-glucosidase was determined in vitro. The results
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23 suggested that all compounds have significant inhibitory activity,
24 especially compounds 3-5 and 8, the IC50 values of which were 2.66 ±
25 0.84, 1.01 ± 0.44, 3.26 ± 0.22, and 3.24 ± 0.21 μM. These compounds
26 were more potent than acarbose (positive control) against α-glucosidase.
27 Kinetic studies were performed to determine the mechanism of inhibition
28 by compounds 3-5 and 8. The kinetic inhibition studies indicated that
29 compound 3 was a non-competitive inhibitor, and the inhibition constant
30 Ki was calculated to be 2.67 ± 0.19 μM. Moreover, the kinetic inhibition
31 studies of compounds 4, 5 and 8 demonstrated that they were mixed-type
32 inhibitors. Furthermore, the actual pharmacological potentials of
33 synthesized compounds 3 and 4 were demonstrated by the reduction of
34 postprandial blood glucose levels in normal Kunming mice. The
35 hypoglycemic effects of these compounds were more evident 30 and 60
36 min after maltose ingestion (P<0.05), which was similar to the effect
37 displayed by the positive control, acarbose.
38 Keywords: Ursolic acid derivatives; Synthesis; α-glucosidase inhibition;
39 Anti-diabetic
40 1. Introduction
41
Diabetes mellitus is a chronic disease that occurs when the pancreas
42 does not produce sufficient insulin or when the body cannot use it
43 effectively, thereby leading to an increased blood glucose concentration
44 [1]. Over the last century, the ever-changing lifestyle of the population
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45 has resulted in a dramatic increase in the incidence of diabetes. The chief
46 form of the disease, type 2 diabetes, is associated with „diabesity‟ and
47 „metabolic syndrome‟. In conjunction with genetic susceptibility in
48 certain ethnic groups, type 2 diabetes is influenced by environmental and
49 other factors such as a sedentary lifestyle, overly rich nutrition and
50 obesity [2]. Treatment of diabetes involves lowering blood glucose
51 through different mechanisms, including insulin secretion, glucose
52 absorption, and metabolism adjustment [3,4].
53
Pentacyclic triterpenes and their derivatives are ubiquitous in the
54 plant kingdom and possess interesting bioactivities, such as antitumor
55 [5,6,7,8], anti-HIV [9], antibacterial [10], antimalarial [11], protein
56 tyrosine phosphatase 1B inhibition [4,12], etc. Ursolic acid (UA,
57 3β-hydroxy-urs-12-en-28-oic acid, 1) is a well-known pentacyclic
58 triterpene that has been reported to possess a wide range of biological
59 activities. It serves as one of the major effective components of many
60 traditional Chinese herbal medicines [13]. Studies have shown that UA
61 has a positive effect on lowing blood glucose levels and curing diabetic
62 complications in diabetic mice [14,15,16,17]. However, UA has a very
63 low water solubility, which limits its bioavailability and therapeutic
64 applications in clinical medicine [18]. To improve its activity and
65 bioavailability, chemical modification of UA at the 3-OH or 17-COOH
66 positions has recently been widely investigated. It has been reported that
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67 UA and its derivatives esterified at the C-3 and/or C-28 positions exhibit
68 significant cytotoxicity against several tumor cell lines [5,19,20].
69 Moreover, a series of UA derivatives were synthesized by condensation
70 of UA and 1,4-bis(3-aminopropyl) piperazine pharmacophore, resulting
71 in promising in vitro anti-malarial activity [12,21]. However, there are
72 few studies focused on the anti-diabetic properties of UA derivatives.
73
In an attempt to explore the activity and bioavailability of UA and its
74 derivatives, and study their structure-activity relationships and
75 mechanisms, structural modifications were made to the 3-OH or
76 17-COOH positions of UA. A series of UA derivatives, particularly new
77 halogen-containing derivatives were synthesized using previously
78 reported methods [5,19,20] with slight modifications and different
79 reactants. The hypoglycemic activity of UA and its derivatives was
80 investigated in vitro and in vivo. In the present study, the results of
81 experiments measuring the activity of UA and its derivatives toward
82 inhibiting alpha-glucosidase and lowering blood glucose in a normal mice
83 model were reported.
84 2. Results and discussion
85 2.1. Chemistry
86
To obtain a series of UA derivatives, structural modifications were
87 made at the 3-OH or 17-COOH positions, beginning with UA as the
88 parent compound. The synthetic routes are shown in Scheme 1 and
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89 Scheme 2.
Ursolic acid (1) was esterified in anhydrous pyridine with acetic
90
91 anhydride to give its 3-O-acetate (2). Compounds 3-5 were synthesized
92 following the same procedure used to prepare the 3-O-acetate, but with
93 different anhydrides (Scheme 1). Compound 2 was treated with oxalyl
94 chloride to give the intermediate 28-acyl chloride [7,20], which is highly
95 reactive and was coupled with small molecules containing reactive amine
96 groups to produce compounds 6-12 (Scheme 2). The target compounds
97 were purified by column chromatography using petroleum ether/ethyl
98 acetate and/or chloroform/methanol as the eluent. Their structures were
99 confirmed by electrospray ionization mass spectrometry (ESI-MS),
100 high-resolution mass spectrometry (HRMS), 1H NMR and 13C NMR.
101
102
103
104
105
106
Scheme 1. Synthesis of compounds 2-5 from ursolic acid. Reagents and conditions: (a)
anhydride/Pyr/DMAP, r.t.
Scheme 2. Synthesis of compounds 6-12 from compound 2. Reagents and conditions: (a) (COCl)2,
CH2Cl2, r.t.; (b) CH2Cl2, Et3N, RNH2, r.t.
107 2.2. In vitro α-glucosidase inhibition assay
To investigate the inhibitory ability of each synthesized UA derivative,
108
109 an in vitro experiment using α-glucosidase from baker‟s yeast, which is
110 widely used in screens for compounds with anti-diabetic activity, was
111 conducted [22]. A stock solution of each derivative (dissolved in DMSO
112 at concentrations of 0.1 μM to 1 mM) was diluted with 0.1 M phosphate
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113 buffer solution (pH=6.8) containing an appropriate concentration of
114 enzyme solution (0.1 U/mL). After a 10 min pre-incubation at 37 ℃, the
115 reactions were initiated by adding the substrate (1 mM
116 p-nitrophenyl-α-D-glucopyranoside) and incubated at 37 ℃ for 30 min.
117 The reactions were then terminated by adding 1 M Na2CO3, and their
118 optical density values were measured using a Multimodel Plate Reader
119 (Infinite 200). Table 1 displays the preliminary results. The inhibited
120 enzyme activity of all compounds was firstly evaluated at two
121 concentrations (5 and 10 μM), which was a guidance for future selection
122 of inhibitor concentrations. Table 1 clearly shows that the inhibition
123 effects were more obvious at higher concentrations.
124
To determine the IC50 value of each derivative, the enzyme activity
125 was measured at a fixed substrate concentration, in which a series of
126 different inhibitor concentrations were tested. The IC50 value was
127 calculated based on the curve fit to the series of concentrations versus the
128 corresponding inhibition activities (data not shown). All tested
129 compounds had lower IC50 values than both positive control and UA
130 against α-glucosidase from baker‟s yeast except compound 11 which had
131 a low solubility and could not disperse well at higher concentrations.
132
133
134
135
Table 1
Inhibitory effects of compounds 1-12 on α-glucosidase from baker‟s yeast.
Each experiment was performed in quadruplicate. The data reported represent the means (n=4) ±
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136
137
138
139
140
SD.
a Percent inhibition of α-glucosidase treated with a 5 μM concentration of each compound.
b Percent inhibition of α-glucosidase treated with a 10 μM concentration of each compound.
c IC50 value representing the concentration that caused a 50% loss of activity.
d Acarbose, positive control.
141
To obtain insight into the mechanism by which the derivatives
142 inhibited α-glucosidase, kinetic studies was carried out. Lineweaver-Burk
143 plots of the initial velocity versus the enzyme concentrations in the
144 presence of different concentrations of the synthesized compounds gave a
145 series of straight lines. As illustrated in Figs. 1-4, compound 3 was
146 determined to be a non-competitive inhibitor because increasing substrate
147 concentrations resulted in a series of lines with a common intercept on the
148 -[I] axis, but different slopes (Fig. 1). The equilibrium constant for
149 inhibitor binding, Ki, was obtained from the -[I] value at the intersection
150 of the four straight lines. The family of lines for compounds 4 and 5
151 intersected in the second quadrant (Figs. 2-3), while the family of lines
152 for compound 8 intersected in the third quadrant (Fig. 4). The inhibition
153 constant Ki and Ki' values for compounds 3-5 and 8 were calculated using
154 the appropriate equations, and the results are presented in Table 2. It was
155 implied that compounds 4, 5 and 8 inhibit α-glucosidase in two different
156 ways: compounds 4, 5 and 8 could retard enzyme function by not only
157 directly binding to α-glucosidase (EI) but also interfering the formation of
158 α-glucosidase-PNPG (ES) intermediate through forming an
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159 α-glucosidase-PNPG-inhibitor (ESI) complex in a noncompetitive
160 manner [23]. According to the inhibition type of tested compounds, it
161 could not dismiss the possibility that an α-glucosidase inhibitor attaches
162 to a wide region including the binding site of α-glucosidase, or attaches to
163 a different region incurring structural modification [24].
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
Figure 1. Compound 3. Double reciprocal plot (1/V versus 1/[S]) (A), the compound concentrations were: 0 μM ■;
2.0 μM ●; 5.0 μM ▲; 9.0 μM ▼. All assays were performed in quadruplicate. The data reported in the figure
represent the means ± S.E.M.
Figure 2. Compound 4. Double reciprocal plot (1/V versus 1/[S]) (A), the compound concentrations were: 0 μM ■;
2.85 μM ●; 4.0 μM ▲; 6.0 μM ▼. All assays were performed in quadruplicate. The data reported in the figure
represent the means ± S.E.M.
Figure 3. Compound 5. Double reciprocal plot (1/V versus 1/[S]) (A), the compound concentrations were: 0 μM ■;
1.6 μM ●; 2.8 μM ▲; 4.5 μM ▼. All assays were performed in quadruplicate. The data reported in the figure
represent the means ± S.E.M.
Figure 4. Compound 8. Double reciprocal plot (1/V versus 1/[S]) (A), the compound concentrations were: 0 μM ■;
2.5 μM ●; 5.0 μM ▲; 8.0 μM ▼. All assays were performed in quadruplicate. The data reported in the figure
represent the means ± S.E.M.
Scheme 3. The linear mixed-type inhibition of compounds 3-5 and 8. When the constant Ki=Ki',
the model describes non-competitive inhibition.
Table 2
Inhibition constants (Ki and Ki') of compound 3-5 and 8 for α-glucosidase. The data shown
represent the mean Ki and Ki' values ± S.E.M.
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187 2.3 Hypoglycemic Effect in Normal Mice
To investigate the pharmacological potential of the most active
188
189 α-glucosidase inhibitors among the synthesized UA derivatives as lead
190 compounds for the development of new drugs to treat diabetes,
191 compounds 3 and 4 were synthesized on a larger scale to obtain enough to
192 perform assays of their hypoglycemic activity in normal mouse. With oral
193 administration at a 50 mg/kg dose, acarbose, UA, compounds 3 and 4
194 were able to reduce glucose uptake in normal mice fed with maltose. The
195 results were presented in Figure 5 and Table 3. It could be concluded that
196 the hypoglycemic effect was more distinct at 30 and 60 min (P<0.05)
197 after maltose ingestion (Figure 5). Compounds 3, 4, UA and acarbose had
198 hypoglycemic effects, and postprandial blood glucose levels were all
199 lower than those in control. The delta area under the curve of the
200 evaluated compounds in figure 5 which could reflect their hypoglycemic
201 effects was calculated in Table 3. It revealed that acarbose (positive drug)
202 had the best potential to postprandial blood glucose levels. Moreover,
203 compound 3 and 4 had similar effects according to the delta area under
204 the curve in Table 3.
205
206
207
208
Figure 5. Effect of compounds 3, 4 and acarbose after a single oral administration of 3 g/kg
maltose in normal mice with a 50 mg/kg dose of each compound. After 5 min, a 3 g/kg maltose
solution was administered to each mouse. The control group was administered the same volume of
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209
210
211
maltose solution without an inhibitor. Blood samples were collected and immediately subjected to
blood glucose level assays using a disposable glucose sensor at 0, 30, 60, 90 and 120 min. Data
were calculated as the means (mg/dL of plasma) ± SD. Statistical significances were evaluated
212
213
214
using Student's t-test (n=5, (*) P < 0.05).
Table 3
Delta area under the curve of evaluated compounds in figure 5.
215
216
217
a
Delta area under the curve △S = Scontrol (The area under the curve of control) - Ssample (The area
under the curve of the sample).
218 3. Conclusions
219
We have designed and synthesized a series of UA derivatives and
220 have demonstrated their potential activity for inhibiting α-glucosidase. All
221 tested compounds exhibited greater potency than acarbose in the
222 α-glucosidase inhibition assay, except compound 11, which is observed
223 because compound 11 had a low solubility under the assay conditions and
224 could not disperse well at higher concentrations. From the IC50 values of
225 compounds 2, 3, 4 and 5, it can be concluded that, the longer the ester
226 group that is conjugated to the 3-OH group, the more active the inhibitor.
227 These results also demonstrated that side chains on the ester group could
228 decrease activity. Compounds containing a free carboxyl in this position
229 might be the most active. The replacement of aniline in the p-position
230 possessed higher potency than in the o-position. Furthermore, as
231 elaborated in the section detailing the in vitro enzyme activity studies,
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232 compound 3 exhibited non-competitive inhibition mechanism; compound
233 4, 5 and 8 presented mixed-type inhibition mechanism. Moreover,
234 postprandial glucose levels could be reduced by compounds 3 and 4, with
235 especially distinct effects at 30 and 60 min after administration (P<0.05).
236 Compounds 3 and 4 might interfere with or delay the absorption of
237 dietary carbohydrates in small intestine, leading to a suppression of
238 plasma glucose increase after a meal. It is worth noting that the
239 modification of UA was performed at the 3-OH and/or 17-COOH
240 positions. This is the first report of UA derivatives with α-glucosidase
241 inhibition activity. The introduction of straight-chain acyl groups at the
242 3-OH position preserved the α-glucosidase activity at the low micromolar
243 level, and while the bioavailability is unknown, these compounds were
244 found to reduce blood glucose in vivo. Further research examining the
245 activities of these derivatives in Caco-2 cells for intestinal absorption
246 study and in the streptozocin-induced diabetic rat model are now
247 underway in our laboratory.
248 4. Experimental
249 4.1. General Remarks
250
Ursolic acid was purchased from Nanjing Zelang Medical Technology
251 Co., Ltd., with over 98% purity. Silica gel (100-200 or 200-300 mesh)
252 used in column chromatography was supplied by Tsingtao Marine
253 Chemistry Co., Ltd. Further purification was carried out by column
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254 chromatography using Sephadex columns (LH-20). Other reagents were
255 purchased from commercial suppliers in their chemically or analytically
256 pure forms.
257
1H NMR spectra were recorded on Bruker AVANCE 400 or Mercury
258 －Plus 300 NMR spectrometers using TMS as an internal standard in
259 DMSO-d6. Electrospray ionization (ESI) mass spectra were measured on
260 an LC-MS-2010A or Thermo Fisher LCQ Fleet and are reported as m/z.
261 High-resolution mass spectra of compounds 6-12 were measured on a
262 Bruker maXis impact. The enzyme inhibition activity was measured
263 using a Multimodel Plate Reader (Infinite 200).
264 4.2. Synthesis
265 4.2.1. General procedure for the preparation of compounds (2-5)
266
Ursolic acid (6 mmol) and 60 mg DMAP were added to 75 mL of
267 anhydrous pyridine in a 125 mL flask and stirred at room temperature
268 until they were completely dissolved. Next, the anhydride (24 mmol) was
269 slowly added into the mixture dropwise. After stirring overnight, the
270 reaction mixture was concentrated under reduced pressure, dispersed in
271 100 mL of distilled water and filtered. The filtration procedure was
272 repeated several times. Next, the mixture was treated with 2 M HCl and
273 filtered when the pH was 3-4. The solid crude product was washed until
274 neutral and the pH remained constant. The resulting mixture was
275 concentrated to dryness. The crude product was purified on a silica gel
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276 column with petroleum ether/ethyl acetate (v/v 10:1) as the eluent to
277 produce compounds 2-5.
278 4.2.1.1. (2) According to the general procedure, UA was treated with
279 acetic anhydride, and then purified on a silica gel column with petroleum
280 ether/ethyl acetate (v/v 10:1) as the eluent to obtain compound 2
281 (Rf=0.55).
1
282
Yield: 81%; white powder; H NMR (300 MHz, DMSO) δ 11.89 (s,
283 1H, -COOH), 5.12 (s, 1H), 4.40 (dd, J = 11.2, 4.5 Hz, 1H), 2.11 (d, J =
284 11.3 Hz, 1H), 2.00 (s, 3H, -OCOCH3), 1.97 – 1.71 (m, 4H), 1.68 – 1.38
285 (m, 10H), 1.30 (t, J = 14.2 Hz, 4H), 1.10 – 0.96 (m, 5H), 0.91 (s, 7H),
286 0.84 (d, J = 13.2 Hz, 10H), 0.76 (s, 3H); 13C NMR (100 MHz, DMSO) δ
287 178.3, 170.1 (-OCOCH3), 138.2, 124.4, 79.9, 54.5, 52.3, 46.8, 46.8, 41.6,
288 38.5, 38.4, 37.7, 37.2, 36.4, 36.3, 32.5, 30.2, 27.8, 27.5, 23.8, 23.2, 23.2,
289 22.8, 22.5 (-OCOCH3), 21.1, 21.0, 17.8, 17.0, 16.9, 16.7, 15.1; ESI-MS
290 m/z 497.77(M-H)-.
291 4.2.1.2. (3) According to the general procedure, UA was treated with
292 propionic anhydride, and then purified on a silica gel column with
293 petroleum ether/ethyl acetate (v/v 10:1) as the eluent to obtain compound
294 3 (Rf=0.56).
1
295
Yield: 73%; white powder; H NMR (400 MHz, DMSO) δ 11.96 (s,
296 1H, -COOH), 5.13 (s, 1H), 4.42 (dd, J = 11.3, 4.5 Hz, 1H), 2.39 – 2.21 (m,
297 2H, -OCOCH2CH3), 2.11 (d, J = 11.2 Hz, 1H), 2.00 – 1.74 (m, 4H), 1.66
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298 – 1.43 (m, 10H), 1.30 (dd, J = 22.7, 11.5 Hz, 4H), 1.04 (dd, J = 13.2, 5.6
299 Hz, 7H, -OCOCH2CH3), 0.91 (s, 7H), 0.88 – 0.79 (m, 11H), 0.75 (s, 3H);
300 13C NMR (100 MHz, DMSO) δ 178.3, 173.2 (-OCOCH2CH3), 138.2,
301 124.4, 79.7, 54.5, 52.3, 46.8, 46.8, 41.6, 38.5, 38.4, 37.7, 37.3, 36.4, 36.3,
302 32.5, 30.2, 27.8, 27.5, 27.2 (-OCOCH2CH3), 23.8, 23.2, 23.2, 22.8, 21.1,
303 21.1, 17.8, 17.0, 16.9, 16.7, 15.1, 9.2 (-OCOCH2CH3); ESI-MS m/z
304 511.80(M-H)-.
305 4.2.1.3. (4) According to the general procedure, UA was treated with
306 butyric anhydride, and then purified on a silica gel column with
307 petroleum ether/ethyl acetate (v/v 10:1) as the eluent to obtain compound
308 4 (Rf=0.55).
1
309
Yield: 63%; white powder; H NMR (400 MHz, DMSO) δ 11.96 (s,
310 1H, -COOH), 5.13 (s, 1H), 4.42 (dd, J = 11.3, 4.6 Hz, 1H), 2.34 – 2.19 (m,
311 2H, -OCOCH2CH2CH3), 2.11 (d, J = 11.2 Hz, 1H), 2.00 – 1.74 (m, 4H),
312 1.62 – 1.47 (m, 10H), 1.30 (dd, J = 22.8, 11.4 Hz, 4H), 1.08 – 0.97 (m,
313 5H), 0.97 – 0.85 (m, 12H, -OCOCH2CH2CH3), 0.82 (s, 10H), 0.75 (s, 3H);
314 13C NMR (100 MHz, DMSO) δ 178.2, 172.4 (-OCOCH2CH2CH3), 138.2,
315 124.4, 79.7, 54.5, 52.3, 46.8, 46.9, 41.6, 38.5, 38.4, 37.7, 37.3, 36.4, 36.3,
316 35.8 (-OCOCH2CH2CH3), 32.5, 30.2, 27.8, 27.5, 23.8, 23.2, 23.2, 22.8,
317 21.0, 21.0, 18.1 (-OCOCH2CH2CH3), 17.8, 17.0, 16.8, 16.7, 15.1, 13.4
318 (-OCOCH2CH2CH3); ESI-MS m/z 525.83(M-H)-.
319 4.2.1.4. (5) According to the general procedure, UA was treated with
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320 isobutyl anhydride, and then purified on a silica gel column with
321 petroleum ether/ethyl acetate (v/v 10:1) as the eluent to obtain compound
322 5 (Rf=0.57).
1
323
Yield: 61%; white powder; H NMR (400 MHz, DMSO) δ 11.96 (s,
324 1H, -COOH), 5.13 (s, 1H), 4.40 (dd, J = 11.4, 4.6 Hz, 1H), 2.11 (d, J =
325 11.2 Hz, 1H), 2.00 – 1.74 (m, 4H), 1.63 – 1.44 (m, 10H), 1.30 (dd, J =
326 23.0, 11.6 Hz, 4H), 1.09 (dd, J = 10.9, 7.0 Hz, 7H, -OCOCH(CH3)2), 1.06
13
327 – 0.97 (m, 5H), 0.91 (s, 7H), 0.82 (d, J = 8.0 Hz, 10H), 0.76 (s, 3H); C
328 NMR (100 MHz, DMSO) δ 178.2, 175.6, (-OCOCH(CH3)2), 138.2, 124.4,
329 79.5, 54.5, 52.3, 46.8, 46.8, 41.6, 38.5, 38.4, 37.6, 37.4, 36.4, 36.3, 33.6
330 (-OCOCH(CH3)2), 32.5, 30.2, 27.8, 27.5, 23.8, 23.2, 23.2, 22.8, 21.0,
331 21.0, 19.0 (-OCOCH(CH3)2), 18.7 (-OCOCH(CH3)2), 17.7, 17.0, 16.9,
332 16.7, 15.1; ESI-MS m/z 525.82(M-H)-.
333 4.2.2. General procedure for the preparation of compounds (6-12)
334
To a solution of compound 2 (0.5 g) in 50 mL of anhydrous CH2Cl2 in
335 a 100 mL round-bottom flask was added oxalyl chloride (0.6 mL). The
336 resulting solution was then stirred at room temperature for 36 h. The
337 reaction was then concentrated to dryness under reduced pressure to
338 obtain crude 3-O-acetylursolyl chloride. This intermediate was dissolved
339 in anhydrous CH2Cl2. The mixture was stirred in the presence of
340 triethylamine, and then treated with an equal amount of the desired amine.
341 The reaction was stirred at room temperature for 6 h. Next, the CH2Cl2
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342 was removed under vacuum to obtain the crude product, which was
343 purified on a silica gel column with petroleum ether and ethyl acetate to
344 yield a white powder. Further purification was performed via column
345 chromatography with
a
Sephadex column (LH-20) using
346 trichloromethane and methanol (v/v 1:1) as the eluent to obtain
347 compounds 6-12.
348 4.2.2.1. (6) According to the general procedure, compound 2 was treated
349 with oxalyl chloride, and then an equal amount of phenylamine was
350 added. The product was purified on a silica gel column with petroleum
351 ether/ethyl acetate (v/v 10:1) as the eluent to obtain compound 6
352 (Rf=0.67).
1
353
Yield: 76%; white powder; H NMR (400 MHz, DMSO) δ 8.86 (s,
354 1H), 7.53 (d, J = 7.7 Hz, 2H), 7.25 (t, J = 7.8 Hz, 2H), 7.00 (t, J = 7.2 Hz,
355 1H), 5.28 (s, 1H), 4.39 (d, J = 7.0 Hz, 1H), 2.37 (d, J = 10.9 Hz, 1H),
356 2.00 (s, 3H, -OCOCH3), 1.76 (dd, J = 39.0, 11.3 Hz, 5H), 1.65 – 1.37 (m,
357 9H), 1.37 – 1.16 (m, 4H), 1.12 – 0.91 (m, 9H), 0.91 – 0.69 (m, 13H), 0.64
358 (s, 3H); 13C NMR (100 MHz, DMSO) δ 175.1 (-CO-NH-), 170.1
359 (-OCOCH3), 139.3, 138.5, 128.3, 128.3, 124.5, 123.0, 120.4, 120.4, 79.9,
360 54.5, 51.6, 47.5, 46.8, 41.6, 38.8, 38.3, 37.7, 37.2, 36.4, 36.3, 32.4, 30.3,
361 27.8, 27.4, 23.4, 23.4, 23.2, 22.9, 21.1, 21.0, 17.7, 17.1, 16.6, 16.6, 16.6,
362 15.1; ESI-MS m/z 612.5(M+K)+; HRMS (ESI) calcd for C38H55NO3
363 [M+H]+=574.4255, found: 574.4253.
16

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364 4.2.2.2. (7) According to the general procedure, compound 2 was treated
365 with oxalyl chloride, and then an equal amount of p-fluoroaniline was
366 added. The product was purified on a silica gel column with petroleum
367 ether/ethyl acetate (v/v 10:1) as the eluent, followed by a Sephadex
368 column (LH-20) with trichloromethane and methanol (v/v 1:1) as the
369 eluent, to obtain compound 7 (Rf=0.43).
1
370
Yield: 51%; white powder; H NMR (300 MHz, DMSO) δ 8.90 (s,
371 1H), 7.57 – 7.47 (m, 2H), 7.08 (t, J = 8.7 Hz, 2H), 5.74 (s, 1H), 5.27 (s,
372 1H), 4.38 (dd, J = 10.7, 4.6 Hz, 1H), 2.36 (d, J = 10.9 Hz, 1H), 1.99 (s,
373 3H), 1.90 – 1.65 (m, 5H), 1.63 – 1.38 (m, 9H), 1.27 (dd, J = 31.3, 11.4 Hz,
374 3H), 1.08 (s, 3H), 0.98 (d, J = 18.6 Hz, 6H), 0.84 (dd, J = 21.7, 5.1 Hz,
375 13H), 0.64 (s, 3H); 13C NMR (100 MHz, DMSO+CDCl3) δ 176.3
376 (-CO-NH-), 172.9 (-OCOCH3), 139.6, 138.3, 127.7, 127.7, 127.1, 127.1,
377 126.2, 124.5, 79.6, 54.6, 52.0, 46.8, 46.6, 42.2, 41.5, 38.8, 38.3, 37.6,
378 37.1, 36.9, 36.3, 32.4, 30.4, 27.7, 27.2, 27.2, 23.5, 23.1, 23.1, 22.7, 20.9,
379 17.6, 16.9, 16.5, 16.4, 15.0; ESI-MS m/z 630.5(M+K)+; HRMS (ESI)
380 calcd for C38H54FNO3 [M+H]+=592.4160, found: 592.4158.
381 4.2.2.3. (8) According to the general procedure, compound 2 was treated
382 with oxalyl chloride, and then an equal amount of p-chloroaniline was
383 added. The product was purified on a silica gel column with petroleum
384 ether/ethyl acetate (v/v 10:1) as the eluent to obtain compound 8
385 (Rf=0.45).
17

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1
386
Yield: 46%; white powder; H NMR (400 MHz, DMSO) δ 9.01 (s,
387 1H), 7.59 (d, J = 8.9 Hz, 2H), 7.31 (d, J = 8.9 Hz, 2H), 5.27 (s, 1H), 4.39
388 (dd, J = 11.3, 4.7 Hz, 1H), 2.36 (d, J = 10.9 Hz, 1H), 1.99 (s, 3H), 1.94 –
389 1.66 (m, 5H), 1.64 – 1.36 (m, 10H), 1.36 – 1.16 (m, 3H), 1.07 (s, 3H),
13
390 1.05 – 0.92 (m, 6H), 0.89 – 0.76 (m, 13H), 0.61 (s, 3H). C NMR (100
391 MHz, DMSO) δ 175.3 (-CO-NH-), 170.0 (-OCOCH3), 138.4, 138.3,
392 128.2, 128.2, 126.6, 124.5, 121.7, 121.7, 79.8, 54.5, 51.6, 47.6, 46.7, 41.5,
393 38.7, 38.3, 37.7, 37.2, 36.3, 36.2, 32.3, 30.2, 27.7, 27.4, 23.4, 23.3, 23.2,
394 22.9, 21.0, 20.9, 17.7, 17.1, 16.6, 16.6, 16.5, 15.1; ESI-MS m/z
395 646.5(M+K)+; HRMS (ESI) calcd for C38H54ClNO3 [M+H]+=608.3865,
396 found: 608.3861.
397 4.2.2.4. (9) According to the general procedure, compound 2 was treated
398 with oxalyl chloride, and then an equal amount of p-bromoaniline was
399 added. The product was purified on a silica gel column with petroleum
400 ether/ethyl acetate (v/v 20:1) as the eluent, followed by a Sephadex
401 column (LH-20) with trichloromethane and methanol (v/v 1:1) as the
402 eluent to obtain compound 9 (Rf=0.47).
1
403
Yield: 76%; white powder; H NMR (300 MHz, DMSO) δ 8.99 (s,
404 1H), 7.52 (d, J = 8.6 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 5.26 (s, 1H), 4.38
405 (dd, J = 10.6, 4.6 Hz, 1H), 2.36 (d, J = 11.3 Hz, 1H), 1.99 (s, 3H), 1.76
406 (dd, J = 35.4, 12.2 Hz, 5H), 1.64 – 1.33 (m, 10H), 1.24 (dd, J = 20.6, 11.8
407 Hz, 3H), 1.12 – 0.91 (m, 9H), 0.86 (d, J = 9.9 Hz, 6H), 0.79 (d, J = 3.2
18

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13
408 Hz, 7H), 0.61 (s, 3H); C NMR (100 MHz, DMSO) δ 175.4 (-CO-NH-),
409 170.1 (-OCOCH3), 138.2, 126.3, 126.1, 125.9, 125.8, 125.7, 125.0, 124.1,
410 115.5, 115.3, 79.9, 54.5, 51.9, 47.7, 46.8, 41.7, 38.8, 38.3, 37.7, 37.2, 36.7,
411 36.4, 32.5, 30.3, 27.8, 27.4, 23.6, 23.2, 22.9, 21.0, 21.0, 17.7, 17.1, 16.6,
412 16.6, 15.1; ESI-MS m/z 692.5(M+K)+; HRMS (ESI) calcd for
413 C38H54BrNO3 [M+H]+=652.3360, found: 652.3356.
414 4.2.2.5. (10) According to the general procedure, compound 2 was treated
415 with oxalyl chloride, and then an equal amount of o-fluoroaniline was
416 added. The product was purified on a silica gel column with petroleum
417 ether/ethyl acetate (v/v 20:1) as the eluent, followed by a Sephadex
418 column (LH-20) with trichloromethane and methanol (v/v 1:1) as the
419 eluent to obtain compound 10 (Rf=0.47).
1
420
Yield: 90%; white powder; H NMR (400 MHz, DMSO) δ 8.51 (s,
421 1H), 7.84 (d, J = 7.2 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.26 (t, J = 7.4 Hz,
422 1H), 7.11 (t, J = 7.0 Hz, 1H), 5.33 (s, 1H), 4.38 (dd, J = 10.9, 5.0 Hz, 1H),
423 2.21 (d, J = 10.7 Hz, 1H), 1.98 (s, 3H), 1.92 – 1.72 (m, 5H), 1.66 – 1.40
424 (m, 9H), 1.40 – 1.19 (m, 4H), 1.09 (s, 3H), 1.03 (d, J = 13.4 Hz, 3H), 0.94
425 (d, J = 6.0 Hz, 3H), 0.87 (d, J = 8.5 Hz, 6H), 0.80 (t, J = 8.0 Hz, 7H),
13
426 0.66 (s, 3H); C NMR (100 MHz, DMSO+CDCl3) δ 175.3 (-CO-NH-),
427 169.8 (-OCOCH3), 137.8, 135.0, 129.0, 127.2, 125.6, 125.3, 125.2, 124.6,
428 124.3, 124.1, 79.8, 54.5, 52.3, 48.1, 46.7, 41.6, 38.2, 37.6, 37.1, 36.7,
429 36.3, 32.2, 30.3, 27.7, 27.2, 23.9, 23.2, 23.1, 22.7, 20.9, 20.8, 17.6, 16.9,
19

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430 16.5, 16.5, 15.0; ESI-MS; m/z 630.5(M+K)+; HRMS (ESI) calcd for
431 C38H54FNO3 [M+H]+=592.4160, found: 592.4160.
432 4.2.2.6. (11) According to the general procedure, compound 2 was treated
433 with oxalyl chloride, and then an equal amount of o-chloroaniline was
434 added. The product was purified on a silica gel column with petroleum
435 ether/ethyl acetate (v/v 20:1) as the eluent, followed by a Sephadex
436 column (LH-20) with trichloromethane and methanol (v/v 1:1) as the
437 eluent to obtain compound 11 (Rf=0.52).
1
438
Yield: 87%; white powder; H NMR (400 MHz, DMSO) δ 8.29 (s,
439 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.25 (t, J = 7.7 Hz,
440 1H), 6.98 (t, J = 7.7 Hz, 1H), 5.35 (s, 1H), 4.36 (dd, J = 9.6, 6.2 Hz, 1H),
441 2.15 (d, J = 10.6 Hz, 1H), 1.96 (s, 3H), 1.82 (dd, J = 40.4, 9.3 Hz, 4H),
442 1.66 – 1.38 (m, 10H), 1.38 – 1.18 (m, 4H), 1.11 – 0.96 (m, 6H), 0.93 (d, J
443 = 6.0 Hz, 3H), 0.89 – 0.82 (m, 6H), 0.78 (d, J = 8.2 Hz, 7H), 0.64 (s, 3H).
444 13C NMR (100 MHz, DMSO) δ 175.2 (-CO-NH-), 169.7 (-OCOCH3),
445 137.6, 136.0, 132.0, 127.6, 125.9, 125.2, 123.6, 115.1, 79.8, 54.5, 52.6,
446 48.2, 46.7, 41.6, 39.0, 38.3, 37.6, 37.1, 36.7, 36.2, 32.1, 30.3, 27.6, 27.2,
447 24.0, 23.2, 23.0, 22.7, 20.8, 20.8, 17.6, 16.8, 16.5, 16.5, 16.3, 15.0;
448 ESI-MS m/z 646.5(M+K)+; HRMS (ESI) calcd for C38H54ClNO3
449 [M+H]+=608.3865, found: 608.3865.
450 4.2.2.7. (12) According to the general procedure, compound 2 was treated
451 with oxalyl chloride, and then an equal amount of o-bromoaniline was
20

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452 added. The product was purified on a silica gel column with petroleum
453 ether/ethyl acetate (v/v 20:1) as the eluent, followed by a Sephadex
454 column (LH-20) with trichloromethane and methanol (v/v 1:1) as the
455 eluent to obtain compound 12 (Rf=0.55).
1
456
Yield: 80%; white powder; H NMR (400 MHz, DMSO) δ 9.00 (s,
457 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.54 – 7.50 (m, 1H), 7.47 – 7.43 (m, 1H),
458 7.42 (d, J = 2.0 Hz, 1H), 5.27 (s, 1H), 4.38 (dd, J = 11.4, 4.7 Hz, 1H),
459 2.36 (d, J = 10.8 Hz, 1H), 1.99 (s, 3H), 1.94 – 1.66 (m, 5H), 1.66 – 1.35
460 (m, 10H), 1.35 – 1.17 (m, 3H), 1.07 (s, 3H), 1.04 – 0.92 (m, 6H), 0.89 –
461 0.82 (m, 6H), 0.79 (d, J = 5.4 Hz, 7H), 0.61 (s, 3H); 13C NMR (100 MHz,
462 DMSO) δ 175.3 (-CO-NH-), 170.1 (-OCOCH3), 138.7, 138.4, 131.1,
463 131.1, 124.5, 122.1, 122.1, 114.6, 79.9, 54.5, 51.6, 47.6, 46.7, 41.5, 38.7,
464 38.3, 37.7, 37.2, 36.3, 36.2, 32.3, 30.2, 27.7, 27.3, 23.4, 23.3, 23.2, 22.9,
465 21.0, 20.9, 17.6, 17.1, 16.6, 16.6, 16.5, 15.1; ESI-MS m/z 692.5(M+K)+;
466 HRMS (ESI) calcd for C38H54BrNO3 [M+H]+=652.3360, found:
467 652.3359.
468 4.3. α-glucosidase inhibitory activity
469 4.3.1. Baker’s yeast α-glucosidase inhibitory activity
470
The α-glucosidase inhibition assay was performed according to the
471 method of Worawalai et al. with slight modification [25]. The
472 α-glucosidase enzyme (0.1 U/mL) and substrate (1 mM
473 p-nitrophenyl-α-D-glucopyranoside) were dissolved in 0.1 M phosphate
21

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474 buffer, pH 6.8. 10 μL of each synthesized compound (1 mg/mL in DMSO)
475 was pre-incubated with 8 μL of α-glucosidase at 37 ℃ for 10 min. A 100
476 μL aliquot of substrate solution was then added to the reaction mixture,
477 which was further incubated at 37 ℃ for 30 min. Then, the reaction was
478 terminated by adding 100 μL of 1 M Na2CO3. Enzymatic activity was
479 quantified by measuring the absorbance at 405 nm using a Multimodel
480 Plate Reader (Infinite 200). The percentage of inhibition was calculated
481 using [(A0-A1)/A0]×100%, where A0 was the absorbance without the
482 compound, and A1 was the absorbance with the compound. The IC50
483 value was determined from a plot of the percentage of inhibition versus
484 the sample concentration. Acarbose was used as standard control and the
485 experiment was performed in duplicate.
486 4.3.2. Measurement of kinetic constants [25]
487
For the kinetic analysis of α-glucosidase inhibition by the active
488 compounds, the enzyme and active compounds were incubated with
489 increasing concentrations of p-nitrophenyl-α-D-glucopyranoside (0-10
490 μM). The modes of inhibition for each compound were determined based
491 on Lineweaver-Burk plots.
492 4.4. Hypoglycemic effect in normal mice [24,26]
493
Kunming mice (18-20 g body weight, 6-8 weeks old) were
494 maintained under identical laboratory conditions. Animals were deprived
495 of food for 16 h and allocated into three groups of five mice each by
22

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496 plasma glucose level. Food was given 20 min after oral administration of
497 0.5% CMC (control) acarbose and the active compounds (50 mg/kg).
498 Blood samples were collected by tail bleeding at 0, 30, 60, 90, and 120
499 min, and plasma glucose was assayed using a disposable glucose sensor
500 (kit). Data are expressed as the means (mg/dL of plasma) ± standard error
501 of the means (n=5).
502 Acknowledgments
503 This research was supported by National Natural Science Foundation of
504 China (Grant No. 21172046). The authors are also grateful to the
505 combination research projects of Guangdong Province and Ministry of
506 Education for financial support (project No. 2011B090600033),
507 Guangdong Province Higher Education “Qianbaishi Engineering”, and
508 Guangzhou Science and Technology Plan (project No. 3013Y2-00081).
509 The author would like to thank Dr. Ying Zhang of the School in
510 Guangdong University of Technology for help with language editing.
511 References:
512
513
514
515
516
517
[1] WHO Fact sheet # 312 October 2013 [on line January 15th 2014], http://www.
who.int/mediacentre/factsheets/fs312/en/index.html.
[2] P. Zimmet, K. Alberti, J. Shaw, Global and societal implications of the diabetes
epidemic, Nature 414 (2001) 782-787.
[3] I. M. Siminialayi, P. C. Emem-Chioma, Type 2 diabetes mellitus: a review of
pharmacological treatment, Niger. J. Med. 15 (2006) 207-214.
23

ACCEPTED MANUSCRIPT
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
[4] J. J. Ramírez-Espinosa, M. Y. Rios, S. López-Martínez, F. López-Vallejo, J. L.
Medina-Franco, P. Paoli, G. Camici, G. Navarrete-Vázquez, R. Ortiz-Andrade, S.
Estrada-Soto, Antidiabetic activity of some pentacyclic acid triterpenoids, role of
PTP–1B: In vitro, in silico, and in vivo approaches, Eur. J. Med. Chem. 46 (2011)
2243-2251.
[5] C. M. Ma, S. Q. Cai, J. R. Cui, R. Q. Wang, P. F. Tu, M. Hattori, M. Daneshtalab,
The cytotoxic activity of ursolic acid derivatives, Eur. J. Med. Chem. 40 (2005)
582-589.
[6] K. Bai, F. Chen, Z. Yu, Y. Zheng, Y. Li,Y. Guo, Synthesis of
[3β-acetoxy-urs-12-en-28-oyl]-1-monoglyceride and investigation on its anti tumor
effects against BGC-823, Bioorg. Med. Chem. 19 (2011) 4043-4050.
[7] J. W. Shao, Y. C. Dai, J. P. Xue, J. C. Wang, F. P. Lin, Y. H. Guo, In vitro and in
vivo anticancer activity evaluation of ursolic acid derivatives, Eur. J. Med. Chem. 46
(2011) 2652-2661.
[8] G. Chadalapaka, I. Jutooru, A. McAlees, T. Stefanac, S. Safe, Structure-dependent
inhibition of bladder and pancreatic cancer cell growth by 2-substituted glycyrrhetinic
and ursolic acid derivatives, Bioorg. Med. Chem. Lett. 18 (2008) 2633-2639.
[9] Y. Zhu, J. Shen, H. Wang, L. M. Cosentino, K. Lee, Synthesis and anti-HIV
activity of oleanolic acid derivatives, Bioorg. Med. Chem. Lett. 11 (2001) 3115-3118.
[10] M. Miyazawa, Y. Okuno, K. Imanishi, Suppression of the SOS-Inducing Activity
of Mutagenic Heterocyclic Amine, Trp-P-1, by Triterpenoid from Uncaria sinensis in
the Salmonella typhimurium TA1535/pSK1002 Umu Test, J. Agr. Food Chem. 53
24

ACCEPTED MANUSCRIPT
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
(2005) 2312-2315.
[11] F. Traore-Keita, M. Gasquet, C. Di Giorgio, E. Ollivier, F. Delmas, A. Keita, O.
Doumbo, G. Balansard, P. Timon-David, Antimalarial activity of four plants used in
traditional medicine in Mali, Phytother. Res. 14 (2000) 45-47.
[12] W. Zhang, D. Hong, Y. Zhou, Y. Zhang, Q. Shen, J. Y. Li, L. H. Hu, J. Li,
Ursolic acid and its derivative inhibit protein tyrosine phosphatase 1B, enhancing
insulin receptor phosphorylation and stimulating glucose uptake, Biochim Biophys
Acta 1760 (2006) 1505-1512.
[13] J. Liu, Pharmacology of oleanolic acid and ursolic acid, J. Ethnopharmacol. 49
(1995) 57-68.
[14] S. L. Ullevig, Q. Zhao, D. Zamora, R. Asmis, Ursolic acid protects diabetic mice
against monocyte dysfunction and accelerated atherosclerosis, Atherosclerosis 219
(2011) 409-416.
[15] J. Lee, S. Yee, J. Kim, M. Choi, E. Kwon, K. Seo, M. Lee, Ursolic acid
ameliorates
thymic
atrophy
and
hyperglycemia
in
streptozotocin–nicotinamide-induced diabetic mice, Chem-Biol. Interact. 188 (2010)
635-642.
[16] S. Jang, S. Yee, J. Choi, M. Choi, G. Do, S. Jeon, J. Yeo, M. Kim, K. Seo, M.
Lee, Ursolic acid enhances the cellular immune system and pancreatic β-cell function
in streptozotocin-induced diabetic mice fed a high-fat diet, Int. Immunopharmacol. 9
(2009) 113-119.
[17] S. Jang, M. Kim, M. Choi, E. Kwon, M. Lee, Inhibitory effects of ursolic acid on
25

ACCEPTED MANUSCRIPT
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
hepatic polyol pathway and glucose production in streptozotocin-induced diabetic
mice, Metabolism Clinical and Experimental 59 (2010) 512-519.
[18] J. Liu, Oleanolic acid and ursolic acid: research perspectives, J. Ethnopharmacol.
100 (2005) 92-94.
[19] K. K. Bai, F. L. Chen, Z. Yu, Y. Q. Zheng, Y. N. Li, Y. H. Guo, Synthesis of [3
beta-acetoxy-urs-12-en-28-oyl]-1-monoglyceride and investigation on its anti tumor
effects against BGC-823, Bioorg. Med. Chem. 19 (2011) 4043-4050.
[20] Y. Q. Meng, D. Liu, L. L. Cai, H. Chen, B. Cao, Y. Z. Wang, The synthesis of
ursolic acid derivatives with cytotoxic activity and the investigation of their
preliminary mechanism of action, Bioorg. Med. Chem. 17 (2009) 848-854.
[21] S. C. B. Gnoatto, S. Susplugas, L. D. Vechia, T. B. Ferreira, A.
Dassonville-Klimpt, K. R. Zimmer, C. Demailly, S. D. Nascimento, J. Guillon, P.
Grellier, H. Verli, G. Gosmann, P. Sonnet, Pharmacomodulation on the
3-acetylursolic acid skeleton: Design, synthesis, and biological evaluation of novel
N-{3-[4-(3-aminopropyl)piperazinyl]propyl}-3-O-acetylursolamide derivatives as
antimalarial agents, Bioorg. Med. Chem. 16 (2008) 771-782.
[22] A. Schafer, P. Hogger, Oligomeric procyanidins of French maritime pine bark
extract (Pycnogenol (R)) effectively inhibit alpha-glucosidase, Diabetes Res. Clin. Pr.
77 (2007) 41-46.
[23] A. Wikul, T. Damsud, K. Kataoka, P. Phuwapraisirisan, (+)-Pinoresinol is a
putative hypoglycemic agent in defatted sesame (Sesamum indicum) seeds though
inhibiting α-glucosidase, Bioorg. Med. Chem. Lett. 22 (2012) 5215-5217.
26

ACCEPTED MANUSCRIPT
584
585
586
587
588
589
590
591
592
593
[24] Y. Kim, Y. Jeong, M. Wang, W. Lee, H. Rhee, Inhibitory effect of pine extract
on α-glucosidase activity and postprandial hyperglycemia, Nutrition 21 (2005)
756-761.
[25] W. Worawalai, S. Wacharasindhu, P. Phuwapraisirisan, Synthesis of new
N-substituted aminoquercitols from naturally available (+)-proto-quercitol and their
alpha-glucosidase inhibitory activity, Med. Chem. Comm. 3 (2012) 1466-1470.
[26] S. B. Ferreira, A. C. Sodero, M. F. Cardoso, E. S. Lima, C. R. Kaiser, F. P. Silva,
V. F. Ferreira, Synthesis, biological activity, and molecular modeling studies of
1H-1,2,3-triazole derivatives of carbohydrates as alpha-glucosidases inhibitors, J.
Med. Chem. 53 (2010) 2364-2375.
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
Figure 1. Compound 3. Double reciprocal plot (1/V versus 1/[S]) (A), the compound
concentrations were: 0 μM ■; 2.0 μM ●; 5.0 μM ▲; 9.0 μM ▼. All assays were performed in
quadruplicate. The data reported in the figure represent the means ± S.E.M.
Figure 2. Compound 4. Double reciprocal plot (1/V versus 1/[S]) (A), the compound
concentrations were: 0 μM ■; 2.85 μM ●; 4.0 μM ▲; 6.0 μM ▼. All assays were performed in
quadruplicate. The data reported in the figure represent the means ± S.E.M.
Figure 3. Compound 5. Double reciprocal plot (1/V versus 1/[S]) (A), the compound
concentrations were: 0 μM ■; 1.6 μM ●; 2.8 μM ▲; 4.5 μM ▼. All assays were performed in
quadruplicate. The data reported in the figure represent the means ± S.E.M.
Figure 4. Compound 8. Double reciprocal plot (1/V versus 1/[S]) (A), the compound
concentrations were: 0 μM ■; 2.5 μM ●; 5.0 μM ▲; 8.0 μM ▼. All assays were performed in
quadruplicate. The data reported in the figure represent the means ± S.E.M.
27