The conversion of oridonin to spirolactone-type or enmein-type diterpenoid: Synthesis and biological evaluation of ent-6,7-seco-oridonin derivatives as novel potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2012.03.024

Starting from commercial available natural product oridonin (1), a practical synthesis of ent-6,7-seco-oridonin derivatives (2, 3, 5, and 9) was accomplished and their biological activities were evaluated. The conversion of spirolactone-type diterpenoid to enmein-type was first completed. The results demonstrated that all synthesized ent-6,7-seco-oridonin derivatives could markedly inhibit the proliferation of cancer cells. Compared with Taxol, the most cytotoxic compound 5 has similar potency in A549 cell and slightly less cytotoxicity in Bel-7402 cell. Compound 5 was also more potent than parent compound oridonin in mice with MGC-803 gastric cancer in vivo. Then a series of novel 14-O-derivatives of 5 were further designed and synthesized, which showed better activity than 5 and similar activity as Taxol in vitro. The structure-activity relationships of oridonin derivatives were also discussed in the present investigations.

Full text of DOI:10.1016/j.ejmech.2012.03.024

European Journal of Medicinal Chemistry 52 (2012) 242e250
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
The conversion of oridonin to spirolactone-type or enmein-type diterpenoid:
Synthesis and biological evaluation of ent-6,7-seco-oridonin derivatives as novel
potential anticancer agents
,
,
,
c
Lei Wang a 1, Dahong Li a 1, Shengtao Xu a, Hao Cai a, Hequan Yao a, Yihua Zhang b , Jieyun Jiang ,
**
,
Jinyi Xu a
*
a Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing 210009, PR China
b Center of Drug Discovery, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing 210009, PR China
c Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 40536, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Starting from commercial available natural product oridonin (1), a practical synthesis of ent-6,7-seco-
oridonin derivatives (2, 3, 5, and 9) was accomplished and their biological activities were evaluated. The
conversion of spirolactone-type diterpenoid to enmein-type was first completed. The results demon-
strated that all synthesized ent-6,7-seco-oridonin derivatives could markedly inhibit the proliferation of
cancer cells. Compared with Taxol, the most cytotoxic compound 5 has similar potency in A549 cell and
slightly less cytotoxicity in Bel-7402 cell. Compound 5 was also more potent than parent compound
oridonin in mice with MGC-803 gastric cancer in vivo. Then a series of novel 14-O-derivatives of 5 were
further designed and synthesized, which showed better activity than 5 and similar activity as Taxol
in vitro. The structureeactivity relationships of oridonin derivatives were also discussed in the present
investigations.
Received 14 January 2012
Accepted 12 March 2012
Available online 23 March 2012
Keywords:
Spirolactone-type diterpenoid
Enmein-type diterpenoid
ent-6,7-seco-Oridonin derivatives
Cytotoxicity
Anti-tumor activity
Ó 2012 Elsevier Masson SAS. All rights reserved.
Structureeactivity relationship
1. Introduction
NF-kB activation [4e6], induction of G2/M phase arrest and
apoptosis [7]. However, in many cases, the potency of 1 is relatively
weak. Therefore it is needed to search for novel derivatives of 1 via
structural modification and bioassay-guided systematic drug
design and synthesis.
Natural products (NP) have been the major sources of chemical
diversity for starting materials while driving pharmaceutical
discovery over the past century. A total of 19 NP based drugs were
approved for marketing worldwide in between the year
2005e2010, among which 7 were classified as NPs, 10 as semi-
synthetic NPs, and 2 as NP derived drugs [1]. But the limited
source of plants as well as the difficulty of chemical synthesis and
modification restricted the development of natural medicines.
Meanwhile, in the last few years, a growing number of publi-
cations have reported the use of kaurane diterpenes for the
synthesis of novel antimicrobial, cytotoxic, and trypanocidal agents,
and this synthetic approach is still far from being fully exploited by
the current interest in the chemistry of natural products [2,3].
Oridonin (1) is a widely distributed ent-kaurene in the Rabdosia
plants and has recently attracted much attention because of its
anti-tumor activity with novel mechanism of inhibition effect on
6,7-seco-Oridonin derivatives (especially enmein-type and
spirolactone-type diterpenoids, Fig. 1) have been shown to have
stronger cytotoxicity than oridonin [8e11] but they were much
more difficult to be isolated from natural plants source. So the
conversion of commercial available oridonin to 6,7-seco-oridonin
derivatives may provide ample materials for intensive study.
Furthermore, in previous studies we found that a series of novel 1-
O- and 14-O-derivatives of 1 exhibited stronger cytotoxicity against
six cancer cell lines (BGC-7901, SW-480, HL-60, Bel-7402, A549, and
B16) in vitro and some of them have stronger anti-tumor activity
than parent compound 1 and positive control cyclophosphamide in
mice with H22 liver tumor in vivo [12,13]. It has been believed that
the 6,7-dihydroxyl-15-oxo-16-methylene moiety of those
compounds is indispensable for the biological activity of most
natural ent-kaurene diterpenoids [14,15]. In this regard, it is inter-
esting to examine whether the 6,7-seco-15-oxo-16-methylene
derivatives of ent-kaurene diterpenoids with enmein- or
spirolactone-type structures (Fig. 2) would remain or further
* Corresponding author. Tel.: þ86 025 83271445.
** Corresponding author. Tel.: þ86 025 83271015.
E-mail addresses: zyhtgd@sohu.com (Y. Zhang), jinyixu@china.com (J. Xu).
Co-first authors.
1
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.024

L. Wang et al. / European Journal of Medicinal Chemistry 52 (2012) 242e250
243
Fig. 1. Some enmein-type or spirolactone-type diterpenoids isolated from natural plants and their cytotoxicity.
improve anti-tumor activity of oridonin. So the synthesis and bio-
logical evaluation of ent-6,7-seco-oridonin derivatives were
accomplished, and the conversion of spirolactone-type diterpenoid
to enmein-type was first completed. Based on the observed phar-
macological data, structureeactivity relationships have been
established.
Compound 6 upon reaction with Ac2O/DMAP/TEA led to acetylated
compound 7 in the yield of 95%. Deprotection of 7 with 10% HCl/THF
(1:1) gave the corresponding alcohol 8 in almost quantitative yield
[18]. Interestingly, compounds 4 and 8 without a hydroxyl group at
C-1 could be converted to spirolactone aldehydes 5 and 9 instead of
enmein-type compounds by oxidation using lead tetraacetate in
the presence of sodium carbonate in THF in yields of 95% and 97%,
respectively [19].
2. Results and discussion
Hydrolyzed the spirolactone-type compound 9 with 1 mol/l
NaOH, and then enmein-type compound 2 was got again (Scheme
3). We assumed that the conversion mechanism of kaurene-type to
enmein-type or spirolactone-type diterpenoid was shown in
Scheme 4. The above convenient conversion made enmein-type or
spirolactone-type diterpenoids available for our further research to
find new chemical entities for anticancer agents.
2.1. Chemistry
The conversion of kaurene-type to 6,7-seco-kaurene-type
compounds were achieved by periodate or lead tetraacetate
oxidation [14,16,17]. The products were either enmein-type or
spirolactone-type. When the starting material has a hydroxyl group
at C-1, the former was obtained; otherwise, the latter formed.
The synthesis of enmein-type derivatives is outlined in Schemes
1 and 2. Treatment of 1 with sodium periodate in water gave ester 2
in 91% yield. Oxidation of 1 and 2 with Jones reagent at 0 ꢀC
afforded corresponding ketones 3 and 4 in yields of 87% and 98%,
respectively. Treatment of 1 with 2,2-dimethoxypropane in the
presence of TsOH in acetone provided ketal 6 in 85% yield.
Since 14-O-derivatives of 1 exhibited stronger cytotoxicity than
parent compound 1 [12,13], we continued to explore whether 14-O-
derivatives of
5 could also improve anticancer potency of
compound 5. So a series of novel 14-O-derivatives of 5 were further
designed and synthesized. As shown in Scheme 5, treatment of 5
with corresponding acids in the presence of DMAP/EDC in DCM
gave corresponding 14-O-derivatives (compounds 10e20).
Fig. 2. Structures of oridonin (1), enmein-type derivatives (2, 3), spirolactone-type derivatives (5, 9).

244
L. Wang et al. / European Journal of Medicinal Chemistry 52 (2012) 242e250
Scheme 1. Synthesis of ent-6,7-seco-oridonin derivatives 2, 3, and 5. Reagents and conditions: (a) NaIO4, H2O, rt; (b) Jones reagent, acetone, isopropanol, 0 ꢀC; (c) Pb(OAc)4, Na2CO3,
THF, rt.
2.2. Biological evaluation of 6,7-seco-oridonin derivatives in vitro
2.3. The anti-tumor activity of 6,7-seco-oridonin derivative 5
in vivo
The synthesized enmein- and spirolactone-type derivatives (2,
3, 5, and 9) were firstly evaluated for their cytotoxicity against six
cancer cell lines (K562, MCF-7, CaEs-17, Bel-7402, Hela and A549)
in vitro. As shown in Table 1, noticeably, in comparison with ori-
donin, these compounds exhibited significant cytotoxicity, espe-
Based on the in vitro evaluated results, we further tested anti-
tumor activity of compound 5 in vivo by performing the assay in
mice with MGC-803 gastric cancer. As shown in Table 2, compound
5 has stronger anti-tumor activity than parent compound oridonin
in mice with MGC-803 gastric cancer. Thus the structure of 5 was
selected for further modification to find novel candidate for cancer
therapeutic agents [20,21].
cially against K562 cell with IC50 values varied from 2.18 to 9.56 mM.
The most cytotoxic compound 5 showed stronger cytotoxicity
against five cancer cell lines than oridonin, among which it is about
2-fold more potent in K562, CaEs-17 and Bel-7402 cells, 8-fold more
potent in Hela cell and 6-fold more potent in A549 cell. It has
similar cytotoxicity as Taxol in A549 cell and slightly less than Taxol
in Bel-7402 cell. The results have revealed that 1-oxo-6,7-seco-
oridonin 5 had more cytotoxic activity than the other derivatives of
oridonin. So compound 5 was regarded as the most promising
compound.
2.4. Cytotoxicity of 14-O-derivatives of compound 5
The synthesized novel 14-O-derivatives of 5 were further tested
against four cancer cell lines (K562, MGC-803, CaEs-17 and Bel-
7402). Taxol was selected as positive control. These results were
summarized in Table 3 and presented as the concentration of drug
Scheme 2. Synthesis of ent-6,7-seco-oridonin derivative 9. Reagents and conditions: (a) 2,2-Dimethoxypropane, acetone, TsOH, 56 ꢀC; (b) Ac2O, TEA, DMAP, rt; (c) 10% HCl, THF, rt;
(d) Pb(OAc)4, Na2CO3, THF, rt.

L. Wang et al. / European Journal of Medicinal Chemistry 52 (2012) 242e250
245
aromatic groups (compounds 12e20), it could be found that the
cytotoxicity activity was more potent, especially when aromatic
ring was substituted by electron donating groups (compounds 13
and 14). These derivatives showed better activity than that with
electron withdrawing groups (compounds 15 and 16). Changing the
position of fluorine on benzene ring (compounds 17 and 18), the
IC50 values in four cancer cell lines were almost no change. While
substituted with chlorine at ortho-position (compound 20), the
cytotoxicity was stronger than that at para-position (compound
19). Nevertheless, a broader range of substitution patterns of ent-
6,7-seco-oridonin would have to be synthesized and tested before
comprehensive SAR could be obtained.
Scheme 3. The conversion of spirolactone-type derivative 9 to enmein-type derivative
2. Reagents and conditions: (a) 10% NaOH, MeOH, rt.
3. Conclusion
inhibiting 50% cell growth (IC50). To our delight, most of the 14-O-
derivatives exhibited stronger cytotoxicity than parent compound 5
and similar cytotoxicity as Taxol in the four cancer cell lines.
Starting from commercial available natural product oridonin,
a practical synthesis of ent-6,7-seco-oridonin derivatives can be
accomplished and the conversion of oridonin to spirolactone-type
or enmein-type diterpenoid as well as spirolactone-type diterpe-
noid to enmein-type are accessible. The present study demon-
strated that all synthesized ent-6,7-seco-oridonin derivatives could
markedly inhibit the proliferation of cancer cells. Among which, 1-
oxo-6,7-seco-oridonin 5 exhibited stronger cytotoxicity than ori-
donin, and it was also more potent than oridonin in mice with
MGC-803 gastric cancer in vivo. Furthermore, most of the 14-O-
derivatives of compound 5 showed stronger cytotoxicity than
parent compound 5 and similar cytotoxicity as Taxol in the four
cancer cell lines (K562, MGC-803, CaEs-17 and Bel-7402). Based on
the evaluated results, the SAR was discussed, which showed that
the 6,7-seco-15-oxo-16-methylene derivatives of ent-kaurene
diterpenoid with enmein- or spirolactone-type structures could
improve the anti-tumor activity of oridonin. As the results, the
potential of these derivatives as cancer therapeutic agents invite us
2.5. Structureeactivity relationship (SAR)
The present results demonstrated that all synthesized ent-6,7-
seco-oridonin derivatives could markedly inhibit the proliferation
of cancer cells. The 6,7-seco-15-oxo-16-methylene derivatives of
ent-kaurene diterpenoids with enmein-type structure (compounds
2 and 3) or spirolactone-type structure (compounds 5 and 9) can
improve the anti-tumor activity of oridonin. Among which, 1-oxo-
6,7-seco-oridonin 5 might be more cytotoxic than oridonin and its
spirolactone-type analogue 1-O-acetyl-6,7-seco-oridonin 9.
Furthermore, the SAR showed that in the derivatives of ent-6,7-
seco-oridonin 5, when R3 were alkyl groups substitution
(compounds 10 and 11), the IC50 values slightly changed with
enhancing the length of carbon chain. As for substitution of
Scheme 4. The mechanism of conversion: from kaurene-type to spirolactone-type or enmein-type diterpenoid.

246
L. Wang et al. / European Journal of Medicinal Chemistry 52 (2012) 242e250
Scheme 5. Synthesis of 14-O-derivatives of ent-6,7-seco-oridonin derivative 5. Reagents and conditions: (a) Corresponding acids, EDC, DMAP, DCM, rt.
to continue exploring more and more this family of interesting and
promising compounds. And it would also provide valuable infor-
mation in the field of structure modification of natural lead
compounds and anticancer drugs development.
100.73, 75.16, 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; ESIMS m/z 747.4
[2M þ Na]þ; HR-MS (ESI, M þ H) m/z: calcd for C20H27O6: 363.1802,
found 363.1799.
4.1.2. ent-14a-Hydroxy-1,7-epoxy-6,7,15-trioxo-6,20-epoxy-6,7-
4. Experimental
seco-16-kaurene (3)
Compound 2 (72 mg, 0.2 mmol) was dissolved in acetone. To this
solution Jones reagent was added dropwise until a red color per-
sisted. The mixture was stirred at 0 ꢀC for 15 min and isopropanol
was added. Then the mixture was diluted with water and extracted
with dichloromethane. The extract was washed with brine, dried
over anhydrous Na2SO4, filtered, and evaporated to give compound
3 (63 mg, 87%) as a white powder: mp 248e250 ꢀC; IR (KBr) ymax
4.1. Chemistry
All commercially available solvents and reagents were used
without further purification. Melting points were taken on XT-4
micro melting point apparatus and uncorrected. Optical rotations
were measured using a JASCO P-1020 digital polarimeter, and
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
3508, 1785, 1751, 1645 cmꢁ1; ½a D20
ꢂ
ꢁ68.5 (c 0.23 CHCl3); 1H NMR
(CDCl3)
d 5.95 (1H, s, 17-CH2), 5.57 (1H, s, 17-CH2), 5.57 (1H, d,
J ¼ 3.5 Hz,14-OH), 4.77 (1H, m,14-CH), 4.72 (1H, m,1-CH), 4.51, 3.69
(each 1H, dd, JA ¼ JB ¼ 9.9 Hz, 20-CH2), 3.00 (1H, d, J ¼ 9.6 Hz, 13-
CH), 1.08 (3H, s, 18-CH3), 0.95 (3H, s, 19-CH3); 13C NMR (DMSO-
shifts are expressed in
in Hz. Mass spectra were obtained using FTMS-2000.
d values (ppm) and the coupling constants (J)
d6)
d 200.25, 175.45, 166.31, 151.48, 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; ESIMS m/z 361.1 [M þ H]þ; HR-MS (ESI, M þ H)
m/z: calcd for C20H25O6: 361.1646, found 361.1647.
4.1.1. ent-6b,14a-Dihydroxy-1,7-epoxy-7,15-dioxo-6,20-hemiketal-
6,7-seco-16-kaurene (2)
Compound 1 (364 mg, 1 mmol) was dissolved in water (10 ml).
To this solution was added sodium periodate (3.16 g, 14.8 mmol).
The mixture was stirred at room temperature for 24 h and then
extracted with dichloromethane. The dichloromethane layer was
washed with brine, dried over anhydrous Na2SO4, filtered, and
evaporated to give compound 2 (360 mg, 99%) as a white powder:
4.1.3. ent-6b,7b,14b-Trihydroxy-1,15-dioxo-7,20-epoxy-16-kaurene
(4)
Following the procedure described for preparation of compound
3, compound 4 was prepared from 1 as a white solid (87.8%): mp
215e217 ꢀC; IR (KBr) ymax 3387, 1702, 1643 cmꢁ1; ½a D20
ꢂ
115.1 (c 0.27
6.25 (1H, s,17-CH2), 5.84 (1H, d, J ¼ 12.0 Hz,
mp 183e185 ꢀC; IR (KBr) ymax 3450, 1754, 1645 cmꢁ1; ½a D20
ꢁ80.3 (c
ꢂ
CHCl3); 1H NMR (CDCl3)
d
0.44 CHCl3); 1H NMR (DMSO-d6)
d
6.17 (1H, d, J ¼ 3.3 Hz, 6-OH),
6-OH), 5.65 (1H, s, 17-CH2), 5.30 (1H, s, 14-OH), 4.89 (1H, s, 14-CH),
4.29, 4.02 (each 1H, dd, JA ¼ JB ¼ 10.5 Hz, 20-CH2), 3.79 (1H, m, 6-CH),
3.07 (1H, d, J ¼ 9.3 Hz, 13-CH), 1.19 (3H, s, 18-CH3), 0.99 (3H, s, 19-
5.92 (1H, s, 17-CH2), 5.52 (1H, s, 17-CH2), 5.38 (1H, d, J ¼ 2.1 Hz, 14-
OH), 5.17 (1H, d, J ¼ 2.1 Hz, 6-CH), 4.67 (1H, s,14-CH), 4.65 (1H, m,1-
CH), 3.87, 3.58 (each 1H, dd, JA ¼ JB ¼ 9.1 Hz, 20-CH2), 2.95 (1H, d,
J ¼ 9.1 Hz, 13-CH), 2.49 (2H, m, 12-CH2), 1.67 (2H, m, 2-CH2), 1.36
(2H, m, 11-CH2), 1.26 (1H, m, 9-CH), 0.93 (3H, s, 18-CH3), 0.88 (3H, s,
CH3); 13C NMR (DMSO-d6)
d 212.75, 206.79, 151.49, 121.03, 98.15,
72.31, 72.11, 64.07, 60.86, 59.68, 48.20, 47.92, 42.78, 38.10, 35.40,
32.55, 30.55, 29.48, 23.40, 18.44,; ESIMS m/z 363.2 [M þ H]þ.
19-CH3); 13C NMR (DMSO-d6)
d 200.30, 166.88, 150.42, 118.59,
Table 1
IC50 Values of ent-6,7-seco-oridonin derivatives (2, 3, 5, 9) for human tumor cell lines.a
Compd.
K562
MCF-7
CaEs-17
Bel-7402
Hela
A549
Taxolb
Oridonin
2
3
5
9
0.41 ꢃ 0.02
4.76 ꢃ 0.32
8.11 ꢃ 0.76
2.64 ꢃ 0.19
2.18 ꢃ 0.17
9.56 ꢃ 0.53
4.77 ꢃ 0.36
14.60 ꢃ 1.29
68.20 ꢃ 4.37
20.30 ꢃ 1.98
24.00 ꢃ 1.27
76.70 ꢃ 6.32
0.43 ꢃ 0.03
11.03 ꢃ 1.02
30.84 ꢃ 2.09
23.67 ꢃ 1.78
5.85 ꢃ 0.27
72.55 ꢃ 5.38
1.89 ꢃ 0.09
7.48 ꢃ 0.53
32.96 ꢃ 2.19
15.98 ꢃ 1.08
5.03 ꢃ 0.37
75.44 ꢃ 4.96
0.89 ꢃ 0.06
3.46 ꢃ 0.23
26.15 ꢃ 1.78
50.86 ꢃ 4.26
43.10 ꢃ 3.17
4.58 ꢃ 0.25
25.99 ꢃ 1.33
32.70 ꢃ 2.98
60.70 ꢃ 4.77
39.70 ꢃ 2.98
4.63 ꢃ 0.29
79.35 ꢃ 6.28
a
Results are expressed as IC50 values in mM. Cell lines: K562 human leukemia cell line; MCF-7 human breast cancer cell line; CaEs-17 human esophageal cancer cell line; Bel-
7402 human hepatoma cell line; Hela human cervical cancer; A549 human lung carcinoma.
b
Taxol was used a positive control. Data are means of three experiments.

L. Wang et al. / European Journal of Medicinal Chemistry 52 (2012) 242e250
247
Table 2
The anti-tumor activity of oridonin derivative 5 in mice with MGC-803 gastric cancer.
Compd.
Injection
Number of
mice
Weight of mice (g)
Weight of tumor X ꢃ SD (g)
Ratio of inhibition (%)
P-Value
Start
End
Start
End
Normal saline
Taxola
5
ip
iv
ip
ip
8
8
8
8
8
8
8
8
15.26 ꢃ 0.92
15.43 ꢃ 0.93
15.63 ꢃ 1.19
15.16 ꢃ 0.99
23.16 ꢃ 1.10
24.10 ꢃ 1.58
19.81 ꢃ 1.45
20.06 ꢃ 1.88
1.17 ꢃ 0.13
0.39 ꢃ 0.30
0.80 ꢃ 0.18
0.86 ꢃ 0.30
66.56%
31.41%
26.28%
< 0.01
< 0.01
< 0.05
Oridonin
a
Taxol was used as a positive control.
4.1.4. ent-1,6,7,15-Tetraoxo-7,20-epoxy-6,7-seco-16-kaurene (5)
Compound 4 (100 mg, 0.28 mmol) was dissolved in THF (10 ml),
and sodium carbonate (240 mg, 2.26 mmol) was added. Lead tet-
raacetate (425 mg, 1 mmol) was added as three portions with
intervals of 5 min. The reaction mixture was stirred at room
temperature for 15 min, and then insoluble matter was filtered. The
mixture was diluted with water and extracted with dichloro-
methane. The extract was washed with brine, dried over anhydrous
Na2SO4, filtered, and evaporated to give compound 5 (95 mg, 95%) as
a white powder: mp 153e155 ꢀC; IR (KBr) ymax 3444, 1711,
72.74, 72.40, 70.04, 62.76, 58.78, 56.33, 50.52, 40.47, 38.73, 33.39,
30.38, 29.20, 25.75, 22.38, 19.62; ESIMS m/z 405.2 [M þ H]þ.
4.1.6. ent-1a-O-Acetyl-6b-hydroxy-7,14-isopropylidene ketal-15-
oxo-7,20-epoxy-16-kaurene (7)
Compound 6 (90 mg, 0.22 mmol) was dissolved in dichloro-
methane, DMAP and TEA (0.2 ml) were added. The mixture was
stirred at room temperature for 2.5 h, and diluted with water and
extracted with dichloromethane. The extract was washed with
saturated NaHCO3 solution and brine, dried over anhydrous
Na2SO4, filtered, and evaporated to give compound 7 (94 mg, 95%)
as a white powder: mp 112e114 ꢀC; IR (KBr) ymax 3367, 1737,
1646 cmꢁ1; ½a D
ꢂ
20 74.1 (c 0.20 CHCl3); 1H NMR (CDCl3)
d 9.84 (1H, s, 6-
CHO), 6.21 (1H, s, 17-CH2), 5.62 (1H, s, 17-CH2), 5.41 (1H, s, 14-OH),
4.98 (1H, s,14-CH), 4.98, 4.77 (each 1H, dd, JA ¼ JB ¼ 11.7 Hz, 20-CH2),
3.11 (1H, d, J ¼ 9.3 Hz, 13-CH), 1.22 (3H, s, 18-CH3), 1.08 (3H, s, 19-
1643 cmꢁ1; ½a 2D0
ꢂ
ꢁ3.0 (c 0.47 CHCl3); 1H NMR (CDCl3)
d 6.16 (1H, s,
17-CH2), 6.12 (1H, d, J ¼ 9.0 Hz, 6-OH), 5.80 (1H, s, 14-CH), 5.51 (1H,
s, 17-CH2), 4.64 (1H, m, 1-CH), 4.27, 4.20 (each 1H, dd,
JA ¼ JB ¼ 10.8 Hz, 20-CH2), 3.80 (1H, m, 6-CH), 3.20 (1H, d, J ¼ 9.9 Hz,
CH3); 13C NMR (DMSO-d6)
d 210.23, 204.46, 199.83, 169.68, 149.67,
120.53, 71.87, 67.52, 61.22, 59.88, 52.37, 44.46, 42.94, 36.61, 36.57,
33.61, 30.53, 29.35, 24.81, 17.96; ESIMS m/z 361.2 [M þ H]þ; HR-MS
(ESI, M þ H) m/z: calcd for C20H25O6: 361.1646, found 361.1646.
13-CH), 2.06 (3H, s, eCH3), 2.00 (3H, s, eCH3), 1.13 (6H, s, eCH3); 13
C
NMR (DMSO-d6)
d 207.37, 169.73, 169.24, 150.67, 119.68, 95.76,
74.71, 74.01, 73.31, 62.40, 61.67, 59.22, 52.24, 41.48, 37.56, 33.34,
4.1.5. ent-1
a
,6b
-Dihydroxy-7,14-isopropylidene ketal-15-oxo-7,20-
32.41, 30.08, 24.94, 21.53, 21.34, 17.89; ESIMS m/z 447.2 [M þ H]þ.
epoxy-16-kaurene (6)
Compound 1 (100 mg, 0.27 mmol) was dissolved in acetone
(10 ml). TsOH and 2,2-dimethoxypropane (1 ml) were added to this
solution. The mixture was stirred at 56 ꢀC for 15 min, and diluted
with water and extracted with dichloromethane. The extract was
washed with saturated NaHCO3 solution and brine, dried over
anhydrous Na2SO4, filtered, and evaporated to afford compound 6
(94 mg, 85%) as a white powder: mp 204e206 ꢀC; IR (KBr) ymax
4.1.7. ent-1a-O-Acetyl-6b,7b,14b-trihydroxy-15-oxo-7,20-epoxy-16-
kaurene (8)
Ketal deprotection of 7 (50 mg, 0.11 mmol) was added to 10 ml
of 10% HCl/THF (1:1) and the solution was stirred at room
temperature for 1 h. Then the mixture was diluted with water and
extracted with dichloromethane. The extract was washed with
brine, dried over anhydrous Na2SO4, filtered, and evaporated to give
compound 8 (45 mg, 99%) as a white powder: mp 222e224 ꢀC; IR
3414, 1713, 1641 cmꢁ1; ½a D20
ꢂ
ꢁ13.2 (c 0.14 CHCl3); 1H NMR (CDCl3)
d
6.15 (1H, s, 17-CH2), 5.77 (1H, d, J ¼ 11.7 Hz, 6-OH), 5.56 (1H, s, 17-
(KBr) ymax 3449, 1721, 1646 cmꢁ1; ½a D20
ꢂ
ꢁ16.3 (c 0.14 CHCl3); 1H
CH2), 4.79 (1H, s, 14-CH), 4.24, 4.04 (each 1H, dd, JA ¼ JB ¼ 9.9 Hz,
20-CH2), 3.90 (1H, m, 1-CH), 3.47 (1H, m, 6-CH), 3.06 (1H, d,
J ¼ 9.0 Hz, 13-CH), 1.65 (6H, s, eCH3), 1.15 (3H, s, eCH3), 1.12 (3H, s,
NMR (CDCl3)
d
6.34 (1H, d, J ¼ 11.4 Hz, 6-OH), 6.18 (1H, s, 17-CH2),
5.56 (1H, s, 17-CH2), 4.86 (1H, s, 14-CH), 4.45 (1H, m, 1-CH), 4.26,
4.18 (each 1H, dd, JA ¼ JB ¼ 10.2 Hz, 20-CH2), 3.80 (1H, m, 6-CH),
3.06 (1H, d, J ¼ 9.3 Hz, 13-CH), 1.98 (3H, s, eCH3), 1.15 (6H, s, eCH3);
eCH3); 13C NMR (DMSO-d6)
d 206.28, 151.46, 119.68, 100.10, 94.60,
13C NMR (DMSO-d6)
d 208.60, 169.67, 151.80, 119.62, 96.98, 74.86,
73.11, 72.59, 62.56, 61.32, 59.42, 51.52, 42.76, 39.20, 37.06, 33.34,
Table 3
IC50 Values of 14-O-derivatives (10e20) of ent-6,7-seco-oridonin 5 for human tumor
32.46, 29.83, 24.94, 21.64, 21.36, 17.51; ESIMS m/z 407.2 [M þ H]þ.
cell lines.a
4.1.8. ent-1
a-O-Acetyl-14b-hydroxy-6,7,15-trioxo-7,20-epoxy-6,7-
Compd.
K562
MGC-803
CaEs-17
Bel-7402
Taxolb
Oridonin
10
11
12
13
14
15
16
17
18
19
20
0.41 ꢃ 0.02
4.76 ꢃ 0.32
1.39 ꢃ 0.17
1.31 ꢃ 0.12
1.48 ꢃ 0.22
1.27 ꢃ 0.34
1.65 ꢃ 0.39
2.82 ꢃ 0.11
1.33 ꢃ 0.52
2.70 ꢃ 0.42
2.86 ꢃ 0.24
1.38 ꢃ 0.27
1.22 ꢃ 0.48
0.85 ꢃ 0.06
5.69 ꢃ 0.39
1.66 ꢃ 0.15
1.78 ꢃ 0.89
1.92 ꢃ 0.70
2.24 ꢃ 0.18
1.87 ꢃ 0.95
4.93 ꢃ 0.24
3.33 ꢃ 0.38
3.96 ꢃ 0.19
3.94 ꢃ 0.67
3.12 ꢃ 0.16
2.66 ꢃ 0.44
0.43 ꢃ 0.03
11.03 ꢃ 1.02
1.35 ꢃ 0.27
1.40 ꢃ 0.31
1.71 ꢃ 0.08
1.05 ꢃ 0.90
2.98 ꢃ 0.16
8.96 ꢃ 0.23
2.68 ꢃ 0.13
8.31 ꢃ 0.46
8.55 ꢃ 0.50
2.55 ꢃ 0.32
2.01 ꢃ 0.07
1.89 ꢃ 0.09
7.48 ꢃ 0.53
1.73 ꢃ 0.65
1.65 ꢃ 0.82
1.85 ꢃ 0.53
1.54 ꢃ 0.90
2.80 ꢃ 0.72
3.99 ꢃ 0.33
3.30 ꢃ 1.00
3.75 ꢃ 0.33
3.95 ꢃ 1.08
3.84 ꢃ 0.91
2.89 ꢃ 0.34
seco-16-kaurene (9)
Following the procedure described for preparation of
compound 5, compound 9 was prepared from compound 8 as
a white solid (97%): mp 154e156 ꢀC; IR (KBr) ymax 3422, 1740,
1712 cmꢁ1; ½a 2D0
ꢂ
36.9 (c 0.37 CHCl3); 1H NMR (CDCl3)
d 9.76 (1H, d,
J ¼ 4.2 Hz, 6-CHO), 6.23 (1H, s, 17-CH2), 5.71 (1H, s, 14-OH), 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), 1.19 (3H, s, eCH3), 1.04 (3H, s, eCH3); 13C
NMR (DMSO-d6)
d 205.37, 199.34, 171.86, 169.40, 149.60, 120.99,
74.76, 71.47, 67.26, 60.82, 60.12, 44.18, 42.93, 42.61, 38.44, 33.73,
32.08, 29.40, 24.34, 23.32, 21.05, 16.89; ESIMS m/z 405.1 [M þ H]þ;
HR-MS (ESI, M þ H) m/z: calcd for C22H29O7: 405.1908, found
405.1911.
a
Results are expressed as IC50 values in
m
M. Cell lines: MGC-803 human gastric
cancer cell line.
b
Taxol was used a positive control. Data are means of three experiments.

248
L. Wang et al. / European Journal of Medicinal Chemistry 52 (2012) 242e250
4.1.9. ent-1,6,7,15-Tetraoxo-7,20-epoxy-(14b-O-acetyl)-6,7-seco-16-
4.1.13. ent-1,6,7,15-Tetraoxo-7,20-epoxy-(14b-O-p-methoxybenzoyl)-
kaurene (10)
6,7-seco-16-kaurene (14)
Compound 5 (72 mg, 0.2 mmol) was dissolved in dichloro-
methane, then EDC, DMAP and acetic acid (15 mg, 0.24 mmol) were
added. The reaction mixture was stirred at room temperature for
about 8 h. Then the mixture was washed with 10% HCl. The organic
layer was washed with brine, dried over anhydrous Na2SO4. After
flash chromatography (dichloromethane/methanol 300:1), 10 was
got as white solid (48 mg, 69%): mp 96e97 ꢀC; IR (KBr) ymax 2962,
Following the procedure described for preparation of compound
10, compound 14 was prepared from compound 5 as a white solid
(23 mg, 35%): mp 108e110 ꢀC; IR (KBr) ymax 2958, 1768, 1714, 1606,
1512, 1466, 769, 695 cmꢁ1 1H NMR (CDCl3)
; d 9.81 (1H, s, eCHO),
7.78 (2H, d, J ¼ 8.4 Hz, AreH), 6.82 (2H, d, J ¼ 8.4 Hz, AreH), 6.19
(1H, s, 17-CH2), 6.15 (1H, s, 14-CH), 5.77 (1H, s, 17-CH2), 4.94, 4.55
(each 1H, dd, JA ¼ JB ¼ 8.4 Hz, 20-CH2), 3.74 (3H, s, eOCH3), 3.32 (1H,
d, J ¼ 7.2 Hz, 13-CH), 1.17 (3H, s, eCH3), 1.14 (3H, s, eCH3); 13C NMR
1771, 1732, 1648, 1463, 1373, 1176 cmꢁ1; 1H NMR (CDCl3)
d 9.85 (1H,
s, eCHO), 6.21 (1H, s, 17-CH2), 5.72 (1H, s, 14-CH), 5.62 (1H, s, 17-
CH2), 4.95, 4.73 (each 1H, dd, JA ¼ JB ¼ 11.7 Hz, 20-CH2), 3.14 (1H,
d, J ¼ 9.3 Hz, 13-CH), 1.22 (3H, s, eCH3), 1.08 (3H, s, eCH3), 1.06 (3H,
(DMSO-d6) d 210.26, 204.17, 198.26, 166.04, 163.35, 147.16, 132.11,
131.32, 114.01, 74.21, 67.40, 60.97, 59.91, 55.52, 52.12, 45.17, 41.58,
36.68, 33.24, 30.04, 29.12, 24.05, 17.99; ESIMS m/z 495.0 [M þ H]þ,
512.1 [M þ NH4]þ, 529.2 [M þ Cl]ꢁ; HR-MS (ESI, M þ H) m/z: calcd
for C28H31O8: 495.2013, found 495.2011.
s, eCH3); 13C NMR (DMSO-d6)
d 210.23, 204.13, 198.18, 169.78,
166.22, 146.94, 120.62, 73.79, 67.37, 61.12, 59.77, 51.99, 45.11, 41.36,
36.68, 36.14, 33.15, 29.95, 29.07, 28.92, 23.86, 17.89; ESIMS m/z
403.1 [M þ H]þ, 420.2 [M þ NH4]þ, 437.5 [M þ Cl]ꢁ; HR-MS (ESI,
M þ H) m/z: calcd for C22H27O7: 403.1751, found 403.1759.
4.1.14. ent-1,6,7,15-Tetraoxo-7,20-epoxy-(14b-O-p-trifloromethylbe-
nzoyl)-6,7-seco-16-kaurene (15)
Following the procedure described for preparation of compound
10, compound 15 was prepared from compound 5 as a white solid
(40 mg, 37%): mp 252e254 ꢀC; IR (KBr) ymax 2968, 1761, 1732, 1710,
1412, 1383, 1325, 1291, 1261, 1281, 1127, 1101, 1065, 1051, 1018, 768,
4.1.10. ent-1,6,7,15-Tetraoxo-7,20-epoxy-(14b-O-propionyl)-6,7-seco-
16-kaurene (11)
Following the procedure described for preparation of compound
10, compound 11 was prepared from compound 5 as a white solid
(43 mg, 62%): mp 132e133 ꢀC; IR (KBr) ymax 2960, 1765, 1725, 1647,
701 cmꢁ1 1H NMR (CDCl3)
; d 9.85(1H, s, eCHO), 8.04 (1H, d,
J ¼ 8.1 Hz, AreH), 7.80 (1H, d, J ¼ 8.1 Hz, AreH), 7.65 (1H, d, J ¼ 8.1 Hz,
AreH), 7.17 (1H, d, J ¼ 8.1 Hz, AreH), 6.29 (1H, s,17-CH2), 6.22 (1H, s,
14-CH), 5.61 (1H, s,17-CH2), 4.88, 4.50 (each 1H, dd, JA ¼ JB ¼ 12.5 Hz,
20-CH2), 3.32 (1H, d, J ¼ 7.5 Hz, 13-CH), 2.62 (1H, m, 12-CH2), 2.10
(1H, m,12-CH2),1.91 (2H, m,11-CH2),1.72 (1H, m, 9-CH),1.69 (2H, m,
2-CH2), 1.57 (2H, m, 3-CH2), 1.27 (1H, m, 5-CH2), 1.25 (3H, s, 18-CH3),
1467, 1374, 1244 cmꢁ1 1H NMR (CDCl3)
; d 9.85 (1H, s, eCHO), 6.20
(1H, s, 17-CH2), 5.74 (1H, s, 14-CH), 5.63 (1H, s, 17-CH2), 4.97, 4.72
(each 1H, dd, JA ¼ JB ¼ 11.4 Hz, 20-CH2), 3.10 (1H, d, J ¼ 8.7 Hz, 13-
CH), 1.24 (3H, s, eCH3), 1.08 (3H, s, eCH3), 0.86 (3H, s, eCH3); 13C
NMR (DMSO-d6)
d 210.23, 204.08, 198.16, 173.02, 166.14, 146.96,
120.52, 73.59, 67.34, 61.11, 59.78, 51.97, 45.09, 41.36, 36.66, 36.12,
33.14, 29.92, 29.06, 26.75, 23.89, 17.88, 8.59; ESIMS m/z 417.2
[M þ H]þ, 434.2 [M þ NH4]þ, 451.4 [M þ Cl]ꢁ; HR-MS (ESI, M þ H)
m/z: calcd for C23H29O7: 417.1908, found 417.1903.
1.24 (3H, s, 19-CH3); 13C NMR (DMSO-d6)
d 210.14, 204.16, 198.08,
166.10, 163.78, 133.12, 132.76, 130.14, 129.95, 125.81, 121.32, 75.00,
67.45, 60.49, 59.97, 52.29, 45.32, 41.49, 36.52, 33.27, 30.36, 29.27,
23.85, 17.94; ESIMS m/z 533.1 [M þ H]þ, 550.3 [M þ NH4]þ; HR-MS
(ESI, M þ H) m/z: calcd for C28H28F3O7: 533.1782, found 533.1784.
4.1.11. ent-1,6,7,15-Tetraoxo-7,20-epoxy-(14b-O-benzoyl)-6,7-seco-
16-kaurene (12)
4.1.15. ent-1,6,7,15-Tetraoxo-7,20-epoxy-(14b-O-p-nitrobenzoyl)-6,
Following the procedure described for preparation of compound
10, compound 12 was prepared from compound 5 as a white solid
(45 mg, 62%): mp 102e104 ꢀC; IR (KBr) ymax 2959, 1768, 1717, 1646,
7-seco-16-kaurene (16)
Following the procedure described for preparation of compound
10, compound 16 was prepared from compound 5 as a white solid
(62 mg, 83%): mp 268e270 ꢀC; IR (KBr) ymax 2956, 1758, 1729, 1647,
1452, 1274, 762, 713 cmꢁ1 1H NMR (CDCl3)
; d 9.82 (1H, s, eCHO),
7.79 (2H, d, J ¼ 5.7 Hz, AreH), 7.63 (1H, t, J ¼ 5.2 Hz, AreH), 7.49 (2H,
t, J ¼ 5.2 Hz, Ar-H), 6.22 (1H, s, 17-CH2), 6.16 (1H, s, 14-CH), 5.78 (1H,
s, 17-CH2), 4.94, 4.55 (each 1H, dd, JA ¼ JB ¼ 7.5 Hz, 20-CH2), 3.33
1533, 1467, 846 cmꢁ1 1H NMR (CDCl3)
; d 9.84 (1H, s, eCHO), 8.18
(2H, d, J ¼ 8.4 Hz, AreH), 8.02 (2H, d, J ¼ 8.4 Hz, AreH), 6.19 (1H, s,
17-CH2), 6.15 (1H, s, 14-CH), 5.75 (1H, s, 17-CH2), 4.95, 4.53 (each
1H, dd, JA ¼ JB ¼ 8.1 Hz, 20-CH2), 3.25 (1H, d, J ¼ 7.2 Hz, 13-CH), 1.16
(1H, d, J ¼ 7.5 Hz, 13-CH), 1.19 (3H, s, eCH3), 1.13 (3H, s, eCH3); 13
C
NMR (DMSO-d6)
d
210.18, 204.16, 198.15, 166.02, 164.81, 147.11,
(3H, s, eCH3), 1.13 (3H, s, eCH3); 13C NMR (DMSO-d6)
d 210.15,
133.46, 129.12, 128.65, 120.95, 74.47, 67.38, 60.76, 59.89, 52.14,
45.19, 41.51, 36.59, 36.31, 33.22, 30.03, 29.14, 23.95, 17.95; ESIMS m/
z 465.1 [M þ H]þ, 482.2 [M þ NH4]þ, 499.3 [M þ Cl]ꢁ; HR-MS (ESI,
M þ H) m/z: calcd for C27H29O7: 465.1908, found 465.1906.
204.22, 198.07, 166.11, 163.40, 150.31, 147.07, 134.78, 130.53, 123.94,
121.53, 75.22, 67.46, 60.37, 59.98, 52.31, 45.35, 41.44, 36.48, 33.28,
30.21, 29.30, 23.84, 17.92; ESIMS m/z 510.1 [M þ H]þ, 527.3
[M þ NH4]þ, 544.3 [M þ Cl]ꢁ; HR-MS (ESI, M þ NH4) m/z: calcd for
C27H31N2O9: 527.2024, found 527.2021.
4.1.12. ent-1,6,7,15-Tetraoxo-7,20-epoxy-(14b-O-p-methylbenzoyl)-6,
7-seco-16-kaurene (13)
4.1.16. ent-1,6,7,15-Tetraoxo-7,20-epoxy-(14b-O-p-florobenzoyl)-6,
Following the procedure described for preparation of compound
10, compound 13 was prepared from compound 5 as a white solid
(41 mg, 62%): mp 208e210 ꢀC; IR (KBr) ymax 2957, 1763, 1718, 1612,
7-seco-16-kaurene (17)
Following the procedure described for preparation of compound
10, compound 17 was prepared from compound 5 as a white solid
(12 mg, 12%): mp 244e246 ꢀC; IR (KBr) ymax 2963, 1763, 1724, 1604,
1505, 1283, 1261, 1151, 1093, 1050, 1016, 854, 802, 761, 686,
1465,1289, 748 cmꢁ1; 1H NMR (CDCl3)
d 9.82 (1H, s, eCHO), 7.68 (2H,
d, J ¼ 3.4 Hz, AreH), 7.29 (2H, d, J ¼ 3.4 Hz, AreH), 7.45 (1H, t,
J ¼ 1.8 Hz, AreH), 6.19 (1H, s,17-CH2), 6.15 (1H, s, 14-CH), 5.77 (1H, s,
17-CH2), 4.92, 4.53 (each 1H, dd, JA ¼ JB ¼ 7.5 Hz, 20-CH2), 3.31 (1H, d,
J ¼ 7.5 Hz, 13-CH), 2.36 (3H, s, eCH3), 1.19 (3H, s, eCH3), 1.13 (3H, s,
612 cmꢁ1; 1H NMR (CDCl3)
d 9.90 (1H, s, eCHO), 7.97 (2H, m, AreH),
7.07 (2H, m, AreH), 6.31 (1H, s, 17-CH2), 6.16 (1H, s, 14-CH), 5.63
(1H, s, 17-CH2), 4.88, 4.50 (each 1H, dd, JA ¼ JB ¼ 12.5 Hz, 20-CH2),
3.34 (1H, d, J ¼ 7.5 Hz, 13-CH), 2.62 (1H, m, 12-CH2), 2.10 (1H, m, 12-
CH2), 1.91 (2H, m, 11-CH2), 1.72 (1H, m, 9-CH), 1.69 (2H, m, 2-CH2),
1.57 (2H, m, 3-CH2), 1.45 (1H, m, 5-CH2), 1.31 (3H, s, 18-CH3), 1.27
eCH3); 13C NMR (DMSO-d6)
d 210.22, 204.16, 198.21, 166.01, 164.85,
147.13,143.90,129.21,126.56,120.87, 74.34, 67.39, 60.87, 59.89, 52.13,
45.19, 41.54, 36.64, 36.27, 33.24, 30.06, 29.15, 24.02, 21.15, 17.98;
ESIMSm/z 479.2 [Mþ H]þ, 496.3 [Mþ NH4]þ, 513.6 [M þ Cl]ꢁ;HR-MS
(ESI, M þ H) m/z: calcd for C28H31O7: 479.2064, found 479.2059.
(3H, s, 19-CH3); 13C NMR (DMSO-d6)
d
210.12, 204.03, 198.11, 166.15,
163.87, 147.10, 132.05, 127.31, 125.90, 120.98, 115.72, 74.61, 67.41,

L. Wang et al. / European Journal of Medicinal Chemistry 52 (2012) 242e250
249
60.70, 59.92, 52.20, 45.23, 41.53, 36.56, 33.21, 30.34, 29.16, 23.90,
17.94; ESIMS m/z 483.1 [M þ H]þ, 500.1 [M þ NH4]þ; HR-MS (ESI,
M þ H) m/z: calcd for C27H28FO7: 483.1814, found 483.1811.
(150 ml/well). After 10 min at room temperature, the OD of each
well was measured on a Microplate Reader (BIO-RAD instruments
Inc NO.550) at a wavelength of 490 nm. In these experiments, the
negative reference agents was 0.1% DMSO, and Taxol was used as
4.1.17. ent-1,6,7,15-Tetraoxo-7,20-epoxy-(14
b
-O-o-florobenzoyl)-6,
the positive reference substance with concentration of 10 mg/ml.
7-seco-16-kaurene (18)
The same method was used in cytotoxic testing against MCF-7,
CaEs-17, Bel-7402, Hela, A549 and MGC-803 cell lines.
Following the procedure described for preparation of compound
10, compound 18 was prepared from compound 5 as a white solid
(13 mg, 13%): mp 206e208 ꢀC; IR (KBr) ymax 2924, 2853, 1701, 1686,
4.3. Anti-tumor activity in vivo
1655, 1615, 1561, 1466, 1384, 1257, 1090, 775, 649 cmꢁ1 1H NMR
;
(CDCl3)
d 9.87 (1H, s, eCHO), 1.25 (3H, s, 19-CH3), 1.26 (3H, s, 18-
Institute of Cancer Research (ICR) female mice with body weight
of 12e16 g was transplanted with MGC-803 cell subcutaneously
into the right oxter according to protocols of tumor transplant
research. After 7 d of tumor transplantation, mice in MGC-803
group was weighed and divided into four groups at random. The
groups with oridonin and 5 were administered intraperitoneally
10 mg/kg in a vehicle of 1% DMSO/2% poloxamer/97% saline,
respectively. The positive control group was treated with Taxol
(10 mg/kg) through intravenous injection in a vehicle of 1% DMSO/
2% poloxamer/97% saline. The negative control group received 0.9%
normal saline through intraperitoneal injection. All of the test
compounds were given through injections after 7 d of tumor
transplantation (or inoculation). Treatments were done at
a frequency of intravenous or intraperitoneal injection one dose per
day for a total of 25 consecutive days. After the treatments, all of the
mice were killed and weighed simultaneously, and then tumors
were segregated and weighed. Ratio of inhibition of tumor
(%) ¼ (1 ꢁ average tumor weight of treated group/average tumor
weight of control group) ꢄ 100%.
CH3), 1.48 (1H, m, 5-CH2), 1.68 (2H, m, 3-CH2), 1.86 (2H, m, 2-
CH2), 7.85 (1H, m, AreH), 7.50 (1H, m, AreH), 7.15 (1H, m, AreH),
7.07 (1H, m, AreH), 6.29 (1H, s, 17-CH2), 6.19 (1H, s, 14-CH), 5.62
(1H, s, 17-CH2), 4.90, 4.50 (each 1H, dd, JA ¼ JB ¼ 12.3 Hz, 20-CH2),
3.33 (1H, d, J ¼ 7.5 Hz, 13-CH), 2.64 (1H, m, 12-CH2), 2.53 (1H, m, 12-
CH2), 2.46 (2H, m, 11-CH2), 1.89 (1H, m, 9-CH); 13C NMR (DMSO-d6)
d
210.16, 204.13,198.13,166.07,163.66,162.76,160.81,147.08,131.68,
125.35, 121.23, 120.61, 115.64, 74.90, 67.44, 60.58, 59.93, 52.23,
45.26, 41.49, 36.46, 33.26, 30.14, 29.19, 23.93, 17.94; ESIMS m/z
483.1 [M þ H]þ, 500.3 [M þ NH4]þ; HR-MS (ESI, M þ H) m/z: calcd
for C27H28FO7: 483.1814, found 483.1807.
4.1.18. ent-1,6,7,15-Tetraoxo-7,20-epoxy-(14b-O-p-chlorobenzoyl)-6,
7-seco-16-kaurene (19)
Following the procedure described for preparation of compound
10, compound 19 was prepared from compound 5 as a white solid
(46 mg, 62%): mp 234e236 ꢀC; IR (KBr) ymax 2937, 1760, 1726, 1643,
1593, 1485, 849 cmꢁ1 1H NMR (CDCl3)
; d 9.82 (1H, s, eCHO), 7.65
(1H, t, J ¼ 1.8 Hz, AreH), 7.55 (2H, m, J ¼ 3.0 Hz, AreH), 7.45 (1H, t,
J ¼ 1.8 Hz, AreH), 6.26 (1H, s, 17-CH2), 6.12 (1H, s, 14-CH), 5.76 (1H,
s, 17-CH2),1.13 (3H, s, eCH3), 4.94, 4.58 (each 1H, dd, JA ¼ JB ¼ 7.5 Hz,
20-CH2), 3.31 (1H, d, J ¼ 7.5 Hz, 13-CH), 1.19 (3H, s, eCH3); 13C NMR
Acknowledgment
This study was supported by grant from National Natural
Science Fund (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 (2011, No. 800), the Fundamental Research Funds
for the Central Universities (JKY2011030) and Key Fund of Ministry
of Education of China (No. 108069) for financial assistance.
(DMSO-d6)
d 210.12, 204.12, 198.08, 166.00, 163.98, 147.07, 138.42,
130.91,128.91, 128.16,121.13, 74.70, 67.39, 60.59, 59.90, 52.19, 45.24,
41.46, 36.41, 33.22, 30.11, 29.19, 23.90, 17.91; ESIMS m/z 499.2
[M þ H]þ, 516.2 [M þ NH4]þ, 533.6 [M þ Cl]ꢁ; HR-MS (ESI, M þ NH4)
m/z: calcd for C27H31ClNO7: 516.1784, found 516.1785.
4.1.19. ent-1,6,7,15-Tetraoxo-7,20-epoxy-(14b-O-o-chlorobenzoyl)-6,
7-seco-16-kaurene (20)
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Following the procedure described for preparation of compound
10, compound 20 was prepared from compound 5 as a white solid
(41 mg, 63%): mp 217e219 ꢀC; IR (KBr) ymax 2956, 1760, 1725, 1643,
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