Enmein-type diterpenoid analogs from natural kaurene-type oridonin: Synthesis and their antitumor biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2013.04.012

A series of enmein-type diterpenoid analogs (11-20) derived from natural kaurene-type diterpenoid oridonin were synthesized and biologically evaluated. All target compounds showed improved anti-proliferative activities against four human cancer cell lines compared with natural oridonin and parent compound 10. Some compounds were more potent than positive control Taxol. Furthermore, mechanistic investigation showed that the representative compound 17 affected cell cycle and induced apoptosis at low micro-molar level in human hepatoma Bel-7402 cells, via an oxidative stress triggered mitochondria-related caspase-dependent pathway.

Full text of DOI:10.1016/j.ejmech.2013.04.012

European Journal of Medicinal Chemistry 64 (2013) 215e221
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Enmein-type diterpenoid analogs from natural kaurene-type
oridonin: Synthesis and their antitumor biological evaluation
,
b
,
,
b
,
,
,
Dahong Li a 1, Shengtao Xu a 1, Hao Cai a b, Lingling Pei a, Hengyuan Zhang a b, Lei Wang a,
Hequan Yao a b, Xiaoming Wu a b, Jieyun Jiang c, Yijun Sun d, Jinyi Xu a
,
,
,b,
*
a Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China
b State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China
c Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 40536, USA
d Drug Screening Center, Nanjing KeyGen Biotech. Co. Ltd., Nanjing 210012, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of enmein-type diterpenoid analogs (11e20) derived from natural kaurene-type diterpenoid
oridonin were synthesized and biologically evaluated. All target compounds showed improved anti-
proliferative activities against four human cancer cell lines compared with natural oridonin and
parent compound 10. Some compounds were more potent than positive control Taxol. Furthermore,
mechanistic investigation showed that the representative compound 17 affected cell cycle and induced
apoptosis at low micro-molar level in human hepatoma Bel-7402 cells, via an oxidative stress triggered
mitochondria-related caspase-dependent pathway.
Received 24 January 2013
Received in revised form
3 April 2013
Accepted 3 April 2013
Available online 11 April 2013
Keywords:
Enmein-type diterpenoid
Oridonin
Ó 2013 Elsevier Masson SAS. All rights reserved.
Anti-proliferative activity
Cell cycle analysis
Apoptosis
1. Introduction
diterpenoids. In this regard, a practical and convenient synthetic
methodology for enmein-type diterpenoid analogs is of great sig-
Diterpenoids have attracted considerable attention because of
their unique structural scaffold and interesting biological proper-
ties [1e9]. Enmein (1), the active ent-kaurene diterpenoid, was
firstly isolated from Japanese folk medicine ‘enmeiso’. Recently, a lot
of enmein-type diterpenoids were isolated and characterized from
Isodon species, such as isodocarpin (2), nodosin (3), epinodosin (4),
carpalasionin (5), sculponeatin A (6), sculponeatin N (7), and
macrocalyxoformin B (8) (Fig. 1) [10e15]. Most of these diterpe-
noids were found to exhibit anti-inflammatory, antibacterial,
antiviral, and more importantly, selective antitumor activities with
very low toxicity to normal cells or tissues [15e17]. Given the
intricate ring systems and many chiral centers, total synthesis or
isolation and purification from natural plants with low yields could
not meet the need for the medicinal and pharmaceutical studies for
nificance. We used a strategy that combined traditional isolation
with the de novo synthetic method to prepare complex target
natural product from commercially available natural products with
relevant skeletons. In our previous studies, we disclosed some
enmein-type and spirolactone-type diterpenoid analogs synthe-
sized from oridonin (9) [18], which exhibited potent antitumor
activities against several human cancer cell lines in vitro and in vivo.
The activity of enmein-type diterpenoid compound 10 (Table 1)
was less potent than spirolactone-type diterpenoid derivatives,
which were firstly selected for further structural modification [19].
Notably, natural enmein-type diterpenoids showed potent anti-
proliferative activities against human tumor cells [10e15], which
were promising for further investigation. Since the 14-O-de-
rivatives of 9 (Scheme 1) exhibited stronger cytotoxicity than their
parent compound 9 [20e22], we wonder whether the similar
modification on enmein-type diterpenoid could improve the anti-
cancer activity of enmein-type diterpenoid. Herein, a series of
novel 14-O-derivatives of enmein-type diterpenoid were designed
and synthesized. The anti-proliferative activities of these com-
pounds were tested by MTT assay. Furthermore, the mechanisms of
* Corresponding author. Department of Medicinal Chemistry, China Pharma-
ceutical University, 24 Tong Jia Xiang, Nanjing 210009, China. Tel.: þ86 25
83271445; fax: þ86 25 83302827.
E-mail address: jinyixu@china.com (J. Xu).
Co-first authors.
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.012

216
D. Li et al. / European Journal of Medicinal Chemistry 64 (2013) 215e221
O
O
O
O
O
H
O
O
O
H
O
O
O
O
H
H
H
H
O
O
O
O
OH
OH
HO
H
H
OH
OH
OH
OH
2 isodocarpin
3 nodosin
1 enmein
4 epinodosin
O
O
O
O
O
O
O
O
O
H
O
H
O
H
O
H
O
O
OH
O
O
OH
OH
HO
H
H
OH
H
O
H
O
O
AcO
5 carpalasionin
6 sculponeatin A
7 sculponeatin N 8 macrocalyxoformin B
Fig. 1. Several enmein-type diterpenoids (1e8) isolated from natural plants.
influence on cell cycle progression, induction of apoptosis and ef-
fect on apoptosis-related proteins by representative compound 17
were investigated.
yields. Flash chromatography was used for purification of target
compounds.
2.2. MTT assay and SAR study
2. Results and discussion
The anti-proliferative activities of enmein-type diterpenoid
analogs 11e20 in vitro against four different kinds of human cancer
cell lines were determined by MTT assay and compared with ori-
donin, parent compound 10 and positive control Taxol (Table 1). All
derivatives exhibited much more potent anti-proliferative activities
than oridonin and parent compound 10; and some were even su-
perior to Taxol in vitro. For example, compounds 13, 14 and 16e19
showed stronger anti-proliferative activities in K562 cell line, and
2.1. Chemistry
As shown in Scheme 1, starting from commercially available
kaurene-type diterpenoid oridonin (9), enmein-type diterpenoid
analog 10 was obtained in nearly quantitative yield using the
method previously well established [23,24] and reported by our
research group [18]. Compound 10 could be a good building block
to construct libraries of enmein-type diterpenoid analogs. The
structure of compound 10 was confirmed by the key HMBC corre-
lations of H-20 with C-6, C-1, C-9, C-10 and C-5 (Fig. 2).
compound 14 was the most potent one with IC50 value of 0.14 mM.
Compounds 14, 16 and 18 exhibited more promising activities
against MGC-803 cancer cells in vitro with IC50 values of 0.61, 0.64
Treatment of compound 10 with corresponding acids in the
presence of DMAP/EDCI in DCM at room temperature for 8e12 h
gave enmein-type diterpenoid analogs 11e20 in moderate to good
and 0.80
showed the strongest activity (IC50, 0.28
enmein-type diterpenoid analogs 11e20 against Bel-7402 cells
ranged from 0.85 to 1.97 M. The above results demonstrated that
mM, respectively. For CaEs-17 cell line, compound 17
mM). The IC50 values of
m
the esterification of 14-hydroxy of enmein-type diterpenoid could
significantly improve the anti-proliferative activities of the parent
compounds. It would be an important SAR in the research of
modification of diterpenoids.
Table 1
a
IC50 values (
m
M) of enmein-type diterpenoid derivatives (11e20) against human
cancer cell lines.b
Compd.
K562
MGC-803
CaEs-17
Bel-7402
Among derivatives 11e20, the SAR showed that when the sub-
stitutions R were alkyl groups (compounds 11 and 12), their IC50
values slightly declined with the extension of the length of R in all
the four tested cell lines. Stronger anti-proliferative activities were
observed in aromatic substituted derivatives (compounds 13e20),
suggesting that the lipophilic moiety may be beneficial for the
cytotoxicity of derivatives. Different from spirolactone-type diter-
penoid derivatives [18,19], no obvious SAR could be concluded from
the substituent species and the position on phenyl ring. Compound
17 with chloro at para-position of benzene ring showed promising
anti-proliferative activities against all the four tested cell lines with
Taxol
Oridonin
10
11
12
13
14
15
16
17
18
19
20
0.41 ꢀ 0.02
4.76 ꢀ 0.32
8.11 ꢀ 0.76
1.11 ꢀ 0.15
1.02 ꢀ 0.26
0.21 ꢀ 0.08
0.14 ꢀ 0.12
0.69 ꢀ 0.08
0.26 ꢀ 0.05
0.24 ꢀ 0.10
0.22 ꢀ 0.02
0.37 ꢀ 0.10
1.04 ꢀ 0.02
0.85 ꢀ 0.06
5.69 ꢀ 0.39
14.21 ꢀ 1.22
1.99 ꢀ 1.08
1.56 ꢀ 0.13
0.93 ꢀ 0.09
0.61 ꢀ 0.18
3.33 ꢀ 0.27
0.64 ꢀ 0.02
1.22 ꢀ 0.19
0.80 ꢀ 0.31
0.96 ꢀ 0.24
1.24 ꢀ 0.68
0.43 ꢀ 0.03
11.03 ꢀ 1.02
30.84 ꢀ 2.09
1.59 ꢀ 0.43
1.21 ꢀ 0.52
0.54 ꢀ 0.04
0.45 ꢀ 0.13
2.71 ꢀ 0.77
0.58 ꢀ 0.04
0.28 ꢀ 0.06
0.60 ꢀ 0.15
0.76 ꢀ 0.18
1.89 ꢀ 0.44
1.89 ꢀ 0.09
7.48 ꢀ 0.53
32.96 ꢀ 2.19
1.97 ꢀ 0.12
1.53 ꢀ 1.00
1.42 ꢀ 0.03
1.01 ꢀ 0.22
1.79 ꢀ 0.15
1.01 ꢀ 0.09
0.87 ꢀ 0.02
0.85 ꢀ 0.04
1.27 ꢀ 0.06
0.89 ꢀ 0.03
IC50 values of 0.24
mM against K562 cell line, 0.28
mM against CaEs-
a
IC50: concentration that inhibits 50% of cell growth.
Results are expressed as the mean ꢀ S.D. of three independent experiments. Cell
17 cell line, 1.22 M against MGC-803 cell line and 0.87
m
mM against
b
Bel-7402 cell line. Based on the above results, compound 17 was
selected for further pharmacological study.
lines: K562 human leukemia cell line; MGC-803 human gastric cancer cell line;
CaEs-17 human esophageal cancer cell line; Bel-7402 human hepatoma cell line.

D. Li et al. / European Journal of Medicinal Chemistry 64 (2013) 215e221
217
12
H
11
20
13
17
OH
O
OH
16
OH
O
H
H
1
14
a
2
3
9
O
6
OH
O
OH
O
OH
O
OH
O
10
5
8
O
O
15
4
OH
OH
O
OH
7
H
H
H
H
OH
OH
O
O
18
19
9
O
O
R
O
O
O
11 R = methyl
12 R = ethyl
O
OH
O
H
OH
H
O
7
13 R = phenyl
14
H
O
O
O
20
14 R = p-methylphenyl
15 R = p-methoxylphenyl
16 R = p-nitrophenyl
H
b
1
O
OH
O
6
17 R = p-chlorophenyl
18 R = o-chlorophenyl
H
H
OH
10
O
OH
11-20
19 R = p-trifluoromethylphenyl
20 R = 2,3,4,5-tetrafluorophenyl
Scheme 1. Synthetic route of enmein-type diterpenoid analogs. Reagents and conditions: (a) NaIO4, H2O, rt; (b) Corresponding acid, EDCI, DMAP, DCM, rt.
2.3. Cell cycle studies of compound 17
2.5. Induction of mitochondrial depolarization by compound 17
In this assay Bel-7402 cells were treated with different con-
centrations of compound 17 (0.15, 0.3 and 0.6 mM, respectively).
Mitochondria play an essential role in the propagation of
apoptosis. Bel-7402 cells were incubated with different concen-
Controls were treated with drug vehicle DMSO. In order to deter-
mine the effects of enmein-type diterpenoid analogs on the cell
cycle progression, we detected the DNA content of cell nuclei by
flow cytometry (Fig. 3). As can be seen, compound 17 could influ-
ence cell cycle progression at low micro-molar concentrations. Cells
at G2/M phase resulted in accumulation of 15.10%, 20.48% and
28.76%, respectively (Table 2), and apoptotic cells increased from
2.07% to 31.97%.
trations (0, 0.15, 0.3, 0.6 mM) of compound 17 for 48 h prior to
staining with the fluorescent dye JC-1. Then the numbers of cells
with collapsed mitochondrial membrane potentials in different cell
groups were determined by flow cytometric analysis (Fig. 5). The
percentage of apoptotic cells increased in a dose-dependent
fashion (0, 8.5%, 17.6% and 45.6%, respectively, Table 3). These
studies showed that incubation with compound 17 increased the
number of cells with collapsed mitochondrial membrane potentials
and induced apoptosis.
2.4. Induction of apoptosis by compound 17
2.6. Effect on the expression of apoptosis-related proteins
Bel-7402 cells were treated with vehicle alone or different
concentrations (0.15, 0.3 and 0.6 mM) of enmein-type diterpenoid
analog 17 for 48 h and stained with FITC-Annexin V and propi-
dium iodide (PI). The percentages of apoptotic Bel-7402 cells
were determined by flow cytometry. As shown in Fig. 4, com-
pound 17 caused significant induction of apoptosis in a dose-
During the process of apoptosis, apoptotic signals can result in
the loss of mitochondrial membrane potential and the release of
cytochrome C from mitochondria to cytoplasm, a down-regulation
of anti-apoptotic protein Bcl2, and an up-regulation of pro-
apoptotic protein Bax, leading to activation of caspase 3 and cas-
pase 9. Further Western blotting analysis revealed that incubation
with compound 17 dramatically increased the relative levels of pro-
apoptotic cytochrome C, Bax, caspase 3, caspase 8, caspase 9 and Fas
expression, but reduced the levels of anti-apoptotic Bcl2 expression
(Fig. 6) in a concentration-dependent manner.
dependent manner. When treated with 0.15, 0.3 and 0.6 mM of
compound 17 for 2 days, the percentages of apoptotic cells were
14.3%, 33.3% and 50.5% (early and late, Q2 þ Q4), respectively,
compared to 4.8% of vehicle control. These findings prompted us
to further investigate the apoptotic process after treatment of
cells with compound 17.
3. Conclusion
In summary, a series of enmein-type diterpenoid analogs (11e20)
with potent anti-proliferative activities were synthesized from
relevant commercially available natural product. And the pre-
liminary SAR for this series of compounds was also discussed.
Furthermore, it was found that representative compound 17 could
induce apoptosis in Bel-7402 cells and lower mitochondrial mem-
brane potentials and expression level of anti-apoptotic Bcl2. Mean-
while, increased cytochrome C release and up-regulation of pro-
apoptotic Bax, Fas, caspase 3, 8 and 9 expression by 17 were
observed. Collectively, compound 17 induced apoptosis via oxidative
stress triggered mitochondria-related caspase-dependent pathway.
It is expected that the remarkable biological profile of the synthetic
O
15
17
16
13
12
O
H
O
7
O
14
H
9
11
20
8
1
2
3
10
O
5
4
6
H
H
O
18
19
,
-
Key HMB
H
1H 1H
Y
COS
(
C
C
)
.
)
(
Fig. 2. Key HMBC and 1He1H COSY of compound 10.

218
D. Li et al. / European Journal of Medicinal Chemistry 64 (2013) 215e221
Fig. 3. Influence of cell cycle by enmein-type compound 17 in Bel-7402 cells.
enmein-type diterpenoid analogs make them promising candidates
for the development of novel anti-cancer agents.
micro melting point apparatus and uncorrected. Infrared (IR)
spectra (KBr) were recorded on a Nicolet Impact 410 instrument
(KBr pellet). NMR spectra were recorded with a Bruker AV-300 or
ACF 500 spectrometer in the indicated solvents (TMS as internal
standard): the values of the chemical shifts are expressed in
4. Experimental
d
values (ppm) and the coupling constants (J) in Hz. Mass spectra
4.1. Chemistry
were obtained using FTMS-2000. HR-MS were carried out with
Agilent QTOF 6520.
All commercially available solvents and reagents were used
without further purification. Melting points were taken on an XT-4
4.2. General procedure to synthesize compounds 10e20
Table 2
The influence of cell cycle progression in Bel-7402 cells by enmein-type diterpenoid
analog 17 at different concentrations.
The synthetic method and physicochemical data of compound
10 were disclosed in our previous report [18].
Bel-7402 cells
Compound 10 (72 mg, 0.2 mmol) was mixed with corresponding
acid (0.24 mmol), EDCI and DMAP in 15 mL of dichloromethane and
stirred at room temperature for 8e12 h. The mixture was poured
into 15 mL of 10% HCl, and extracted with dichloromethane
(10 mL ꢁ 3). The organic layer was combined, washed with water
and saturated NaCl solution sequentially, dried over anhydrous
Group
G1 (%)
S (%)
G2 (%)
Apoptosis (%)
Negative control
Compound 17
48.23
46.18
50.28
51.07
39.17
38.72
29.24
20.17
12.60
15.10
20.48
28.76
1.67
2.07
14.86
31.97
0.15 mM
0.3
0.6
m
m
M
M

D. Li et al. / European Journal of Medicinal Chemistry 64 (2013) 215e221
219
Fig. 4. Flow cytometric analysis: compound 17 induces apoptosis in Bel-7402 cells.
Na2SO4, and concentrated in vacuo. The crude product was purified
by column chromatography (MeOH/CH2Cl2 1:150 v/v) to give the
title compounds.
5.73 (1H, s, 14-CH), 5.60 (1H, s, 17-CH2), 4.58 (1H, m, 1-CH), 4.12,
3.97 (each 1H, dd, JA ¼ JB ¼ 9.6 Hz, 20-CH2), 3.19 (1H, d, J ¼ 9.3 Hz,
13-CH), 1.96 (3H, s, eCH3), 1.06 (3H, s, eCH3), 1.01 (3H, s, eCH3); 13
C
NMR (DMSO-d6)
d 197.84, 172.96, 169.71, 148.64, 121.76, 101.87,
4.2.1. Compound 11
74.80, 72.13, 62.08, 53.64, 49.22, 46.87, 40.58, 37.26, 33.05, 32.15,
31.67, 29.58, 23.36, 23.23, 22.91, 20.32, 19.77, 14.33; MS(ESI) m/z:
419.2 [M þ H]þ, 436.2 [M þ NH4]þ; HR-MS (ESI, M þ H) m/z: calcd
for C23H31O7: 419.2064, found 419.2072.
White solid, 79% yield: m.p. 117e119 ꢂC; IR (KBr) ymax 3478,
2954, 2025, 1756, 1647, 1456, 1374, 1246, 1045 cmꢃ1 1H NMR
;
(CDCl3, 300 MHz)
d (ppm) 6.25 (1H, s, 17-CH2), 6.13 (1H, s, 6-CH),
5.73 (1H, s, 14-CH), 5.60 (1H, s, 17-CH2), 4.58 (1H, m, 1-CH), 4.12,
3.97 (each 1H, dd, JA ¼ JB ¼ 9.6 Hz, 20-CH2), 3.19 (1H, d, J ¼ 9.3 Hz,
4.2.3. Compound 13
13-CH), 1.96 (3H, s, eCH3), 1.06 (3H, s, eCH3), 1.01 (3H, s, eCH3); 13
C
White solid, 79% yield: m.p. 152e154 ꢂC; IR (KBr) ymax 3466,
NMR (DMSO-d6)
d 198.22, 171.00, 167.27, 148.00, 120.52, 102.00,
2953, 2025, 1756, 1707, 1649, 1452, 730, 716 cmꢃ1; 1H NMR (CDCl3,
74.80, 74.11, 60.39, 54.05, 50.09, 48.70, 40.89, 37.30, 33.13, 31.36,
31.23, 30.01, 29.78, 23.44, 23.26, 21.18, 20.01; MS(ESI) m/z: 405.2
[M þ H]þ, 422.2 [M þ NH4]þ, 439.3 [M þ Cl]ꢃ; HR-MS (ESI, M þ H)
m/z: calcd for C22H29O7: 405.1908, found 405.1913.
300 MHz)
d
(ppm) 7.95 (2H, d, J ¼ 7.2 Hz, Ar-H), 7.53 (1H, t,
J ¼ 7.5 Hz, Ar-H), 7.38 (2H, t, J ¼ 7.5 Hz, Ar-H), 6.25 (1H, s, 17-CH2),
5.87 (1H, s, 14-CH), 5.56 (1H, s, 17-CH2), 5.34 (1H, m, 6-CH), 5.30
(1H, s, 6-CH), 4.61 (1H, m, 1-CH), 3.99, 4.09 (each 1H, dd,
JA ¼ JB ¼ 9.6 Hz, 20-CH2), 3.38 (1H, d, J ¼ 9.3 Hz, 13-CH), 1.03 (3H,
4.2.2. Compound 12
s, eCH3), 0.97 (3H, s, eCH3); 13C NMR (DMSO-d6)
d 198.53, 166.82,
White solid, 76% yield: m.p. 160e161 ꢂC; IR (KBr) ymax 3478,
166.19, 147.93, 133.74, 130.26, 129.29, 128.64, 120.66, 101.98, 74.82,
74.72, 60.33, 54.10, 50.37, 48.79, 41.16, 37.30, 33.12, 31.25, 29.91,
23.51, 23.27, 20.00; MS(ESI) m/z: 467.1 [M þ H]þ, 484.1 [M þ NH4]þ,
2954, 2025, 1756, 1647, 1456, 1374, 1246, 1045 cmꢃ1 1H NMR
;
(CDCl3, 300 MHz)
d (ppm) 6.25 (1H, s, 17-CH2), 6.13 (1H, s, 6-CH),
Fig. 5. Effect of compound 17 on the mitochondrial membrane potentials of Bel-7402 cells.

220
D. Li et al. / European Journal of Medicinal Chemistry 64 (2013) 215e221
13-CH), 1.25 (3H, s, eCH3), 0.97 (3H, s, eCH3); 13C NMR (DMSO-d6)
197.77, 164.20, 160.74, 153.55, 147.82, 135.35, 132.78, 124.36, 120.84,
Table 3
Effect of compound 17 on mitochondrial membrane potentials in Bel-7402 cells.
d
116.97, 102.88, 75.61, 74.75, 60.04, 53.60, 50.06, 47.92, 40.98, 37.25,
33.10, 31.76, 30.00, 29.93, 23.37, 19.98; MS(ESI) m/z: 512.2 [M þ H]þ,
529.2 [M þ NH4]þ, 546.5 [M þ Cl]ꢃ; HR-MS (ESI, M þ NH4) m/z: calcd
for C27H30NO9: 512.1915, found 512.1922.
Bel-7402 72 h
Group
Q2 (%)
Normal cells
Q4 (%)
Apoptotic cells
Negative control
100
0
Compound 17
0.15
m
M
M
M
91.5
82.4
54.4
8.5
17.6
45.6
0.3
0.6
m
m
4.2.7. Compound 17
White solid, 73% yield: m.p. 163e165 ꢂC; IR (KBr) ymax 3431,
2950, 2025, 1754, 1723, 1630, 1490, 1277, 855 cmꢃ1; 1H NMR (CDCl3,
300 MHz)
d
(ppm) 7.88 (2H, d, J ¼ 8.4 Hz, Ar-H), 7.36 (2H, d,
501.3 [M þ Cl]ꢃ; HR-MS (ESI, M þ H) m/z: calcd for C27H31O7:
J ¼ 8.4 Hz, Ar-H), 6.25 (1H, s,17-CH2), 5.85 (1H, s,14-CH), 5.57 (1H, s,
17-CH), 5.34 (1H, s, 6-CH), 4.60 (1H, m, 1-CH), 4.10, 3.99 (each 1H,
dd, JA ¼ JB ¼ 9.3 Hz, 20-CH2), 3.32 (1H, d, J ¼ 9.6 Hz,13-CH),1.03 (3H,
467.2064, found 467.2071.
s, eCH3), 0.97 (3H, s, eCH3); 13C NMR (DMSO-d6)
d 198.39, 168.05,
4.2.4. Compound 14
White solid, 69% yield: m.p. 159e161 ꢂC; IR (KBr) ymax 3443,
166.86, 165.18, 164.67, 147.83, 132.86, 120.76, 116.00, 115.70, 102.00,
76.46, 74.94, 74.70, 60.32, 54.06, 50.36, 48.76, 41.13, 37.29, 33.11,
31.25, 23.50, 23.25, 19.98; MS(ESI) m/z: 501.2 [M þ H]þ, 535.5
[M þ Cl]ꢃ; HR-MS (ESI, M þ H) m/z: calcd for C27H30ClO7: 501.1675,
found 501.1679.
2950, 2025, 1755, 1719, 1612, 1453, 1279, 839 cmꢃ1; 1H NMR (CDCl3,
300 MHz)
d
(ppm) 7.87 (2H, d, J ¼ 7.8 Hz, Ar-H), 7.17 (2H, t,
J ¼ 7.8 Hz, Ar-H), 6.24 (1H, s, 17-CH2), 5.85 (1H, s,14-CH), 5.50 (1H, s,
17-CH2), 5.34 (1H, m, 6-CH), 4.59 (1H, m, 1-CH), 4.10, 4.00 (each 1H,
dd, JA ¼ JB ¼ 9.3 Hz, 20-CH2), 3.01 (1H, d, J ¼ 9.0 Hz,13-CH),1.03 (3H,
s, eCH3), 0.97 (3H, s, eCH3); 13C NMR (DMSO-d6)
d 198.60, 166.74,
4.2.8. Compound 18
White solid, 74% yield: m.p. 164e166 ꢂC; IR (KBr) ymax 3442,
166.24,148.00,144.55,130.31, 129.69, 129.35,126.55,120.55,101.99,
74.79, 74.61, 60.31, 54.08, 50.39, 48.80, 41.17, 37.31, 33.13, 31.25,
29.90, 23.51, 23.27, 21.93, 20.00; MS(ESI) m/z: 503.3 [M þ Na]þ; HR-
MS (ESI, M þ H) m/z: calcd for C28H33O7: 481.2221, found 481.2228.
2953, 2025, 1757, 1647, 1591, 1471, 1251, 749 cmꢃ1; 1H NMR (CDCl3,
300 MHz)
d
(ppm) 7.84 (1H, d, J ¼ 7.5 Hz, Ar-H), 7.39 (2H, m, Ar-H),
7.26 (1H, m, Ar-H), 6.25 (1H, s,17-CH2), 5.95 (1H, s,14-CH), 5.58 (1H,
s, 17-CH2), 5.35 (1H, m, 6-CH), 4.63 (1H, m, 1-CH), 4.10, 3.99 (each
1H, dd, JA ¼ JB ¼ 8.7 Hz, 20-CH2), 3.31 (1H, d, J ¼ 8.7 Hz, 13-CH), 1.04
4.2.5. Compound 15
(3H, s, eCH3), 0.97 (3H, s, eCH3); 13C NMR (DMSO-d6)
d 198.31,
White solid, 53% yield: m.p. 219e222 ꢂC; IR (KBr) ymax 3453,
2964, 2827, 1760, 1716, 1606, 1512, 1168, 1373, 1257, 771 cmꢃ1 1H
;
167.11, 164.83, 147.85, 134.21, 133.32, 132.66, 131.05, 127.13, 120.83,
102.00, 75.01, 74.74, 60.37, 54.10, 50.25, 48.85, 41.03, 37.28, 33.12,
31.25, 29.98, 23.46, 23.27, 20.04; MS(ESI) m/z: 501.2 [M þ H]þ, 518.2
[M þ NH4]þ, 535.5 [M þ Cl]ꢃ; HR-MS (ESI, M þ H) m/z: calcd for
NMR (CDCl3, 300 MHz)
d
(ppm) 7.90 (2H, d, J ¼ 9.0 Hz, Ar-H), 6.86
(2H, d, J ¼ 8.7 Hz, Ar-H), 6.24 (1H, s, 17-CH2), 5.83 (1H, s, 14-CH),
5.55 (1H, s, 17-CH2), 5.34 (1H, s, 6-CH), 4.61 (1H, m, 1-CH), 4.10, 3.99
(each 1H, dd, JA ¼ JB ¼ 9.9 Hz, 20-CH2), 3.82 (3H, s, eOCH3),1.03 (3H,
C27H30ClO7: 501.1675, found 501.1674.
s, eCH3), 0.97 (3H, s, eCH3); 13C NMR (DMSO-d6)
d 198.66, 166.79,
4.2.9. Compound 19
165.84, 164.09, 148.05, 132.40, 121.68, 120.50, 113.93, 102.00, 74.75,
74.50, 60.33, 55.67, 54.06, 50.40, 48.78, 41.19, 37.31, 33.12, 32.15,
31.24, 29.92, 29.88, 29.58, 23.51, 23.38, 22.91; MS(ESI) m/z: 497.1
[M þ H]þ, 514.2 [M þ NH4]þ, 531.3 [M þ Cl]ꢃ; HR-MS (ESI, M þ H)
m/z: calcd for C28H33O8: 497.2170, found 497.2173.
White solid, 35% yield: m.p. 146e148 ꢂC; IR (KBr) ymax 3482,
2955, 1758, 1727, 1648, 1326, 1279, 1170, 1132, 1066, 999, 773,
702 cmꢃ1 1H NMR (CDCl3, 300 MHz)
; d (ppm) 8.07 (2H, m, Ar-H),
7.65 (2H, m, Ar-H), 6.27 (1H, s, 17-CH2), 5.90 (1H, s, 14-CH), 5.59
(1H, s, 17-CH2), 5.36 (1H, s, 6-CH), 4.58 (1H, m, 1-CH), 4.10, 4.00
(each 1H, dd, JA ¼ JB ¼ 8.7 Hz, 20-CH2), 3.33 (1H, d, J ¼ 8.7 Hz, 13-
CH), 1.05 (3H, s, eCH3), 0.86 (3H, s, eCH3); 13C NMR (DMSO-d6)
4.2.6. Compound 16
Yellow solid, 89% yield: m.p. 216e218 ꢂC; IR (KBr) ymax 3450,
2964, 2901, 2874, 1752, 1721, 1533, 1421, 1251, 1129, 1066, 842,
d
198.22, 166.91, 164.98, 147.70, 132.54, 130.87, 130.22, 125.69,
719 cmꢃ1; 1H NMR (CDCl3, 300 MHz)
d
(ppm): 8.23 (2H, d, J ¼ 8.7 Hz,
125.64, 120.87, 101.98, 75.18, 74.58, 60.34, 54.06, 50.32, 48.79, 41.09,
37.25, 33.07, 31.23, 29.57, 23.49, 22.90; MS(ESI) m/z: 535.2
[M þ H]þ, 552.2 [M þ NH4]þ, 557.2 [M þ Na]þ; HR-MS (ESI, M þ H)
m/z: calcd for C28H30F3O7: 535.1938, found 535.1941.
Ar-H), 8.11 (2H, d, J ¼ 8.7 Hz, Ar-H), 6.28 (1H, s,17-CH2), 5.90 (1H, s, 6-
CH), 5.61 (1H, s, 17-CH2), 5.35 (1H, s, 14-CH), 4.59 (1H, m, 1-CH), 4.10,
3.99 (each 1H, dd, JA ¼ JB ¼ 9.0 Hz, 20-CH2), 3.33 (1H, d, J ¼ 9.0 Hz,
Fig. 6. Effect of compound 17 on the expression of apoptosis-related proteins in Bel-7402 cells (1: control; 2: 0.15
compound 17). *P < 0.05 vs. the vehicle control, **P < 0.01 vs. the vehicle control.
mM of compound 17; 3: 0.3 mM of compound 17; 4: 0.6 mM of

D. Li et al. / European Journal of Medicinal Chemistry 64 (2013) 215e221
221
4.2.10. Compound 20
White solid, 24% yield: m.p. 218e220 ꢂC; IR (KBr) ymax 3465,
2955, 1758, 1528, 1488, 1377, 1220, 1043, 921, 775, 696 cmꢃ1 1H
NMR (CDCl3, 300 M Hz) (ppm) 7.52 (1H, m, Ar-H), 6.29 (1H, s, 17-
4.7. Western blot analysis
;
Bel-7402 cells were incubated in triplicate with different doses
of test compound for 48 h. After the protein concentrations were
d
CH2), 5.92 (1H, s, 14-CH), 5.63 (1H, s, 17-CH2), 5.36 (1H, s, 6-CH),
4.60 (1H, m, 1-CH), 4.09, 3.97 (each 1H, dd, JA ¼ JB ¼ 9.3 Hz, 20-CH2),
3.31 (1H, d, J ¼ 9.3 Hz, 13-CH), 1.04 (3H, s, eCH3), 0.98 (3H, s, eCH3);
determined, individual cell lysates (50 mg per lane) were separated
by sodium dodecyl sulfate polyacrylamide gel electrophoresis (10%
gel, SDS-PAGE) and transferred onto nitrocellulose membranes.
After being blocked with 5% fat-free milk, the target proteins in the
membranes were probed with monoclonal anti-Bax (KGA714),
13C NMR (DMSO-d6)
d 197.66, 147.48, 121.06, 113.88, 113.56, 101.99,
75.87, 74.66, 60.31, 54.42, 54.10, 50.11, 48.79, 40.97, 37.28, 33.11,
31.25, 29.93, 23.46, 23.24, 19.99; MS(ESI) m/z: 539.2 [M þ H]þ,
556.2 [M þ NH4]þ; HR-MS (ESI, M þ NH4) m/z: calcd for
anti-Bcl2 (KGA715), anti-caspase
(KGA720), anti-cyto (KGA723) and anti-
(KGA731, KeyGEN Biotech, Nanjing, China), respectively. The rela-
3
(KGA717), anti-caspase
9
C
b-actin antibodies
C
27H30F4NO8: 556.1953, found 556.1959.
tive levels of each signaling event to control
mined by densimetric scanning.
b-actin were deter-
4.3. MTT assay in vitro
Acknowledgments
The MTT assay was performed in 96-well plates. K562 cells
(CaEs-17, Bel-7402 and MGC-803 cells) at the log phase of their
growth cycle (5 ꢁ 104 cell/mL) were added to each well (100
mL/
This study was supported by a grant from the National Natural
Science Foundation (No. 30973610), Specialized Research Fund for
the Doctoral Program of Higher Education (No. 20100096110001),
Project for Research and Innovation of Graduates in Universities of
Jiangsu Province (CXZZ11-0800), the Fundamental Research Funds
for the Central Universities (JKY2011030) and Key Fund of Ministry
of Education of China (No. 108069) for financial assistance.
well), then treated in the presence or absence of test compounds,
and incubated for 24 h at 37 ꢂC in a humidified atmosphere of 5%
CO2. After 72 h, 20
added to each cultured medium, which was incubated for another
4 h. Then, DMSO was added to each well (150 L/well). After 10 min
mL of MTT solution (5 mg/mL) per well was
m
at room temperature, the OD of each well was measured on a
Microplate Reader (BIO-RAD Instruments Inc NO. 550) at the
wavelength of 490 nm. In these experiments, the negative refer-
ence agent was 0.1% DMSO, and Taxol was used as the positive
Appendix A. Supplementary data
reference with the concentration of 10 mg/mL.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.04.012.
4.4. Cell cycle study
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with 70% ethanol, treated with RNase, and stained with PI. Cellular
DNA content, for cell cycle distribution analysis, was measured
using a flow cytometer (FACS Calibur BectoneDickinson).
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