Library Construction and Biological Evaluation of Enmein-Type Diterpenoid Analogues as Potential Anticancer Agents

Article abstract of DOI:10.1002/cmdc.201200559

A library of promising enmein-type 14-O-diterpenoid derivatives was constructed from a commercially available kaurene-type oridonin by practical and efficient synthetic methods. These synthetic derivatives were evaluated for their antiproliferative activities against a set of four human cancer cell lines. The IC₅₀ values are similar to or improved over those of the parent molecule and paclitaxel, the latter of which was used as a positive control. Compound 29 was further investigated for its apoptotic properties against human hepatocarcinoma Bel-7402 cells to better understand its mode of action. Moreover, compound 29 was shown to have potent antitumor activity invivo in studies with a murine model of gastric cancer (MGC-803 mice). These results warrant further preclinical investigations of these diterpenoid-based analogues as potential novel anticancer chemotherapeutics. Dosing with diterpenoids: A synthetic library of enmein-type diterpenoid derivatives with potent antitumor activities was constructed by using commercially available starting materials. The analogue shown here exhibits potent invitro and invivo activities and suitable chemical properties similar to or exceeding those of the parent compound, warranting further preclinical investigations for potential diterpenoid-based anticancer agents. Copyright

Full text of DOI:10.1002/cmdc.201200559

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DOI: 10.1002/cmdc.201200559
Library Construction and Biological Evaluation of Enmein-
Type Diterpenoid Analogues as Potential Anticancer
Agents
Dahong Li,[a, b] Shengtao Xu,[a, b] Hao Cai,[a, b] Lingling Pei,[a] Lei Wang,[a] Xiaoming Wu,[a, b]
Hequan Yao,[a, b] Jieyun Jiang,[c] Yijun Sun,[d] and Jinyi Xu*[a, b]
A library of promising enmein-type 14-O-diterpenoid deriva-
tives was constructed from a commercially available kaurene-
type oridonin by practical and efficient synthetic methods.
These synthetic derivatives were evaluated for their antiproli-
ferative activities against a set of four human cancer cell lines.
The IC50 values are similar to or improved over those of the
parent molecule and paclitaxel, the latter of which was used as
a positive control. Compound 29 was further investigated for
its apoptotic properties against human hepatocarcinoma Bel-
7402 cells to better understand its mode of action. Moreover,
compound 29 was shown to have potent antitumor activity
in vivo in studies with a murine model of gastric cancer (MGC-
803 mice). These results warrant further preclinical investiga-
tions of these diterpenoid-based analogues as potential novel
anticancer chemotherapeutics.
Introduction
Nature is an untapped source of unique and desirable scaffolds
for library construction and subsequent drug discovery.[1] Natu-
ral products (NPs) have been the major sources of chemical di-
versity as starting materials, driving pharmaceutical discovery
over the past century. A total of 19 NP-based drugs were ap-
proved for marketing worldwide from 2005 to 2010, of which
seven compounds were classified as NPs, 10 compounds as
semisynthetic NPs, and two compounds as NP-derived drugs.[2]
Indeed, numerous NP scaffolds have led to approved drugs or
drug candidates for a range of diseases, especially anticancer
agents (paclitaxel, rapamycins, epothilones).[3] The success of
NPs and their semisynthetic derivatives as therapeutic agents
is intrinsically linked to the fact that NPs are often referred to
as evolutionarily selected “privileged structures.”[1,4]
skeletons have been isolated and characterized.[6] Among
these, enmein-type ent-kaurane diterpenoids (Figure 1) with
unique chemical skeletons have exhibited promising antitumor
activities.[5,7] However, the structures are generally quite com-
plex, incorporating large numbers of stereogenic centers and
intricate ring systems. Therefore, preparations of NP-based li-
braries inevitably involve rather sophisticated and laborious
synthetic sequences. Furthermore, therapeutic development of
these leads is significantly impeded by the problem of large-
scale compound supply.
The genus Isodon is famous for producing bioactive diterpe-
noids with diverse skeletons, especially ent-kaurane diterpe-
noids.[5,6] Approximately 600 new diterpenoids with diverse
[a] Dr. D. Li, Dr. S. Xu, H. Cai, L. Pei, Dr. L. Wang, Prof. X. Wu, Prof. H. Yao,
Prof. J. Xu
Department of Medicinal Chemistry
China Pharmaceutical University, 24 Tongjia St., Nanjing 210009 (China)
E-mail: jinyixu@china.com
[b] Dr. D. Li, Dr. S. Xu, H. Cai, Prof. X. Wu, Prof. H. Yao, Prof. J. Xu
State Key Laboratory of Natural Medicines
China Pharmaceutical University, 24 Tongjia St., Nanjing 210009 (China)
[c] Prof. J. Jiang
Department of Microbiology, Immunology and Molecular Genetics
University of Kentucky College of Medicine
800 Rose St., Lexington, KY 40536 (USA)
[d] Y. Sun
Drug Screening Center, Nanjing KeyGen Biotech. Co. Ltd.
439 Changhong Road, Nanjing 210012 (China)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200559.
Figure 1. Reported enmein-type diterpenoids isolated from natural plant
sources and their antiproliferative activities in cancer cells.
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10–12), improved IC50 values were observed against K562 (the
lowest of which was 0.31 mm for 11) and Bel-7402 cells (from
0.78 to 1.37 mm). Compounds with an aromatic group generally
showed more potent antiproliferative activities. In particular,
a pronounced inhibitory effect was observed for those com-
pounds with an electron-withdrawing nitro (16) or chloro (17
and 18) group. Although fluorinated compounds comprise
a substantial proportion of therapeutic drugs and impart a vari-
ety of properties to certain medicines,[11] compounds 19–22 ex-
hibited only moderate IC50 values in vitro. Adding a linkage be-
tween the enmein-type skeleton and an aromatic group (23–
27) maintained good activity in Bel-7402 cells. A similar effect
was achieved in compounds for which the R group was a heter-
ocyclic ring (27–30).
Results and Discussion
Chemistry
While total synthesis of the required diterpenoid scaffold is
a possible solution to the supply issue,[8] the structural com-
plexity and need for multistep syntheses to make some of the
templates can be prohibitive.[9] Our approach (Scheme 1) in-
Compound 29, with a 3-pyridyl R moiety, could react with
various acids to form salts, therefore solving the critical prob-
lem of poor solubility of the diterpenoids. Thus, compound 29
was chosen for further investigation. In the MTT assay, 17 of 21
derivatives showed more promising IC50 values than paclitaxel
against Bel-7402 cells. Consequently, this cell line was selected
for intensive mechanistic studies.
Cell-cycle analysis
Scheme 1. Synthesis of enmein-type diterpenoid building block compound
9 and a library of 14-O-derivatives (10–30). Reagents and conditions:
a) NaIO4, H2O, 24 h, RT; b) Jones reagent, acetone, 15 min, 08C; c) corre-
sponding acid, EDCI, DMAP, CH2Cl2, 8–12 h, RT.
To determine whether the suppression of cell growth by
enmein-type diterpenoids is caused by a cell-cycle effect, we
detected the DNA content of cell nuclei by flow cytometry
volves the use of relevant commercially available re-
Table 1. Cytotoxicity of synthetic enmein-type diterpenoid analogues 10–30 against
various human cancer cell lines.
sources to acquire target NP scaffolds, thus bypassing
the de novo synthetic strategy for scaffold produc-
tion. Starting from commercially available oridonin
(7), enmein-type diterpenoid analogue 8 was ob-
tained by treatment with sodium periodate in water.
Oxidation of 8 with Jones reagent at 08C afforded
corresponding ketone 9 in total yields of 87%. The
procedure is well-established, and the mechanism of
structure conversion has also been studied.[10] Com-
pound 9 provided a good building block to construct
a library of 14-O-derivatives (10–30) of enmein-type
diterpenoids with diverse chemical properties and
potential anticancer activities.
Compd
R
IC50 [mm][a]
Bel-7402 CaEs-17
K563
MGC-803
Paclitaxel
Oridonin
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
0.41Æ0.02 1.89Æ0.09 0.43Æ0.03 0.85Æ0.06
4.76Æ0.32 7.48Æ0.53 11.03Æ1.02 5.69Æ0.39
0.35Æ0.05 1.37Æ1.03 0.69Æ0.12 1.27Æ0.10
0.31Æ0.07 0.99Æ0.04 0.47Æ0.09 1.13Æ0.02
1.82Æ0.17 0.78Æ0.06 3.77Æ0.13 1.23Æ0.19
methyl
ethyl
cyclopentyl
phenyl
4-methylphenyl
4-methoxylphenyl
4-nitrophenyl
4-chlorophenyl
2-chlorophenyl
4-fluorophenyl
3-fluorophenyl
0.33Æ0.02 0.79Æ0.11
0.81Æ0.05 0.46Æ0.08
0.52Æ0.09 1.58Æ0.22 1.78Æ0.17 1.54Æ0.19
2.18Æ0.11 3.93Æ0.62 3.83Æ0.42 3.47Æ0.50
0.18Æ0.10 0.82Æ0.07 0.27Æ0.02 0.66Æ0.11
0.14Æ0.08 0.89Æ0.09 0.34Æ0.03 0.34Æ0.01
0.26Æ0.03 0.72Æ0.01 0.28Æ0.16 0.82Æ0.07
2.26Æ0.17 1.95Æ1.06 4.66Æ0.53 1.93Æ0.14
2.03Æ0.21 1.25Æ0.44 4.47Æ0.53 1.68Æ0.45
In vitro antiproliferative activities and structure–-
activity relationships
2,3,4,5-tetraflrorophenyl 2.15Æ0.27 1.98Æ0.80 4.07Æ0.02 3.43Æ0.94
4-trifluoromethylphenyl 0.69Æ0.07 1.85Æ0.12 0.93Æ0.10 1.44Æ0.19
4-fluorophenylvinyl
2-phenylethyl
4-fluorophenylmethyl
1-naphthylmethyl
3-(1H-indolyl)methyl
2-quinolyl
2.01Æ0.35 1.08Æ0.14 4.33Æ0.53 1.62Æ0.11
1.90Æ0.09 0.89Æ0.40 3.90Æ0.21 1.38Æ0.31
0.46Æ0.03 1.72Æ0.07 0.30Æ0.10 0.87Æ0.16
Antiproliferative activities of enmein-type diterpenoid
analogues 10–30 were evaluated by MTT assay
against four cancer cell lines (K562, Bel-7402, CaEs-17,
and MGC-803). The results are summarized in Table 1.
All of the derivatives exhibited stronger antiprolifera-
tive activities than parent compound oridonin in the
four cancer cell lines, and some were even more
active than positive control paclitaxel (in red), espe-
cially against human hepatoma Bel-7402 cells. When
the R substituent was an alkyl group (compounds
25
26
27
28
29
30
1.65Æ0.07 0.77Æ0.11
3.81Æ0.09 1.47Æ0.16
1.60Æ0.03 0.68Æ0.20 3.16Æ0.13 1.12Æ0.08
1.41Æ0.18 0.63Æ0.07 2.69Æ0.11 0.76Æ0.15
1.74Æ0.21 0.71Æ0.36 3.54Æ0.27 1.16Æ0.23
2.72Æ0.26 1.93Æ1.04 5.41Æ0.06 2.86Æ0.55
3-pyridyl
2-tetrahydrofuryl
[a] IC50: concentration required to inhibit cell growth by 50% relative to untreated
control cells; results are expressed as the mean ÆSD of three independent experi-
ments. Human cell lines: leukemia (K562); hepatoma (Bel-7402); esophageal cancer
(CaEs-17); gastric cancer (MGC-803).
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were stained with annexin V–
FITC and PI. In the early stages
of apoptosis, phosphatidylserine
was translocated from within the
cell membrane to outside of it.
Annexin V, a calcium-dependent
phospholipid-binding
protein
characterized by a high affinity
for phosphatidylserine, was used
to detect early apoptotic cells. PI
is a red fluorescent dye that
stains cells that have lost their
membrane
integrity.
Cells
stained with annexin V–FITC and
PI were classified as necrotic
cells (annexinÀ/PI+), late apop-
totic cells (annexin+/PI+), intact
cells (annexinÀ/PIÀ), or early
apoptotic cells (annexin+/PIÀ).
Percentages of apoptotic Bel-
7402 cells were determined by
flow cytometry. As shown in
Figure 3, compound 29 exhibit-
ed potent dose-dependent activ-
ity in the induction of apoptosis.
Treatment of Bel-7402 cells with
compound 29 at 0.25, 0.5, and
1.0 mm for two days resulted in
16.6, 24.9, and 33.5% apoptotic
cells (early and late), as com-
pared with 8.3% in an untreated
Figure 2. Influence of the Bel-7402 cell cycle by enmein-type compound 29. Bel-7402 cells were incubated with
the indicated concentrations of 29 for 48 h before staining with PI. Cellular DNA content for cell-cycle distribution
analysis was measured by flow cytometry. The diagrams show the distribution of cells according to their DNA con-
tent; inserts give the percentages of cells in various cell-cycle phases.
(Figure 2). Bel-7402 cells were treated with compound 29 at
concentrations of 0.25, 0.5, and 1.0 mm, which resulted in accu-
mulation of 12.81, 22.12, and 26.89% of cells at the G2/M
phase, respectively, compared with the untreated cells. The in-
fluence of cell-cycle progression
vehicle control, indicating that compound 29 was able to
induce apoptotic cell death in Bel-7402 cells. During the pro-
cess of apoptosis, apoptotic signals can result in the loss of mi-
tochondrial membrane potential. To determine plausible path-
at low micromolar concentra-
tions of enmein-type diterpenoid
analogues makes these com-
pounds promising agents for
use in combination with anti-
cancer drugs acting at different
stages of the cell cycle.
Induction of apoptosis
To clarify whether the loss of
cancer cell viability promoted by
enmein-type diterpenoids is as-
sociated with apoptosis, an an-
nexin V–FITC/propidium iodide
(PI) binding assay was per-
formed. Bel-7402 cells were
treated with vehicle alone (con-
trols) or with various concentra-
tions (0.25, 0.5, or 1.0 mm) of
Figure 3. Apoptosis ratio detection of compound 29. Bel-7402 cells were incubated with the indicated concentra-
compound 29 for 48 h, then tions of 29 for 48 h before staining with annexin V–FITC and PI, followed by flow cytometric analysis.
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48 h treatment with compound
29 in a concentration-dependent
manner. Further western blotting
analysis revealed that incubation
with 29 dramatically increased
the relative levels of pro-apop-
totic cytochrome c, Bax, and Fas
expression, but decreased the
levels of anti-apoptotic Bcl2 ex-
pression (Figure 5), thus suggest-
ing the mitochondrial pathway
for activation of this important
mechanism of tumor cell death.
Antitumor activity in vivo
Based on the in vitro results and
intensive mechanistic studies,
we further tested the antitumor
activity of compound 29 in vivo
Figure 4. Effect of compound 29 on the mitochondrial membrane potentials of Bel-7402 cells. Bel-7402 cells were
incubated with the indicated concentrations of 29 for 48 h prior to staining with JC-1.
ways by which 29 triggered cell
apoptosis, Bel-7402 cells were in-
cubated with different concen-
trations (0, 0.25, 0.5, 1.0 mm) of
29 for 48 h prior to staining with
the lipophilic mitochondrial
probe
5,5’,6,6’-tetrachloro-
1,1’,3,3’-tetraethylbenzimidazol-
carbocyanine iodide (JC-1). The
number of cells with collapsed
mitochondrial membrane poten-
tials in different groups of cells
were determined by flow cytom-
etry analysis (Figure 4), yielding
0, 8.5, 17.6, and 45.6% apoptotic
cells, respectively. These studies
showed that incubation with 29
increased the number of cells
with collapsed mitochondrial
membrane potentials.
Effect of apoptosis-related
proteins
In general, all apoptotic path-
ways depend on activation of
caspases for the final execution
of apoptosis. Therefore, it might
be reasonable to first look into
caspases as a sign of apoptosis.
Within this context, the assess-
ment of the activities of caspas-
es 3, 8, and 9 in Bel-7402 cells
Figure 5. a) Western blot analysis of compound 29 on the expression of apoptosis-related proteins in Bel-7402
cells. Lane 1: negative control, 2: 0.25 mm 29, 3: 0.5 mm 29, 4: 1.0 mm 29; b) quantitative analysis showing the
showed strong activation after levels of each protein relative to control b-actin: *p<0.05, **p<0.01 vs. vehicle control.
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with an FTMS-2000 instrument. HRMS data were collected with an
Agilent QTOF 6520.
by performing an assay in mice with gastric cancer (MGC-803).
Oridonin and compound 29 were administered intraperitoneal-
ly 40 mgkgÀ1 in a vehicle of 1% DMSO, 2% poloxamer, and
97% saline. The positive control group was treated with pacli-
taxel (10 mgkgÀ1) through intravenous injection. The negative
control group received 0.9% normal saline through intraperito-
neal injection. Results are shown in Table 2. Compound 29 ex-
Compound 8: Compound 7 (364 mg, 1 mmol) was dissolved in
H2O (10 mL). NaIO4 (3.16 g, 14.8 mmol) was added to this solution,
and the mixture was stirred at room temperature for 24 h and ex-
tracted with CH2Cl2. The CH2Cl2 layer was washed with brine, dried
over anhydrous Na2SO4, filtered, and evaporated to give compound
8 (360 mg, 99%) as a white powder; mp: 183–1858C; 1H NMR
([D6]DMSO, 500 MHz): d=6.18 (1H, d, J=3.3 Hz, 6-OH), 5.92 (1H, s,
17-CH2), 5.52 (1H, s, 17-CH2), 5.38 (1H, d, J=2.1 Hz, 14-OH), 5.17
(1H, d, J=2.1 Hz, 6-CH), 4.67 (1H, s, 14-CH), 4.65–4.63 (1H, m, 1-
CH), 3.87, 3.58 (each 1H, dd, JA =JB =9.1 Hz, 20-CH2), 2.95 (1H, d,
J=9.1 Hz, 13-CH), 2.49–2.40 (2H, m, 12-CH2), 1.70–1.67 (2H, m, 2-
CH2), 1.40–1.36 (2H, m, 11-CH2), 1.29–1.25 (1H, m, 9-CH), 0.93 (3H,
s, 18-CH3), 0.88 ppm (3H, s, 19-CH3); 13C NMR ([D6]DMSO, 300 MHz):
d=200.30, 166.88, 150.42, 118.59, 100.73, 72.81, 71.28, 62.18, 54.99,
53.78, 49.86, 47.67, 43.07, 36.44, 32.67, 30.70, 29.55, 23.22, 22.82,
18.75 ppm; IR (KBr): v˜max =3450, 1754, 1645 cmÀ1; MS(ESI) m/z:
747.4 [2M+Na]+; HRMS (ESI, M+H) m/z calcd for C20H27O6:
363.1802, found: 363.1799.
Table 2. Antitumor activity of compound 29 in mice with MGC-803 gas-
tric cancer.[a]
Compd
Mouse weight [g]
Tumor weight [g]
Inhib. [%]
–
Start
End
Saline
15.41Æ0.55
23.45Æ1.19
1.26Æ0.24
(control)
Paclitaxel
Oridonin
29
15.37Æ0.47
15.49Æ0.35
15.45Æ0.89
23.98Æ1.87
22.69Æ1.35
23.27Æ1.23
0.36Æ0.15
0.79Æ0.09
0.45Æ0.11
71.43*
37.30**
64.28*
[a] Number of mice in each group was 10; all mice completed treatment;
weights given are the mean ÆSD. *p<0.01, **p<0.05.
Compound 9: Compound 8 (72 mg, 0.2 mmol) was dissolved in
acetone. Jones reagent was added dropwise to this solution until
a red color persisted. The mixture was stirred at 08C for 15 min.
The mixture was then diluted with H2O and extracted with CH2Cl2.
The extract was washed with brine, dried over anhydrous Na2SO4,
filtered, and evaporated to give compound 9 (63 mg, 87%) as
a white powder; mp: 248–2508C; 1H NMR ([D6]DMSO, 300 MHz):
d=5.95 (1H, s, 17-CH2), 5.57 (1H, s, 17-CH2), 5.53 (1H, d, J=3.5 Hz,
14-OH), 4.79–4.76 (1H, m, 14-CH), 4.76–4.72 (1H, m, 1-CH), 4.51,
3.69 (each 1H, dd, JA =JB =9.9 Hz, 20-CH2), 3.00 (1H, d, J=9.6 Hz,
13-CH), 1.08 (3H, s, 18-CH3), 0.95 ppm (3H, s, 19-CH3); 13C NMR
([D6]DMSO, 300 MHz): d=200.25, 175.45, 166.31, 149.84, 119.45,
73.78, 71.03, 70.98, 61.66, 50.19, 47.42, 45.77, 42.87, 35.50, 32.66,
31.83, 29.09, 23.42, 22.87, 18.15 ppm; IR (KBr): v˜max =3508, 1785,
1751, 1645 cmÀ1; MS(ESI) m/z: 361.1 [M+H]+; HRMS (ESI, M+H)
m/z calcd for C20H25O6: 361.1646, found: 361.1647.
hibited a stronger antitumor activity (tumor inhibitory ratio of
64.28%) than that of parent compound oridonin (37.30%) and
only slightly less potent than that of paclitaxel (71.43%) in
MGC-803 mice. Thus, compound 29 is worthy of further inves-
tigation as a potential anticancer drug candidate.
Conclusions
The findings arising from the studies described above open
a possible approach to the development of novel enmein-type
diterpenoid analogues as potential anticancer agents. In this
effort, a novel semisynthetic library of enmein-type diterpenoid
derivatives with potent in vitro and in vivo antitumor activities
was prepared from commercially available starting materials.
Furthermore, the molecular mode of action revealed that de-
rivative 29 caused cell-cycle arrest and induced apoptosis in
Bel-7402 cells through oxidative-stress-triggered mitochondria-
related caspase 3-, 8-, and 9-dependent pathways. It is expect-
ed that the mechanistic and biological studies described, to-
gether with our previous reports of different kinds of diterpe-
noids,[10,12] could expedite the development of new diterpe-
noid-based therapeutic agents for clinical cancer intervention.
General procedure for the synthesis of enmein-type diterpe-
noids 10–30: Compound 9 (72 mg, 0.2 mmol) was mixed with the
corresponding acid (0.24 mmol), EDCI (0.4 mmol), and DMAP
(0.02 mmol) in 15 mL CH2Cl2. After stirring at room temperature for
8–12 h, the mixture was poured into 15 mL 10% HCl and extracted
with CH2Cl2 (3ꢁ10 mL). The organic layer was combined, washed
sequentially with H2O and saturated NaCl solution, dried over an-
hydrous Na2SO4, and concentrated in vacuo. The crude product
was purified by column chromatography (MeOH/CH2Cl2 1:100 v/v)
to give the title compounds. Data for compounds 10–28 and 30
are listed in the Supporting Information.
Compound 29: Yellow powder (41 mg, 46%): mp: 150–1528C;
1H NMR (CDCl3, 300 MHz): d=9.12 (1H, s, Ar-H), 8.77 (1H, s, Ar-H),
8.19 (1H, d, J=7.9 Hz, Ar-H), 7.39–7.28 (1H, m, Ar-H), 6.25 (1H, s,
17-CH2), 6.09 (1H, s, 14-CH), 5.68 (1H, s, 17-CH2), 4.66–4.62 (1H, m,
1-CH), 4.38, 4.11 (each 1H, dd, JA =JB =10.6 Hz, 20-CH2), 3.37 (1H,
d, J=9.3 Hz, 13-CH), 1.22 (3H, s, 18-CH3), 1.07 ppm (3H, s, 19-CH3);
13C NMR (CDCl3): d=196.92, 175.11, 165.63, 164.53, 153.87, 151.00,
146.65, 137.38, 124.87, 123.33, 121.74, 74.58, 74.31, 71.20, 59.37,
50.72, 47.53, 45.99, 40.41, 36.28, 32.99, 32.14, 29.49, 23.54, 23.01,
19.04 ppm; IR (KBr): v˜max =2925, 1762, 1731, 1647, 1591, 1282,
1188, 1023, 916, 736, 701 cmÀ1; MS(ESI) m/z: 446.2 [M+H]+; HRMS
(ESI, M+H) m/z calcd for C26H28NO7: 466.1860, found: 466.1856.
Experimental Section
Chemistry
General: 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. 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 in-
ternal standard); chemical shifts (d) are expressed in ppm, and cou-
pling constants (J) are reported in Hz. Mass spectra were obtained
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ministered intraperitoneally 40 mgkgÀ1 to two different groups in
a vehicle of 1% DMSO, 2% poloxamer, and 97% saline. The posi-
tive control group was treated with paclitaxel (10 mgkgÀ1) through
intravenous injection in a vehicle of 1% DMSO, 2% poloxamer, and
97% saline. The negative control group received 0.9% normal
saline through intraperitoneal injection. All test compounds were
given through injections after 7 d of tumor transplantation (or in-
oculation). Treatments were given at a frequency of one dose of in-
travenous or intraperitoneal injection per day for a total of 25 con-
secutive days. After the treatments, all of the mice were killed and
weighed simultaneously, and the tumors were segregated and
weighed. Ratio of inhibition of tumor (%)=(1Àaverage tumor
weight of treated group/average tumor weight of control group)ꢁ
100%. Female Balb/c mice (12–16 g) were purchased from Shang-
hai SLAC Laboratory Animals Co. Ltd. (China). All procedures were
performed following institutional approval in accordance with the
NIH Guide for the Care and Use of Laboratory Animals.
Biological evaluations
In vitro MTT assays: The MTT assay was employed in an in vitro cy-
totoxicity assay, which was performed in 96-well plates. K562 cells
at the log phase of their growth cycle (5ꢁ10 cellsmLÀ1) were
added to each well (100 mL per well) and treated in three replicates
at various concentrations of the samples (0.39–100 mgmLÀ1). The
cells were then incubated for 24 h at 378C in a humidified atmos-
phere of 5% CO2. After 72 h, 20 mL of MTT solution (5 mgmLÀ1) per
well was added to each cultured medium before incubating for an-
other 4 h. DMSO was then added to each well (150 mL per well).
After 10 min at room temperature, the OD of each well was mea-
sured using a microplate reader (Bio-Rad 550) at a wavelength of
490 nm. In these experiments, the negative reference agent was
0.1% DMSO, and paclitaxel was used as the positive reference at
a concentration of 10 mgmLÀ1. The same method was used for
CaEs-17, Bel-7402, and MGC-803 cell lines.
Cell-cycle analyses: Bel-7402 cells were plated in six-well plates
(5.0ꢁ103 cells per well) and incubated at 378C for 24 h. Exponen-
tially growing cells were then incubated with test compound at
various concentrations. Untreated cells and cells treated with
DMSO alone were included as controls. After 48 h treatment, cells
were centrifuged and fixed in 70% EtOH at 48C overnight, and
were subsequently resuspended in PBS containing 100 mL RNase A
and 400 mL PI. Cellular DNA content was measured for cell-cycle
distribution analysis using a flow cytometer (FACSCalibur, Becton–
Dickinson).
Acknowledgements
This study was supported by grants from the National Natural
Science Foundation (No. 30973610), the Specialized Research
Fund for the Doctoral Program of Higher Education (No.
20100096110001), the Project for Research and Innovation of
Graduates in Universities of Jiangsu Province (CXZZ11-0800), the
Fundamental Research Funds for the Central Universities
(JKY2011030) and the Key Fund of Ministry of Education of China
(No. 108069).
Analyses of cellular apoptosis: Bel-7402 cells were incubated with
the compounds as described above. Cells were treated with tested
compound for 48 h, and apoptosis was analyzed using annexin V
and PI double staining by flow cytometry, according to the manu-
facturer’s instructions, in order to detect apoptotic cells.
Keywords: antitumor agents · apoptosis · cell cycle · enmein-
type diterpenoids · oridonin
Mitochondrial membrane potential assays: Bel-7402 cells were cul-
tured overnight and incubated in triplicate with test compounds
(0.25, 0.5, and 1.0 mm) or vehicle for 48 h. The cells were stained
with JC-1 dye, according to the manufacturer’s instructions
(KGA601, Keygen). The percentage of cells with healthy or col-
lapsed mitochondrial membrane potentials was monitored by flow
cytometry analysis (lex =488 nm, lem =530 nm).
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Received: December 3, 2012
Revised: February 26, 2013
Published online on March 20, 2013
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ChemMedChem 2013, 8, 812 – 818 818