Synthesis and anti-tumor evaluation of novel 25-hydroxyprotopanaxadiol analogs incorporating natural amino acids

Article abstract of DOI:10.1016/j.steroids.2012.09.012

In the current study, derivatives of 25-hydroxyprotopanaxadiol (25-OH-PPD) were prepared and their in vitro anti-tumor activities were tested on six different human tumor cell lines by standard MTT assay. The results showed that combining an ester group combined with the presence of an amino acid moiety led to a 10-fold improved anti-tumor activity. Compound 1c exhibited the best anti-tumor activity in the in vitro assays. Compounds 2c, 3c, 4c, 5c, 6c and 8b showed better anti-tumor activities compared to the parent compound 25-OH-PPD. The current results may provide useful data for researching and developing new anti-cancer agents.

Full text of DOI:10.1016/j.steroids.2012.09.012

Steroids 78 (2013) 203–209
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and anti-tumor evaluation of novel 25-hydroxyprotopanaxadiol analogs
incorporating natural amino acids
a,
Peng Wang a,b, Xiu-Li Bi c, Jing Xu a, Hao-Nan Yuan a,b, Hu-Ri Piao b, , Yu-Qing Zhao
⇑
⇑
a School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, PR China
b Key Laboratory of Natural Resources of Changbai Mountain & Functional Molecules, Ministry of Education, Yanbian University College of Pharmacy, Yanji 133002, PR China
c School of Life Science, Liaoning University, Shenyang 110036, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 June 2012
Received in revised form 18 September 2012
Accepted 25 September 2012
Available online 22 November 2012
In the current study, derivatives of 25-hydroxyprotopanaxadiol (25-OH-PPD) were prepared and their
in vitro anti-tumor activities were tested on six different human tumor cell lines by standard MTT assay.
The results showed that combining an ester group combined with the presence of an amino acid moiety
led to a 10-fold improved anti-tumor activity. Compound 1c exhibited the best anti-tumor activity in the
in vitro assays. Compounds 2c, 3c, 4c, 5c, 6c and 8b showed better anti-tumor activities compared to the
parent compound 25-OH-PPD. The current results may provide useful data for researching and develop-
ing new anti-cancer agents.
Keywords:
25-OH-PPD derivatives
Amino acids
Ó 2012 Elsevier Inc. All rights reserved.
Anti-tumor activity
MTT
1. Introduction
has a higher absolute oral bioavailability and can be obtained in
a higher amount [8,9].
Ginsenosides and their aglycones have long been used for med-
ical purposes in treatment of cancer, diabetes, heart problems, etc.
[1]. There is increasing interest in developing ginseng products as
cancer preventive or therapeutic agents [2–4]. The main ginseno-
sides are glycosides that contain an aglycone with a dammarane
skeleton and include protopanaxadiol-type saponins, such as gin-
senosides Rb1, Rb2, Rb3, Rc and Rd, and protopanaxatriol-type sap-
onins such as ginsenosides Re and Rg1, Rg2, Rh1. Among the
saponin ginsenosides are compounds with a dammarane structure,
of which there are two main classes: panaxadiols (PPD) and pana-
xatriols (PPT). 25-Hydroxyprotopanaxadiol (20(R)-dammarane-3b,
12b, 20, 25-tetrol, 25-OH-PPD, structure shown in Fig. 1), is a
promising anti-tumor natural compound isolated from the fruits
of Panax ginseng [5]. In our previous study, 25-OH-PPD was found
to inhibit cancer cell proliferation, cause cell cycle arrest in vitro
and inhibit the growth of xenograft tumors without obvious host
toxicity in vivo [5]. Compared with ginsenoside-Rg3, an agent al-
ready being marketed for cancer therapy [6], 25-OH-PPD exhibits
a stronger effect on cancer cell growth, which IC50 values (20–
Previous studies on the structure–activity relationships (SAR) of
ginsenosides suggested that the anti-cancer activities were af-
fected by structural types of dammarane saponin, and numbers
of sugar moieties and substituent groups in the side chain of the
aglycone also played crucial roles for their anti-cancer activities
[10–13]. 25-OCH3-PPD, a derivative of 25-OH-PPD, has subtle dis-
tinction on structure from the latter, but shows better anti-tumor
activity. The biological activities of 25-OCH3-PPD and its known
analogs were evaluated in 12 human cancer cell lines. In all cell
lines, the order of anti-tumor activities of the test compounds
was 25-OCH3-PPD > 25-OH-PPD > PPD > Rg3. 25-OCH3-PPD also in-
duced apoptosis, cell cycle arrest in the G1 phase and inhibited
proliferation in breast cancer cell lines, which demonstrating its
potent biological effects. The IC50 values of 25-OCH3-PPD for most
cell lines were in the lower lM range, a 5- to 15-fold greater cyto-
toxicity relative to PPD and a 10- to 100-fold increase over Rg3 [14–
16].
Improving the anti-tumor activity of 25-OH-PPD without for-
feiting its apoptotic effect is one of the big challenges when dealing
with the derivatization process of 25-OH-PPD. Changing the polar-
ity pattern of the molecule is a good option, in which introducing
an extra amino group (for gain of polarity) and a protected amino
group (for gain of lipophilicity) to 25-OH-PPD. Assuming that the
amino acid derivatives should especially benefit the solubility in-
dex of these compounds even if esterases cleave the ester prodrug
in vivo it will still fall apart into the desired triterpane and the
60 lM) were 5- to 15-fold lower than those of ginsenoside-Rg3
[7]. Impressively, compared with other ginsenosides, 25-OH-PPD
⇑
Corresponding authors. Tel.: +86 433 2435003; fax: +86 433 2435004 (H.-R.
Piao), tel./fax: +86 24 23986521 (Y.-Q. Zhao).
E-mail addresses: piaohuri@yahoo.com.cn (H.-R. Piao), zyq4885@126.com (Y.-Q.
Zhao).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.09.012

204
P. Wang et al. / Steroids 78 (2013) 203–209
Fig. 1. 25-OH-PPD and its amino acid derivatives.
non-hazardous amino acids. In order to find more potent, higher
selectivity compounds, we synthesized 20 derivatives of 25-OH-
PPD, esterified for the protection of the amino acids at C(3) and
C(12) and varied by coupling with amino acids at C(3), and the
structures of these compounds were identified using IR, MS, 1H
NMR and 13C NMR spectroscopy. Meanwhile, their biological activ-
ities (IC50 values) were compared using a MTT assay with six hu-
man cancer cell lines including human breast cancer cell line
MCF-7, human lung cancer cell line A549, human colorectal cancer
cell line Lovo, HCT-116, HT-29 and human glioblastoma U-87 cells.
25-OH-PPD and 5-Fu were used as references.
Table 1
Numbering, structure of the 25-OH-PPD protected and deprotected esters with amino
acids.
Compound
R1
R2
Compound
R1
R2
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
H
Boc-Ala
H
H
Boc-Val
H
H
Boc-Gly
H
H
4b
4c
5a
5b
5c
6a
6b
6c
7b
8b
Boc-Pro
Pro
H
Boc-Met
Met
H
Boc-Phe
Phe
Boc-Lys(Boc)
Boc-Arg
H
H
Boc-Ala
Ala
H
Boc-Val
Val
H
Boc-Gly
Gly
H
Boc-Met
H
H
Boc-Phe
H
H
H
H
Boc-Pro
2. Experimental
2.1. Chemistry
alanine (1 equiv) in dry dichloromethane (50 mL) and the reaction
mixtures were shaken for 24 h. The solvent was removed under re-
duced pressure to give a white solid. The white solid was dissolved
in ethyl ether and washed with NaHCO3 (5%), dried (MgSO4) and
concentrated under reduced pressure to give the crude product.
The crude products were chromatographed using silica gel and
eluted with petroleum ether/ethyl acetate (1:2) to give the pure
products 1a and 1b. Using the same procedure described above
for compounds 2a–8b.
2.1.1. General
The compounds 1a–6c were synthesized by reacting 25-OH-
PPD with a series of natural amino acids in the presence of DCC
(N,N0-dicyclohexylcarbodiimide) and DMAP (4-dimethylaminopyr-
idine) mediated esterifications of N-Boc protected amino acids
(alanine, valine, proline, glycine, methionine, phenylalanine, lysine
and arginine) in dry dichloromethane [17–21]. Deprotection of the
Boc group was achieved using silica gel catalyzed in toluol for 6 h
(Scheme 1 and Table 1). All the products were purified from the
reaction mixture by silica gel column chromatography and the
structures were confirmed by IR, 1H NMR, 13C NMR and mass spec-
tra. Melting points were determined in open capillary tubes and
are uncorrected. IR spectra were recorded (in KBr) on a FT-IR
1730 (PerkineElmer, USA). 1H NMR and 13C NMR spectra were
measured on a Bruker AV-300 spectrometer using tetramethylsil-
ane as the internal standard. The major chemicals were purchased
from Sigma–Aldrich and Fluka.
2.1.2.1. (20R)-12b-O-(Boc- -alanyl)-dammarane-3b, 20, 25-triol (1a).
L
Yield: 11%. Mp: 175–177 °C. 1H NMR (300 Hz, CDCl3): d 4.72 (1H,
td, J = 4.7, 10.4 Hz; H-12), d 3.47 (1H, dd; H-3), d 2.30 (2H, q;
CH2), d 1.16 (3H, t; J = 7.5 Hz; CH3), d 1.22 (6H, s, 2ꢀCH3), d 1.15
(3H, s, CH3), d 1.02 (3H, s, CH3), d 0.98 (3H, s, CH3), d 0.97 (3H, s,
CH3), d 0.86 (3H, s, CH3), d 0.77 (3H, s; CH3); 13C NMR (75 MHz,
CDCl3): 38.9 (C-1), 27.9 (C-2), 78.7 (C-3), 38.8 (C-4), 55.8 (C-5),
18.2 (C-6), 34.5 (C-7), 39.7 (C-8), 48.7 (C-9), 37.1 (C-10), 28.3 (C-
11), 76.5 (C-12), 45.4 (C-13), 52.8 (C-14), 31.7 (C-15), 27.1 (C-16),
50.1 (C-17), 16.2 (C-18), 16.5 (C-19), 73.7 (C-20), 23.1 (C-21),
42.5 (C-22), 17.9 (C-23), 44.4 (C-24), 71.1 (C-25), 29.1 (C-26),
29.4 (C-27), 28.9 (C-28), 15.4 (C-29), 17.6 (C-30), 172.9 (Ala-
2.1.2. General procedure for the protection of the amino acids 1a–8b
The 25-OH-PPD (1 equiv, 46 mg, 0.1 mmol) was added to a solu-
tion of DCC (2 equiv, 25 mg), DMAP (2 equiv, 41 mg) and N-Boc-
Scheme 1. Synthesis of the esters of 25-OH-PPD with amino acids and their subsequent deprotection. R-side chain of amino acid for compounds 1a–6c.

P. Wang et al. / Steroids 78 (2013) 203–209
205
COO), 28.3 (Boc-CH3), 79.7 (Boc-C-CO), 155.0 (Boc-CO), 48.2 (NH-
CH), 17.4 (Ala-CH3); IR (cmꢁ1): 3382 (OH), 2964 (CH3), 1385
(CH3), 1738 (carbonyl group); HR-ESIMS: m/z 650.4990 [M+H]+
(Calcd for C38H67NO7, 649.4918).
29.4 (C-27), 28.9 (C-28), 15.4 (C-29), 17.6 (C-30), 28.3 (Boc-CH3),
79.9 (Boc-C-CO), 155.7 (Boc-CO-NH), 42.7 (NH-CH-), 170.3 (Gly-
COO), 33.9 (Gly-CH), 19.3 (2ꢀCH3); IR (cmꢁ1): 3378 (OH), 2925
(CH3), 1381 (CH3), 1737 (carbonyl group); HR-ESIMS: m/z
658.4652 [M+Na]+ (Calcd for C37H65NO7, 635.4761).
2.1.2.2. (20R)-3b-O-(Boc- -alanyl)-dammarane-12b, 20, 25-triol (1b).
L
Yield: 82%. Mp: 188–191 °C. 1H NMR (300 Hz, CDCl3): d 5.13(1H,
dd; CH-CO), d 4.53 (1H, t, J = 8.1 Hz; H-3), d 3.58 (1H, td; H-12), d
1.39 (9H, s; 3ꢀCH3), d 1.23 (6H, s; 2ꢀCH3), d 1.12 (3H, s; CH3), d
0.99 (3H, s, CH3), d 0.91 (3H, s, CH3), d 0.88 (3H, s, CH3), d 0.87
(6H, s, 2ꢀCH3); 13C NMR (75 MHz, CDCl3): 38.5 (C-1), 23.6(C-2),
81.8 (C-3), 38.0 (C-4), 55.8 (C-5), 18.1 (C-6), 34.6 (C-7), 39.7 (C-
8), 49.5 (C-9), 36.9 (C-10), 31.0 (C-11), 71.0 (C-12), 49.2 (C-13),
51.5 (C-14), 31.0 (C-15), 26.2 (C-16), 49.9 (C-17), 16.1 (C-18),
16.5 (C-19), 74.1 (C-20), 21.6 (C-21), 42.8 (C-22), 17.5 (C-23),
44.1 (C-24), 70.6 (C-25), 29.4 (C-26), 29.4 (C-27), 27.9 (C-28),
15.6 (C-29), 17.0 (C-30), 173.0 (Ala-COO), 28.3 (Boc-CH3), 79.7
(Boc-C-CO), 155.0 (Boc-CO-NH), 48.3 (NH-CH), 17.5 (Ala-CH3); IR
(cmꢁ1): 3382 (OH), 2964 (CH3), 1385 (CH3), 1735 (carbonyl group);
HR-ESIMS: m/z 650.4990 [M+H]+ (Calcd for C38H67NO7, 649.4918).
2.1.2.6. (20R)-3b-O-(Boc-L-glycyl)-dammarane-12b, 20, 25-triol (3b).
Yield: 76%. Mp: 218–219 °C. 1H NMR (300 Hz, CDCl3): d 4.54 (1H,
dd, J = 8.7 Hz; H-3), d 3.89 (1H, m; CH-CO), d 3.59 (1H, td; H-12),
d 1.38 (9H, s; 3ꢀCH3), d 1.23 (6H, s; 2ꢀCH3), d 1.12 (3H, s; CH3),
d 0.99 (3H, s, CH3), d 0.91 (3H, s, CH3), d 0.88 (3H, s, CH3), d 0.87
(6H, s, 2ꢀCH3); 13C NMR (75 Hz, CDCl3): 38.6 (C-1), 23.6 (C-2),
82.1 (C-3), 38.0 (C-4), 55.9 (C-5), 18.1 (C-6), 34.7 (C-7), 39.7 (C-
8), 49.9 (C-9), 37.0 (C-10), 31.0 (C-11), 71.0 (C-12), 49.1 (C-13),
51.6 (C-14), 31.2 (C-15), 26.3 (C-16), 50.2 (C-17), 16.2 (C-18),
16.5 (C-19), 74.2 (C-20), 21.7 (C-21), 42.9 (C-22), 17.6 (C-23),
44.1 (C-24), 70.6 (C-25), 29.4 (C-26), 29.5 (C-27), 28.0 (C-28),
15.7 (C-29), 17.1 (C-30), 28.3 (Boc-CH3), 79.9 (Boc-C-CO), 155.7
(Boc-CO-NH), 42.7 (NH-CH-), 170.2 (Gly-COO), 33.9 (Gly-CH),
19.3 (2ꢀCH3); IR (cmꢁ1): 3378 (OH), 2925 (CH3), 1381 (CH3),
1736 (carbonyl group); HR-ESIMS: m/z 658.4652 [M+Na]+ (Calcd
for C37H65NO7, 635.4761).
2.1.2.3. (20R)-12b-O-(Boc- -valyl)-dammarane-3b, 20, 25-triol (2a).
L
Yield: 9%. Mp: 182–183 °C. 1H NMR (300 Hz, CDCl3): d 4.72 (1H, td,
J = 4.7, 10.4 Hz; H-12), d 3.47 (1H, dd; H-3), d 2.30 (2H, q; CH2), d
1.16 (3H, t; J = 7.5 Hz; CH3), d 1.22 (6H, s, 2ꢀCH3), d 1.15 (3H, s,
CH3), d 1.02 (3H, s, CH3), d 0.98 (3H, s, CH3), d 0.97 (3H, s; CH3), d
0.86 (3H, s; CH3), d 0.77 (3H, s; CH3); 13C NMR (75 MHz, CDCl3):
38.9 (C-1), 27.9 (C-2), 78.7 (C-3), 38.8 (C-4), 55.8 (C-5), 18.2 (C-
6), 34.5 (C-7), 39.7 (C-8), 48.7 (C-9), 37.1 (C-10), 28.3 (C-11), 76.5
(C-12), 45.4 (C-13), 52.8 (C-14), 31.7 (C-15), 27.1 (C-16), 50.1 (C-
17), 16.2 (C-18), 16.5 (C-19), 73.7 (C-20), 23.1 (C-21), 42.5 (C-22),
17.9 (C-23), 44.4 (C-24), 71.1 (C-25), 29.1 (C-26), 29.4 (C-27),
28.9 (C-28), 15.4 (C-29), 17.6 (C-30), 28.3 (Boc-CH3), 79.6 (Boc-C-
CO), 155.8 (Boc-CO-NH), 58.9 (NH-CH), 172.0 (Val-COO), 33.9
(Val-CH), 19.3 (Val-CH3); IR (cmꢁ1): 3420 (OH), 2923 (CH3), 1388
(CH3), 1728 (carbonyl group); HR-ESIMS: m/z 678.5303 [M+H]+
(Calcd for C40H71NO7, 677.5231).
2.1.2.7. (20R)-12b-O-(Boc- -prolyl)-dammarane-3b, 20, 25-triol (4a).
L
Yield: 13%. Mp: 176–178 °C. 1H NMR (300 Hz, CDCl3): d 4.73 (1H,
td, J = 4.7, 10.4 Hz; H-12), d 3.46 (1H, dd; H-3), d 2.30 (2H, q;
CH2), d 1.16 (3H, t; J = 7.5 Hz; CH3), d 1.22 (6H, s, 2ꢀCH3), d 1.15
(3H, s, CH3), d 1.02 (3H, s, CH3), d 0.98 (3H, s, CH3), d 0.97 (3H, s;
CH3), d 0.86 (3H, s; CH3), d 0.77 (3H, s; CH3); 13C NMR (75 MHz,
CDCl3): 38.9 (C-1), 27.9 (C-2), 78.7 (C-3), 38.8 (C-4), 55.8 (C-5),
18.2 (C-6), 34.5 (C-7), 39.7 (C-8), 48.7 (C-9), 37.1 (C-10), 28.3 (C-
11), 76.5 (C-12), 45.4 (C-13), 52.8 (C-14), 31.7 (C-15), 27.1 (C-16),
50.1 (C-17), 16.2 (C-18), 16.5 (C-19), 73.7 (C-20), 23.1 (C-21),
42.5 (C-22), 17.9 (C-23), 44.4 (C-24), 71.1 (C-25), 29.1 (C-26),
29.4 (C-27), 28.9 (C-28), 15.4 (C-29), 17.6 (C-30), 28.4 (Boc-CH3),
79.6 (Boc-C), 153.9 (Boc-CO-NH), 59.4 (Pro-NH-CH-CO), 53.6
(Pro-CH), 172.3 (Pro-COO); IR (cmꢁ1): 3415 (OH), 2931 (CH3),
1385 (CH3), 1736 (carbonyl group); HR-ESIMS: m/z 676.5147
[M+H]+ (Calcd for C40H69NO7, 675.5074).
2.1.2.4. (20R)-3b-O-(Boc- -valyl)-dammarane-12b, 20, 25-triol (2b).
L
Yield: 78%. Mp: 231–232 °C. 1H NMR (300 Hz, CDCl3): d 4.52 (1H,
dd, J = 8.1 Hz; H-3), d 4.21 (1H, m; CH-CO), d 3.59 (1H, td; H-12),
d 1.39 (9H, s; 3ꢀCH3), d 1.23 (6H, s; 2ꢀCH3), d 1.12 (3H, s; CH3),
d 0.99 (3H, s, CH3), d 0.91 (3H, s, CH3), d 0.88 (3H, s, CH3), d 0.87
(6H, s, 2ꢀCH3); 13C NMR (75 Hz, CDCl3): 38.6 (C-1), 23.7 (C-2),
82.1 (C-3), 37.9 (C-4), 55.9 (C-5), 18.1 (C-6), 34.7 (C-7), 39.7 (C-
8), 49.9 (C-9), 37.0 (C-10), 31.0 (C-11), 71.0 (C-12), 49.2 (C-13),
51.6 (C-14), 31.2 (C-15), 26.3 (C-16), 50.1 (C-17), 16.2 (C-18),
16.6 (C-19), 74.2 (C-20), 21.8 (C-21), 42.9 (C-22), 17.6 (C-23),
44.1 (C-24), 70.7 (C-25), 29.3 (C-26), 29.5 (C-27), 28.1 (C-28),
15.7 (C-29), 17.1 (C-30), 28.4 (Boc-CH3), 79.6 (Boc-C-CO), 155.8
(O-CO-NH), 58.9 (NH-CH), 172.1 (–COO–), 33.9 (Val-CH), 19.3
(Val-CH3); IR (cmꢁ1): 3420 (OH), 2923 (CH3), 1388 (CH3), 1725
(carbonyl group); HR-ESIMS: m/z 678.5303 [M+H]+ (Calcd for
2.1.2.8. (20R)-3b-O-(Boc- -prolyl)-dammarane-12b, 20, 25-triol (4b).
L
Yield: 73%. Mp: 196–197 °C. 1H NMR (300 Hz, CDCl3): d 4.49 (1H,
dd, J = 8.7 Hz; H-3), d 4.23 (1H, m; CH-CO), d 3.59 (1H, td; H-12),
d 1.39 (9H, s; 3ꢀCH3), d 1.23 (6H, s; 2ꢀCH3), d 1.12 (3H, s; CH3),
d 0.99 (3H, s, CH3), d 0.91 (3H, s, CH3), d 0.88 (3H, s, CH3), d 0.87
(6H, s, 2ꢀCH3); 13C NMR (75 Hz, CDCl3): 38.6 (C-1), 23.7 (C-2),
81.3 (C-3), 37.9 (C-4), 55.9 (C-5), 18.1 (C-6), 34.7 (C-7), 39.8 (C-
8), 49.9 (C-9), 37.0 (C-10), 31.0 (C-11), 71.0 (C-12), 49.1 (C-13),
51.6 (C-14), 31.1 (C-15), 26.3 (C-16), 50.0 (C-17), 16.2 (C-18),
16.5 (C-19), 74.1 (C-20), 21.7 (C-21), 42.9 (C-22), 17.7 (C-23),
44.1 (C-24), 70.7 (C-25), 29.2 (C-26), 29.5 (C-27), 28.1 (C-28),
15.7 (C-29), 17.1 (C-30), 28.4 (Boc-CH3), 79.6 (Boc-C-CO), 153.9
(Boc-CO-NH), 59.4 (Pro-NH-CH), 53.6 (Pro-CH), 172.3 (–COO–); IR
(cmꢁ1): 3414 (OH), 2925 (CH3), 1385 (CH3), 1738 (carbonyl group);
HR-ESIMS: m/z 676.5147 [M+H]+, 698.4966 [M+Na]+ (Calcd for
C40H71NO7, 677. 5231).
2.1.2.5. (20R)-12b-O-(Boc-
L
-glycyl)-dammarane-3b, 20, 25-triol (3a).
C40H69NO7, 675.5074).
Yield: 16%. Mp: 112–114 °C. 1H NMR (300 Hz, CDCl3): d 4.73 (1H,
td, J = 4.7, 10.4 Hz; H-12), d 3.46 (1H, dd; H-3), d 2.30 (2H, q;
CH2), d 1.16 (3H, t; J = 7.5 Hz; CH3), d 1.22 (6H, s, 2ꢀCH3), d 1.15
(3H, s, CH3), d 1.02 (3H, s, CH3), d 0.98 (3H, s, CH3), d 0.97 (3H, s;
CH3), d 0.86 (3H, s; CH3), d 0.77 (3H, s; CH3); 13C NMR (75 MHz,
CDCl3): 38.9 (C-1), 27.9 (C-2), 78.7 (C-3), 38.8 (C-4), 55.8 (C-5),
18.2 (C-6), 34.5 (C-7), 39.7 (C-8), 48.7 (C-9), 37.1 (C-10), 28.3 (C-
11), 76.5 (C-12), 45.4 (C-13), 52.8 (C-14), 31.7 (C-15), 27.1 (C-16),
50.1 (C-17), 16.2 (C-18), 16.5 (C-19), 73.7 (C-20), 23.1 (C-21),
42.5 (C-22), 17.9 (C-23), 44.4 (C-24), 71.1 (C-25), 29.1 (C-26),
2.1.2.9. (20R)-12b-O-(Boc-
L
-methinyl)-dammarane-3b, 20, 25-triol
(5a). Yield: 18%. Mp: 136–138 °C. 1H NMR (300 Hz, CDCl3): d
4.71 (1H, td, J = 4.7, 10.4 Hz; H-12), d 3.47 (1H, dd; H-3), d 2.30
(2H, q; CH2), d 1.16 (3H, t; J = 7.5 Hz; CH3), d 1.22 (6H, s, 2ꢀCH3),
d 1.15 (3H, s, CH3), d 1.02 (3H, s, CH3), d 0.98 (3H, s, CH3), d 0.97
(3H, s; CH3), d 0.86 (3H, s; CH3), d 0.77 (3H, s; CH3); 13C
NMR(75 MHz, CDCl3): 38.9 (C-1), 27.9 (C-2), 78.7 (C-3), 38.8 (C-
4), 55.8 (C-5), 18.2 (C-6), 34.5 (C-7), 39.7 (C-8), 48.7 (C-9), 37.1
(C-10), 28.3 (C-11), 76.5 (C-12), 45.4 (C-13), 52.8 (C-14), 31.7 (C-

206
P. Wang et al. / Steroids 78 (2013) 203–209
15), 27.1 (C-16), 50.1 (C-17), 16.2 (C-18), 16.5 (C-19), 73.7 (C-20),
23.1 (C-21), 42.5 (C-22), 17.9 (C-23), 44.4 (C-24), 71.1 (C-25),
29.1 (C-26), 29.4 (C-27), 28.9 (C-28), 15.4 (C-29), 17.6 (C-30),
28.3 (Boc-CH3), 79.9 (Boc-C-CO), 155.3 (Boc-CO-NH), 53.5 (NH-
CH-), 172.0 (Met-COO), 30.0, 29.5 (Met-CH), 15.4 (Met-CH3); IR
(cmꢁ1): 3393 (OH), 2962 (CH3), 1383 (CH3), 1730 (carbonyl group);
HR-ESIMS: m/z 710.5024 [M+H]+ (Calcd for C40H71NO7S, 709.4951).
3), 37.9 (C-4), 55.9 (C-5), 18.1 (C-6), 34.6 (C-7), 39.7 (C-8), 49.9
(C-9), 36.9 (C-10), 30.9 (C-11), 71.1 (C-12), 49.3 (C-13), 51.5 (C-
14), 31.0 (C-15), 26.2 (C-16), 50.0 (C-17), 16.1 (C-18), 16.6 (C-19),
74.1 (C-20), 21.6 (C-21), 42.7 (C-22), 17.6 (C-23), 44.1 (C-24),
70.7 (C-25), 29.3 (C-26), 29.4 (C-27), 28.0 (C-28), 15.6 (C-29),
17.1 (C-30), 28.4 (Boc-CH3), 28.3 (Boc-CH3), 79.7, 79.0 (Boc-C-
CO), 156.1, 155.5 (Boc-CO), 60.4 (Lys-NH-CH-CO), 40.1 (NH-CH),
175.1 (Lys-COO), 22.4–29.0 (Lys-CH2); IR (cmꢁ1): 3266 (OH),
2951 (CH3), 1387 (CH3), 1735 (carbonyl group); HR-ESIMS: m/z
807.5913 [M+H]+ (Calcd for C46H82N2O9, 806.6020).
2.1.2.10. (20R)-3b-O-(Boc-L-methinyl)-dammarane-12b, 20, 25-triol
(5b). Yield: 75%. Mp: 157–159 °C. 1H NMR (300 Hz, CDCl3): d 4.53
(1H, dd, J = 8.1 Hz; H-3), d 4.33 (1H, m; CH-CO), d 3.62 (1H, td; H-
12), d 1.38 (9H, s; 3ꢀCH3), d 1.23 (6H, s; 2ꢀCH3), d 1.12 (3H, s; CH3),
d 0.99 (3H, s, CH3), d 0.91 (3H, s, CH3), d 0.88 (3H, s, CH3), d 0.87 (6H,
s, 2ꢀCH3); 13C NMR (75 Hz, CDCl3): 38.5 (C-1), 23.6 (C-2), 82.3 (C-
3), 38.0 (C-4), 55.9 (C-5), 18.1 (C-6), 34.7 (C-7), 39.7 (C-8), 49.9 (C-
9), 37.0 (C-10), 31.0 (C-11), 71.1 (C-12), 49.1 (C-13), 51.6 (C-14),
31.2 (C-15), 26.3 (C-16), 50.0 (C-17), 16.2 (C-18), 16.5 (C-19),
74.3 (C-20), 21.7 (C-21), 42.9 (C-22), 17.6 (C-23), 44.1 (C-24),
70.7 (C-25), 29.4 (C-26), 29.5 (C-27), 28.0 (C-28), 15.7 (C-29),
17.1 (C-30), 28.3 (Boc-CH3), 79.9 (Boc-C-CO), 155.3 (Boc-CO-NH),
53.5 (NH-CH), 172.0 (Met-COO), 30.0, 29.5 (Met-CH), 15.4 (Met-
CH3); IR (cmꢁ1): 3392 (OH), 2963 (CH3), 1385 (CH3), 1733 (car-
bonyl group); HR-ESIMS: m/z 710.5024 [M+H]+ (Calcd for C40H71-
NO7S, 709.4951).
2.1.2.14. (20R)-3b-O-(Boc-L-arginyl)-dammarane-12b, 20, 25-triol
(8b). Yield: 63%. Mp: 179–181 °C. 1H NMR (300 Hz, CDCl3): d
7.70 (2H, NH2), d 7.28 (H, -NH), d 4.53 (1H, t, J = 8.1 Hz; H-3), d
4.28 (1H, m; CH-CO), d 3.58 (1H, td; H-12), d 1.38 (9H, s; 3ꢀCH3),
d 1.23 (6H, s; 2ꢀCH3), d 1.12 (3H, s; CH3), d 0.99 (3H, s, CH3), d
0.91 (3H, s, CH3), d 0.88 (3H, s, CH3), d 0.87 (6H, s, 2ꢀCH3); 13C
NMR (75 Hz, CDCl3): 38.5 (C-1), 23.7 (C-2), 82.6 (C-3), 37.9 (C-4),
55.9 (C-5), 18.1 (C-6), 34.6 (C-7), 39.7 (C-8), 49.9 (C-9), 37.0 (C-
10), 29.6 (C-11), 71.0 (C-12), 49.4 (C-13), 51.5 (C-14), 30.8 (C-15),
26.8 (C-16), 50.0 (C-17), 16.1 (C-18), 16.5 (C-19), 74.2 (C-20),
21.7 (C-21), 42.8 (C-22), 17.6 (C-23), 44.1 (C-24), 70.7 (C-25),
29.3 (C-26), 29.4 (C-27), 28.0 (C-28), 15.6 (C-29), 17.1 (C-30),
80.5 (Boc-C-CO), 159.5 (Boc-CO-NH), 60.3 (Arg-NH-CH), 172.1
(Arg-COO), 40.4 (NH-CH), 28.3 (Boc-CH3); IR (cmꢁ1): 3264 (OH),
2968 (CH3), 1367 (CH3), 1716 (carbonyl group); HR-ESIMS: m/z
733.5630 [MꢁH]+ (Calcd for C41H74N4O7, 734.5558).
2.1.2.11. (20R)-3b-O-(Boc-L-phenylalanyl)-dammarane-12b, 20, 25-
triol (6a). Yield: 14%. Mp: 171–172 °C. 1H NMR (300 Hz, CDCl3): d
4.69 (1H, td, J = 4.7, 10.4 Hz; H-12), d 3.45 (1H, dd; H-3), d 2.30
(2H, q; CH2), d 1.16 (3H, t; J = 7.5 Hz; CH3), d 1.22 (6H, s, 2ꢀCH3),
d 1.15 (3H, s, CH3), d 1.02 (3H, s, CH3), d 0.98 (3H, s, CH3), d 0.97
(3H, s; CH3), d 0.86 (3H, s; CH3), d 0.77 (3H, s; CH3); 13C NMR
(75 MHz, CDCl3): 38.9 (C-1), 27.9 (C-2), 78.7 (C-3), 38.8 (C-4),
55.8 (C-5), 18.2 (C-6), 34.5 (C-7), 39.7 (C-8), 48.7 (C-9), 37.1 (C-
10), 28.3 (C-11), 76.5 (C-12), 45.4 (C-13), 52.8 (C-14), 31.7 (C-15),
27.1 (C-16), 50.1 (C-17), 16.2 (C-18), 16.5 (C-19), 73.7 (C-20),
23.1 (C-21), 42.5 (C-22), 17.9 (C-23), 44.4 (C-24), 71.1 (C-25),
29.1 (C-26), 29.4 (C-27), 28.9 (C-28), 15.4 (C-29), 17.6 (C-30),
28.3 (Boc-CH3), 79.9 (Boc-C-CO), 155.0 (Boc-CO-NH), 60.3 (Phe-
NH-CH), 171.8 (Phe-COO), 136.1, 129.3, 128.5, 126.9 (Phe-CH); IR
(cmꢁ1): 3266 (OH), 2956 (CH3), 1388 (CH3), 1731 (carbonyl group);
HR-ESIMS: m/z 726.5303 [M+H]+ (Calcd for C44H71NO7, 725.5231).
2.1.3. General procedure for deprotection compounds 1c–6c
To a solution of the Boc-protected compounds 1b–6b (50 mg) in
toluol (30 mL), silica gel (100 mg) was added. The mixture was al-
lowed to stir at 115 °C for 6 h. After completion of the reaction (as
monitored by TLC) the solution was removed under reduced pres-
sure. The residue was washed with methanol until no target sub-
stance could be detected and concentrated under reduced
pressure to give the crude product. The crude products were chro-
matographed using silica gel and eluted with ethyl acetate/metha-
nol (15:1) to afford corresponding products 1c–6c. The yield of this
step was between 65% and 76%.
2.1.3.1. (20R)-3b-O-( -alanyl)-dammarane-12b, 20, 25-triol (1c).
L
Yield: 73%. Mp: 260–262 °C. 1H NMR (300 Hz, CDCl3): d 5.76 (1H,
m; NH-C), d 4.42 (1H, dd, J = 8.1 Hz; H-3), d 3.53 (1H, td; H-12), d
1.23 (6H, s; 2ꢀCH3), d 1.12 (3H, s; CH3), d 0.99 (3H, s, CH3), d
0.91 (3H, s, CH3), d 0.88 (3H, s, CH3), d 0.86 (6H, s, 2ꢀCH3); 13C
NMR (75 Hz, CDCl3): 40.2 (C-1), 23.5 (C-2), 80.2 (C-3), 38.1 (C-4),
55.1 (C-5), 18.2 (C-6), 34.3 (C-7), 40.4 (C-8), 49.5 (C-9), 36.6 (C-
10), 30.8 (C-11), 69.8 (C-12), 49.2 (C-13), 51.1 (C-14), 31.2 (C-15),
26.6 (C-16), 49.8 (C-17), 16.0 (C-18), 16.5 (C-19), 72.3 (C-20),
22.4 (C-21), 42.9 (C-22), 17.6 (C-23), 44.6 (C-24), 69.0 (C-25),
29.4 (C-26), 29.5 (C-27), 27.8 (C-28), 15.4 (C-29), 17.0 (C-30),
48.7 (Ala-CH), 175.4 (Ala-COO), 15.0 (Ala-CH3); IR (cmꢁ1): 3382
(OH), 2964 (CH3), 1385 (CH3), 1735 (carbonyl group); HR-ESIMS:
m/z 550.4466 [M+H]+ (Calcd for C33H59NO5, 549.4393).
2.1.2.12. (20R)-3b-O-(Boc-L-phenylalanyl)-dammarane-12b, 20, 25-
triol (6b). Yield: 66%. Mp: 186–187 °C. 1H NMR (300 Hz, CDCl3):
7.14 (1H, t, J = 7.6 Hz), 7.26–7.29 (m, 5H), d 4.50 (1H, t, J = 8.1 Hz;
H-3), d 4.10 (1H, m; CH-CO), d 3.57 (1H, td; H-12), d 1.38 (9H, s;
3ꢀCH3), d 1.23 (6H, s; 2ꢀCH3), d 1.12 (3H, s; CH3), d 0.99 (3H, s,
CH3), d 0.91 (3H, s, CH3), d 0.88 (3H, s, CH3), d 0.87 (6H, s,
2ꢀCH3); 13C NMR (75 Hz, CDCl3): 38.5 (C-1), 23.5 (C-2), 82.3 (C-
3), 37.8 (C-4), 55.9 (C-5), 18.1 (C-6), 34.7 (C-7), 39.7 (C-8), 50.0
(C-9), 37.0 (C-10), 31.0 (C-11), 71.0 (C-12), 49.9 (C-13), 51.5 (C-
14), 31.1 (C-15), 26.4 (C-16), 50.6 (C-17), 16.1 (C-18), 16.4 (C-19),
74.2 (C-20), 21.6 (C-21), 42.7 (C-22), 17.6 (C-23), 44.1 (C-24),
70.6 (C-25), 29.4 (C-26), 29.5 (C-27), 27.9 (C-28), 15.6 (C-29),
17.0 (C-30), 28.3 (Boc-CH3), 79.9 (Boc-C-CO), 155.0 (Boc-CO-NH),
60.3 (Phe-NH-CH), 171.8 (Phe-COO), 136.1, 129.3, 128.5, 126.9
(Phe-CH); IR (cmꢁ1): 3266 (OH), 2956 (CH3), 1388 (CH3), 1733 (car-
bonyl group); HR-ESIMS: m/z 726.5303 [M+H]+ (Calcd for
2.1.3.2. (20R)-3b-O-( -valyl)-dammarane-12b, 20, 25-triol (2c).
L
Yield: 68%. Mp: 267–268 °C. 1H NMR (300 Hz, CDCl3): d 4.60 (1H,
dd, J = 7.8 Hz; H-3), d 3.66 (1H, m; CH-CO), d 3.57 (1H, td; H-12),
d 1.39 (9H, s; 3ꢀCH3), d 1.23 (6H, s; 2ꢀCH3), d 1.12(3H, s; CH3), d
0.99 (3H, s, CH3), d 0.91 (3H, s, CH3), d 0.88 (3H, s, CH3), d 0.87
(6H, s, 2ꢀCH3); 13C NMR (75 Hz, CDCl3): 39.0 (C-1), 23.7 (C-2),
84.3 (C-3), 38.2 (C-4), 57.2 (C-5), 18.9 (C-6), 35.8 (C-7), 39.7 (C-
8), 50.9 (C-9), 37.0 (C-10), 32.0 (C-11), 71.9 (C-12), 49.2 (C-13),
52.6 (C-14), 31.2 (C-15), 27.1 (C-16), 51.2 (C-17), 16.2 (C-18),
16.7 (C-19), 74.7 (C-20), 22.4 (C-21), 42.9 (C-22), 17.8 (C-23),
44.0 (C-24), 71.5 (C-25), 29.1 (C-26), 29.4 (C-27), 28.8 (C-28),
C44H71NO7, 725.5231).
2.1.2.13. (20R)-3b-O-(Boc-L-lysyl)-dammarane-12b, 20, 25-triol (7b).
Yield: 51%. Mp: 131–133 °C. 1H NMR(300 Hz, CDCl3): d 4.64 (1H, t,
J = 8.1 Hz; H-3), d 4.44 (1H, m; CH-CO), d 3.52 (1H, td; H-12), d 1.39
(18H, s; 6ꢀCH3), d 1.23 (6H, s; 2ꢀCH3), d 1.12 (3H, s; CH3), d 0.99
(3H, s, CH3), d 0.91 (3H, s, CH3), d 0.88 (3H, s, CH3), d 0.87 (6H, s,
2ꢀCH3); 13C NMR (75 Hz, CDCl3): 38.5 (C-1), 23.6 (C-2), 82.0 (C-

P. Wang et al. / Steroids 78 (2013) 203–209
207
15.8 (C-29), 17.2 (C-30), 60.4 (Val-NH-CH), 172.7 (Val-COO), 33.9
2.2. Cytotoxicity evaluation
(Val-CH), 19.2 (Val-CH3); IR (cmꢁ1): 3420 (OH), 2923 (CH3), 1388
(CH3), 1725 (carbonyl group); HR-ESIMS: m/z 578.4779 [M+H]+
(Calcd for C35H63NO5, 577.4706).
Cytotoxicities of these derivatives were evaluated on six differ-
ent human tumor cell lines (MCF-7 cell line, A549 cell line, Lovo
cell line, HCT-116 cell line, HT29 cell line and U87 cell line) using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) as-
say. 25-OH-PPD and 5-Fu were used as references. In short,
1 ꢀ 106 cells/well were seeded into 96-well plates, 24 h later, the
cells were treated with serial dilutions of the compounds (0–
2.1.3.3. (20R)-3b-O-( -glycyl)-dammarane-12b, 20, 25-triol (3c).
L
Yield: 76%. Mp: 227–228 °C. 1H NMR (300 Hz, CDCl3):d 4.46 (1H,
dd, J = 8.1 Hz; H-3), d 3.62 (1H, m; NH-C), d 3.53 (1H, td; H-12), d
1.23 (6H, s, 2ꢀCH3), d 1.12 (3H, s, CH3), d 0.99 (3H, s, CH3), d 0.91
(3H, s, CH3), d 0.88 (3H, s, CH3), d 0.86 (6H, s, 2ꢀCH3); 13C NMR
(75 Hz, CDCl3): 40.2 (C-1), 23.6 (C-2), 82.0 (C-3), 38.1 (C-4), 55.8
(C-5), 18.1 (C-6), 34.6 (C-7), 40.4 (C-8), 49.9 (C-9), 36.9 (C-10),
31.0 (C-11), 70.9 (C-12), 49.2 (C-13), 51.4 (C-14), 31.1 (C-15),
26.7 (C-16), 50.4 (C-17), 16.2 (C-18), 16.5 (C-19), 74.1 (C-20),
22.6 (C-21), 42.7 (C-22), 17.5 (C-23), 44.1 (C-24), 70.4 (C-25),
29.3 (C-26), 29.6 (C-27), 28.0 (C-28), 15.6 (C-29), 17.1 (C-30),
47.6 (Gly-CH), 171.1 (Gly-COO); IR (cmꢁ1): 3378 (OH), 2925
(CH3), 1381 (CH3), 1737 (carbonyl group); HR-ESIMS: m/z
558.4129 [M+Na]+ (Calcd for C32H57NO5, 535.4237).
100 lM) for another 48 h. Ten microliters of MTT solution (5 mg/
mL) was added to each well, and the tumor cells were incubated
at 37 °C in a humidified atmosphere of 5% CO2 air for 4 h. At the
end of incubation, the growth medium was removed and replaced
with 100 lL of DMSO (at room temperature). After agitating on a
vortex for 10 min, the absorbance was determined at 492 nm as
reference on a Bio-Rad (model 550) microplate reader to calculate
50% inhibition concentration (IC50). DMSO and MTT were pur-
chased from Sigma Chemical Co., Ltd., USA.
2.1.3.4. (20R)-3b-O-(L-prolyl)-dammarane-12b, 20, 25-triol (4c).
3. Results and discussion
Yield: 75%. Mp: 243–245 °C. 1H NMR: d 4.71(1H, dd, J = 8.1 Hz;
H-3), d 4.23 (1H, m; CH-CO), d 3.58 (1H, td; H-12), d 1.23 (6H, s;
2ꢀCH3), d 1.12 (3H, s; CH3), d 0.99 (3H, s, CH3), d 0.91 (3H, s,
CH3), d 0.88 (3H, s, CH3), d 0.87 (6H, s, 2ꢀCH3); 13C NMR: 38.6
(C-1), 23.7 (C-2), 82.2 (C-3), 37.9 (C-4), (C-5), 18.1 (C-6), 34.7 (C-
7), 39.8 (C-8), 49.9 (C-9), 37.0 (C-10), 31.0 (C-11), 70.9 (C-12),
49.1 (C-13), 51.5 (C-14), 31.1 (C-15), 26.3 (C-16), 50.4 (C-17),
16.2 (C-18), 16.5 (C-19), 74.0 (C-20), 21.7 (C-21), 42.9 (C-22),
17.7 (C-23), 44.1 (C-24), 70.4 (C-25), 29.2 (C-26), 29.5 (C-27),
28.1 (C-28), 15.7 (C-29), 17.1 (C-30), 59.6 (Pro-NH-CH), 46.3 (NH-
CH), 172.3 (Pro-COO), 25.0, 30.2 (Pro-CH2); IR (cmꢁ1): 3414 (OH),
2925 (CH3), 1385 (CH3), 1730 (carbonyl group); HR-ESIMS: m/z
576.4623 [M+H]+ (Calcd for C35H61NO5, 575.4550).
The results of cytotoxic studies are shown in Table 2, which is
expressed by the IC50 values. The results showed that most of the
derivatives displayed potent or similar cytotoxic activity in vitro
compared with 25-OH-PPD. Cell growth inhibition ability of these
synthesized analogs on MCF-7, A549, Lovo, HCT-116, HT-29 and U-
87 cell lines are shown in Fig. 2.
Screening the cytotoxicity of the tested compounds on A549
cell, where 25-OH-PPD was used as standard drug (IC50 = 34.25 -
lM), the results showed that the proliferation of A549 cells was
suppressed dose-dependently after treatment with 1a–6c for
48 h, and compounds 1c, 2c, 3c, 4c, 5c, 6c and 8b had more potent
anti-tumor activities than 25-OH-PPD (53.3%) with cell viabilities
of 6.1%, 15.2%, 13.1%, 20.5%, 25.4%, 19.2% and 12.6% at 30 lM,
2.1.3.5. (20R)-3b-O-(
L
-methinyl)-dammarane-12b, 20, 25-triol (5c).
respectively. The results indicated that most amino acid com-
pounds showed stronger activity than the protection of the amino
acid derivatives. The differences were also distinguishable between
the two kinds of derivatives for the protection of the amino acids at
C(3) and C(12). Compounds 1a–6a showed weak activity
Yield: 69%. Mp: 169–170 °C. 1H NMR (300 Hz, CDCl3): d 4.53 (1H,
dd, J = 8.1 Hz; H-3), d 4.33 (1H, m; CH-CO), d 3.62 (1H, td; H-12),
d 1.38 (9H, s; 3ꢀCH3), d 1.23 (6H, s; 2ꢀCH3), d 1.12 (3H, s; CH3),
d 0.99 (3H, s, CH3), d 0.91 (3H, s, CH3), d 0.88 (3H, s, CH3), d 0.87
(6H, s, 2ꢀCH3); 13C NMR (75 Hz, CDCl3): 38.8 (C-1), 23.4 (C-2),
80.2 (C-3), 38.0 (C-4), 55.1 (C-5), 18.1 (C-6), 34.7 (C-7), 39.7 (C-
8), 49.9 (C-9), 37.0 (C-10), 31.0 (C-11), 71.0 (C-12), 49.1 (C-13),
51.6 (C-14), 31.2 (C-15), 26.3 (C-16), 50.0 (C-17), 16.2 (C-18),
16.5 (C-19), 74.3 (C-20), 21.7 (C-21), 42.9 (C-22), 17.6 (C-23),
44.1 (C-24), 70.6 (C-25), 29.4 (C-26), 29.5 (C-27), 28.0 (C-28),
15.7 (C-29), 17.1 (C-30), 53.3 (Met-NH-CH), 175.2 (Met-COO),
29.8, 34.0 (Met-CH), 15.4 (Met-CH3); IR (cmꢁ1): 3393 (OH), 2962
(CH3), 1383 (CH3), 1730 (carbonyl group); HR-ESIMS: m/z
610.4500 [M+H]+ (Calcd for C35H63NO5S, 609.4427).
(IC50 > 50
lM) but compounds 1b–8b exhibited better activities
than 25-OH-PPD with IC50 values in the range of 6.21–24.73
l
M.
For the human breast cancer cells MCF-7, compounds 1b–7b,
4c, 6c exhibited more potent anti-tumor activity than 25-OH-PPD
(IC50, 48.96
showed excellent anti-tumor activity with 15.03%, 10.45% and
11.37% cell viabilities remaining (25-OH-PPD 51.35%) at 30 M,
lM). As shown in Fig. 2, compounds 8b, 1c and 5c
l
respectively. The data from anti-tumor tests of the other four cells
showed similar results as in A549 and MCF-7 cells. Compounds 1c–
6c and 8b exhibited weaker anti-tumor activities than parent com-
pound 25-OH-PPD. In conclusion, a series of amino acid esters of
25-OH-PPD derivatives were investigated in their ability to inhibit
the proliferation of the human breast cancer MCF-7 cell line, hu-
man lung tumor cells A549 and three human colorectal cancer cells
in the current study. Some derivatives demonstrated good antipro-
liferative activities, in some cases are better than 25-OH-PPD. The
most promising compounds in this series of synthetic compounds
were 1c, 2c, 3c, 4c and 8b, which showed strong antiproliferative
activities.
The conclusion we could get from the current data is, for all es-
ters, activity increases when C(3) is substituted with the protected
amino acids and decreases when C(12) is substituted. Data sug-
gested that hydroxyl substitutions of ginseng sapogenin were cru-
cial and a subtle change of 25-OH-PPD could result in a large
variability of anti-tumor activity (Fig. 3). Previous studies on the
structure–activity relationships (SAR) of ginsenosides suggested
2.1.3.6. (20R)-3b-O-(L-phenylalanyl)-dammarane-12b, 20, 25-triol
(6c). Yield: 65%. Mp: 181–182 °C. 1H NMR (300 Hz, CDCl3): 7.14
(t, J = 7.6 Hz, 1H), 7.26–7.29 (m, 5H), d 4.43 (1H, t, J = 8.1 Hz; H-
3), d 4.10 (1H, m; CH-CO), d 3.75 (1H, td; H-12), d 1.23 (6H, s;
2ꢀCH3), d 1.12 (3H, s; CH3), d 0.99 (3H, s, CH3), d 0.91 (3H, s,
CH3), d 0.88 (3H, s, CH3), d 0.87 (6H, s, 2ꢀCH3); 13C NMR (75 Hz,
CDCl3): 38.5 (C-1), 23.6 (C-2), 81.8 (C-3), 37.9 (C-4), 55.9 (C-5),
18.1 (C-6), 34.6 (C-7), 39.7 (C-8), 50.0 (C-9), 36.9 (C-10), 31.0 (C-
11), 70.9 (C-12), 49.9 (C-13), 51.5 (C-14), 31.1 (C-15), 26.4 (C-16),
50.2 (C-17), 16.1 (C-18), 16.4 (C-19), 74.1 (C-20), 21.6 (C-21),
42.7 (C-22), 17.6 (C-23), 44.1 (C-24), 70.5 (C-25), 29.4 (C-26),
29.5 (C-27), 27.9 (C-28), 15.6 (C-29), 17.0 (C-30), 55.1 (Phe-NH-
CH), 174.2 (Phe-COO), 137.1, 129.3, 128.5, 126.8 (Phe-CH); IR
(cmꢁ1): 3266 (OH), 2956 (CH3), 1388(CH3), 1731(carbonyl group);
HR-ESIMS: m/z 626.4779 [M+H]+ (Calcd for C39H63NO5, 625.4706).

208
P. Wang et al. / Steroids 78 (2013) 203–209
Table 2
Antiproliferative activity (IC50 SD values,
l
M, 48 h) of 25-OH-PPD and synthetic derivatives substituted with amino acids in six different human tumor cell lines.
Compound
A549
MCF-7
Lovo
HCT-116
HT29
U87
1a
>100
>50
>100
>50
>50
>50
2a
>50
>50
>100
>50
>50
>50
3a
>50
>100
>50
>50
>50
>50
4a
>50
>50
>50
>50
>50
>50
5a
>100
>50
>100
>50
>50
>50
6a
>50
>50
>100
>50
>50
>50
1b
2b
3b
4b
5b
6b
7b
8b
1c
2c
3c
4c
5c
6c
37.81 1.06
15.43 0.51
16.62 0.26
19.13 0.31
21.64 0.70
19.85 0.54
24.73 0.75
6.21 0.12
2.77 0.09
8.23 0.56
6.11 0.31
11.82 0.56
12.97 0.63
16.35 1.15
12.83 0.36
34.25 1.96
25.43 0.93
15.71 0.38
20.54 0.33
21.02 0.51
18.94 0.62
28.42 0.45
40.31 1.55
6.82 0.24
2.93 0.11
9.18 0.88
8.94 0.21
12.85 0.88
4.72 0.21
14.13 0.46
16.28 0.65
48.91 2.36
30.41 1.38
32.23 0.77
22.76 0.46
21.62 0.43
41.27 1.56
29.58 1.56
13.64 0.22
0.53 0.06
5.91 0.16
11.63 0.64
10.74 0.28
9.21 0.91
19.26 0.87
11.62 0.72
12.73 0.51
45.21 2.33
34.39 1.15
24.05 0.74
21.81 0.91
22.03 0.35
29.16 1.33
23.63 1.16
36.82 1.23
4.94 0.11
6.83 0.16
10.71 0.61
9.56 0.24
11.51 0.82
26.13 1.13
14.32 0.53
36.17 1.33
32.33 2.11
32.17 1.16
17.32 0.39
22.38 0.41
20.54 0.34
25.97 0.81
10.61 0.57
21.53 0.28
10.25 0.86
6.57 0.26
13.34 0.58
9.88 0.11
1.88 0.06
16.83 1.36
5.26 0.18
18.83 1.06
26.31 1.88
28.93 0.73
24.96 0.35
13.34 0.22
17.39 0.25
43.13 1.81
5.54 0.16
22.83 0.31
10.44 0.77
3.79 0.08
5.93 0.12
8.59 0.15
14.74 0.23
20.75 1.86
2.43 0.11
38.15 1.26
26.61 0.96
5-Fu
25-OH-PPD
Fig. 2. Antiproliferative activity of synthetic derivatives 1c–6c and 8b in six different human tumor cell lines (A549, MCF-7, Lovo, HCT-116, HT-29 and U-87) at 10, 30 and
100 M in vitro.
l
that the anti-tumor activities were affected by structural types of
dammarane saponin, and the numbers of sugar moieties and sub-
stituent groups in the side chain of the aglycone also played crucial
roles for their anti-tumor activities. Obviously, the derivatives of
deprotected amino acids show the highest cytotoxic activity. We
also could observe a strong correlation between the type of ester

P. Wang et al. / Steroids 78 (2013) 203–209
209
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derivatives (C(12) is substituted with the protected amino acids: 1a–6a, P < 0.001;
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Acknowledgements
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This work was financially supported by the ‘‘11th Five-Year’’
State Plan on Technology Major Projects (2009ZX09102-114),
Technology Platform of Industrialization Chromatographic Prepa-
ration for Standard Extract of Traditional Chinese Medicine
(2010ZX09401-304-105B) and the National Science Foundation of
China (Grant number 81273389). We are grateful to the Analytical
Center of Shenyang Pharmaceutical University for identification of
the measurements of NMR, IR and HR-ESIMS.
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triterpenoid (lupeol) derivatives and their in vivo antihyperglycemic and
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