Synthesis of spirolactone-type diterpenoid derivatives from kaurene-type oridonin with improved antiproliferative effects and their apoptosis-inducing activity in human hepatoma Bel-7402 cells

Article abstract of DOI:10.1016/j.ejmech.2012.11.002

A series of novel spirolactone-type diterpenoid derivatives of oridonin (12a-j) were designed and synthesized. All the target compounds showed improved anti-proliferative activity against a panel of human cancer cell lines and the most effective compound 12j was more potent than positive control Taxol in K562 and Bel-7402 cells with IC₅₀ values of 0.39 μM and 1.39 μM, respectively. The cellular mechanisms showed that compound 12j induced apoptosis at low micromolar concentrations in human hepatoma Bel-7402 cells. These results demonstrate that the spirolactone-type diterpenoid derivatives of oridonin have optimized growth inhibitory activity against cancer cells and interesting apoptosis-inducing ability.

Full text of DOI:10.1016/j.ejmech.2012.11.002

European Journal of Medicinal Chemistry 59 (2013) 322e328
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis of spirolactone-type diterpenoid derivatives from kaurene-type
oridonin with improved antiproliferative effects and their apoptosis-inducing
activity in human hepatoma Bel-7402 cells
,
,
,b
Dahong Li a b, Hao Cai a b, Bowen Jiang a, Guyue Liu c, Yuetong Wang c, Lei Wang a, Hequan Yao a
,
Xiaoming Wu a , Yijun Sun , Jinyi Xu
,
b,
d
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 Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China
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 novel spirolactone-type diterpenoid derivatives of oridonin (12aej) were designed and
synthesized. All the target compounds showed improved anti-proliferative activity against a panel of
human cancer cell lines and the most effective compound 12j was more potent than positive control
Received 3 August 2012
Received in revised form
9 October 2012
Accepted 5 November 2012
Available online 21 November 2012
Taxol in K562 and Bel-7402 cells with IC50 values of 0.39 mM and 1.39 mM, respectively. The cellular
mechanisms showed that compound 12j induced apoptosis at low micromolar concentrations in human
hepatoma Bel-7402 cells. These results demonstrate that the spirolactone-type diterpenoid derivatives of
oridonin have optimized growth inhibitory activity against cancer cells and interesting apoptosis-
inducing ability.
Keywords:
Spirolactone-type diterpenoid
Oridonin
Ó 2012 Elsevier Masson SAS. All rights reserved.
Anti-proliferative activity
Apoptosis
Structureeactivity relationship
1. Introduction
ludongnin A (3), maoesin F (4), laxiflorin A (5), laxiflorin B (6),
isolongirabdiol (7), and others have exhibited anti-tumor activities
Diterpenoids, with diverse structures and interesting biological
properties, have attracted considerable attention [1]. Since 1976, as
part of the search for novel natural products as useful leads for the
discovery of therapeutic agents to treat cancer, more than 60
species of Isodon genus from mainland China have been investi-
gated phytochemically. About 600 new diterpenoids with diverse
skeletons have been isolated and characterized. Some of these ent-
kauranoids, such as commercially available kaurene-type diterpe-
noid oridonin (1, see Fig. 1), possess potential anti-tumor activity
with low toxicity [2,3].
(Fig. 2) [4e10]. But arduous isolation work with low yields was far
from being satisfying for the development of new drugs. For these
reasons, a practical and convenient synthesis of spirolactone-type
diterpenoid analogs could provide us with enough samples for
structureeactivity relationship (SAR) and pharmacological studies.
In our previous studies, we disclosed that some novel 1-O-and
14-O-derivatives of oridonin [11e13] and ent-6,7-seco-oridonin
derivatives [14] exhibited excellent cytotoxicity against several
human cancer cell lines in vitro and in vivo. Since some 1-O-acetyl-
14-O-derivatives of oridonin exhibited stronger anti-tumor activity
than the corresponding 14-O-oridonin derivatives [11,15], a series
of 1-O-acetyl-14-O-derivatives of spirolactone-type diterpenoid
(6,7-seco-oridonin skeleton) were designed and synthesized. The
anti-proliferative activity of these compounds was tested by MTT
assay against four kinds of human cancer cell lines (K562, MGC-
803, CaEs-17 and Bel-7402). Furthermore, the mechanism of
induction of apoptosis and influence of cell cycle progression in
human hepatoma Bel-7402 cells by these spirolactone-type diter-
penoid analogs were investigated.
Spirolactone-type diterpenoids with unique chemical skeletons
and interesting activities have attracted our attention and aroused
our curiosity for intensive study; for example, shikodonin (2),
* Corresponding authors. Department of Medicinal Chemistry, China Pharma-
ceutical University, 24 Tong Jia Xiang, Nanjing 210009, China. Tel.: þ86 025
83271445.
E-mail addresses: xmwu@cpu.edu.cn (X. Wu), jinyixu@china.com (J. Xu).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.002

D. Li et al. / European Journal of Medicinal Chemistry 59 (2013) 322e328
323
novel structure of the synthetic spirolactone-type diterpenoid
analogs make them possible as promising candidates for the
development of anti-cancer agents.
The SAR shows that all the synthesized derivatives of
spirolactone-type diterpenoid analogs can improve the anti-
proliferative activity of oridonin. Among the tested compounds
12aej, when the substitution R were alkyl groups (compounds 12a,
12b and 12c), their IC50 values slightly declined with the extension
12
H
11
20
13
17
OH
16
1
14
9
O
6
OH
O
10
5
8
15
3
4
OH
7
H
OH
18
19
1
of the length of R in MGC-803 (from 3.47 to 2.49 mM) cell line and
had no significant change in the other three tested cell lines. While
in the substitution of aromatic groups (compounds 12de12j), it
could be found that their anti-proliferative activity was more
potent than that of alkyl groups, especially the aromatic ring was
substituted by chloro. For example, compound 12j with chloro
substitution at ortho-position of benzene ring was the most potent
Fig. 1. The structure and atom numbering of oridonin (1).
2. Results and discussion
2.1. Chemistry
with the IC50 values of 0.39
than Taxol and 12-fold more potent than 1), 1.28
803 cell line (4-fold more potent than 1), 0.60 M against CaEs-17
cell line (18-fold more potent than 1) and 1.39 M against Bel-
mM against K562 cell line (slightly better
mM against MGC-
AsshowninScheme1, treatmentof 1 with2,2-dimethoxypropane
(DMP) inthe presence ofTsOH inanhydrous acetone afforded 7,14-(1-
methylethylene)-dioxy-oridonin derivative 8, which, upon reaction
with Ac2O/Et3N in CH2Cl2, yielded 1-O-acetyl derivative 9. Depro-
tection of 9 with 10% HCl gave compound 10 in quantitative yields,
which could be converted to spirolactone-type diterpenoid 11 by
oxidation using lead tetraacetate in the presence of sodium carbonate
in THF in high yields. Compounds 12aej were prepared in high yields
(up to 80% of the total yield from the first step) by reaction of 11 and
corresponding acids in the presence of DMAP/EDCI in CH2Cl2, over-
night at room temperature. Flash chromatography was taken only at
the last step of the synthetic route of each target compound.
m
m
7402 cell line (slightly potent than Taxol and 5-fold more potent
than 1), respectively. In contrast, compound 12g with tri-
fluoromethyl substitution at para-position of benzene ring dis-
played the weakest anti-proliferative activity in all tested cell lines.
Then, other aromatic group-substituted derivatives showed IC50
values between compounds 12g and 12j. So the substitution of
aromatic moiety is favorable for the activity, especially o-chlor-
ophenyl is optimal.
2.3. GSTp inhibition activity
Glutathione transferases (GSTs) (EC 2.5.1.18), a family of phase II
detoxification enzymes in plants and animals, can catalyze the
conjugation of glutathione (GSH) to a wide range of endogenous
and exogenous electrophilic compounds to generate less toxicity
and to make more water-soluble products easily eliminated.
Among these enzymes, GSTp is an abundant enzyme which regu-
lates the signaling pathway of cell death and proliferation, protects
cells from death and detoxifies chemotherapeutic agents in cancer
2.2. Anti-proliferative activity and SAR study
The anti-proliferative activities of the target compounds were
determined by MTT assay in vitro against four kinds of different
cancer cell lines: K562 (human leukemia), MGC-803 (human
gastric cancer cell line), CaEs-17 (human esophageal cancer cell
line) and Bel-7402 (human hepatoma). The activities of these
derivatives were compared with their parent compound oridonin
and positive control Taxol in each panel. As shown in Table 1, all the
synthesized derivatives were more potent than oridonin in vitro,
and compound 12j had stronger anti-proliferative activity than
cells [16e18]. Ethacrynic acid (EA) and its derivatives have GST
inhibition activity as reported earlier by our pharmacological co-
workers [19e21]. They inhibited the activity of GST by binding
p
p
directly to the substrate-binding site of the isozyme and by
depleting GSH via conjugation of the Michael addition to the thiol
group of GSH, due to the chemical structure of a, b-unsaturated
carbonyl group which also exists in the synthesized compounds
12aej. We wondered if spirolactone-type diterpenoid derivatives
Taxol (0.41
synthetic series with IC50 value of 0.39
and 12j exhibited stronger activity than Taxol (1.89
7402 cancer cell line with IC50 values of 1.74, 1.51 and 1.39
respectively. Consequently, the remarkable biological profile and
m
M) in K562 cell line, which was the most potent in the
M. Compounds 12h, 12i
M) against Bel-
M,
m
m
m
12aej could also inhibit the activity of GST
12j were selected to test their inhibitory effects on GST
When EA was used as the positive control with the inhibition rate of
78.34% at the concentration of 10 M, unfortunately, almost no
inhibitory effect of either compound on GST activity (7.08% and 0,
respectively) was observed at the concentration of 40 M. These
results suggest that GST is not a target of spirolactone-type
p
, so compounds 12a and
p
activity.
HO
HO
m
AcO
H
H
p
H
H
H
H
m
O
O
AcO
p
OAc
O
O
O
O
O
O
CHO O
O
O
OH
diterpenoid analogs and other mechanisms of action may be
probably involved in the anti-proliferative effects of these
compounds.
ludongnin A
0.0001% yield
2
3
shikodonin
0.00005% yield
O
4
manesin F
0.00004% yield
O
H
H
H
H
H
2.4. Induction of apoptosis
H
O
O
OH
On the basis of the anti-proliferative data, the most potent
compound 12j was selected for further mechanistic studies. Bel-
7402 cells were not only treated with vehicle alone as controls
O
O
O
O
O
O
OH
laxiflorin A
OH
laxiflorin B
0.00061% yield
OH
7
5
6
isolongirabdiol
0.0098% yield
0.01% yield
but were dealt with different concentrations (0.6, 0.8 or 1.0 mM) of
12j for 48 h and stained with FITC-Annexin V and propidium iodide
Fig. 2. Some reported spirolactone-type diterpenoids isolated from natural plants and
their isolation yields.
(PI). The percentages of apoptotic Bel-7402 cells were determined

324
D. Li et al. / European Journal of Medicinal Chemistry 59 (2013) 322e328
OAc
OAc
OH
OH
OH
b
O
c
O
OH
a
O
O
O
O
O
O
O
O
OH
O
O
OH
H
H
H
H
OH
10
OH
9
OH
8
OH
1
12a R = methyl
12b R = ethyl
12c R = -butyl
n
OAc
12d R = phenyl
12e R = -methylphenyl
OAc
p
e
d
H
12f R = p-methoxylphenyl
H
O
O
OH
O
12g R = p-trifluoromethylphenyl
12h R = p-nitrophenyl
O
R
CHO O
12
O
CHO O
11
O
12i R = p-chlorophenyl
12j R = o-chlorophenyl
aReagents and conditions: (a) 2,2-Dimethoxypropane, acetone, TsOH, reflux; (b)
Ac2O, DMAP, rt; (c) 10% HCl, THF, rt; (d) Pb(OAc)4, Na2CO3, THF, rt; (e)
corresponding acids, DMAP, EDCI, DCM, rt.
Scheme 1. Synthetic route of 1-O-acetyl-14-O-derivatives of spirolactone-type diterpenoida.
by flow cytometry. As shown in Fig. 3, compound 12j has potent
3. Conclusion
activity in the induction of apoptosis in a dose-dependent manner.
Treatment of the Bel-7402 cells by 0.6, 0.8 and 1.0
m
M of compound
In summary, a series of novel spirolactone-type diterpenoid
derivatives were synthesized from commercially available oridonin
(1) and tested for their anti-proliferative activity against four kinds
of human cancer cell lines by MTT assay in vitro, and preliminary
SAR was concluded based on the biological evaluation. Within this
series of compounds, compound 12j with chloro substitution at
ortho-position of benzene ring was found to exhibit the most
potent inhibition with IC50 values similar to those of positive
control Taxol against each tested cell line. The 12j mediated
apoptosis in Bel-7402 cells may contribute to its anti-proliferative
effects. Furthermore, the determination of PK (pharmacokinetics)/
PD (pharmacodynamics) properties and further mechanistic
studies of these derivatives were undergoing and the results would
be reported in due course.
12j for 3 days results in 12.6%, 18.9% and 33.4% of apoptotic cells
(early and late), as compared to 6.9% in an untreated vehicle
control. Apparently, the 12j-mediated apoptosis in Bel-7402 cells,
at least in part, contributes to its anti-proliferative effects.
To know the function of apoptosis-inducing and effects of
spirolactone-type diterpenoid analogs on the cell cycle progression,
we detected the DNA content of cell nuclei by flow cytometry
(Fig. 4.). Bel-7402 cells were treated with compound 12j at the
concentrations of 0.6 mM, 0.8 mM and 1.0 mM, which resulted in
accumulation of 23.20%, 53.30% and 92.16% of cells at G2/M phase,
respectively, compared with the untreated cells. The influence of
cell cycle progression at low micromolar concentrations of
spirolactone-type diterpenoid analogs and the property to induce
cell cycle arrest could make them possible as promising agents in
combination with anticancer drugs acting at different stages of cell
cycle.
4. Experimental
4.1. General methods
All commercially available solvents and reagents were used
without further purification. Melting points were taken on an XT-4
micro melting point apparatus and uncorrected. Infrared (IR)
spectra (KBr) were recorded on a Nicolet Impact 410 instrument
(KBr pellet). 1H and 13C 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
Table 1
a
IC50 values (
m
M) of synthetic spirolactone-type diterpenoid derivatives (12aej)
against human cancer cell linesb.
Compd.
K562
MGC-803
CaEs-17
Bel-7402
Taxol
Oridonin
12a
12b
12c
12d
12e
12f
0.41 ꢀ 0.02
4.76 ꢀ 0.32
1.74 ꢀ 0.60
1.50 ꢀ 0.11
1.56 ꢀ 0.73
1.40 ꢀ 0.06
1.31 ꢀ 0.30
1.92 ꢀ 0.29
3.15 ꢀ 0.22
1.14 ꢀ 0.83
0.87 ꢀ 0.14
0.39 ꢀ 0.08
0.85 ꢀ 0.06
5.69 ꢀ 0.39
3.47 ꢀ 0.71
3.22 ꢀ 0.13
2.49 ꢀ 0.58
2.18 ꢀ 0.33
1.90 ꢀ 0.41
2.53 ꢀ 0.66
5.54 ꢀ 0.86
1.98 ꢀ 0.30
1.73 ꢀ 0.11
1.28 ꢀ 0.42
0.43 ꢀ 0.03
11.03 ꢀ 1.02
2.80 ꢀ 0.46
2.77 ꢀ 0.23
2.85 ꢀ 0.19
1.99 ꢀ 0.04
1.83 ꢀ 0.51
1.96 ꢀ 0.03
9.16 ꢀ 0.62
1.60 ꢀ 0.29
1.25 ꢀ 0.12
0.60 ꢀ 0.05
1.89 ꢀ 0.09
7.48 ꢀ 0.53
3.96 ꢀ 1.03
3.78 ꢀ 0.42
3.64 ꢀ 1.11
3.05 ꢀ 1.09
2.09 ꢀ 0.62
2.47 ꢀ 0.52
5.09 ꢀ 1.10
1.74 ꢀ 0.44
1.51 ꢀ 0.92
1.39 ꢀ 0.77
in d values (ppm) and the coupling constants (J) in Hz. Mass spectra
were obtained using FTMS-2000. HR-MS were carried out with
Agilent QTOF 6520. Compounds 8e11 were synthesized as previ-
ously described, but without flash chromatography [14].
12g
12h
12i
4.2. General procedure to synthesize compounds 12
12j
a
Crude compound 11 (80 mg, 0.2 mmol) in 15 mL of CH2Cl2 was
mixed with corresponding acid (0.24 mmol), EDCI and DMAP by
IC50: Concentration that inhibits 50% of cell growth.
Results are expressed as the mean ꢀ S.D. of three independent experiments.
b

D. Li et al. / European Journal of Medicinal Chemistry 59 (2013) 322e328
325
Fig. 3. Compound 12j induces apoptosis in Bel-7402 cells. A: Flow cytometry analysis of apoptotic Bel-7402 cells; B: Apoptotic ratio of control group and different concentrations of
12j.
stirring at room temperature for 8e12 h. The mixture was poured
into 15 mL of 10% HCl, and extracted with CH2Cl2 (10 mL ꢁ 3). The
organic layer was combined, washed with water and saturated NaCl
solution sequentially, dried over anhydrous Na2SO4, and concen-
trated in vacuo. The crude product was purified by column chro-
matography (MeOH/CH2Cl2 1:100 v/v) to give the title compounds
(yields 25e80%, from commercially available starting material 1).
4.2.2. ent-1
6,7-seco-16-kaurene (12b)
White solid, 67% yield: m.p.134e136 ꢂC; IR (KBr) ymax 2954, 2025,
1756, 1462, 1372, 1256, 1168 cmꢃ1 1H NMR (CDCl3, 300 MHz),
(ppm) 9.76 (1H, s, eCHO), 6.08 (1H, s, 17-CH2), 5.74 (1H, s, 14-CH),
a-O-Acetyl-14b-O-propionyl-6,7,15-trioxo-7,20-epoxy-
;
d
5.71(1H, s,17-CH2), 4.99, 4.73 (each 1H, dd, JA ¼ JB ¼ 7.5 Hz, 20-CH2),
4.66 (2H, m, eCH2), 3.10 (1H, d, J ¼ 3.9 Hz, 13-CH), 2.03 (3H, s, e
COCH3), 1.62 (3H, t, eCH3), 1.19 (3H, s, eCH3), 1.13 (3H, s, eCH3); 13
C
4.2.1. ent-1
16-kaurene (12a)
White solid, 69% yield: m.p. 128e130 ꢂC; IR (KBr) ymax 2956,
2025, 1746, 1455, 1372, 1239 cmꢃ1 1H NMR (CDCl3, 300 MHz),
a
-O, 14
b
-O-Diacetyl-6,7,15-trioxo-7,20-epoxy-6,7-seco-
NMR (CDCl3, 300 MHz) d 201.88, 198.36, 172.94, 169.83, 148.53,
122.34, 75.17, 71.92, 67.75, 61.39, 60.70, 44.83, 43.39, 42.39, 39.89,
32.91, 29.15, 21.16, 17.05; MS(ESI) m/z: 478.2 [M þ NH4]þ; HR-MS
(ESI, M þ NH4) m/z: calcd for C25H36NO8: 478.2435, found 478.2443.
;
d
(ppm) 9.79 (1H, d, J ¼ 4.2 Hz, eCHO), 6.25 (1H, s, 17-CH2), 5.90
(1H, s, 14-CH), 5.63 (1H, s, 17-CH2), 5.22, 5.12 (each 1H, dd,
JA ¼ JB ¼ 12.3 Hz, 20-CH2), 4.66 (1H, m,1-CH), 3.09 (1H, d, J ¼ 9.0 Hz,
13-CH), 2.04 (3H, s, eCH3),1.17 (3H, s, eCH3), 1.09 (3H, s, eCH3),1.03
4.2.3. ent-1a-O-Acetyl-14b-O-valeryl-6,7,15-trioxo-7,20-epoxy-6,7-
seco-16-kaurene (12c)
White solid, 71% yield: m.p. 192e193 ꢂC; IR (KBr) ymax 2962,
(3H, s, eCH3); 13C NMR (CDCl3, 300 MHz)
d
202.43, 197.25, 170.43,
2877, 1746, 1722, 1463, 1386, 1267, 1227, 1171, 1131, 1083, 1055, 951,
166.14, 146.68, 121.50, 74.46, 73.17, 66.32, 62.75, 59.96, 45.76, 43.62,
42.35, 39.75, 32.86, 29.66, 21.44, 17.40; MS(ESI) m/z: 447.2
[M þ H]þ, 464.3 [M þ NH4]þ; HR-MS (ESI, M þ H) m/z: calcd for
C24H31O8: 447.2013, found 447.2022.
913 cmꢃ1 1H NMR (CDCl3, 300 MHz),
; d (ppm) 9.76 (1H, d,
J ¼ 4.5 Hz, eCHO), 6.23 (1H, s, 17-CH2), 5.62 (1H, s, 17-CH2), 5.24,
5.14 (each 1H, dd, JA ¼ JB ¼ 12.6 Hz, 20-CH2), 4.66 (1H, m, 1-CH),
4.43 (1H, s, 14-CH), 3.09 (1H, d, J ¼ 9.0 Hz, 13-CH), 2.04 (3H, s, e

326
D. Li et al. / European Journal of Medicinal Chemistry 59 (2013) 322e328
Fig. 4. Influence of Bel-7402 cell cycle by spirolactone-type compound 12j.
CH3), 1.17 (3H, s, eCH3), 1.05 (3H, s, eCH3), 1.02 (3H, s, eCH3); 13C
NMR (CDCl3, 300 MHz) 201.89, 198.36, 172.96, 169.86, 148.51,
122.37, 75.18, 71.92, 67.75, 61.38, 60.69, 44.82, 43.38, 42.38, 39.88,
31.85, 29.15, 23.77, 21.15, 17.04, 14.04; MS(ESI) m/z: 489.2 [M þ H]þ,
506.3 [M þ NH4]þ; HR-MS (ESI, M þ H) m/z: calcd for C27H37O8:
489.2483, found 489.2489.
AreH), 6.23 (1H, s, 17-CH2), 5.92 (1H, s, 14-CH), 5.63 (1H, s, 17-CH2),
5.22, 5.12 (each 1H, dd, JA ¼ JB ¼ 12.3 Hz, 20-CH2), 4.66 (1H, m, 1-
CH), 3.07 (1H, d, J ¼ 9.0 Hz, 13-CH), 2.03 (3H, s, eCH3), 1.17 (3H, s,
d
eCH3), 1.02 (3H, s, eCH3); 13C NMR (CDCl3, 300 MHz)
d 202.52,
170.43, 132.06, 122.45, 113.65, 75.21, 71.97, 67.78, 61.46, 55.47,
44.90, 42.43, 39.94, 29.19, 21.20,17.09; MS(ESI) m/z: 509.1 [M þ H]þ,
526.1 [M þ NH4]þ; HR-MS (ESI, M þ NH4) m/z: calcd for C29H36NO8:
526.2435, found 526.244.
4.2.4. ent-1
a
-O-Acetyl-14
b
-O-benzoyl-6,7,15-trioxo-7,20-epoxy-
6,7-seco-16-kaurene (12d)
ꢂ
White solid, 62% yield: m.p. 249e252 C; IR (KBr) ymax 2965,
4.2.5. ent-1a-O-Acetyl-14b-O-p-methylbenzoyl-6,7,15-trioxo-7,20-
epoxy-6,7-seco-16-kaurene (12e)
2025, 1746, 1711, 1643, 1455, 738, 713 cmꢃ1
;
1H NMR (CDCl3,
300 MHz),
d
(ppm) 9.76 (1H, d, J ¼ 4.2 Hz, eCHO), 7.95 (2H, d,
White solid, 61% yield: m.p. 176e178 ꢂC; IR (KBr) ymax 2956,
J ¼ 7.2 Hz, AreH), 7.53 (1H, t, J ¼ 7.5 Hz, AreH), 7.38 (2H, t, J ¼ 7.5 Hz,
2025, 1740, 1647, 1455, 1373, 1233, 803 cmꢃ1 1H NMR (CDCl3,
;

D. Li et al. / European Journal of Medicinal Chemistry 59 (2013) 322e328
327
300 MHz),
d
(ppm) 9.76 (1H, d, J ¼ 4.2 Hz, eCHO), 7.87 (2H, d,
23.82, 21.40; MS(ESI) m/z: 543.2 [M þ H]þ, 560.3 [M þ NH4]þ, 577.4
[M þ Cl]ꢃ; HR-MS (ESI, M þ H) m/z: calcd for C29H32ClO8: 543.178,
found 543.1792.
J ¼ 7.8 Hz, AreH), 7.17 (2H, t, J ¼ 7.8 Hz, AreH), 6.23 (1H, s, 17-CH2),
5.63 (1H, s, 17-CH2), 5.22, 5.12 (each 1H, dd, JA ¼ JB ¼ 12.3 Hz, 20-
CH2), 4.66 (1H, m, 1-CH), 4.40 (1H, s, 14-CH), 3.07 (1H, d,
J ¼ 9.0 Hz, 13-CH), 2.03 (3H, s, eCH3), 1.28 (3H, s, eCH3), 1.19 (3H, s,
4.2.10. ent-1a-O-Acetyl-14b-O-o-chlorobenzoyl-6,7,15-trioxo-7,20-
eCH3), 1.04 (3H, s, eCH3); 13C NMR (CDCl3, 300 MHz)
d
202.66,
epoxy-6,7-seco-16-kaurene (12j)
170.64, 122.13, 74.58, 71.86, 67.17, 64.55, 61.34, 37.92, 34.39, 30.41,
29.65, 23.66, 21.97, 18.05; MS(ESI) m/z: 523.2 [M þ H]þ, 540.3
[M þ NH4]þ; HR-MS (ESI, M þ NH4) m/z: calcd for C30H38NO8:
540.2592, found 540.2596.
White solid, 64% yield: m.p. 144e146 ꢂC; IR (KBr) ymax 3066,
2959, 1736, 1649, 1473, 1437, 1374, 1235, 1121, 1052, 748, 703 cmꢃ1
;
1H NMR (CDCl3, 300 MHz),
d
(ppm) 9.76 (1H, d, J ¼ 4.2 Hz, eCHO),
7.80 (1H, d, J ¼ 7.5 Hz, AreH), 7.36 (2H, m, AreH), 7.25 (1H, m, Are
H), 6.22 (1H, s, 17-CH2), 5.92 (1H, s, 14-CH), 5.63 (1H, s, 17-CH2),
5.12, 5.22 (each 1H, dd, JA ¼ JB ¼ 11.4 Hz, 20-CH2), 4.66 (1H, m, 1-
CH), 3.06 (1H, d, J ¼ 7.8 Hz, 13-CH), 2.02 (3H, s, eCH3), 1.17 (3H, s,
4.2.6. ent-1a-O-Acetyl-14b-O-p-methoxybenzoyl-6,7,15-trioxo-
7,20-epoxy-6,7-seco-16-kaurene (12f)
White solid, 25% yield: m.p.120e122 ꢂC; IR (KBr) ymax 2959, 2025,
eCH3), 1.05 (3H, s, eCH3); 13C NMR (CDCl3, 300 MHz)
d 201.89,
1739, 1606, 1512, 1462, 1373, 1257, 849 cmꢃ1
;
1H NMR (CDCl3,
171.28, 146.79, 131.24, 121.36, 74.39, 73.40, 69.59, 59.25, 45.98,
40.19, 36.21, 32.94, 30.46, 29.56, 25.30; MS(ESI) m/z: 543.1
[M þ H]þ, 560.2 [M þ NH4]þ, 577.3 [M þ Cl]ꢃ; HR-MS (ESI, M þ NH4)
m/z: calcd for C29H35ClNO8: 560.2046, found 560.2057.
300 MHz),
d
(ppm) 9.76 (1H, d, J¼ 4.5Hz, eCHO), 7.90(2H, d,J¼ 9.0Hz,
AreH), 6.88 (2H, d, J ¼ 8.7 Hz, AreH), 6.23 (1H, s,17-CH2), 5.92 (1H, s,
14-CH), 5.63 (1H, s,17-CH2), 5.22, 5.12 (each 1H, dd, JA ¼ JB ¼ 12.3 Hz,
20-CH2), 4.66(1H, m,1-CH), 3.82(3H, s,eOCH3),3.03(1H, d,J¼ 8.7Hz,
13-CH), 2.03 (3H, s, eCH3), 1.19 (3H, s, eCH3), 1.02 (3H, s, eCH3); 13
C
4.3. MTT assay in vitro
NMR (CDCl3, 300 MHz) d 202.39,170.53,146.73,131.14,129.25,121.43,
74.42, 73.98, 66.28, 62.62, 47.85, 45.66, 43.55, 42.07, 39.35, 32.84,
31.74, 29.31, 28.15, 24.85, 21.38, 20.63, 17.41; MS(ESI) m/z: 539.1
[M þ H]þ, 556.3 [M þ NH4]þ, 573.3 [M þ Cl]ꢃ; HR-MS (ESI, M þ NH4)
m/z: calcd for C30H38NO9: 556.2541, found 556.2546.
The MTT assay was employed in cytotoxicity assay in vitro,
which was performed in 96-well plates. K562 cells at the log phase
of their growth cycle (5 ꢁ 104 cell/mL) were added to each well
(100
trations of the samples (0.39e100
37 ꢂC in a humidified atmosphere of 5% CO2. After 72 h, 20
m
mL/well), then treated in three replicates at various concen-
mg/mL), and incubated for 24 h at
4.2.7. ent-1a-O-Acetyl-14
b-O-p-trifluoromethylbenzoyl-6,7,15-
L of
trioxo-7,20-epoxy-6,7-seco-16-kaurene (12g)
MTT solution (5 mg/mL) per well was added to each cultured
medium, which was incubated for another 4 h. Then, DMSO was
added to each well (150 mL/well). After 10 min at room tempera-
ture, 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 reference agent was 0.1% DMSO,
and Taxol was used as the positive reference with the concentration
Yellow solid, 28% yield: m.p. 226e228 ꢂC; IR (KBr) ymax 2927,
2855, 1729, 1466, 1412, 1375, 1326, 1278, 1129, 1066, 863, 772 cmꢃ1
;
1H NMR (CDCl3, 300 MHz),
d
(ppm): 9.79 (1H, s, eCHO), 8.06 (2H, m,
AreH), 7.67 (2H, m, AreH), 6.32 (1H, s, 17-CH2), 5.98 (1H, s, 14-CH),
5.66 (1H, s, 17-CH2), 5.14, 4.76 (each 1H, dd, JA ¼ JB ¼ 12.0 Hz, 20-
CH2), 3.13 (1H, d, J ¼ 8.4 Hz, 13-CH), 1.26 (3H, s, 18-CH3), 1.22 (3H,
s, 19-CH3); 13C NMR (CDCl3, 300 MHz)
d
202.36, 170.44, 131.35,
of 10
mg/mL. The same method was used in the test against CaEs-17,
128.71, 121,78, 74.55, 73.29, 66.39, 62.59, 45.78, 42.29, 39.45, 32.90,
29.68, 21.43, 17.46; MS(ESI) m/z: 577.1 [M þ H]þ; HR-MS (ESI,
M þ NH4) m/z: calcd for C30H35F3NO8: 594.2309, found 594.2309.
Bel-7402 and MGC-803 cell lines.
4.4. GSTp activity inhibition assay
4.2.8. ent-1
a
-O-Acetyl-14
b
-O-p-nitrobenzoyl-6,7,15-trioxo-7,20-
Bel-7402 cells were cultured in RPMI-1640 medium supple-
mented with 100 U/mL penicillin,100 g/mL streptomycin, 1 mmol/
-glutamine, and 10% heat-inactivated fetal bovine serum in
a humidified atmosphere of 95% air and 5% CO2 at 37 ꢂC. Cell lysates
epoxy-6,7-seco-16-kaurene (12h)
m
White solid, 80% yield: m.p. 138e140 ꢂC; IR (KBr) ymax 2959,
L L
2025, 1731, 1606, 1529, 1464, 1349, 843 cmꢃ1 1H NMR (CDCl3,
;
300 MHz),
d
(ppm) 9.79 (1H, d, J ¼ 4.2 Hz, eCHO), 8.24 (2H, d,
were prepared as reported before [19e21] and the GSTp activity
J ¼ 8.7 Hz, AreH), 8.11 (2H, d, J ¼ 8.7 Hz, AreH), 6.23 (1H, s, 17-CH2),
5.78 (1H, s, 14-CH), 5.63 (1H, s, 17-CH2), 5.22, 5.12 (each 1H, dd,
JA ¼ JB ¼ 11.7 Hz, 20-CH2), 4.66 (1H, m, 1-CH), 3.07 (1H, d, J ¼ 9.0 Hz,
was measured spectrophotometrically at 25 ꢂC using 1-cholro-2,4-
dinitrobenzene (CDNB) and GSH as substrates. The CDNB-GSH
complex has a strong absorptivity at 340 nm and the extinction
13-CH), 2.03 (3H, s, eCH3), 1.17 (3H, s, eCH3), 1.05 (3H, s, eCH3); 13
C
coefficient of 9.6 mMꢃ1 cmꢃ1 was used to calculate GST
p
activity.
activity was defined as nanomoles of product per minute per
milligram of protein. The GST activity inhibition rate was calcu-
activity of control
activity of treated group.
NMR (CDCl3, 300 MHz)
d
202.41, 170.44, 166.48, 146.72, 121.23,
GSTp
74.39, 72.14, 66.27, 62.78, 45.66, 43.57, 42.17, 39.33, 32.85, 27.61,
p
23.87, 21.42, 17.38; MS(ESI) m/z: 553.8 [M þ H]þ, 571.3 [M þ NH4]þ,
lated as (VceVt)/Vc ꢁ 100%. Vc refers to GST
p
588.4 [M þ Cl]ꢃ; HR-MS (ESI, M þ NH4) m/z: calcd for C29H35N2O10
:
group; Vt refers to GSTp
571.2286, found 571.2294.
4.5. Cell cycle analysis
4.2.9. ent-1a-O-Acetyl-14b-O-p-chlorobenzoyl-6,7,15-trioxo-7,20-
epoxy-6,7-seco-16-kaurene (12i)
Bel-7402 cells were plated in 6-well plates (5.0 ꢁ 103 cells/well)
and incubated at 37 ꢂC for 24 h. Exponentially growing cells were
then incubated with tested compound at a certain concentration.
Untreated cells (control) or cells treated with the solvent (DMSO) of
the compound were included. After 48 h treatment, cells were
centrifuged and fixed in 70% ethanol at 4 ꢂC overnight and subse-
White solid, 63% yield: m.p. 118e120 ꢂC; IR (KBr) ymax 2956,
2025, 1726, 1632, 1594, 1272, 821 cmꢃ1; 1H NMR (CDCl3, 300 MHz),
d
(ppm) 9.73 (1H, d, J ¼ 4.2 Hz, eCHO), 7.85 (2H, d, J ¼ 8.1 Hz, AreH),
7.32 (2H, d, J ¼ 8.1 Hz, AreH), 6.23 (1H, s, 17-CH2), 5.92 (1H, s, 14-
CH), 5.63 (1H, s, 17-CH2), 5.27, 5.15 (each 1H, dd, JA ¼ JB ¼ 9.3 Hz,
20-CH2), 4.64 (1H, m,1-CH), 3.02 (1H, d, J ¼ 9.0 Hz,13-CH), 2.10 (3H,
s, eCH3), 1.24 (3H, s, eCH3), 1.05 (3H, s, eCH3); 13C NMR (CDCl3,
quently resuspended in PBS containing 100 mL RNase A and 400 mL
propidium iodide (PI). Cellular DNA content, for cell cycle distri-
bution analysis, was measured using a flow cytometer (FACS Cal-
ibur BectoneDickinson).
300 MHz)
d 202.29, 170.47, 146.71, 131.08, 123.48, 122.28, 74.69,
73.72, 66.49, 62.24, 45.79, 43.63, 42.16, 39.56, 32.93, 29.93, 24.01,

328
D. Li et al. / European Journal of Medicinal Chemistry 59 (2013) 322e328
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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.
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