Synthesis and anti-cancer cell activity of pseudo-ginsenoside Rh2

Article abstract of DOI:10.1016/j.steroids.2014.08.021

β-d-Glucopyranoside,(3β,12β,20E)-12,25-dihydroxydammar-20(22)-en-3-yl (pseudo-ginsenoside Rh2) and its 20Z-isomer were synthesized from ginsenoside Rh2 under a mild condition, via a simple three-step called acetylation, elimination-addition and saponification. In addition, their activities were evaluated by eight different human tumor cells, compared with ginsenoside Rh2 group. Results indicated that the reaction in the side chain might greatly enhance the anti-proliferative activity of ginsenosides.

Full text of DOI:10.1016/j.steroids.2014.08.021

Steroids 92 (2014) 1–6
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and anti-cancer cell activity of pseudo-ginsenoside Rh2
Guangtao Qian a, Zhicai Wang a, Jinyu Zhao a, Dandan Li a, Wei Gao a, Baohui Wang a, Dayuan Sui b,
Xiangru Qu b, Yanping Chen a,
⇑
a Department of Chemistry, Jilin University, Changchun, Jilin 130021, China
b Department of Pharmaceutical Sciences, Jilin University, Changchun, Jilin 130021, China
a r t i c l e i n f o
a b s t r a c t
Article history:
b-D-Glucopyranoside,(3b,12b,20E)-12,25-dihydroxydammar-20(22)-en-3-yl (pseudo-ginsenoside Rh2)
Received 23 February 2014
Received in revised form 9 August 2014
Accepted 17 August 2014
and its 20Z-isomer were synthesized from ginsenoside Rh2 under a mild condition, via a simple three-
step called acetylation, elimination–addition and saponification. In addition, their activities were evalu-
ated by eight different human tumor cells, compared with ginsenoside Rh2 group. Results indicated that
the reaction in the side chain might greatly enhance the anti-proliferative activity of ginsenosides.
Ó 2014 Elsevier Inc. All rights reserved.
Available online 10 September 2014
Keywords:
Steroid derivatives
Human tumor cells
Ginsenoside
Synthesis
1. Introduction
2. Experimental
Ginseng, which is well-known as the king of herbs, has been an
important medicinal resource all over the world [1]. A large number
of experimental studies have shown this herb to have antidiabetic,
anti-HIV protease, and antitumor activities [2,3]. Ginsenosides are
considered as the major pharmacologically active ingredient of gin-
seng, which are defined as types of protopanaxadiol (PPD), proto-
panaxatriol (PPT), and oleanolic acid according to aglycone
skeleton [4–6]. Among them, ginsenoside Rh2, which is synthesized
via glycosylation of protopanaxadiol derivatives, has been proved
to have a measurable effect on various cancer cells [7].
2.1. General
Column chromatography (CC) was performed with silica gel
(300–400 mesh, Qingdao Haiyang Chemical Co., Ltd., China) and
spots were visualized by spraying the plates with 10% H2SO4 etha-
nol solution, followed by heating. Preparative HPLC was carried out
on a LC-8A pump, a SPA-10A UV detector (Shimadzu) and a C18
column (20 mm ꢀ 200 mm), at an elution flow rate of 5.0 ml/min.
The 1D, 2D and ROESY NMR spectroscopy were performed on a
Bruker AVANCE-500 NMR spectrometer in Pyridine-d5 and Metha-
nol-d4 using standard Bruker pulse programs. Optical rotations
were obtained on a WZZ-15 polarimeter (Na 589 nm); ultraviolet
(UV) spectra were determined on a Shimadzu UV-3600 spectro-
photometer; infrared (IR) spectra were recorded with a PerkinEl-
mer Spectrum One Fourier transform infrared (FTIR)
spectrometer (KBr); and HRESI mass spectra were performed on
an Agilent1290-micrOTOF Q II mass instrument. The absorbance
was recorded on a Microplate Reader (SpectraMaxÒ Plus384,
Molecular Devices, USA) at a wavelength of 570 nm.
In the previous study, many dammarane-type derivatives have
been prepared and investigated for their activities [8]. In current,
we have described the synthesis of two dammarane-type deriva-
tives, namely, b-
dammar-20(22)-en-3-yl (3a, Pseudo-G-Rh2) and b-
D
-Glucopyranoside,(3b,12b,20E)-12,25-dihydroxy-
-Glucopyrano-
D
side,(3b,12b,20Z)-12,25-dihydroxydammar-20(22)-en-3-yl (3b),
both of which bear a different side chain at C-17 by comparing
with ginsenoside Rh2 (Scheme 1). Moreover, we have obtained
the detailed structure elucidation of the new compounds by 1D,
2D and ROESY NMR, HRESIMS and their inhibitory effects on
several tumor cells.
2.2. Plant material
The whole extraction of ginseng was purchased from Hongjiv
Biotech Company, Jilin province, China, and identified by Prof. Yan-
ping Chen. The material 20(S)-Rh2 was prepared and isolated from
the crude product of ginseng in our laboratory [9].
⇑
Corresponding author. Tel./fax: +86 0431 85619803.
E-mail address: Ypch2196@sina.com (Y. Chen).
http://dx.doi.org/10.1016/j.steroids.2014.08.021
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

2
G. Qian et al. / Steroids 92 (2014) 1–6
Scheme 1. Chemical transformation pathways of dammarane-type derivatives. aThe yield of compound 1 is 93.4%; bIsolated yields.
2.3. Synthesis and isolation
2.3.2. b-
dammar-20(22)-en-3-yl,2,3,4,6-tetraacetate (2a)
Colorless amorphous solid; yield 67.7%; [
D-Glucopyranoside,(3b,12b,20E)-12-(acetyloxy)-25-hydroxy-
25
Ginsenoside Rh2 (3.3 g) and acetic anhydride (24.3 ml) were
dissolved and stirred in pyridine (25 ml) for 24 h at room temper-
ature. Terminated by methanol (12.5 ml), the residue was taken up
in ethyl acetate (25 ml) and water (200 ml). The organic phase was
washed with brine (20 ml), water (20 ml), dried with anhydrous
Na2SO4 and concentrated under reduced pressure. The dried
(4.3 g) was rechromatographed on normal silica gel CC eluted with
Et2O/cyclohexane (4:1 ? 3:1 ? 2:1) to give compound 1 as a white
solid (4.12 g).
To a solution of sulfuric acid/acetic acid solution (2%, 0.72 ml,
36 ml) in methanol (36 ml), compound 1 (4.0 g) was added in
one portion which was stirred for 4 h at ꢁ10 °C. After being diluted
with water (210 ml), the reaction mixture was extracted with an
equal volume of ethyl acetate for three times. The combine organic
layer was washed with a saturated aqueous solution of sodium car-
bonate and water, and then evaporated in vacuo to yield a white
solid (3.89 g). The ethyl acetate extraction was submitted to silica
gel CC eluted with Et2O/cyclohexane (5:1 ? 4:1) to yield a fraction.
The fraction was further purified by preparative RP C18 HPLC
[MeOH/H2O (9:1), 5 ml/min, monitored at 203 nm] to obtain com-
pounds 2a (2.71 g) and 2b (1.17 g), respectively.
a]
+15.2 (c 0.05,
D
MeOH); UV (MeOH) kmax nm 204; IR (KBr) mmax cmꢁ1 3327
(C–OH), 1758 (C@O), 1633 (C@C), 1036 (C–H); HR ESI-MS m/z
833.5045 [M+H]+, (calcd for 833.5046); for 1H and 13C NMR spec-
tral data, see Table 1.
2.3.3. b-
D-Glucopyranoside,(3b,12b,20Z)-12-(acetyloxy)-25-hydroxy-
dammar-20(22)-en-3-yl,2,3,4,6-tetraacetate (2b)
25
Colorless amorphous solid; yield 29.2%; [
a
]
ꢁ18.1 (c 0.05,
D
MeOH); UV (MeOH) kmax nm 203; IR (KBr) mmax cmꢁ1 3349
(C–OH), 1760 (C@O), 1638 (C@C), 1037 (C–H); HR ESI-MS m/z
833.5029 [M+H]+ (calcd for 833.5046); for 1H and 13C NMR spectral
data, see Table 1.
2.3.4. b-D-Glucopyranoside,(3b,12b,20E)-12,25-dihydroxydammar-20
(22)-en-3-yl (3a)
25
Colorless amorphous solid; yield 90.0%; [
a
]
ꢁ23.9 (c 0.03,
D
MeOH); UV (MeOH) kmax nm 201; IR (KBr) mmax cmꢁ1 3374
(C–OH), 1655 (C@C), 1051 (C–H); HR ESI-MS m/z 623.4511
[M+H]+ (calcd for 623.4517); for 1H, 13C and HMBC NMR spectral
data, see Table 2.
Sodium hydroxide (2.7 g) was added to the solution of com-
pound 2a (2.7 g) in methanol (15 ml) and dioxane (15 ml). The
resulting mixture was stirred for 4 h under reflux at 90 °C. Then
the solution was diluted with water (90 ml) and extracted with an
equal volume of n-Butanol for three times. The organic layer was
washed with water until neutral and dried over anhydrous sodium
sulfate. After evaporation of the solvent under reduced pressure and
recrystallization from methanol, the desired white solid compound
3a (1.82 g) was obtained. At the same time, compound 3b (0.79 g)
was prepared using the same procedure as described for the prepa-
ration of compound 3a with the above reactants and solvents.
2.3.5. b-D-Glucopyranoside,(3b,12b,20Z)-12,25-dihydroxydammar-20
(22)-en-3-yl (3b)
25
Colorless amorphous solid; yield 90.8%; [
a]
+23.9 (c 0.03,
D
MeOH); UV (MeOH) kmax nm 206; IR (KBr) mmax cmꢁ1 3362
(C–OH), 1656 (C@C), 1044 (C–H); HR ESI-MS m/z 623.4515
[M+H]+ (calcd for 623.4517); for 1H, 13C and HMBC NMR spectral
data, see Table 2.
2.4. Cytotoxicity evaluation
Cytotoxicities of these derivatives were evaluated on eight
different human tumor cell lines (SGC cell line, A549 cell line,
HT1080 cell line, Hela cell line, A375 cell line, K562 cell line,
HL-60 cell line and MCF-7 cell line) using 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium (MTT) assay [10]. Briefly, after being
seeded in a 96-well plate at a density of 1.2 ꢀ 104 cells/well and
incubated for 24 h, these cells were treated with the tested com-
2.3.1. b-D-Glucopyranoside,(3b,12b,20S)-12-(acetyloxy)-20-hydroxy-
dammar-24(25)-en-3-yl,2,3,4,6-tetraacetate (1)
25
Colorless amorphous solid; yield 93.4%; [
a
]
ꢁ19.0 (c 0.05,
D
MeOH); UV (MeOH) kmax nm 202; IR (KBr) mmax cmꢁ1 3340
(C–OH), 1753 (C@O), 1620 (C@C), 1037 (C–H); ESI-MS m/z 833.6
[M+H]+, 855.7 [M+Na]+, 871.6 [M+K]+; for 1H and 13C NMR spectral
data, see Table 1.
pounds (0–150 lM) for 24 h. The cell viability was determined

G. Qian et al. / Steroids 92 (2014) 1–6
3
Table 1
1H and 13C NMR spectroscopic data for compounds 1, 2a and 2b in methanol-d4.a
Position
1
2a
2b
dH (J in Hz)
dC, mult
dH (J in Hz)
dC, mult
dH (J in Hz)
dC, mult
1
2
3
4
5
6
7
8
1.61 (1H, m), 1.01 (1H, m)
1.71 (1H, m), 1.32 (1H, m)
3.18 (1H,dd, J = 11.7, 4.4 Hz)
39.95, CH2
27.77, CH2
91.21, CH
41.06, qC
57.39, CH
19.37, CH2
35.77, CH2
38.14, qC
51.41, CH
40.21, qC
29.69, CH2
76.92, CH
46.77, CH
53.81, qC
32.43, CH2
26.06, CH2
53.40, CH
16.84, CH3
16.76, CH3
75.39, qC
27.08, CH3
38.99, CH2
23.59, CH2
126.35, CH
132.16, qC
26.05, CH3
18.11, CH3
28.39, CH3
16.21, CH3
17.89, CH3
103.83, CH
74.48, CH
70.20, CH
73.41, CH
72.76, CH
63.38, CH2
1.61 (1H, m), 1.07 (1H, m)
1.77 (1H, m), 1.39 (1H, m)
3.22 (1H,dd, J = 11.5, 4.5 Hz)
40.10, CH2
29.58, CH2
91.17, CH
41.35, qC
57.39, CH
19.37, CH2
36.08, CH2
38.28, qC
51.83, CH
40.22, qC
29.72, CH2
76.03, CH
48.3, CH
52.34, qC
33.19, CH2
27.12, CH2
51.00, CH
16.91, CH3
16.91, CH3
138.79, qC
12.54, CH3
125.86, CH
24.23, CH2
44.95, CH2
71.38, qC
29.30, CH3
29.24, CH3
28.44, CH3
16.31, CH3
17.29, CH3
103.84, CH
74.45, CH
70.21, CH
73.39, CH
72.74, CH
63.40, CH2
1.67 (1H, m), 1.03 (1H, m)
1.73 (1H, m), 1.37 (1H, m)
3.22 (1H,dd, J = 11.5, 4.5 Hz)
40.10, CH2
29.39, CH2
91.17, CH
41.40, qC
57.38, CH
19.37, CH2
36.12, CH2
38.29, qC
51.88, CH
40.22, qC
29.41, CH2
76.14, CH
48.43, CH
52.52, qC
33.23, CH2
27.12, CH2
41.32, CH
16.90, CH3
16.90, CH3
138.92, qC
19.81, CH3
126.55, CH
23.57, CH2
45.49, CH2
71.48, qC
29.14, CH3
29.14, CH3
28.44, CH3
16.34, CH3
17.21, CH3
103.83, CH
74.46, CH
70.20, CH
73.40, CH
72.75, CH
63.39, CH2
0.82 (1H,d, J = 10.7 Hz m)
1.57 (1H, m), 1.54 (1H, m)
1.61 (1H, m), 1.32 (1H, m)
0.86 (1H,d, J = 9.7 Hz m)
1.57 (1H, m), 1.54 (1H, m)
1.61 (1H, m), 1.35 (1H, m)
0.86 (1H,d, J = 9.9 Hz m)
1.56 (1H, m), 1.53 (1H, m)
1.61 (1H, m), 1.34 (1H, m)
9
1.57 (1H, m)
1.57 (1H, m)
1.47 (1H, m)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
3-glc-1’
2’
1.71 (1H, m), 1.30 (1H, m)
4.81 (1H, m)
2.11 (1H, m)
1.92 (1H, m), 1.45 (1H, m)
4.94 (1H, m)
2.13 (1H, m)
1.76 (1H, m), 1.34 (1H, m)
4.93 (1H, m)
2.11 (1H, m)
1.65 (1H, m), 1.09 (1H, m)
1.92 (1H, m), 1.61 (1H, m)
2.14b
0.74 (3H, s)
0.95 (3H, s)
1.74 (1H, m),1.16 (1H, m)
1.92 (1H, m), 1.71 (1H, m)
2.55 (1H,dd, J = 10.7, 6.5 Hz)
0.79 (3H, s)
1.73 (1H, m), 1.18 (1H, m)
1.89 (1H, m), 1.67 (1H, m)
3.06 (1H,dd, J = 17.3, 10.3 Hz)
0.78 (3H, s)
0.96 (3H, s)
0.95 (3H, s)
1.13 (3H, s)
1.65 (3H, s)
1.70 (3H, s)
1.98 (1H, m),1.57(1H, m)
2.11 (1H, m), 1.92 (1H, m)
5.14 (1H,t, J = 7.1 Hz)
5.10 (1H,t, J = 6.6 Hz)
2.14 (1H, m), 1.98 (1H, m)
1.50 (2H, m)
5.11 (1H,t, J = 7.0 Hz)
2.20 (1H, m), 2.07 (1H, m)
1.45 (2H, m)
1.69 (3H, s)
1.63 (3H, s)
1.03 (3H, s)
0.98 (3H, s)
0.90 (3H, s)
4.72 (1H,d, J = 8.0 Hz)
5.25 (1H, m)
4.98 (1H, m)
4.90 (1H, m)
1.22 (3H, s)
1.22 (3H, s)
0.99 (3H, s)
1.11 (3H, s)
1.02 (3H, s)
4.75 (1H,d, J = 8.0 Hz)
5.29 (1H, m)
5.01 (1H, m)
4.97 (1H, m)
1.21 (3H, s)
1.21 (3H, s)
0.99 (3H, s)
1.10 (3H, s)
1.06 (3H, s)
4.76 (1H,d, J = 8.0 Hz)
5.28 (1H, m)
5.01 (1H, m)
4.97 (1H, m)
3’
4’
5’
6’
3.86 (1H, m)
4.29 (1H, m), 4.11 (1H, m)
3.91 (1H, m)
4.33 (1H, m), 4.14 (1H, m)
3.90 (1H, m)
4.32 (1H, m), 4.14 (1H, m)
1H and 13C NMR data were measured at 500 and 126 MHZ, respectively.
Overlapping signals.
a
b
by adding MTT (5 mg/ml) to each well followed by incubation at
37 °C in a humidified incubator containing 5% CO2 gas for 4 h. After
careful removal of the medium, all tested compounds were dis-
The structures of the synthesized compounds (2a, 2b and 3a,
3b) were determined by NMR and HR ESI-MS measurements. Their
characteristic signals of 20(22)-en-25-ol of the side chain at C-17
were observed at the 13C NMR signals at C-20, C-22, C-24, C-25,
C-26, C-27 (Tables 1 and 2) [12]. The stereochemistry of the alkene
bond was determined by ROESY, in which the proton signal of H-22
was correlated with signal of H-17 or H-21, suggesting that 2a, 3a
had the E-configuration and 2b, 3b were in the Z-configuration
(Fig. 1), which was also supported by the chemical shift of the C-
21 [13]. Analysis of the above NMR signals indicated 2a was
assigned to be 1 with a different side chain, and 2b was elucidated
as the 20(Z)-isomer of 2a (Table 1), in agreement with the molec-
ular formula C46H72O13 established by HR ESI-MS. Same as above,
the NMR data of 3a and 3b were closely resemble those of ginseno-
side Rh2 except for the significant difference of signals appeared at
the side chain, reflecting the similar features in the molecular
structure (Table 2) [9,11]. From these data, the structure of 3a
solved in 200 ll DMSO. The absorbance was recorded on a Micro-
plate Reader at a wavelength of 570 nm. The inhibition efficiency
of the compounds on cell proliferation was calculated as
100% ꢀ (1 ꢁ absorption of well treated with compounds/absorp-
tion of control treated with vehicle).
2.5. Statistical analysis
The count data expressed as a percentage and the measurement
data expressed as SD. IC50 (median inhibitory concentration)
were determined by multiple regression, with p < 0.05 considered
to be statistically significant.
was deduced to be b-D-Glucopyranoside,(3b,12b,20E)-12,25-
3. Results and discussion
dihydroxydammar-20(22)-en-3-yl, and 3b was described as the
20(Z)-isomer of 3a.
In 1H NMR spectrum, the proton signals at dH 4.72 (H-10), 5.14
(H-24), 3.18 (H-3), 2.05–0.74 (13 singlet methyl signals), and in
13C NMR spectrum, the carbon signals at dC 126.35 (C-24), 132.16
(C-25), 75.39 (C-20), 171.1–172.4 (five carbonyl carbon signals)
(Table 1), their shape and the chemical shift completely deter-
mined 1 as ginsenoside Rh2 with the hydroxyl moiety of the sugar
residue and C-12 being acetylated [11].
Here we proposed a low-temperature synthesis of 3a and its
20Z-isomer (3b) as described in Scheme 1. Through the elimination
and addition at different positions in a one-step reaction, the excel-
lent yields of the title compounds were achieved conveniently. In
addition, we found that if raise the temperature or alter the acid
medium, the reaction was unsatisfactory and often resulted in a

4
G. Qian et al. / Steroids 92 (2014) 1–6
Table 2
1H, 13C and HMBC NMR spectroscopic data for compounds 3a and 3b in pyridine-d5.a
Position
3a
3b
dH (J in Hz)
dC, mult
HMBC
dH (J in Hz)
dC, mult
HMBC
1
2
3
4
0.77 (1H, m), 1.53 (1H, m)
1.80 (1H, m), 2.24 (1H, m)
3.40 (1H, dd, J = 11.8, 4.4 Hz)
39.66 CH2
27.13 CH2
89.14 CH
40.08 qC
C-3,C-5,C-10,C-18
C-1,C-3
C-1’,C-2,C-4
0.84, 1.54 (1H, m)
1.83 (1H, m), 2.25 (1H, m)
3.40 (1H, dd, J = 11.7, 4.2 Hz)
39.66 CH2
27.12 CH2
89.13 CH
40.07 qC
C-3,C-5,C-10,C-18
C-1,C-3
C-1,C-1’
5
6
7
8
0.76 (1H, d, J = 11.6 Hz)
1.41 (1H, m), 1.52 (1H, m)
1.24 (1H, m), 1.51 (1H, m)
56.78 CH
18.84 CH2
35.73 CH2
40.65 qC
C-4,C-6,C-10,C-28
C-5,C-7
C-6,C-8,C-9,C-19
0.76 (1H, d, J = 11.8 Hz)
1.38 (1H, m), 1.52 (1H, m)
1.25 (1H, m), 1.49 (1H, m)
56.76 CH
18.83 CH2
35.72 CH2
40.64 qC
51.21 CH
37.46 qC
33.05 CH2
72.79 CH
50.91 CH
51.57 qC
32.97 CH2
28.8 CH2
41.28 CH
16.82 CH3
17.42 CH3
139.53 qC
20.45 CH3
126.35 CH2
23.65 CH2
45.42 CH2
69.98 qC
30.41 CH3
30.27 CH3
28.51 CH3
17.18 CH3
16.22 CH3
107.31 CH
76.17 CH
79.14 CH
72.25 CH
78.73 CH
63.45 CH2
C-3,C-6,C-10,C-28
C-5,C-7
C-6,C-8,C-9,C-19
9
1.41 (1H, s)
51.16 CH
37.46 qC
33.03 CH2
72.96 CH
50.93 CH
51.26 qC
32.62 CH2
16.22 CH3
51.36 CH
16.85 CH3
17.44 CH3
139.98 qC
13.45 CH3
125.96 CH2
24.08 CH2
44.66 CH2
69.89 qC
30.39 CH3
30.15 CH3
28.53 CH3
17.17 CH3
17.44 CH3
107.36 CH
76.18 CH
79.15 CH
72.27 CH
78.77 CH
63.48 CH2
C-8,C-10,C-19
1.44 (1H, s)
C-8,C-10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
3-glc-1’
2’
1.09 (1H, m), 1.68 (1H, m)
3.93 (1H, td, J = 12.9, 5.0 Hz)
2.00 (1H, m)
C-8,C-9
C-13,C-17
C-12,C-14,C-15,C-20
1.10 (1H, m), 1.69 (1H, m)
3.92 (1H,td, J = 7.4 Hz)
2.08 (1H, m)
C-8,C-9
C-13,C-17
C-11,C-12,C-14,C-17
1.44 (1H, m), 1.94 (1H, m)
1.03 (3H, s)
2.82 (1H, dd, J = 10.6, 4.4 Hz)
0.83 (3H, s)
C-14,C-17,C-30
C-15,C-17
C-16,C-20,C-21,C-22
C-1,C-5,C-9,C-10
C-7,C-8,C-9
1.46 (1H, m), 1.91 (1H, m)
1.51 (1H, m), 2.01 (1H, m)
3.42b
0.83 (3H, s)
1.00 (3H, s)
C-14,C-17,C-30
C-15,C-17,C-14,
C-16
C-1,C-9,C-10
C-8,C-9,C-14
0.99 (3H, s)
1.84 (3H, s)
5.62 (1H,t, J = 6.8 Hz)
2.38 (2H, m)
C-17,C-20,C-22
1.95 (3H, s)
5.35 (1H,t, J = 7.0 Hz)
2.57 (2H, m)
C-17,C-20,C-22
C-17,C-21,C-23,C-24
C-20,C-22,C-24,C-25
C-22,C-23,C-25,C-27
C-17,C-21,C-23,C-24
C-20,C-22,C-24,C-25
C-22,C-23,C-25,C-27
1.75 (2H, m)
1.82 (2H, m)
1.37 (3H, s)
1.37 (3H, s)
1.33 (3H, s)
1.01 (3H, s)
0.99 (3H, s)
4.96 (1H,d, J = 7.5 Hz)
4.07 (1H, m)
4.27 (1H, m)
4.23 (1H, m)
C-24,C-25,C-27
C-24,C-25,C-26
C-3,C-4,C-5
C-3,C-4,C-5,C-28
C-8,C-14,C-15
C-3,C-3’
C-1’,C-3’
C-2’,C-4’
C-5’,C-6’
C-4’,C-6’
1.41 (3H, s)
1.41 (3H, s)
1.33 (3H, s)
1.02 (3H, s)
1.04 (3H, s)
4.96 (1H,d, J = 7.8 Hz)
4.06 (1H, m)
4.27 (1H, m)
4.25 (1H, m)
C-24,C-25,C-27
C-24,C-25,C-26
C-3,C-4,C-5
C-3,C-4,C-5,C-28
C-7,C-8,C-14
C-2’,C-3,C-5’
C-1’,C-3’
C-2’,C-4’
C-5’,C-6’
C-4’,C-6’
C-4’,C-5’
3’
4’
5’
6’
4.03 (1H, m)
4.42 (1H, m), 4.62 (1H, m)
4.03 (1H, m)
4.42 (1H, m), 4.61 (1H, m)
C-4’,C-5’
a
1H and 13C NMR data were measured at 500 and 126 MHZ, respectively.
Overlapping signals.
b
Fig. 1. The key ROESY correlations of compound 2a, 3a and 2b, 3b.
complicated mixture of products, which indirectly indicated the
irreplaceability of the low-temperature and the acid catalysis.
Screening the inhibitory rate of the title compound on eight dif-
ferent human tumor cells (Fig. 2), the results showed that the pro-
liferation of these human tumor cells was suppressed dose-
dependently after treatment with 3a for 24 h. Moreover the results
of cytotoxic studies were shown in Fig. 3, which was expressed by
the IC50 values. The testing indicated that 3b showed a weaker
cytotoxic activity in vitro, but 3a had much stronger activity, com-
pared with ginsenoside Rh2.
ginsenoside Rh2 could result in a large variability of anti-tumor
activity (Fig. 3). Previous studies on the structure–activity relation-
ships of ginsenosides suggested that the anti-tumor activities were
affected by the positions of the hydroxyl group and double band in
the side chain, and the stereochemistry of the double bond also
played a crucial role for their anti-tumor activities. For ginsenoside
Rh2, with an appropriate change of the side chain at these atomic
positions could increase the bioactivity, such as for 3a. Currently,
we are in the process of synthesizing and testing a much broader
group of these types of molecules. Further analysis on these new
artificial ginsenosides and their activities toward a broader spec-
trum of cancers will be published in a separate report in due course.
The conclusion we could get from the current data was that the
side chain of ginseng sapogenin was crucial and a subtle change of

G. Qian et al. / Steroids 92 (2014) 1–6
5
Fig. 2. The inhibitory rate of compound 3a on different tumor cells. The cells were plated in 96-well plate, and treated with different doses of compound 3a for 24 h, and then
measured by MTT assay. It showed that compound 3a inhibited SGC cell growth in a dose-dependent manner, and the inhibitory effect on SGC cells was more obvious than on
the others. The experiment was repeated for at least three times. Each column represents the mean SD.
Fig. 3. The IC50 values of compound 3a, compound 3b and ginsenoside Rh2 on different tumor cells. The cells were plated in 96-well plate, and treated with different doses of
compound 3a, compound 3b or ginsenoside Rh2 for 24 h, and then measured by MTT assay. The experiment was repeated for at least three times. Each column represents the
mean SD.
Acknowledgements
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Appendix A. Supplementary data
Spectral data including 1D, 2D and ROESY NMR, HRESIMS data
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Supplementary data associated with this article can be found, in
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