Design and preparation of derivatives of oleanolic and glycyrrhetinic acids with cytotoxic properties

Article abstract of DOI:10.2147/DDDT.S166051

Background: The structural modification of natural products with the aim to improve the anticancer activity is a popular current research direction. The pentacyclic triterpenoid compounds oleanolic acid (OA) and glycyrrhetinic acid (GA) are distributed widely in nature. Methods: In this study, various oleanolic acids and glycyrrhetinic acids were designed and synthesized by using the combination principle. The in vitro anticancer activities of new OA and GA derivatives were tested by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method with SGC-7901 (gastric cancer), MCF-7 (breast cancer), Eca-109 (esophageal cancer), HeLa (cervical cancer), Hep-G2 (hepatoma cancer) and HSF (normal human skin fibroblast) cells. Results and conclusion: The screening results showed that the compound 3m presented the highest inhibitory activities against SGC-7901, MCF-7 and Eca-109 cell lines with IC₅₀ values of 7.57±0.64 μM, 5.51±0.41 μM and 5.03±0.56 μM, respectively. In addition, this compound also showed effective inhibition of Hep-G2 cells with an IC₅₀ value of 4.11±0.73 μM. Moreover, compound 5b showed the strongest inhibitory activity against Hep-G2 cells with an IC₅₀ value of 3.74±0.18 μM and compound 3l showed strong selective inhibition of the HeLa cells with the lowest IC₅₀ value of 4.32±0.89 μM. A series of pharmacology experiments indicated that compound 5b could induce Hep-G2 cells autophagy and apoptosis. These compounds will expand the structural diversity of anti-cancer targets and confirm the prospects for further research.

Full text of DOI:10.2147/DDDT.S166051

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O r i g i n a l r e s e a r c h
Design and preparation of derivatives of oleanolic
and glycyrrhetinic acids with cytotoxic properties
This article was published in the following Dove Press journal:
Drug Design, Development andTherapy
rui Wang1,*
Background: The structural modification of natural products with the aim to improve the anti-
cancer activity is a popular current research direction. The pentacyclic triterpenoid compounds
oleanolic acid (OA) and glycyrrhetinic acid (GA) are distributed widely in nature.
Yang li2,*
Xu-Dong huai3
Qing-Xuan Zheng1
Wei Wang1
hui-Jing li4
Qi-Yong huai1
1Marine college, shandong University,
Weihai, china; 2Zhong Yuan academy
of Biological Medicine, liaocheng
People’s hospital/affiliated liaocheng
hospital,Taishan Medical University,
liaocheng, china; 3school of
chemistry and Molecular engineering,
Qingdao University of science and
Technology, Qingdao, china; 4school
of Marine science and Technology,
harbin institute of Technology at
Weihai,Weihai, china
Methods: In this study, various oleanolic acids and glycyrrhetinic acids were designed and syn-
thesized by using the combination principle. The in vitro anticancer activities of new OA and GA
derivatives were tested by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)
method with SGC-7901 (gastric cancer), MCF-7 (breast cancer), Eca-109 (esophageal cancer), HeLa
(cervical cancer), Hep-G2 (hepatoma cancer) and HSF (normal human skin fibroblast) cells.
Results and conclusion: The screening results showed that the compound 3m presented the
highest inhibitory activities against SGC-7901, MCF-7 and Eca-109 cell lines with IC50 values
of 7.57±0.64 μM, 5.51±0.41 μM and 5.03±0.56 μM, respectively. In addition, this compound
also showed effective inhibition of Hep-G2 cells with an IC50 value of 4.11±0.73 μM. Moreover,
compound 5b showed the strongest inhibitory activity against Hep-G2 cells with an IC50 value
of 3.74±0.18 μM and compound 3l showed strong selective inhibition of the HeLa cells with
the lowest IC50 value of 4.32±0.89 μM. A series of pharmacology experiments indicated that
compound 5b could induce Hep-G2 cells autophagy and apoptosis. These compounds will expand
the structural diversity of anti-cancer targets and confirm the prospects for further research.
Keywords: oleanolic acid, glycyrrhetinic acid, cytotoxic properties, synthesis, apoptosis
*These authors contributed equally to
this work
Introduction
Cancer is the world’s main public health problem. It is also a main cause of death in
the whole world. On the basis of GLOBOCAN, about 17.5 million cancer cases were
detected and 8.7 million deaths occurred in 2015 worldwide.1 In the USA, 1,688,780
new cancer cases and 600,920 cancer deaths were projected to occur in 2017.2 Although
these disturbing numbers indicate that we have not won the “war on cancer”, recently
developed anticancer drugs with higher inhibitory activity and strong selectivity by
structural modifications of natural products (such as etoposide, teniposide, vindesine
and vinorelbine, which were derived from vinca alkaloids and epipodophyllotoxin) have
raised hopes for cancer patients to survive.3 Today, we use a lot of drugs indirectly or
directly derived from natural products, and structural modification of natural products
with pharmacological activity to obtain anticancer drugs with high inhibitory activities
and selectivity has become a popular research direction.3–5 Fructus Ligustri Lucidi, a
natural product, is the fruit of Ligustrum lucidum Ait. As a traditional Chinese medicine,
Fructus Ligustri Lucidi was discovered and used for the very early treatment of various
diseases.6 Oleanolic acid (OA), a pentacyclic triterpenoid compound, has been isolated
from Fructus Ligustri Lucidi.7 Researchers showed a strong interest in the investigation
of OA because of its extensive biological activities, such as antivirus,8 antitumor,9,10
correspondence: hui-Jing li
school of Marine science and Technology,
harbin institute of Technology at Weihai,
Weihai 264209, china
Tel +86 1 866 031 8701
email lihuijing@iccas.ac.cn
Qi-Yong huai
Marine college, shandong University,
Weihai 264209, china
Tel +86 1 328 787 5796
email huaiqy01@163.com
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anti-inflammatory11 and other pharmacological activities. Materials and methods
Glycyrrhetinic acid (GA) is a natural product extracted from
Glycyrrhiza uralensis.12 GA and OA are similar in structure,
Materials
Most of the solvents and reagents used in the experiment
were bought from Xinyue Chemical and Glass Co. (Weihai,
China) without further purification. The OA and GA were
bought from Tianjin Heowns Biochemical Technology Co.,
Ltd (Tianjin, China) without further purification. All the cell
lines were bought from Shanghai Institute of Cellular Biology
of Chinese Academy of Sciences. 13C- and 1H-NMR spectra
were obtained with a Bruker-400 instrument (400 MHz) by
using CDCl3 as the solvent. High-resolution-electrospray
ionization-mass spectra (HR-ESI-MS) were recorded with
LTQ Orbitrap mass spectrometer. SGW X-4 micro-melting
point apparatus was used to measure melting points (mp).
Thin-layer chromatography (TLC) and column chromatog-
raphy were performed on silica gel plates and silica gel.
and both are pentacyclic triterpenoid compounds (Figure 1).
GA also has antitumor,13,14 anti-inflammatory,15,16 antiviral,17,18
antiallergic19 and antiulcer activities.20 Some studies have
found that OA and GA have a broad inhibitory effect on the
growth of many tumor cell lines in vitro by inducing tumor cell
autophagy and apoptosis, blocking the cell cycle and inhibit-
ing multidrug resistance of tumors.21–25 To further explore the
physiological activities of OA and GA, researchers performed
a large number of structural modifications and studies of bio-
logical activities on these two natural products.26–30
Through previous reports, we think that through structural
modification to improve the anticancer activity of OA and
GA is appropriate. Chu et al reported the first preparation
of ligustrazine-oleanolic acid and then the introduction of
amino acid fragments to obtain new compounds with high synthesis of compounds
inhibitory activities.31 Li et al reported that first the prepara- general experimental method for the synthesis
tion of GA esters and then the introduction of 3-(1H-benzo[d] of 2a,b
imidazol-2-yl)propanoic acid fragments can greatly improve 1a or 1b (1 mmol) and anhydrous potassium carbonate
the anticancer activity, as compared with GA itself.32 In this (500 mg, 3.62 mmol) in N,N-dimethylformamide (DMF;
research, we use the method of combination to prepare OA and 10 mL) were stirred at 25°C for 30 min. Then, 0.18 mL of
GA esters to increase their lipophilicity and introduce organic benzyl bromide (1.5 mmol) was added and the mixture was
acid groups to increase their polarity. We selected 10 kinds of stirred at 25°C for 8 h. When the reaction was over, first,
organic acids (nicotinic acid,33 indole-3-acetic acid, 3-indole- saturated sodium chloride (10 mL) was added. Then, extrac-
butyric acid, cinnamic acid,34 isonicotinic acid, salicylic acid,35 tion was carried out with ethyl acetate. The organic phase
acetyl 4-hydroxycinnamic acid,36 acetylsalicylic acid, acetyl was washed with water and dried over anhydrous magnesium
3-hydroxybenzoic acid and acetyl 4-hydroxybenzoic acid) sulfate, followed by suction filtration and concentration to
with certain biological activity into the 3-OH position of the obtain the target compound 2a or 2b.
OA ester and GA ester, and finally synthesized 20 new OA and
GA derivatives, of which 18 new compounds were the target general experimental method for the synthesis
products. We used five cancer cell lines (SGC-7901, MCF-7, of 3a–e, 3h–l, 5a,b, 6a,b and 7a,b
Eca-109, HeLa and Hep-G2) to determine the antitumor activ- One millimole of R1OH (or R2OH) and 1-ethyl-(3-(3-dim-
ity and human skin fibroblasts (HSF) as a control.
ethylamino) propyl)-carbodiimide hydrochloride (EDCI)
30
29
CO2H
30
29
20
17
19
20
17
21
22
19
18
21
12
26
18
12
26
13
14
O
25
13
22
28
25
11
11
CO2H
28
H
9
H
9
16
14
16
1
4
1
4
2
10
8
7
15
2
3
10
8
7
15
H
H
27
5
3
5
27
6
6
HO
HO
H
H
23
24
23
24
GA
OA
Figure 1 Oa and ga.
Abbreviations: ga, glycyrrhetinic acid; Oa, oleanolic acid.
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cytotoxic properties of Oa and ga derivatives
were dissolved in anhydrous dichloromethane (15 mL) were 1.62 (m, 2H, H-15 and H-15′), 1.58 (m, 1H, H-1), 1.49 (m,
stirred at 0°C for 1 h, at the same time, to a solution of 2a 3H, H-9, H-6′ and H-6), 1.43 (m, 1H, H-7′), 1.39 (m, 1H,
or 2b (1 mmol) in dry dichloromethane (15 mL) was stirred H-21), 1.29 (m, 1H, H-21′), 1.23 (m, 1H, H-19′), 1.19 (s, 3H,
at room temperatures for 1 h. After 1 h, the two solutions H-27), 1.10 (m, H-1′), 0.98 (s, 3H, H-23), 0.96 (s, 6H, H-30
were mixed and 0.2 mmol of N,N-dimethyl-4-aminopyridine and H-29), 0.93 (s, 3H, H-25), 0.92 (s, 3H, H-24), 0.87 (m,
(DMAP) was added. The mixture was stirred at room 1H, H-5), 0.67 (s, 3H, H-26); 13C-NMR (CDCl3) δ: 177.5,
temperature, and 1 day was needed for the reaction to be 173.6, 143.7, 136.5, 136.4, 128.5, 128.5, 128.0, 128.0, 128.0,
completed. We used TLC to monitor the reaction. Once 127.5, 122.5, 121.9, 121.5, 119.2, 118.9, 115.6, 111.2, 80.8,
the reaction was complete, the mixture was washed with 1 66.0, 55.3, 47.6, 46.8, 45.9, 41.7, 41.4, 39.4, 38.2, 37.8,
M HCl, and then the solvent was distilled off. Lastly, the 37.0, 34.5, 33.9, 33.2, 32.7, 32.4, 30.7, 28.2, 27.7, 27.0,
mixture was purified by column chromatography on silica 25.9, 25.7, 24.6, 23.7, 23.6, 23.4, 23.1, 18.3, 16.9, 16.9,
gel (ethyl acetate/petroleum=1/4) to get target compounds 15.4; HR-ESI-MS: m/z 754.4797 [M+Na]+ (calculated for
3a–e, 3h–l, 5a,b, 6a,b and 7a,b.
C49H65O4NNa, 754.4806).
Benzyl 3beta-(2-(1H-indol-3-yl)acetoxy)olean-12-en-
Benzyl 3beta-cinnamoyloxyolean-12-en-28-oate (3c)
White solid, yield 46%, mp 141.1°C–142.6°C. H-NMR
1
28-oate (3a)
1
White solid, yield 41%, mp 90.5°C–92.1°C. H-NMR (CDCl3) δ: 7.69 (d, 1H, J=15.9 Hz), 7.52 (m, 2H), 7.38 (s,
(CDCl3) δ: 8.12 (s, 1H, NH), 7.64 (d, J=7.6 Hz, 1H), 7.37 2H), 7.35 (s, 6H, Ar-H), 6.46 (d, 1H, J=16.0 Hz), 5.29 (s,
(s, 5H, Ar-H), 7.32 (d, J=8.0 Hz, 1H), 7.17 (dt, J=24, 7.2 Hz, 2H, Ph-CH2), 5.11 (dt, 1H, H-12, J=17.9, 10.9 Hz), 4.67 (t,
2H), 7.12 (s, 1H), 5.27 (s, 2H, Ph-CH2), 5.16 (dd, J=22, 12.8 1H, H-3, J=5.6 Hz), 2.95 (s, 1H, H-18), 2.20 (m, 1H, H-16),
Hz, 1H, H-12), 4.56 (t, J=8.8 Hz, 1H, H-3), 3.80 (s, 2H, 2.02 (m, 1H, H-16′), 1.89 (m, 1H, H-11), 1.77 (m, 1H, H-22),
CH2CO), 2.97 (dd, 1H, H-18, J=13.7, 4.3 Hz), 2.02 (1H, m, 1.71 (m, 1H, H-11′), 1.65 (m, 1H, H-1), 1.63 (m, 1H, H-19),
H-11), 1.88 (m, 2H, H-16 and H-16′), 1.76 (m, 1H, H-7), 1.61 (m, 1H, H-9), 1.59 (m, 1H, H-22), 1.54 (m, 1H, H-6),
1.64 (m, 1H, H-19), 1.62 (m, 3H, H-11′, H-15 and H-15′), 1.41 (m, 1H, H-6′), 1.38 (m, 1H, H-21), 1.35 (m, 1H, H-21′),
1.58 (m, 1H, H-1), 1.53–1.56 (m, 3H, H-9, H-6′ and H-6), 1.30 (m, 1H, H-21′), 1.22 (m, 1H, H-19′), 1.19 (s, 3H, H-27),
1.51 (m, 1H, H-7′), 1.38 (m, 1H, H-21), 1.27 (m, 1H, H-21′), 1.14 (m, 3H, H-23), 1.07 (m, 1H, H-1′), 0.95 (m, 6H, H-29
1.22 (m, 1H, H-19′), 1.18 (s, 3H, H-27), 1.08 (m, H-1′), 1.03 and H-30), 0.94 (m, 6H, H-25 and H-24), 0.78 (m, 1H, H-5),
(s, 3H, H-23), 0.97 (s, 3H, H-30), 0.95 (s, 3H, H-29), 0.92 0.67 (s, 3H), 0.63 (s, 3H, H-26); 13C-NMR (CDCl3) δ: 177.4,
(s, 3H, H-25), 0.82 (s, 3H, H-24), 0.75 (d, J=11.2 Hz, 1H, 166.8, 144.3, 143.7, 136.5, 134.6, 130.2, 128.9, 128.9, 128.4,
H-5), 0.66 (s, 3H, H-26); 13C-NMR (CDCl3) δ: 177.5, 172.1, 128.4, 128.1, 128.1, 128.0, 128.0, 127.9, 122.5, 118.9, 81.0,
143.7, 136.4, 136.2, 128.4, 128.4, 128.0, 128.0, 128.0, 127.3, 65.9, 55.4, 47.6, 46.8, 45.9, 41.7, 41.4, 39.3, 38.2, 38.0, 37.0,
123.2, 121.9, 119.3, 118.9, 111.3, 108.4, 78.9, 66.0, 53.5, 33.9, 33.2, 32.7, 32.4, 30.7, 28.2, 27.7, 25.9, 23.7, 23.5,
47.7, 47.5, 46.8, 45.9, 45.9, 41.7, 41.4, 39.3, 38.1, 37.8, 36.9, 23.1, 18.3, 16.9, 15.4; HR-ESI-MS: m/z 699.4384 [M+Na]+
33.9, 33.1, 32.8, 32.7, 32.4, 31.8, 30.7, 28.2, 28.0, 27.7, 27.2, (calculated for C46H60O4Na, 637.4384).
27.0, 26.0, 25.9, 23.7, 23.7, 23.5, 23.4, 23.1, 18.4, 16.9, 16.8,
15.7, 15.4; HR-ESI-MS: m/z 726.4479 [M+Na]+ (calculated
for C47H61O4Na, 726.4493).
Benzyl 3beta-salicyloyloxyolean-12-en-28-oate (3d)
White solid, yield 46%, mp 165.5°C–166.8°C. H-NMR
(CDCl3) δ: 10.96 (s, 1H, OH), 7.85 (d, 1H, J=8.0 Hz), 7.47
1
Benzyl 3beta-(4-(1H-indol-3-yl)butyroxy)olean-12-en-
(t, 1H, J=7.6 Hz), 7.37 (s, 5H, Ar-H), 7.00 (d, J=8.4 Hz, 1H),
6.90 (t, 1H, J=7.6 Hz), 5.44 (s, 2H, Ph-CH2), 5.32 (d, 1H,
28-oate (3b)
1
White solid, yield 53%, mp 84.5°C–86.1°C. H-NMR H-12, J=1.3 Hz), 4.79 (t, 1H, H-3, J=8.2 Hz), 2.94 (m, 1H,
(CDCl3) δ: 8.17 (s, 1H, NH), 7.66 (d, J=7.9 Hz, 1H), 7.40 H-18), 2.02 (m, 1H, H-11), 1.90 (m, 1H, H-16), 1.80 (m, 1H,
(s, 5H, Ar-H), 7.37 (m, 1H), 7.19 (dt, J=29.0, 7.4 Hz, 2H), H-16′), 1.72 (m, 1H, H-7), 1.67 (m, 1H, H-19), 1.58–1.62 (m,
7.00 (s, 1H), 5.31 (s, 2H, Ph-CH2), 5.18 (dd, J=21.2, 12.8 Hz, 3H, H-11′, H-15 and H-15′), 1.55 (m, 1H, H-1), 1.49–1.53
1H, H-12), 4.59 (t, J=7.8 Hz, 1H, H-3), 2.98 (dd, J=13.9, 4.3 (m, 3H, H-9, H-6 and H-6′), 1.45 (m, 1H, H-7′), 1.34 (m, 1H,
Hz, 1H, H-18), 2.86 (t, J=7.5 Hz, 2H), 2.44 (t, J=7.4 Hz, H-21), 1.20 (m, 1H, H-21′), 1.17 (s, 3H, H-27), 1.12 (m, 1H,
2H), 2.08 (2H, m, H-11 and H-11′), 1.90 (m, 2H, H-16 and H-19′), 1.07 (m, 1H, H-1′), 1.04 (s, 3H, H-23), 0.98 (s, 3H,
H-16′), 1.74 (m, 1H, H-7), 1.67 (m, 2H), 1.64 (m, 1H, H-19), H-30), 0.97 (s, 3H, H-29), 0.95 (s, 3H, H-25), 0.93 (s, 3H,
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H-24), 0.88 (m, 1H, H-5), 0.65 (s, 3H, H-26); 13C-NMR 37.7, 37.0, 32.7, 31.8, 31.8, 31.2, 28.4, 28.3, 28.0, 26.5, 26.4,
(CDCl3) δ: 177.4, 169.9, 161.7, 143.8, 136.5, 135.5, 129.8, 23.6, 23.3, 18.7, 17.3, 16.7, 16.4; HR-ESI-MS: m/z 740.4280
128.4, 128.4, 128.0, 128.0, 127.9, 122.4, 119.1, 117.6, 113.1, [M+Na]+ (calculated for C47H59O5NNa, 740.4285).
82.4, 65.9, 55.4, 53.4, 47.6, 46.8, 45.9, 41.7, 41.4, 39.3, 38.1,
38.1, 37.0, 33.9, 33.1, 32.6, 32.4, 30.7, 28.2, 27.6, 25.9, 23.7, Benzyl 3beta-(4-(1H-indol-3-yl)butyroxy)-11-oxo-olean-
23.6, 23.4, 23.1, 18.2, 17.0, 16.9, 15.4; HR-ESI-MS: m/z 12-en-30-oate (3i)
1
689.4434 [M+Na]+ (calculated for C44H58O5Na, 689.4176).
White solid, yield 58%, mp 207.1°C–209.2°C. H-NMR
(CDCl3) δ: 8.26 (s, 1H, NH), 7.64 (d, 1H, J=7.8 Hz), 7.39 (s,
6H, Ar-H), 7.38 (d, 1H, J=8.0 Hz), 7.21 (m, 1H), 7.16 (m, 1H),
6.99 (s, 1H), 5.60 (s, 1H, H-12), 5.30 (s, 2H, Ph-CH2), 4.58
Benzyl 3beta-(4-acetoxycinnamoyloxy)olean-12-en-
28-oate (3e)
1
White solid, yield 41%, mp 91.1°C–92.9°C. H-NMR (dd, 1H, H-3, J=11.9, 4.6 Hz), 2.84 (t, 2H), 2.42 (t, 2H), 2.36
(CDCl3) δ: 7.65 (d, 1H, J=15.9 Hz), 7.56 (d, 2H, J=8.3 Hz), (s, 1H, H-9), 2.09 (dd, 1H, H-18, J=13.5, 3.67 Hz), 2.02 (ddd,
7.36 (s, 5H, Ar-H), 7.13 (d, 2H, J=8.3 Hz), 6.41 (d, 1H, 1H, H-15, J=13.5, 13.5, 4.47 Hz), 1.98 (m, 1H, H-21), 1.92
J=16.0 Hz), 5.31 (s, 2H, Ph-CH2), 5.13 (m, 1H, H-12), 4.65 (ddd, 1H, H-19, J=13.6, 3.8, 2.97 Hz), 1.82 (ddd, 1H, J=13.7,
(t, 1H, H-3, J=8.4 Hz), 2.92 (m, 1H, H-18), 2.32 (s, 3H, 13.7, 4.77 Hz), 1.71 (m, 1H, H-2), 1.68 (m, 2H), 1.67 (m, 1H,
COCH3), 1.99 (m, 1H, H-16), 1.95 (m, 1H, H-16′), 1.91 (m, H-7), 1.64 (m, 2H), 1.60 (dd, 1H, J=13.6, 13.67 Hz), 1.60
1H, H-11), 1.73 (m, 1H, H-22), 1.71 (m, 1H, H-11′), 1.66 (m, 1H), 1.51 (m, 1H, H-6), 1.48 (m, 1H, H-6′), 1.46 (m, 1H,
(m, 1H, H-1), 1.62 (m, 1H, H-19), 1.56 (m, 1H, H-9), 1.53 H-7′), 1.43 (m, 1H, H-22), 1.38 (s, 3H, H-27), 1.34 (m, 1H,
(m, 1H, H-22), 1.50 (m, 1H, H-6), 1.39 (m, 1H, H-6′), 1.36 H-22′), 1.29 (m, 1H, H-21′), 1.24 (m, 1H, H-16′), 1.20 (s, 6H,
(m, 1H, H-21), 1.32 (m, 1H, H-21′), 1.15 (s, 3H, H-27), 1.08 H-25 and H-28), 1.14 (s, 3H, H-26), 1.09 (m, 1H), 1.01 (m,
(m, 1H, H-19′), 1.05 (m, 1H, H-1′), 0.95 (s, 9H, H-25, H-24 1H, H-15′), 0.92 (s, 6H, H-23 and H-24), 0.85 (m, 1H, H-5),
and H-23), 0.92 (s, 6H, H-30 and H-29), 0.76 (m, 1H, H-5), 0.77 (s, 3H, H-29); 13C-NMR (CDCl3) δ: 200.1, 176.3, 173.7,
0.64 (s, 3H, H-26); 13C-NMR (CDCl3) δ: 177.4, 169.1, 166.7, 169.3, 136.4, 136.1, 128.6, 128.6, 128.4, 128.3, 128.3, 128.3,
152.0, 143.7, 143.2, 136.4, 132.3, 129.2, 129.2, 128.4, 128.4, 121.8, 121.6, 119.1, 118.9, 115.4, 111.2, 80.4, 66.3, 61.7,
128.0, 128.0, 127.9, 122.4, 122.1, 119.0, 81.1, 65.9, 60.4, 55.0, 53.5, 48.3, 45.4, 44.0, 43.2, 41.1, 38.8, 38.1, 37.7, 37.0,
55.3, 53.5, 47.5, 46.7, 45.8, 41.7, 41.4, 39.3, 38.1, 37.9, 36.9, 34.5, 32.7, 31.8, 31.2, 28.5, 28.3, 28.2, 26.5, 26.4, 25.7, 24.6,
33.9, 33.1, 32.7, 32.4, 30.7, 28.1, 27.6, 25.9, 23.7, 23.4, 23.0, 23.7, 23.3, 18.7, 17.4, 16.9, 16.5; HR-ESI-MS: m/z 768.4590
21.1, 21.1, 18.2, 16.9, 16.9, 15.4, 14.2; HR-ESI-MS: m/z [M+Na]+ (calculated for C49H63O5NNa, 768.4598).
757.4439 [M+Na]+ (calculated for C48H62O6Na, 757.4439).
Benzyl 3beta-cinnamoyloxy-11-oxo-olean-12-en-
Benzyl 3beta-(2-(1H-indol-3-yl)acetoxy)-11-oxo-olean-
30-oate (3j)
1
12-en-30-oate (3h)
White solid, yield 48%, mp 229.3°C–231.6°C. H-NMR
1
White solid, yield 55%, mp 259.1°C–260.6°C. H-NMR (CDCl3) δ: 7.69 (d, 2H, J=16.1 Hz), 7.54 (dt, J=7.4, 4.6 Hz,
(CDCl3) δ: 8.38 (s, 1H, NH), 7.66 (d, J=8 Hz, 1H), 7.38 (s, 1H), 7.40 (m, 2H), 7.39 (s, 6H, Ar-H), 6.46 (d, J=16.0 Hz,
6H, Ar-H), 7.34 (m, 1H), 7.18 (m, 2H), 7.13 (m, 1H), 5.57 1H), 5.57 (s, 1H, H-12), 5.31 (s, 2H, Ph-CH2), 4.69 (dd, 1H,
(s, 1H, H-12), 5.29 (s, 2H, Ph-CH2), 4.59 (dt, J=11.4, 5.9 H-3, J=7.6, 4.8 Hz), 2.86 (dt, J=13.7, 3.6 Hz, 1H, H-1), 2.40
Hz, 1H, H-3), 3.80 (s, 2H, CH2CO), 2.82 (m, 1H, H-1), 2.36 (s, 1H, H-9), 2.08 (m, 1H, H-18), 2.01 (m, 1H, H-15), 1.97
(s, 1H, H-9), 2.08 (m, 1H, H-18), 2.01 (m, 1H, H-15), 1.98 (m, 1H, H-21), 1.91 (m, 1H, H-19), 1.82 (m, 1H, H-16), 1.75
(m, 1H, H-21), 1.92 (m, 1H, H-19), 1.80 (ddd, J=34.4, 18.1, (m, 1H, H-2), 1.70 (m, 1H, H-7), 1.67 (m, 1H, H-2′), 1.61
9.9 Hz, 1H, H-16), 1.69 (m, 1H, H-2), 1.65 (m, 1H, H-7), (m, 1H, H-19′), 1.57 (m, 1H, H-6), 1.49 (m, 1H, H-6′), 1.44
1.60 (dd, 1H, H-2′, J=13.6, 13.67 Hz), 1.56 (m, 1H, H-6), (m, 1H, H-7′), 1.41 (m, 1H, H-22), 1.38 (s, 3H, H-27), 1.36
1.49 (m, 1H, H-6′), 1.46 (m, 1H, H-7′), 1.43 (m, 1H, H-22), (m, 1H, H-22′), 1.30 (m, 1H, H-21′), 1.27 (m, 1H, H-16′),
1.36 (s, 3H, H-27), 1.31 (m, 1H, H-22′), 1.29 (m, 1H, H-21′), 1.22 (s, 3H, H-25), 1.19 (s, 3H, H-28), 1.14 (s, 3H, H-26),
1.23 (m, 1H, H-16′), 1.19 (s, 6H, H-25 and H-28), 1.12 (s, 1.09 (m, 1H, H-1′), 1.03 (m, 1H, H-15′), 0.99 (s, 3H, H-23),
3H, H-26), 1.04 (m, 1H, H-1′), 0.93 (m, 1H, H-15′), 0.85 (s, 0.95 (s, 3H, H-24), 0.88 (m, 1H, H-5), 0.76 (s, 3H, H-29);
3H), 0.81 (s, 3H), 0.76 (s, 3H); 13C-NMR (CDCl3) δ: 200.1, 13C-NMR (CDCl3) δ: 99.9, 176.1, 169.0, 166.7, 144.3, 136.2,
176.3, 172.0, 169.2, 136.2, 136.1, 128.6, 128.6, 128.4, 128.3, 134.6, 130.1, 128.8, 128.8, 128.6, 128.6, 128.5, 128.3, 128.2,
128.3, 128.3, 127.3, 123.1, 122.0, 119.5, 118.9, 111.2, 108.6, 128.2, 128.0, 128.0, 118.9, 80.7, 66.2, 61.7, 55.1, 53.4, 48.3,
81.0, 66.3, 61.7, 55.0, 48.3, 45.4, 44.0, 43.2, 41.1, 38.8, 38.1, 45.4, 43.9, 43.2, 41.1, 38.9, 38.3, 37.7, 37.0, 32.8, 31.8, 31.2,
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cytotoxic properties of Oa and ga derivatives
28.4, 28.3, 28.1, 26.9, 26.5, 26.4, 23.7, 23.3, 18.7, 17.4, 16.9, 43.2, 41.1, 38.8, 38.3, 37.7, 37.0, 32.7, 31.8, 31.2, 28.4,
16.4; HR-ESI-MS: m/z 691.4373 [M+H]+ (calculated for 28.3, 28.1, 26.5, 26.4, 23.7, 23.3, 21.2, 18.7, 17.4, 16.9,
C46H59O5, 691.4357).
16.5; HR-ESI-MS: m/z 749.4434 [M+H]+ (calculated for
C48H61O7, 749.4412).
Benzyl 3beta-salicyloyloxy-11-oxo-olean-12-en-30-
oate (3k)
Benzyl 3beta-acetyloxyolean-12-en-28-oate (5a)
1
1
White solid, yield 32%, mp 135.6°C–137.1°C. H-NMR White solid, yield 63%, mp 267.5°C–268.3°C. H-NMR
(CDCl3) δ: 10.94 (s, 1H, OH), 7.85 (d, 1H, J=8.0 Hz), 7.47 (CDCl3) δ: 7.35 (s, 5H, Ar-H), 5.31 (s, 2H, Ph-CH2), 5.10
(t, 1H, J=8.0 Hz), 7.39 (s, 5H, Ar-H), 6.99 (d, 1H, J=8.0 Hz), (m, 1H, H-12), 4.50 (t, 1H, H-3, J=7.9 Hz), 2.92 (dd, J=13.9,
6.90 (t, 1H, J=7.2 Hz), 5.54 (s, 1H, H-12), 5.32 (s, 2H, 4.3 Hz, 1H, H-18), 2.05 (s, 3H, OCH3), 1.96 (m, 1H, H-11),
Ph-CH2), 4.80 (d, 1H, H-3, J=11.6 Hz), 2.88 (dd, 1H, H-1, 1.86 (dd, 2H, H-16 and H-16′, J=8.9, 3.5 Hz), 1.70 (m, 1H,
J=13.6 Hz), 2.40 (s, 1H, H-9), 2.09 (m, 1H, H-18), 2.01 (m, H-7), 1.65 (m, 1H, H-19), 1.63 (m, 3H, H-11′, H-15), 1.58
1H, H-15), 1.98 (m, 1H, H-21), 1.92 (m, 1H, H-19), 1.82 (m, (m, 1H, H-1), 1.55–1.52 (m, 3H, H-9, H-6 and H-6′), 1.51
1H, H-16), 1.71 (m, 1H, H-2), 1.69 (m, 1H, H-7), 1.65 (m, (m, 1H, H-7′), 1.34 (m, 1H, H-21), 1.27 (s, 3H, H-27), 1.21
1H, H-2′), 1.61 (m, 1H, H-19′), 1.56 (m, 1H, H-6), 1.51 (m, (m, 1H, H-21′), 1.18 (m, 1H, H-19′), 1.13 (s, 3H, H-23), 1.03
1H, H-6′), 1.47 (m, 1H, H-7′), 1.41 (m, 1H, H-22), 1.39 (s, (m, 1H, H-1′), 0.90 (s, 6H, H-30 and H-29), 0.89 (s, 6H, H-25
3H, H-27), 1.35 (m, 1H, H-22′), 1.33 (m, 1H, H-21′), 1.30 and H-24), 0.76 (m, 1H, H-5), 0.62 (s, 3H, H-26); 13C-NMR
(m, 1H, H-16′), 1.28 (s, 3H, H-25), 1.18 (s, 3H, H-26), 1.15 (CDCl3) δ: 177.4, 171.0, 143.7, 136.4, 128.4, 128.4, 128.0,
(s, 3H, H-28), 1.06 (m, 1H, H-1′), 1.02 (m, 1H, H-15′), 1.00 128.0, 127.9, 122.4, 80.9, 65.9, 55.3, 47.5, 46.7, 45.8, 41.7,
(s, 3H, H-23), 0.97 (s, 3H, H-24), 0.88 (m, 1H, H-5), 0.76 (s, 41.4, 39.3, 38.1, 37.7, 36.9, 33.9, 33.1, 32.6, 32.4, 30.7,
3H, H-29); 13C-NMR (CDCl3) δ: 199.9, 176.2, 169.9, 169.2, 28.0, 27.6, 26.9, 25.8, 23.6, 23.5, 23.0, 21.3, 18.2, 16.9, 16.7,
161.7, 136.2, 135.5, 129.8, 128.6, 128.6, 128.4, 128.3, 128.2, 15.4; HR-ESI-MS: m/z 611.4147 [M+Na]+ (calculated for
128.2, 119.1, 117.5, 113.0, 82.0, 77.2, 66.2, 61.6, 55.1, 53.5, C39H56O4Na, 611.4071).
48.2, 45.4, 44.0, 43.2, 41.1, 38.8, 38.4, 37.7, 32.7, 31.9, 31.8,
31.2, 29.7, 29.4, 28.4, 28.3, 28.2, 26.5, 26.4, 23.6, 23.3, 22.7, Benzyl 3beta-acetyloxy-11-oxo-olean-12-en-30-oate (5b)
1
18.7, 17.4, 16.9, 16.4; HR-ESI-MS: m/z 703.3973 [M+Na]+ White solid, yield 56%, mp 225.6°C–226.9°C. H-NMR
(calculated for C44H56O6Na, 703.3969).
(CDCl3) δ: 7.35 (s, 5H, Ar-H), 5.53 (s, 1H, H-12), 5.31 (s, 2H,
Ph-CH2), 4.55–4.44 (m, 1H, H-3), 2.76 (m, 1H, H-1), 2.32 (s,
1H, H-9), 2.09 (m, 1H, H-18), 2.05 (m, 1H, H-15), 2.02 (s,
3H, COCH3), 1.91 (m, 1H, H-21), 1.85 (m, 1H, H-19), 1.79
Benzyl 3beta-(4-acetoxycinnamoyloxy)-11-oxo-olean-
12-en-30-oate (3l)
1
White solid, yield 45%, mp 206.1°C–207.8°C. H-NMR (m, 1H, H-16), 1.67 (m, 1H, H-2), 1.63 (m, 1H, H-7), 1.60
(CDCl3) δ: 7.66 (d, 1H, J=15.9 Hz), 7.57 (d, 2H, J=8.5 Hz), (m, 1H, H-2′), 1.58 (m, 1H, H-19′), 1.55 (m, 1H, H-6), 1.46
7.39 (s, 5H, Ar-H), 7.14 (d, J=8.1 Hz, 2H), 6.42 (d, J=16.0 Hz, (m, 1H, H-6′), 1.39 (m, 1H, H-7′), 1.36 (m, 1H, H-22), 1.33
1H), 5.67 (s, 1H, H-12), 5.32 (s, 2H, Ph-CH2), 4.68 (dd, (s, 3H, H-27), 1.30 (m, 1H, H-22′), 1.23 (m, 1H, H-21′), 1.19
J=11.8, 4.8 Hz, 1H, H-3), 2.86 (dd, J=10.8, 7.0 Hz, 1H, (m, 1H, H-16′), 1.14 (s, 6H, H-28 and H-25), 1.10 (s, 3H,
H-1), 2.39 (s, 1H, H-9), 2.33 (s, 3H, COCH3), 2.09 (ddd, H-26), 1.01 (m, 1H, H-15′), 0.97 (m, 1H, H-1′), 0.86 (s, 6H,
1H, H-18, J=13.4, 4.0, 0.87 Hz), 2.02 (m, 1H, H-15), 1.97 H-23 and H-24), 0.78 (m, 1H, H-5), 0.71 (s, 3H, H-29); 13C-
(m, 1H, H-21), 1.91 (m, 1H, H-19), 1.85 (m, 1H, H-16), 1.72 NMR (CDCl3) δ: 199.9, 176.1, 170.8, 169.0, 136.2, 128.6,
(m, 1H, H-2), 1.69 (m, 1H, H-7), 1.65 (m, 1H, H-2′), 1.61 128.6, 128.5, 128.4, 128.2, 128.2, 128.2, 80.6, 66.2, 61.7,
(m, 1H, H-19′), 1.58 (m, 1H, H-6), 1.45 (m, 1H, H-6′), 1.44 55.0, 48.2, 45.3, 45.3, 44.0, 43.2, 43.1, 41.0, 38.7, 38.0, 37.6,
(m, 1H, H-7′), 1.41 (m, 1H, H-22), 1.38 (s, 3H, H-27), 1.33 36.9, 32.6, 31.8, 31.1, 28.4, 28.2, 28.0, 26.4, 23.5, 21.3, 21.2,
(m, 1H, H-22′), 1.28 (m, 1H, H-21′), 1.25 (m, 1H, H-16′), 18.6, 17.4, 16.7, 16.4; HR-ESI-MS: m/z 603.4030 [M+H]+
1.21 (s, 3H, H-25), 1.19 (s, 3H, H-28), 1.14 (s, 3H, H-26), (calculated for C39H55O5, 603.4044).
1.09 (ddd, 1H, H-1′, J=13.7, 13.7, 3.67 Hz), 1.04 (m, 1H,
H-15′), 0.98 (s, 3H, H-23), 0.94 (s, 3H, H-24), 0.87 (m, Benzyl 3beta-(3-acetoxybenzoyloxy)olean-12-en-
1H, H-5), 0.76 (s, 3H, H-29); 13C-NMR (CDCl3) δ: 200.0, 28-oate (6a)
176.2, 169.2, 169.1, 166.7, 152.0, 143.2, 136.12, 132.3, White solid, yield 55%, mp 141.8°C–142.6°C. H-NMR
1
129.2, 129.2, 128.6, 128.6, 128.5, 128.3, 128.3, 128.3, 122.1, (CDCl3) δ: 7.93 (d, J=6.8 Hz, 1H), 7.76 (s, 1H), 7.85 (d, 1H,
122.1, 119.0, 80.8, 66.2, 61.7, 55.0, 53.5, 48.2, 45.4, 44.0, J=8.0 Hz), 7.46 (td, J=8.0, 3.2 Hz, 1H), 7.37 (s, 5H, Ar-H),
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7.30 (d, J=9.2 Hz, 1H), 5.30 (s, 2H, Ph-CH2), 5.10 (dd, 1H, 4.75 (dd, J=10.4, 6.0 Hz, 1H, H-3), 2.94 (dd, J=14.0, 4.4 Hz,
H-12, J=20.8, 12.8 Hz), 4.76 (dt, J=9.9, 4.0 Hz, 1H, H-3), 1H, H-18), 2.35 (s, 3H, COCH3), 2.0 (m, 1H, H-11), 1.90 (dd,
2.94 (dd, J=14.0, 4.2 Hz, 1H, H-18), 2.33 (s, 3H, COCH3), J=9.1, 3.5 Hz, 1H, H-16), 1.85 (m, 1H, H-16′), 1.75 (m, 1H,
2.03 (dd, J=24.1, 8.0 Hz, 1H, H-11), 1.90 (dd, J=9.1, 4.0 Hz, H-7), 1.65 (m, 1H, H-19), 1.58–1.61 (m, 3H, H-11′, H-15
1H, H-16), 1.82 (m, 1H, H-16′), 1.72 (m, 1H, H-7), 1.64 (m, and H-15′), 1.54 (m, 1H, H-1), 1.46–1.51 (m, 3H, H-9, H-6
1H, H-19), 1.58–1.61 (m, 3H, H-11′, H-15 and H-15′), 1.55 and H-6′), 1.41 (m, 1H, H-7′), 1.29 (m, 1H, H-21), 1.24 (m,
(m, 1H, H-1), 1.48–1.51 (m, 3H, H-9, H-6 and H-6′), 1.41 1H, H-21′), 1.17 (s, 3H, H-27), 1.12 (m, 1H, H-19′), 1.09 (m,
(m, 1H, H-7′), 1.29 (m, 1H, H-21), 1.24 (m, 1H, H-21′), 1H, H-1′), 1.03 (s, 3H, H-23), 0.97 (s, 3H, H-30), 0.95 (s,
1.17 (s, 3H, H-27), 1.10 (m, 1H, H-19′), 1.06 (m, 1H, H-1′), 6H, H-29 and H-25), 0.93 (s, 3H, H-24), 0.84 (m, 1H, H-5),
1.02 (s, 3H, H-23), 0.97 (s, 3H, H-30), 0.95 (s, 6H, H-29 0.65 (s, 3H, H-26); 13C-NMR (CDCl3) δ: 177.5, 169.0, 165.5,
and H-25), 0.93 (s, 3H, H-24), 0.82 (m, 1H, H-5), 0.65 (s, 154.1, 143.7, 136.5, 131.1, 131.1, 128.6, 128.4, 128.4, 128.3,
3H, H-26); 13C-NMR (CDCl3) δ: 177.4, 169.3, 165.3, 150.6, 128.0, 128.0, 127.9, 122.4, 121.6, 81.7, 66.3, 65.9, 55.4,
143.7, 136.5, 132.6, 129.4, 128.4, 128.4, 128.0, 128.0, 127.9, 53.4, 47.5, 46.8, 45.9, 41.7, 41.4, 39.3, 38.1, 38.1, 37.0, 33.9,
127.0, 126.2, 122.7, 122.4, 81.9, 65.9, 55.3, 53.5, 47.6, 46.8, 33.1, 32.7, 32.4, 30.7, 29.7, 28.2, 27.6, 25.9, 23.7, 23.6, 23.4,
45.9, 41.7, 41.4, 39.3, 38.1, 38.1, 36.9, 33.9, 33.1, 32.7, 32.4, 23.0, 21.2, 18.2, 16.9, 17.0, 15.4; HR-ESI-MS: m/z 731.4288
30.7, 28.2, 27.6, 25.9, 23.7, 23.6, 23.4, 23.1, 21.1, 18.2, 17.0, [M+Na]+ (calculated for C46H60O6Na, 731.4282).
16.9, 15.4; HR-ESI-MS: m/z 731.4282 [M+Na]+ (calculated
for C46H60O6Na, 731.4282).
Benzyl 3beta-(4-acetoxybenzoyloxy)-11-oxo-olean-
12-en-30-oate (7b)
White solid, yield 4%, mp 164.6°C–165.7°C. H-NMR
1
Benzyl 3beta-(3-acetoxybenzoyloxy)-11-oxo-olean-
12-en-30-oate (6b)
(CDCl3) δ: 8.09 (d, J=8.7 Hz, 2H), 7.39 (s, 5H, Ar-H), 7.18
1
White solid, yield 32%, mp 135.6°C–137.1°C. H-NMR (d, J=8.4 Hz, 2H), 5.58 (s, 1H, H-12), 5.32 (s, 2, Ph-CH2),
(CDCl3) δ: 7.92 (d, J=7.8 Hz, 1H), 7.75 (s, 1H), 7.45 (t, 1H, 4.77 (dd, J=11.2, 4.8 Hz, 1H, H-3), 2.87 (d, J=13.6 Hz, 1H
J=8.0 Hz), 7.37 (s, 5H, Ar-H), 7.31 (d, 1H, J=8.4 Hz), 5.57 H-1), 2.40 (s, 1H, H-9), 2.35 (s, 3H, COCH3), 2.07 (m, 1H,
(s, 1H, H-12), 5.29 (s, 2, Ph-CH2), 4.77 (dd, J=11.7, 4.7 Hz, H-18), 2.03 (m, 1H, H-15), 1.99 (m, 1H, H-21), 1.96 (m,
1H, H-3), 2.86 (dt, J=13.3, 3.4 Hz, 1H H-1), 2.39 (s, 1H, 1H, H-19), 1.87 (m, 1H, H-16), 1.75 (m, 1H, H-2), 1.68 (m,
H-9), 2.32 (s, 3H, COCH3), 2.06 (m, 1H, H-18), 2.04 (m, 1H, H-7), 1.66 (m, 1H, H-2′), 1.61 (m, 1H, H-19′), 1.57 (m,
1H, H-15), 1.98 (m, 1H, H-21), 1.94 (m, 1H, H-19), 1.82 1H, H-6), 1.55 (m, 1H, H-6′), 1.49 (m, 1H, H-7′), 1.46 (m,
(m, 1H, H-16), 1.75 (m, 1H, H-2), 1.68 (m, 1H, H-7), 1.66 1H, H-22), 1.39 (s, 3H, H-27), 1.36 (m, 1H, H-22′), 1.34 (m,
(m, 1H, H-2′), 1.60 (m, 1H, H-19′), 1.58 (m, 1H, H-6), 1.53 1H, H-21′), 1.31 (m, 1H, H-16′), 1.23 (s, 3H, H-25), 1.19 (s,
(m, 1H, H-6′), 1.49 (m, 1H, H-7′), 1.45 (m, 1H, H-22), 1.37 3H, H-26), 1.15 (s, 3H, H-28), 1.12 (m, 1H, H-1′), 1.09 (m,
(s, 3H, H-27), 1.32 (m, 1H, H-22′), 1.28 (m, 1H, H-21′), 1.26 1H, H-15′), 1.05 (s, 3H, H-23), 0.96 (s, 3H, H-24), 0.89 (m,
(m, 1H, H-16′), 1.22 (s, 3H, H-25), 1.18 (s, 3H, H-26), 1.13 1H, H-5), 0.76 (s, 3H, H-29); 13C-NMR (CDCl3) δ: 200.1,
(s, 3H, H-28), 1.11 (m, 1H, H-1′), 1.07 (m, 1H, H-15′), 1.04 176.2, 169.2, 169.0, 165.5, 154.1, 136.1, 131.1, 131.1, 129.6,
(s, 3H, H-23), 0.95 (s, 3H, H-24), 0.89 (m, 1H, H-5), 0.75 (s, 128.6, 128.6, 128.5, 128.3, 128.3, 128.3, 121.6, 121.6, 81.4,
3H, H-29); 13C-NMR (CDCl3) δ: 199.9, 176.2, 169.2, 169.1, 66.2, 61.7, 55.1, 48.3, 45.4, 44.0, 43.2, 41.1, 38.8, 38.4, 37.7,
165.3, 150.6, 136.1, 132.5, 129.4, 128.6, 128.6, 128.4, 128.3, 37.0, 32.7, 31.8, 29.7, 28.4, 28.3, 28.2, 26.5, 26.4, 23.6, 23.3,
128.2, 128.2,127.0, 126.2, 122.7, 81.6, 66.2, 61.7, 55.1, 48.2, 21.2, 18.7, 17.4, 17.0, 16.4; HR-ESI-MS: m/z 723.4262
45.4, 44.0, 43.2, 41.1, 38.8, 38.4, 37.7, 37.0, 32.7, 31.8, 31.2, [M+H]+ (calculated for C46H59O7, 723.4255).
28.4, 28.3, 28.2, 26.5, 26.4, 23.6, 23.3, 21.1, 18.7, 17.4, 17.0,
16.4; HR-ESI-MS: m/z 723.4243 [M+H]+ (calculated for general experimental way for the synthesis of 3f,g
C46H59O7, 723.4255).
and 3m,n
DMF (50 μL) was added to a solution of nicotinic acid or
isonicotinic acid (1.5 mol) in SOCl2 (10 mL) at 0°C for
10 min. The mixture was slowly heated to 78°C, stirred and
Benzyl 3beta-(4-acetoxybenzoyloxy)olean-12-en-
28-oate (7a)
1
White solid, yield 3%, mp 141.8°C–142.6°C. H-NMR refluxed for 3 h, and the excess SOCl2 was distilled off under
(CDCl3) δ: 8.09 (d, J=8.3 Hz, 2H), 7.37 (s, 5H, Ar-H), 7.18 reduced pressure to obtain a white acid chloride product.
(d, J=8.3 Hz, 2H), 5.32 (s, 2H, Ph-CH2), 5.11 (m, 1H, H-12), Then, an anhydrous dichloromethane solution (20 mL) of
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cytotoxic properties of Oa and ga derivatives
2a or 2b (1 mmol) was mixed with the white acid chloride 33.1, 32.6, 32.4, 30.7, 28.2, 27.6, 25.9, 23.7, 23.5, 23.4, 23.1,
product as well as Et3N at 0°C. The mixture was stirred at 18.2, 17.0, 16.9, 15.4; HR-ESI-MS: m/z 652.4434 [M+H]+
room temperature, and TLC was used to monitor the reaction (calculated for C43H58O4N, 652.4360).
progress. After the reaction was over, the mixture was washed
with saturated sodium bicarbonate solution and saturated Benzyl 3beta-nicotinoyloxy-11-oxo-olean-12-en-
sodium chloride solution. The organic layer was dried over 30-oate (3m)
anhydrous Na2SO4 and filtered, and the filtrate was concen- White solid, yield 39%, mp 163.1°C–164.8°C. H-NMR
1
trated to get a yellow solid. The target product was purified (CDCl3) δ: 9.24 (s, 1H), 8.79 (d, J=5.0 Hz, 1H), 8.33 (d,
by column chromatography (ethyl acetate/petroleum=1/4) to J=7.8 Hz, 1H), 7.55 (m, 1H), 7.38 (s, 5H, Ar-H), 5.57 (s, 1H,
obtain 3f,g and 3m,n as white solids.
H-12), 5.31 (s, 2H, Ph-CH2), 4.80 (d, 1H, H-3, J=11.6 Hz),
2.83 (dd, 1H, H-1, J=33.2, 11.2 Hz), 2.39 (s, 1H, H-9), 2.05
(m, 1H, H-18), 2.01 (m, 1H, H-15), 1.97 (m, 1H, H-21), 1.94
Benzyl 3beta-nicotinoyloxyolean-12-en-28-oate (3f)
1
White solid, yield 32%, mp175.6°C–177.2°C. H-NMR (dd, J=13.8, 8.7 Hz, 1H, H-19), 1.85 (m, 1H, H-16), 1.74 (m,
(CDCl3): 9.25 (s, 1H), 8.80 (s, 1H), 8.34 (d, J=7.6 Hz, 1H), 1H, H-2), 1.71 (m, 1H, H-7), 1.66 (m, 1H, H-2′), 1.60 (m,
7.44 (m, 1H), 7.36 (s, 5H, Ar-H), 5.32 (s, 2H, Ph-CH2), 5.10 1H, H-19′), 1.57 (m, 1H, H-6), 1.52 (m, 1H, H-6′), 1.48 (m,
(m, 1H, H-12), 4.78 (t, 1H, H-3, J=8.8 Hz), 2.94 (m, 1H, 1H, H-7′), 1.43 (m, 1H, H-22), 1.38 (s, 3H, H-27), 1.35 (m,
H-18), 2.04 (m, 1H, H-11), 1.88 (m, 1H, H-16), 1.80 (m, 1H, 1H, H-22′), 1.33 (m, 1H, H-21′), 1.30 (m, 1H, H-16′), 1.14
H-16′), 1.73 (m, 1H, H-7), 1.69 (m, 1H, H-19), 1.58–1.62 (m, (s, 3H, H-25), 1.05 (s, 3H, H-26), 1.01 (s, 3H, H-28), 0.97 (s,
3H, H-11′, H-15 and H-15′), 1.55 (m, 1H, H-1), 1.47–1.51 3H, H-23), 0.91 (m, 1H, H-1′), 0.88 (m, 1H, H-15′), 0.81 (s,
(m, 3H, H-9, H-6 and H-6′), 1.44 (m, 1H, H-7′), 1.35 (m, 3H, H-24), 0.74 (s, 3H, H-29), 0.69 (m, 1H, H-5); 13C-NMR
1H, H-21), 1.20 (m, 1H, H-21′), 1.16 (s, 3H, H-27), 1.11 (m, (CDCl3) δ: 176.2, 169.2, 164.8, 152.9, 150.6, 137.3, 136.1,
1H, H-19′), 1.06 (m, 1H, H-1′), 1.03 (s, 3H, H-23), 0.98 (s, 128.6, 128.6, 128.5, 128.4, 128.3, 128.2, 128.2, 123.4, 82.1,
3H, H-30), 0.96 (s, 6H, H-29), 0.94 (s, 3H, H-25), 0.92 (s, 66.2, 61.8, 53.5, 48.2, 45.4, 44.0, 43.2, 41.1, 39.1, 38.8, 38.4,
3H, H-24), 0.86 (m, 1H, H-5), 0.64 (s, 3H, H-26); 13C-NMR 37.6, 37.1, 36.9, 32.8, 32.7, 31.8, 31.2, 28.4, 28.3, 28.2, 28.1,
(CDCl3) δ: 177.4, 164.8, 152.8, 150.5, 143.7, 137.5, 136.4, 27.3, 26.5, 26.4, 23.6, 23.4, 23.3, 18.7, 17.5, 17.4, 17.0, 16.4,
128.4, 128.4, 128.0, 128.0, 127.9, 127.0, 123.5, 122.4, 82.5, 16.4, 15.6; HR-ESI-MS: m/z 666.4147 [M+H]+ (calculated
65.9, 55.3, 47.5, 46.7, 45.9, 41.7, 41.4, 39.3, 38.1, 36.9, 33.9, for C43H56O5N, 666.4153).
33.1, 32.6, 32.4, 30.7, 28.2, 27.6, 25.9, 23.6, 23.6, 23.4, 23.1,
18.2, 17.0, 16.9, 15.4; HR-ESI-MS: m/z 652.4357 [M+H]+ Benzyl 3beta-isonicotinoyloxy-11-oxo-olean-12-en-30-
(calculated for C43H58O4N, 652.4360).
oate (3n)
1
White solid, yield 48%, mp 190.5°C–192.3°C. H-NMR
(CDCl3) δ: 8.78 (s, 2H), 7.86 (d, J=5.1 Hz, 2H), 7.38 (s, 5H,
Ar-H), 5.57 (s, 1H, H-12), 5.31 (s, 2H, Ph-CH2), 4.80 (dd,
Benzyl 3beta-isonicotinoyloxyolean-12-en-
28-oate (3g)
1
White solid, yield 35%, mp 81.5°C–82.7°C. H-NMR J=11.5, 5.2 Hz, 1H, H-3), 2.80 (dd, J=13.4 Hz, 1H, H-1),
(CDCl3): 8.80 (d, J=3.6 Hz, 2H), 7.88 (d, J=4.4 Hz, 2H), 2.39 (s, 1H, H-9), 2.04 (m, 1H, H-18), 1.99 (m, 1H, H-15),
7.36 (s, 5H, Ar-H), 5.32 (s, 2H, Ph-CH2), 5.10 (d, 1H, H-12, 1.96 (m, 1H, H-21), 1.93 (m, 1H, H-19), 1.83 (dd, J=22.2,
J=8.1 Hz), 4.78 (t, 1H, H-3, J=8.1 Hz), 2.93 (dd, J=13.9, 9.9 Hz, 1H, H-16), 1.75 (m, 1H, H-2), 1.70 (m, 1H, H-7),
4.4 Hz, 1H, H-18), 2.02 (m, 1H, H-11), 1.88 (m, 1H, H-16), 1.65 (m, 1H, H-2′), 1.59 (m, 1H, H-19′), 1.56 (m, 1H, H-6),
1.79 (m, 1H, H-16′), 1.73 (m, 1H, H-7), 1.67 (m, 1H, H-19), 1.51 (m, 1H, H-6′), 1.47 (m, 1H, H-7′), 1.41 (m, 1H, H-22),
1.58–1.62 (m, 3H, H-11′, H-15 and H-15′), 1.55 (m, 1H, H-1), 1.38 (s, 3H, H-27), 1.33 (m, 1H, H-22′), 1.28 (m, 1H, H-21′),
1.47–1.51 (m, 3H, H-9, H-6 and H-6′), 1.44 (m, 1H, H-7′), 1.26 (m, 1H, H-16′), 1.22 (s, 3H, H-25), 1.18 (s, 3H, H-26),
1.35 (m, 1H, H-21), 1.20 (m, 1H, H-21′), 1.16 (s, 3H, H-27), 1.14 (s, 3H, H-28), 1.05 (s, 3H, H-23), 0.96 (s, 3H, H-24),
1.13 (m, 1H, H-19′), 1.08 (m, 1H, H-1′), 1.03 (s, 3H, H-23), 0.90 (m, 1H, H-1′), 0.87 (m, 1H, H-15′), 0.81 (m, 1H, H-5),
0.97 (s, 3H, H-30), 0.94 (s, 6H, H-29 and H-25), 0.92 (s, 0.75 (s, 3H, H-29); 13C-NMR (CDCl3) δ: 199.9, 176.2, 169.2,
3H, H-24), 0.87 (m, 1H, H-5), 0.64 (s, 3H, H-26); 13C-NMR 164.7, 150.4, 150.4, 138.2, 136.1, 128.6, 128.6, 128.4, 128.3,
(CDCl3) δ: 177.4, 164.6, 150.2, 150.2, 143.8, 138.4, 136.4, 128.2, 128.2, 122.9, 122.9, 82.5, 66.2, 61.6, 55.0, 53.5, 48.2,
128.4, 128.4, 128.0, 128.0, 127.9, 123.0, 122.3, 82.9, 65.9, 45.4, 44.0, 43.2, 41.1, 38.7, 38.4, 37.6, 36.9, 32.7, 31.8,
55.3, 53.4, 47.5, 46.7, 45.9, 41.7, 41.4, 39.3, 38.1, 36.9, 33.9, 31.2, 28.4, 28.2, 26.5, 26.4, 23.5, 23.3, 18.7, 17.4, 17.0,
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16.4; HR-ESI-MS: m/z 688.3969 [M+Na]+ (calculated for for 24 or 48 h. Cell apoptosis was assayed by using a PE
C43H55O5NNa, 688.3972).
Annexin V Apoptosis Detection Kit I (BD Biosciences)
according to the manufacturer’s instructions. Cells were
digested by 0.25% trypsin-EDTA solution, washed twice
with cold PBS and stained with Annexin V-PE (5 μL) and
7-AAD (5 μL) in binding buffer. After incubation at room
temperature for 15 min, cell apoptosis was analyzed by BD
FACS Aria III flow cytometer.
another experimental way for the synthesis of 5a,b
Compound 2a or 2b (1 mmol) was dissolved in pyridine
(5 mL); then, anhydrous acetic anhydride (10 mL) was
added, which was stirred at room temperature for 24 h.37,38
After the reaction was completed, the mixture was poured
into ice-cold dilute hydrochloric acid and kept overnight.
Afterward, the mixture was filtered and washed with 2 mol/L
hydrochloric acid to remove the pyridine; this was washed
with water to neutralize and dried with anhydrous sodium
sulfate. The organic layer was filtered, and the filtrate was
concentrated to get a yellow solid. The latter was purified
by column chromatography (ethyl acetate/petroleum=1/4) to
give a white solid. The spectroscopic results are consistent
with those of 5a,b.
apoptosis assay by hoechst 33342
staining methods
Hep-G2 cells were seeded into six-well plates, cultured
in DMEM supplemented with 10% FBS and incubated at
37°C in 5% CO2 for 24 h. The medium was replaced with a
medium containing different concentrations of compound 5b
(0, 4 and 8 μM) for 48 h. The medium was removed, washed
with cold PBS and fixed with formalin (4%, w/v). Cell nuclei
were counterstained with Hoechst 33342 at a concentration
of 10 μg/mL in PBS for 10 min in the dark. Finally, the cells
were washed twice with cold PBS and examined under a
fluorescence microscope.
cytotoxic activity assay
The tested compounds were dissolved in a suitable amount
of dimethyl sulfoxide prior to the experiment to obtain a
known concentration of the solution, and then these solu-
tions were diluted to various concentrations with the culture
medium. To evaluate the cytotoxic activity, the SGC-7901
(gastric cancer), MCF-7 (breast cancer), Eca-109 (esopha-
geal cancer), HeLa (cervical cancer) and Hep-G2 (hepatoma
cancer) cell lines (2×104 cells/mL) and HSF (normal HSF;
1×104 cells/mL) were placed in 96-well plates and cultured
for 24 h at 37°C in the presence of 5% CO2 atmosphere. After
24 h of incubation, the culture medium was discarded, the
cells were treated with the test compound of various con-
centrations for 48 h and the control groups were treated with
the medium alone. Then, 20 μL of MTT solution (5 mg/mL)
was added to each well. After incubation for another 4 h,
the medium was aspirated, and the formazan crystals were
dissolved in 100 μL dimethyl sulfoxide for each well. The
absorbance was measured at a test wave length of 490 nm
using a Bio-Rad iMark™ microplate reader. The IC50 values
were obtained by linear regression analysis using GraphPad
Prism (version 5.01).
Determination of autophagy
Hep-G2 cells were seeded into six-well plates, cultured in
DMEM supplemented with 10% FBS and incubated at 37°C
in 5% CO2 for 24 h. After that, the medium was replaced with
a medium containing different concentrations of compound
5b (0, 4 and 8 μM) for 24 or 48 h. The medium was removed
again, and the cells were washed with ice-cold PBS twice.
Then, the cells were stained with monodansylcadaverine
(MDC) solution (50 mM) for 15 min and washed twice with
PBS. The data were obtained by flow cytometry.
Results and discussion
chemistry
As shown in Scheme 1, OA derivatives, namely, 3a–g, were
synthesized. The esterification of OA at C-28 was performed
by treatment with benzyl bromide to produce OA ester 2a.
Organic acids (R1OH) were coupled with the 3-OH position
of 2a to obtain esters 3a–e using the DMAP/EDCI system.
Nicotinic acid or isonicotinic acid was dissolved in SOCl2, and
triethylamine and 2a were added to obtain 3f–g. The synthesis
of GA derivatives 3h–n is described in Scheme 2. Treatment
of GA with benzyl bromide in the presence of K2CO3 gives
annexinV/7-aminoactinomycin D
(7-aaD) assay
Hep-G2 cells were seeded into six-well plates, cultured the GA ester 2b. The 3-OH position of 2b reacts with organic
in DMEM supplemented with 10% fetal bovine serum acids (R2OH) in the EDCI/DMAP system to form compounds
(FBS) and incubated at 37°C in 5% CO2 for 24 h. After 3h–l. Treatment of nicotinic acid or isonicotinic acid with
that, the medium was replaced with a medium containing SOCl2 was followed by the addition of triethylamine and 2b
different concentrations of compound 5b (0, 4 and 8 μM) as the acid-binding agent, using the esterification procedure
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cytotoxic properties of Oa and ga derivatives
ꢀEꢁꢂRUꢂꢀFꢁ
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Scheme 1 general experimental method for the synthesis of 3a–g.
Notes: reagents and conditions: (a) DMF, BnBr, K2cO3, room temperature; (b) anhydrous DCM, DMAP, EDCI, reflux, room temperature; (c) SOCl2, et3N, reflux.
Abbreviations: BnBr, benzyl bromide; DcM, dichloromethane; DMaP, N-dimethyl-4-aminopyridine; DMF, N-dimethylformamide; eDci, 1-ethyl-(3-(3-dimethylamino)
propyl)-carbodiimide hydrochloride; Oa, oleanolic acid.
CO2Bn
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Scheme 2 general experimental method for the synthesis of 3h–n.
Notes: reagents and conditions: (d) DMF, BnBr, K2cO3, room temperature; (e) anhydrous DCM, DMAP, EDCI, reflux, room temperature; (f) SOCl2, et3N, reflux.
Abbreviations: BnBr, benzyl bromide; DcM, dichloromethane; DMaP, N-dimethyl-4-aminopyridine; DMF, N-dimethylformamide; eDci, 1-ethyl-(3-(3-dimethylamino)
propyl)-carbodiimide hydrochloride; ga, glycyrrhetinic acid.
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Wang et al
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to obtain the compounds 3m,n. Compounds 5a,b, 6a,b and followed by removal of DMAP and transformation into the
7a,b were prepared as shown in Schemes 3 and 4. In some desired ester 5a or 5b. We have attempted to obtain 6a,b and
studies, it has been reported that the esters of acetylsalicylic 7a,b by treating 2a,b with acetyl 3-hydroxybenzoic acid and
acid easily undergo ester exchange reactions.21–25,39 Thus, acetyl 4-hydroxybenzoic acid in the EDCI/DMAP system.
when acetylsalicylic acid was reacted with 2a,b in the EDCI/ While the treatment of 2a,b with acetyl 3-hydroxybenzoic
DMAP system, we did not obtain 4a,b, but rather 5a,b, and we acid indeed allows preparing 6a,b, we almost did not get
have shown the possible reaction mechanism in Figure 2. The other by-products. Interestingly, the main product obtained
high nucleophilicity of DMAP contributed to its nucleophilic as a result of the interaction of acetylated 4-hydroxybenzoic
addition to the ester moiety of acetylsalicylic acid to form acid with 2a,b is 5a,b instead of 7a,b.
intermediate I, which was converted to a N-acylpyridinium
intermediate by elimination of a phenoxide anion. Afterward, in vitro cytotoxic activity
the N-acylpyridinium salt was nucleophilically attacked by The compounds 1a,b, 2a,b, 3a–n, 5a,b, 6a,b and 7a,b
the hydroxyl moiety of 2a,b to produce intermediate II, were evaluated for their in vitro cytotoxicity against SGC-
which was neutralized by the in situ formed phenoxide anion, 7901, MCF-7, Eca-109, HeLa, Hep-G2 and HSF cells by
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Scheme 3 general experimental method for the synthesis of 5a, 6a and 7a.
Notes: Reagents and conditions: (g) DCM, DMAP, EDCI, acetylsalicylate, reflux, room temperature; (h) DCM, acetyl 3-hydroxybenzoic acid, DMAP, EDCI, reflux, room
temperature; (i) DCM, acetyl 4-hydroxybenzoic acid, DMAP, EDCI, reflux, room temperature.
Abbreviations: DcM, dichloromethane; DMaP, N-dimethyl-4-aminopyridine; eDci, 1-ethyl-(3-(3-dimethylamino) propyl)-carbodiimide hydrochloride.
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cytotoxic properties of Oa and ga derivatives
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Scheme 4 general experimental method for the synthesis of 5b, 6b and 7b.
Notes: Reagents and conditions: (j) DCM, DMAP, EDCI, acetylsalicylate, reflux, room temperature; (k) DCM, acetyl 3-hydroxybenzoic acid, DMAP, EDCI, reflux, room
temperature; (l) DCM, acetyl 4-hydroxybenzoic acid, DMAP, EDCI, reflux, room temperature.
Abbreviations: DcM, anhydrous dichloromethane; DMaP, N-dimethyl-4-aminopyridine; eDci, 1-ethyl-(3-(3-dimethylamino) propyl)-carbodiimide hydrochloride.
standard MTT assay. Gefitinib and doxorubicin were used
CO2H
CO2H
as positive controls.40 Considering that the literatures related
to this study used both micromolar studies of the cytotoxic-
ity of OA and GA derivatives,31,32,41,42 so this research used
micromolar to study the cytotoxicity of the compounds 1a,b,
2a,b, 3a–n, 5a,b, 6a,b and 7a,b. The antitumor activities
were expressed in terms of IC50 (μM) and are summarized
in Table 1. On the whole, GA and its derivatives had bet-
ter cytotoxicity than OA and its derivatives. The parent
compounds OA and GA both showed low inhibitory activi-
ties against MCF-7 cells, Hep-G2 cells and HSF cells. GA
had better cytotoxicity values than gefitinib for SGC-7901
cells, Eca-109 cells and HeLa cells, especially for SGC-
7901 cells with an IC50 value of 10.20±1.23 μM, but OA
showed a poor inhibitory effect on these three cell lines.
In contrast to OA and GA, esterified compounds 2a and 2b
+
N
NMe2
N
O
O
+
O
O
–
NMe2
DMAP
Acetylsalicylic acid
Intermediate I
+
O
–
O
O
CO2H
CO2H
HOR
H
–
–
+
O
N
+
O
N
+
+
NMe2
Intermediate II
NMe2
N-Acylpyridinium
CO2H
N
O
OH2
+
+
R
O
NMe2
DMAP
Salicylic acid
Compd 5a or 5b
Figure 2 Proposed mechanism of the reaction of 2a (or 2b) with acetylsalicylate.
Abbreviation: DMaP, N-dimethyl-4-aminopyridine.
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Table 1 cytotoxicity of ga and Oa derivatives against sgc-7901, McF-7, eca-109, hela, hep-g2 and hsF cell lines
Compound
IC50 (μmol/L)a
SGC-7901
MCF-7
Eca-109
HeLa
Hep-G2
HSF
1a
52.73±5.69
10.20±1.23
47.35±3.25
18.35±3.19
100
100
78.15±7.26
18.99±1.11
69.16±5.09
6.37±1.48
100
34.76±0.52
17.32±0.66
75.55±1.12
25.81±2.79
31.64±0.22
19.79±0.91
32.17±0.04
17.44±0.36
63.34±3.30
35.56±0.38
32.07±2.80
19.11±0.83
20.29±1.58
28.24±1.44
42.45±0.32
4.32±0.89
20.21±0.29
24.09±0.46
15.46±2.71
12.36±0.45
11.98±0.51
7.82±0.02
15.51±0.73
10.51±0.62
26.41±1.00
0.18±0.03
100
63.59±1.74
48.23±1.99
55.72±2.38
47.84±1.06
96.03±2.54
90.62±0.25
100
1b
2a
100
100
44.10±4.09
17.74±1.79
56.28±0.78
22.65±0.43
58.40±0.92
24.83±0.40
62.17±1.20
18.84±2.11
16.82±1.31
26.92±4.09
67.35±2.11
8.10±0.40
42.26±0.89
25.60±1.36
5.51±0.41
8.63±2.04
45.85±1.40
5.52±0.07
86.63±1.79
53.99±1.82
100
42.52±0.46
12.04±0.27
24.47±1.18
8.97±0.13
21.92±0.64
4.06±0.24
31.95±0.89
10.61±0.37
8.44±0.26
100
2b
3a
3b
3c
39.43±2.72
100
100
100
3d
3e
79.12±4.01
100
95.83±8.99
100
79.94±4.42
100
3f
25.10±2.20
20.02±0.91
74.62±7.21
100
40.34±2.55
32.98±3.88
44.26±3.30
100
41.56±1.26
40.16±0.98
100
3g
3h
3i
100
100
3j
11.46±2.94
98.35±3.24
41.49±1.13
7.57±0.64
10.07±3.26
42.81±2.64
11.29±0.75
100
15.03±2.83
60.65±7.22
30.79±4.46
5.03±0.56
17.12±4.17
22.22±4.29
6.36±0.88
100
44.47±1.73
53.40±0.78
16.38±3.23
4.11±0.73
8.76±1.05
32.25±1.23
3.74±0.18
44.12±1.17
25.78±1.78
58.06±1.65
44.65±0.90
13.77±0.49
0.10±0.01
47.04±2.90
100
3k
3l
57.48±0.57
23.18±0.93
33.82±0.89
100
3m
3n
5a
5b
6a
22.12±1.11
100
6b
7a
100
100
100
100
100
100
7b
Gefitinib
DOX
100
100
100
100
18.05±2.75
0.13±0.04
12.86±0.71
0.09±0.02
23.89±3.13
0.17±0.02
20.54±0.52
0.16±0.01
Notes: aic50 is the drug concentration effective in inhibiting 50% of the cell growth measured by MTT method. ic50 values are given only if they were 100 μM. all values
are given as means±sD.
Abbreviations: DOX, doxorubicin; GA, glycyrrhetinic acid; HSF, human skin fibroblasts; OA, oleanolic acid.
with benzyl group exhibited relatively high inhibitory activi- addition, 3f and 3g with heteroaroyl group at C-3 position of
ties against MCF-7 cells, Eca-109 cells and Hep-G2 cells. 2a also presented the superior activity against Hep-G2 cells.
This indicated that the benzyl group introduced in C-28 posi- Surprisingly, compounds 6a and 7a, bearing the same AcO
tion of OA or C-30 position of GA was beneficial to improve group in different locations, presented a strong inhibitory
the inhibitory activity against these cancer cells. The selec- activity against HeLa cells, and the compound with o-AcO
tive inhibitory activity of the compounds was significantly substitutions (6a, IC50 11.98±0.51 μM) at an aromatic ring
enhanced when OA was further modified. Compounds 3a–g was more active than the compound with p-AcO substitutions
showed strong inhibitory activities against Hep-G2 cells, but (7a, IC50 15.51±0.73 μM). As a result of further modification
a poor inhibitory effect on HSF cells. This result is consistent of GA, the compounds exhibited different anticancer activi-
with the test results of novel ligustrazine-oleanolic acid- ties. Compounds 3h, 3i and 3k displayed a poor inhibitory
amino acid derivatives synthesized by Chu et al.31 The R1 effect on the five kinds of cancer cells, indicating that these
group was 4-(1H-indol-3-yl)butyroxy long-chain-substituted organic acid moieties introduced in C-3 position of 2b were
compound (3b, IC50 8.97±0.13 μM), which had a signifi- not beneficial to enhance their inhibitory activity. Com-
cantly stronger inhibitory activity against Hep-G2 cells than pounds 3j and 3l exhibited a strongly selective inhibitory
2-(1H-indol-3-yl)acetoxy short-chain-substituted compound activity, and in particular, compound 3l showed the strongest
(3a, IC50 24.47±1.18 μM). Introducing salicyloyloxy (3d) to inhibitory activity against HeLa cells with an IC50 value of
C-3 position of 2a led to a significant increase of the selec- 4.32±0.89 μM, suggesting that an AcO substituent might
tive inhibitory activity with a low IC50 value of 4.06±0.24 enhance 3j toward tumor cell lines. Compounds 3m and 3n
μM on Hep-G2 cells, but for HSF cells, it exhibited a poor presented more potential anticancer activity in comparison
inhibitory effect with a high IC50 value of 79.94±4.42 μM. In with gefitinib, with compound 3m being the most active
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cytotoxic properties of Oa and ga derivatives
Hep-G2
one, indicating that the heteroaroyl introduced in C-3 posi-
tion of 2b was beneficial to enhance its inhibitory activity.
The cytotoxicity of the derivatives obtained by the Schwarz
and Csuk introduction of amino acids at the C-3 position of
the GA benzyl ester is consistent with the present results.42
Furthermore, the inhibitory activity of compound 3m on
HSF cells was lower than that of gefitinib (IC50, 20.54±0.52
μM). In addition, 5b with AcO group at C-3 position of
2b showed the superior inhibitory activity against tumor
cell lines, especially for Hep-G2 cells with an IC50 value of
3.74±0.18 μM. Yadav et al43 used the quantitative structure-
activity relationship (QSAR) model to predict that compound
5b has a significant inhibitory effect on MCF-7 cells. Our
experimental results are consistent with the predictions. Like
compounds 6a and 7a, compounds 6b and 7b also presented
strong targeting inhibition of HeLa cells with IC50 values of
7.82±0.02 and 10.51±0.62 μM, respectively.
100
80
60
40
20
0
24 h
48 h
1 2.5
5
10
20
Concentration (µM)
Figure 3 The proliferation inhibition of 5b toward hep-g2 assayed by MTT.
Notes: hep-g2 cells were continuously treated with different concentrations (0, 1,
2.5, 5, 10, 20 μM) of 5b for 24 and 48 h. cell viability was then determined by
MTT assay.
inhibition of hep-g2 cells’ proliferation
In summary, although the structures of OA and GA
were similar, they and their derivatives exhibited different
anticancer activities. Their anticancer activity was differ-
ent even if the same group was introduced. Another important
finding was that the introduction of a pyridine ring with a
lower electron density to the 3-OH position of GA benzyl
ester (compounds 3m and 3n) could greatly increase its
anticancer activity.
by compound 5b
Given that 5b showed high antiproliferative activity on
Hep-G2 cells, we used Hep-G2 cells to further study the
mechanism of action of 5b. As shown in Figure 3, 5b
presented a significant effect on inhibiting Hep-G2 cells’
growth, and it inhibited cell proliferation in a concentra-
tion- and time-dependent manner. Figure 3 also shows
that ~50% of Hep-G2 cells died during treatment with 5
μM 5b for 48 h.
Furthermore, the incorporation of a carbonyl group with
an electron-withdrawing property into the 3-OH position of
GA benzyl ester (compound 5b) also could greatly improve
its anticancer activity. Therefore, we tried to sum up a con-
clusion as follows: GA was first converted into its benzyl
ester of GA, and then an electron-withdrawing group was
introduced to the 3-OH position of the benzyl ester of GA,
which could greatly improve the anticancer activity of GA.
This conclusion also applied to OA because the introduction
of electron-withdrawing groups to the 3-OH position of the
cell apoptosis induced by compound 5b
Apoptosis and necrosis, especially the former, are common
cellular responses to anticancer drugs. To further evaluate
whether the cytotoxic effect of 5b is related to apoptosis, we
detected cell apoptosis by Hoechst 33342 apoptotic stain-
ing kit and detected apoptosis rate by Annexin V/7-AAD
double staining method (Figures 4 and 5). After treatment
with different concentrations (0 μM for control, 4 and 8
benzyl ester of OA (compounds 3f, 3g and 5a) could also μM) of compound 5b for 48 h, the nuclei of Hep-G2 cells
significantly improve the anticancer activity of OA.
were stained by Hoechst 33342 to detect the apoptotic cells.
Control (0 µM)
5b (4 µM)
5b (8 µM)
Figure 4 Fluorescent microscopic analysis of nuclei fragmentation of hoechst 33342 staining.
Notes: Representative photomicrographs of Hep-G2 cells stained with Hoechst 33342 fluorescent dye after exposure to 5b (drug concentrations were 0, 4 and 8 μM)
for 48 h.
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Abbreviation: 7-aaD, 7-aminoactinomycin D.
As shown in Figure 4, compared with the control group, a remarkably with a dose- and time-dependent relation (the
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cated the existence of induced apoptosis caused by the treat- h treatment and 7.2% for the control group, 15.7% for 4 μM
ment of compound 5b. Then, we used Annexin V-PE/7-AAD and 45.8% for 8 μM after 48 h treatment).
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Figure 6 MDC fluorescent intensity of autophagy was determined by flow cytometry, while Hep-G2 cells were coincubated with various concentrations of 5b (drug
concentrations were 0, 4 and 8 μM) for 24 and 48 h.
Abbreviations: M, mean; MDc, monodansylcadaverine.
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cytotoxic properties of Oa and ga derivatives
apoptosis and necrosis, is essential for homeostasis under are grateful for the support of the National Natural Sci-
normal conditions. To determine whether autophagy is truly ence Foundation of China (21202028, 21372054), the
triggered by these derivatives, Hep-G2 cells were treated with Natural Science Foundation of Shandong Province (No
0, 4 and 8 μM of compound 5b for 24 and 48 h. Then, the cells ZR2010BM021) and the project of Weihai Science and
were stained with MDC as the fluorescent probe to detect Technology Bureau (No 1070432121708).
the autophagic activity. As shown in Figure 6, exposure of
Hep-G2 cells to different concentrations of compound 5b for
Disclosure
The authors report no conflicts of interest in this work.
24 h led to an increase in MDC fluorescent intensity, with
a mean (M) of M=1,171 for 0 μM as the control, M=1,458
for 4 μM and M=1,912 for 8 μM. At 48 h, this increase was
maintained, with M=1,224 for 0 μM as the control, M=1,760
for 4 μM and M=2,480 for 8 μM. The increase in MDC
fluorescent intensity demonstrates the increasing number of
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In this study, based on the principle of combination and
through a simple two-step synthetic method, we designed
and synthesized a series of OAs and GAs and tested their
cytotoxicity by MTT assay with SGC-7901, MCF-7, Eca-
109, HeLa, Hep-G2 and HSF cells. Among all the OA and
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HeLa cells (IC50, 4.32±0.89 μM) in comparison with four
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drug, which has a unique mechanism of action and deserves
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