Novel nitric oxide-releasing spirolactone-type diterpenoid derivatives with in vitro synergistic anticancer activity as apoptosis inducer

Article abstract of DOI:10.1016/j.bmcl.2016.07.059

Herein, we reported the cytotoxicity, NO-releasing property, and apoptosis induced ability of two series of novel nitric oxide-releasing spirolactone-type diterpenoid derivatives (10a–f and 15a–f). All the title compounds were more potent than oridonin (7) and parent compound (9 or 14) against human tumor Bel-7402, K562, MGC-803 and CaEs-17 cells. SARs were concluded based on above data. Compound 15d exhibited the strongest antiproliferative activity with the IC₅₀of 0.86, 1.74, 1.16 and 3.75?μM, respectively, and could produce high level (above 25?μM) of NO at the time point of 60?min. Further mechanism evaluation showed that 15d could induce S phase cell cycle arrest and apoptosis at low micromolar concentrations in Bel-7402 cells via mitochondria-related pathways. It was expected that the remarkable biological profile of the synthetic NO-releasing spirolactone-type diterpenoid analogs make them possible as promising candidates for the development of anticancer agents.

Full text of DOI:10.1016/j.bmcl.2016.07.059

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel nitric oxide-releasing spirolactone-type diterpenoid
derivatives with in vitro synergistic anticancer activity as apoptosis
inducer
Dahong Li a, Tong Han a, Kangtao Tian a, Shuang Tang a, Shengtao Xu b, Xu Hu a, Lei Wang b, Zhanlin Li a,
Huiming Hua a, , Jinyi Xu b,
⇑
⇑
a Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, and School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
103 Wenhua Road, Shenyang 110016, China
b Department of Medicinal Chemistry and State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we reported the cytotoxicity, NO-releasing property, and apoptosis induced ability of two series
of novel nitric oxide-releasing spirolactone-type diterpenoid derivatives (10a–f and 15a–f). All the title
compounds were more potent than oridonin (7) and parent compound (9 or 14) against human tumor
Bel-7402, K562, MGC-803 and CaEs-17 cells. SARs were concluded based on above data. Compound
Received 24 April 2016
Revised 20 July 2016
Accepted 25 July 2016
Available online xxxx
15d exhibited the strongest antiproliferative activity with the IC50 of 0.86, 1.74, 1.16 and 3.75
lM, respec-
tively, and could produce high level (above 25 M) of NO at the time point of 60 min. Further mechanism
l
Keywords:
evaluation showed that 15d could induce S phase cell cycle arrest and apoptosis at low micromolar con-
centrations in Bel-7402 cells via mitochondria-related pathways. It was expected that the remarkable
biological profile of the synthetic NO-releasing spirolactone-type diterpenoid analogs make them possi-
ble as promising candidates for the development of anticancer agents.
NO-releasing
Spirolactone-type
Diterpenoid
Oridonin
Ó 2016 Elsevier Ltd. All rights reserved.
Antiproliferative
Nitric oxide (NO) is an important, active, and small molecule.1 It
plays important roles in various physiological processes, including
neuromodulation, vasorelaxation, and biodefence, it may also be
involved in the pathophysiology of diseases such as cancer,
schizophrenia and Alzheimer’s disease. Based on previous research,
high concentration of NO and its metabolic derivatives, the reactive
nitrogen species (RNS) could modify functional proteins by S-nitro-
sylation, nitration, disulfide formation leading to bioregulation,
inactivation, and cytotoxicity particularly to tumor cells by apopto-
sis and blocking cell migration.2–6 Therefore, methods for precisely
controlled release of NO were required for research purposes and
might ultimately be clinically relevant. Since NO was unstable
under ambient conditions, compounds (NO donors) that release
NO in situ have been developed and employed for the research.
NO donors could release a certain amount of NO by the action of
enzymes and/or non-enzymes. Several kinds of NO donors were
developed, such as organic nitrates, metal-NO complexes,
nitrosothiols, and so on.7,8 With the development of NO donors,
NO-donating drugs emerged and developed rapidly. NO donor
hybrid compounds had come into focus in the treatment of tumor
because NO was a key signaling molecule involved in the death and
apoptosis of tumor cells.7–12
Diterpenoids were well-known to produce bioactive mole-
cules.13–15 Among which, spirolactone-type 6,7-seco-kaurane diter-
penoids had unique chemical skeletons and exhibited significant
AcO
OH
O
AcO
O
CHO
H
H
H
O
O
O
H
O
O
O
O
H
O
OH
O
trichorabdal G
OH
maoecrystal V
maoecrystal Z
AcO
H
H
H
H
H
CH3
H
CH3
O
H
O
O
O
O
O
O
O
O
OMe
CHO O
O
O
sculponeatin J
rabdosichuanin A
ludongnin G
⇑
Corresponding authors. Tel.: +86 24 23986465 (H.H.); tel.: +86 25 83271445;
fax: +86 25 83302827 (J.X.).
Figure 1. The structures of natural spirolactone-type 6,7-seco-kaurane
E-mail addresses: huimhua@163.com (H. Hua), jinyixu@china.com (J. Xu).
diterpenoids.
http://dx.doi.org/10.1016/j.bmcl.2016.07.059
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Li, D.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.07.059

2
D. Li et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 1
b
a
SH
SO2CH2COOH
SCH2COOH
Antiproliferative activity of target compounds 10a–f and 15a–f against four human
cancer cell lines (IC50a lM)
1
c
2
3
Compd
K562
MGC-803
CaEs-17
Bel-7402
PhO2S
SO2Ph
HO
O
SO2Ph
R1
7
9
4.59 0.22
2.92 0.23
2.40 0.07
2.04 0.17
2.34 0.23
1.97 0.13
1.99 0.11
1.90 0.06
9.11 0.36
2.48 0.20
2.03 0.12
2.91 0.29
1.74 0.07
2.88 0.18
1.79 0.13
19.65 0.87
0.38 0.02
5.47 0.48
4.27 0.32
2.43 0.12
1.93 0.10
2.89 0.13
1.63 0.12
1.80 0.17
1.87 0.18
28.54 2.58
2.73 0.20
1.68 0.09
3.09 0.15
1.16 0.04
3.03 0.22
1.56 0.14
24.57 1.34
0.83 0.07
12.16 0.67
5.85 0.27
5.21 0.24
4.67 0.10
5.39 0.25
4.12 0.27
4.83 0.33
4.66 0.16
66.94 4.05
5.88 0.32
4.51 0.19
6.26 0.39
3.75 0.08
6.20 0.27
4.40 0.22
22.60 1.41
0.51 0.02
7.56 0.32
5.03 0.37
1.50 0.13
0.96 0.02
1.49 0.05
0.93 0.04
1.24 0.11
1.03 0.08
69.40 3.72
1.53 0.14
0.92 0.06
1.83 0.12
0.86 0.01
1.74 0.16
0.91 0.07
27.86 0.76
1.95 0.17
d
e
N+
N
N+
N
O-
O-
O
4
10a
10b
10c
10d
10e
10f
14
15a
15b
15c
15d
15e
15f
16
O
5a-c
R1=
5a
5b
CH2CH2
CH2CH2CH2
5c CH2CH2CH2CH2
R1=
R2=
HOOCR2COOR1O
SO2Ph
6a
6b
(CH2)2
(CH2)2
6c (CH2)3
6d (CH2)3
6e (CH2)4
CH2CH2
o-C6H4
CH2CH2
o-C6H4
CH2CH2
o-C6H4
N
N+
O-
O
6a-f
6f
(CH2)4
Taxol
OH
O
O
a
H
H
IC50: concentration that inhibits 50% of cell growth. Results are expressed as the
14
g
1
OH
O
f
OH
O
H
mean S.D. of three independent experiments.
O
O
OH
O
OH
OH
H
H
O
CHO O
O
OH
OH
8
9
7
with a lot of stereogenic centers and complex ring systems had
attracted much attention from chemists in the field of total synthe-
sis.22–25 They also aroused our curiosity for further medicinal chem-
istry investigation.
O
R1=
(CH2)2
(CH2)2
R2=
H
10a
10b
CH2CH2
o-C6H4
CH2CH2
o-C6H4
CH2CH2
o-C6H4
O
O
h
R2
O
O
N
O
CHO O
10c (CH2)3
10d (CH2)3
10e (CH2)4
R1
O
On the basis of the above mentioned reasons, as a part of further
development of our research work on the design of new NO
donor/diterpenoid derivatives, two series of spirolactone-type
6,7-seco-kaurane diterpenoid and NO donor motif hybrids were
synthesized. Relevant commercial available resources were used
for acquiring complicated target natural product skeletons by pass-
ing traditional isolation and the de novo synthetic method. Then
the antiproliferative activity of target compounds was tested by
MTT assay. Furthermore, the influence of cell cycle progression
and effects on apoptosis and mitochondrial membrane potential
by representative compound 15d in Bel-7402 cells were disclosed.
The synthesis of substituted furoxans (6a–i) was illustrated in
Scheme 1. Starting from benzenethiol (1), diphenylsulfonylfuroxan
(4) was got and then treated with corresponding diol (ethane-1,2-
diol, propane-1,3-diol, and butane-1,4-diol) to afford monophenyl-
sulfonylfuroxans (5a–c). Key intermediate furoxan-based NO
donors 6a–f were obtained from the condensation of 5a–c and
anhydride (succinic anhydride or phthalic anhydride).10 Selected
oxidation of 7 with Jones Reagent gave 1-oxo derivative 8, and then
lead tetraacetate was added in the presence of sodium carbonate in
THF to get spirolactone-type diterpenoid 9 in high yields. Treat-
ment of 7 with 2,2-dimethoxypropane (DMP) in the presence of
TsOH in anhydrous acetone afforded 11, which, upon reaction with
Ac2O/Et3N in CH2Cl2, yielded 1-O-acetyl derivative 12. Deprotec-
tion with 10% HCl gave compound 13 in quantitative yields, which
could be converted to spirolactone-type diterpenoid 14 by oxida-
tion using lead tetraacetate in the presence of sodium carbonate
in THF in high yields.26,27 9 and 14 with a hydroxyl group at 14-
position were good building blocks to synthesize spirolactone-type
diterpenoid analogs. Target novel NO donor/spirolactone-type 6,7-
seco-ent-kaurane diterpenoid hybrids 10a–f and 15a–f were
designed and synthesized from 9 and 14 accordingly with NO
donors 6a–f in the presence of DMAP/EDCI in DCM at room tem-
perature for 8–12 h. Flash chromatography could only be taken
at the last step of the synthetic route of each target compound.28
6a was reacted with TMSCHN2 to get 16.
N+
O
O-
PhO2S
10f
(CH2)4
10a-f
OH
OAc
OH
H
H
H
j
i
O
O
O
OH
O
O
O
O
O
OH
O
O
H
H
H
OH
OH
OH
11
7
12
OAc
OAc
H
OH
O
h
l
k
H
O
OH
O
OH
H
CHO O
14
O
OH
13
OAc
R1=
(CH2)2
(CH2)2
R2=
H
O
15a
15b
O
O
CH2CH2
o-C6H4
CH2CH2
o-C6H4
CH2CH2
o-C6H4
R2
O
O
N
O
CHO O
R1
O
15c (CH2)3
15d (CH2)3
15e (CH2)4
N+
O
O-
PhO2S
15a-f
15f
(CH2)4
PhO2S
O(CH2)2OCO(CH2)2COOH
PhO2S
O(CH2)2OCO(CH2)2COOCH3
m
N
N
N
N
O
O
O
O
16
6a
Scheme 1. The synthetic routine of target NO releasing spirolactone-type 6,7-seco-
kaurane diterpenoid derivatives. Reagents and conditions: (a) chloroactic acid,
NaOH (aq), 60 °C; (b) 30% H2O2, glacial acetic acid, rt; (c) fuming HNO3, glacial acetic
acid, 60 °C; (d) corresponding diol, NaOH (aq), THF, rt; (e) anhydride, DMAP/TEA,
DCM, rt; (f) Jones reagent, acetone, isopropanol, 0 °C; (g) Pb(OAc)4, Na2CO3, THF, rt;
(h) 6a–f, EDCI, DMAP, DCM, rt; (i) 2,2-dimethoxypropane, acetone, TsOH, 56 °C; (j)
Ac2O, TEA, DMAP, rt; (k) 10% HCl, THF, rt; (l) Pb(OAc)4, Na2CO3, THF, rt; (m)
TMSCHN2, MeOH, 0 °C, 10 min.
activities, such as maoecrystal V,16 maoecrystal Z,17 trichorabdal
G,18 sculponeatin J,19 ludongnin G,20 rabdosichuanin A,21 and so
on (Fig. 1). These hot off the press spirolactone-type diterpenoids
The antiproliferative activity of 10a–f and 15a–f against four
different kinds of human cancer cell lines (K562 leukemia cell line,
MGC-803 gastric cancer cell line, CaEs-17 esophageal cancer cell
Please cite this article in press as: Li, D.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.07.059

D. Li et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
line, and Bel-7402 hepatoma cell line) were determined by MTT
assay and compared with NO donor 16, oridonin (7), parent com-
pounds (9 and 14) and positive control Taxol in each panel
(Table 1). All the synthetic target molecules 10a–f and 15a–f were
more potent than oridonin and the parent compound (9 or 14).
Target hybrid compounds were very sensitive to Bel-7402 cell line
might, at least to some extent, contribute to the strong
cytotoxicity.
Anticancer drugs were known to interact with cells leading to
cell growth arrest or cell death. The cell cycle had four functional
phases: S phase where DNA replication occurred; M phase (mito-
sis) where DNA and cellular components were divided to form
two daughter cells; G2 phase, between S and M, where cells pre-
pared for mitosis; G1 phase after mitosis and before S phase, where
cells committed and prepared for another round of DNA and cellu-
lar replication.29 Many anticancer compounds exerted their growth
inhibitory effect either by arresting the cell cycle at a particular
checkpoint of cell cycle or by induction of apoptosis or a combined
effect of both cycle block and apoptosis. Furthermore the regula-
tion of the cell cycle and apoptosis was considered to be effective
cancer therapeutic methods.30 To shed more light on the mecha-
nism responsible for the anticancer effect of NO donor/spirolac-
tone-type 6,7-seco-ent-kaurane diterpenoid hybrids, 15d was
examined for their influence on the cell cycle progression. In this
with IC50 values ranging from 0.86 to 1.83
Taxol. While, in CaEs-17 cancer cells, the antiproliferative activity
was not so good with IC50 values of 3.75–6.26 M. In K562 and
lM and even superior to
l
MGC-803 cell lines, target compounds showed similar potency
and the IC50 values were between those of Bel-7402 and CaEs-17
cell lines.
The preliminary SARs showed that when R2 was aromatic
groups o-C6H4 (10b, 10d, 10f, 15b, 15d, and 15f), the activity was
stronger than those with alkyl groups (CH2)2 (10a, 10c, 10e, 15a,
15c, and 15e). 10d and 15d, correspondingly, exhibited the most
promising antiproliferative activity of NO donor/1-oxo spirolac-
tone-type 6,7-seco-kaurane diterpenoid hybrids 10a–f and NO
donor/1-OAc spirolactone-type 6,7-seco-kaurane diterpenoid
hybrids 15a–f against all the four tested cell lines. The IC50 values
assay, Bel-7402 cells were treated with 0.25, 0.5 and 1.0 lM of
compound 15d. Controls were treated with vehicle DMSO. In order
to determine the effects on the cell cycle progression, we detected
the DNA content of cell nuclei by flow cytometry (Fig. 3). Treat-
ment with 15d led to a dose-dependent accumulation of cells in
the S phase with a concomitant decrease in the population of cells
in G1 and G2 phases. Cells at S phase resulted in accumulation of
were 1.97 and 1.74
against CaEs-17 cells, 1.63 and 1.16
and 0.93 and 0.86 M against Bel-7402 cells, respectively. Clear
lM against K562 cell line, 4.12 and 3.75
lM
l
M against MGC-803 cell line,
l
morphological changes were observed in cells exposed to the com-
pounds (Fig. 2). Under light microscopy, control cells appeared
with a regular shape, whereas after incubation with 15d, Bel-
7402 cells showed a remarkable reduction in cell number and cell
size. Based on the above results, the most potent NO-releasing
hybrid 15d was selected for further mechanism study in Bel-
Table 2
NO-releasing ability of hybrids 10a–f and 15a–f (lM)
7402 cells and 0.1 lM of Taxol was used as positive control (data
shown in Supplementary material).
Compd
10 min
20 min
30 min
40 min
50 min
60 min
10a
10b
10c
10d
10e
10f
15a
15b
15c
15d
15e
15f
16
6.71
7.66
2.91
3.52
2.80
5.65
8.84
6.88
5.37
6.10
11.91
10.46
12.25
9.14
11.72
3.58
3.47
0.99
6.16
9.14
7.32
6.99
7.15
12.88
8.42
15.57
12.36
14.40
5.68
4.30
4.91
10.54
10.15
12.46
12.46
12.63
18.26
12.74
19.26
13.23
13.28
8.08
9.96
6.92
13.17
11.79
14.83
16.66
16.88
22.02
16.27
24.10
15.81
14.82
12.12
12.45
15.54
16.42
13.44
18.68
24.74
22.09
26.83
21.54
25.89
19.37
16.89
18.82
17.11
20.09
19.87
16.94
22.63
28.92
25.77
31.68
24.67
27.04
The key requirement for any therapeutic effectiveness of the NO
releasing prodrugs was their ability to undergo biotransformation
to release NO, either enzymatically or nonenzymatically. The NO-
releasing ability of the hybrids 10a–f and 15a–f were determined
by Griess assay. The levels of nitrate/nitrite in the lysates were
determined at 100 lM over duration of 1 h and measured at the
time point of 10, 20, 30, 40, 50 and 60 min. As shown in Table 2,
the concentrations of released NO for all synthetic hybrids
increased with time and were more than 15
of 60 min. The most effective compounds 10d and 15d produced
17.11 and 25.77 M of NO, respectively. High NO releasing ability
lM at the time point
l
Figure 2. Effects of 15d on Bel-7402 cell morphology. Cells were treated with compound 15d for 48 h, then morphology was observed under light microscopy at 200
magnification: (a) negative control, (b) positive control (5 M Taxol), (c) cells treated with 5 M 15d, (d) 1.25 M 15d, (e) 0.3125 M 15d.
l
l
l
l
Please cite this article in press as: Li, D.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.07.059

4
D. Li et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Figure 3. Cell cycle effects of 15d on Bel-7402 cells.
30.34%, 43.68% and 48.50%, with more than 18% increase, at the
selected concentrations, respectively.
compound 15d for 48 h, the percentages of apoptotic cells were
18.92%, 34.82% and 60.91% (early and late, Q2 + Q4), respectively,
compared to 8.41% of vehicle control. These findings prompted
us to further investigate the apoptotic process after treatment of
cells with compound 15d.
The maintenance of mitochondrial membrane potential was
significant for mitochondrial integrity and bio-energetic function.
Mitochondrial changes, including loss of mitochondrial membrane
potential, were key events that took place during drug-induced
apoptosis. Since the apoptosis is an energy-dependent process
and can be activated by the intracellular signal from the mitochon-
dria,33 we first examined the change of mitochondrial membrane
potential (MMP) after 15d treatment by JC-1 method. Bel-7402
cells were incubated with different concentrations (0, 0.25, 0.5
Apoptosis was a programmed cell death that occurred in phys-
iological and pathological conditions and also a highly regulated
cellular process, in which the cell actively pursued a course
towards death upon receiving either extrinsic or intrinsic signals.
On the other hand, many compounds exerted their cytotoxic activ-
ity in tumor cells by inducing apoptosis.31 To determine whether
the strong cytotoxicity of 15d was caused by apoptosis to some
extent, Bel-7402 cells were treated with vehicle alone or different
concentrations (0.25, 0.5 and 1.0 lM) of 15d for 48 h and stained
with FITC-Annexin V and propidium iodide (PI). PI was a DNA-
intercalating dye which entered in the cells only when plasma
membrane integrity was lost, a typical event of late apoptotic or
necrotic cells. In the scatter plot of a double variant flow cytome-
try, the lower left (LL) quadrant of the histograms represented
the viable cells, which exclude PI and were negative for FITC-
Annexin V staining. The lower right (LR) quadrant showed the early
apoptotic cells, which were PI negative and annexin V positive
indicating that cell population endowed with cytoplasmic mem-
brane integrity. The upper right (UR) quadrant provided informa-
tion about the non-viable necrotic and late-stage apoptotic cells,
which were positive for annexin V binding and PI uptake. This pop-
ulation had loss of membrane integrity and presents externaliza-
tion of PS. Finally, the upper left (UL) quadrant represented
necrotic cell population, which had loss of cell membrane integrity
(PI positive and Annexin-V negative).32 As shown in Figure 4, com-
pound 15d caused significant induction of apoptosis in a dose-
and 1.0 lM) of compound 15d for 48 h prior to staining with the
fluorescent dye JC-1. Then the numbers of cells with collapsed
mitochondrial membrane potentials in different cell groups were
determined by flow cytometry analysis (Fig. 5). The percentage of
apoptotic cells increased in a dose-dependent fashion (3.69%,
12.18%, 20.49% and 25.54%, respectively). These studies showed
that incubation with compound 15d increased the number of cells
with collapsed mitochondrial membrane potentials and induced
apoptosis.
In summary, twelve NO-releasing spirolactone-type 6,7-seco-
kaurane diterpenoid analogs (10a–f and 15a–f) were designed
and synthesized. The antiproliferative activity was evaluated in
comparison to oridonin and Taxol. All the synthetic derivatives
were more potent than oridonin against four kinds of human can-
cer cell lines (Bel-7402, K562, MGC-803 and CaEs-17), and even
dependent manner. When treated with 0.25, 0.5 and 1.0 lM of
Please cite this article in press as: Li, D.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.07.059

D. Li et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
Figure 4. Apoptosis effects of 15d on Bel-7402 cells.
Figure 5. Mitochondrial membrane potential effects of 15d on Bel-7402 cells.
superior to Taxol against Bel-7402 cells. SARs were also concluded
based on above results. 15d exhibited the most promising antipro-
liferative activity with the IC50 values of 0.86, 1.74, 1.16 and
NO at the time point of 60 min. High NO releasing ability should,
at least to some extent, contribute to the strong cytotoxicity. Based
on the above results, 15d was selected for further mechanism
study in Bel-7402 cells. Furthermore, it was found that incubation
3.75 lM, respectively, and produced high level of 25.77 lM of
Please cite this article in press as: Li, D.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.07.059

6
D. Li et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
12. Ai, Y.; Kang, F.; Huang, Z.; Xue, X.; Lai, Y.; Peng, S.; Tian, J.; Zhang, Y. J. Med.
Chem. 2015, 58, 2452.
13. Sun, H. D.; Huang, S. X.; Han, Q. B. Nat. Prod. Rep. 2006, 23, 673.
14. Lin, Z.; Guo, Y.; Gao, Y.; Wang, S.; Wang, X.; Xie, Z.; Niu, H.; Chang, W.; Liu, L.;
Yuan, H.; Lou, H. J. Med. Chem. 2015, 58, 3944.
15. Wang, L.; Li, D. H.; Wang, C. L.; Zhang, Y. H.; Xu, J. Y. Mini-Rev. Med. Chem. 2011,
11, 910.
16. Li, S. H.; Wang, J.; Niu, X. M.; Shen, Y. H.; Zhang, H. J.; Sun, H. D.; Li, M. L.; Tian,
Q. E.; Lu, Y.; Cao, P.; Zheng, Q. T. Org. Lett. 2004, 6, 4327.
17. Han, Q. B.; Cheung, S.; Tai, J.; Qiao, C. F.; Song, J. Z.; Tso, T. F.; Sun, H. D.; Xu, H.
X. Org. Lett. 2006, 8, 4727.
with representative compound 15d induced apoptosis in Bel-7402
cells and the mitochondrial membrane potentials were lowered.
Apparently, compound 15d induced apoptosis via mitochondria-
related pathways. It was expected that the remarkable biological
profile of the synthetic NO-releasing spirolactone-type diterpenoid
analogs made them possible as promising candidates for the devel-
opment of novel anticancer agents.
Acknowledgments
18. Xiang, W.; Li, R. T.; Wang, Z. Y.; Li, S. H.; Zhao, Q. S.; Zhang, H. J.; Sun, H. D.
Phytochemistry 2004, 65, 1173.
19. Jiang, H. Y.; Wang, W. G.; Zhou, M.; Wu, H. Y.; Zhan, R.; Li, X. N.; Du, X.; Li, Y.;
Pu, J. X.; Sun, H. D. Fitoterapia 2014, 93, 142.
20. Han, Q. B.; Zhao, A. H.; Zhang, J. X.; Lu, Y.; Zhang, L. L.; Zheng, Q. T.; Sun, H. D. J.
Nat. Prod. 2003, 66, 1391.
21. Bai, S. P.; Wei, Q. Y.; Jin, X. L.; Wu, Q. X.; Yang, L. Planta Med. 2005, 71, 764.
22. Yeoman, J. T.; Mak, V. W.; Reisma, B. S. E. J. Am. Chem. Soc. 2013, 135, 11764.
23. Cha, J. Y.; Yeoman, J. T. S.; Reisman, S. E. J. Am. Chem. Soc. 2011, 133, 14964.
24. Pan, Z.; Zheng, C.; Wang, H.; Chen, Y.; Li, Y.; Cheng, B.; Zhai, H. Org. Lett. 2014,
16, 216.
25. Gong, J. X.; Lin, G. A.; Sun, W. B.; Li, C. C.; Yang, Z. J. Am. Chem. Soc. 2010, 132,
16745.
26. Wang, L.; Li, D.; Xu, S.; Cai, H.; Yao, H.; Zhang, Y.; Jiang, J.; Xu, J. Eur. J. Med.
Chem. 2012, 52, 242.
27. Li, D.; Cai, H.; Jiang, B.; Liu, G.; Wang, Y.; Wang, L.; Yao, H.; Wu, X.; Sun, Y.; Xu, J.
Eur. J. Med. Chem. 2013, 59, 322.
This work was financially supported by the National Natural
Science Foundation of China (81373280, 21502121), Project
Funded by China Post Doctoral Science Foundation
(2015M570258), General Scientific Research Projects of Depart-
ment of Education in Liaoning Province (L2014382), Key Labora-
tory for the Chemistry and Molecular Engineering of Medicinal
Resources (Guangxi Normal University), Ministry of Education of
China (CMEMR2015-B07) and Career Development Support Plan
for Young and Middle-aged Teachers in Shenyang Pharmaceutical
University.
Supplementary data
28. Compound 15d: white solid, 39% yield: mp 106–108 °C; IR (KBr) tmax 3441,
2960, 2025, 1730, 1616, 1553, 1451, 1374, 736, 685 cmÀ1 1H NMR (CDCl3,
;
300 MHz), d (ppm) 9.70 (1H, d, J = 4.2 Hz, –CHO), 8.06 (2H, d, J = 7.5 Hz, Ar-H),
7.73 (3H, m, Ar-H), 7.63 (2H, t, J = 7.8 Hz, Ar-H), 7.52 (2H, t, J = 3.6 Hz, Ar-H),
6.23 (1H, s, 17-CH2), 5.73 (1H, s, 14-CH), 5.63 (1H, s, 17-CH2), 5.22, 5.12 (each
1H, dd, JA = JB = 12.3 Hz, 20-CH2), 4.66 (1H, m, 1-CH), 3.07 (1H, d, J = 9.0 Hz, 13-
CH), 2.01 (3H, s, –CH3), 1.18 (3H, s, –CH3), 1.03 (3H, s, –CH3); 13C NMR (CDCl3,
100 MHz), d (ppm) 202.52, 197.23, 170.03, 167.54, 166.44, 166.29, 158.74,
146.56, 136.14, 135.68, 131.60, 129.98, 129.18, 128.81, 127.60, 126.15, 125.47,
123.35, 120.80, 110.48, 74.35, 74.06, 70.67, 67.95, 64.57, 62.71, 59.91, 45.80,
43.66, 42.08, 39.80, 33.58, 32.43, 30.25, 25.78, 25.18, 23.60, 21.56, 18.05; MS
(ESI) m/z: 852.3 [M+NH4]+, 869.2 [M+Cl]À; HR-MS (ESI, M+NH4) m/z: calcd for
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.07.
059.
References and notes
1. Mocellin, S. Curr. Cancer Drug Targets 2009, 9, 214.
2. Pervin, S.; Singh, R.; Chaudhuri, G. Nitric Oxide 2008, 19, 103.
3. Ignarro, L. J.; Buga, G. M.; Wood, K. S.; Byrns, R. E.; Chaudhuri, G. Proc. Natl.
Acad. Sci. U.S.A. 1987, 84, 9265.
4. Sattler, R.; Xiong, Z.; Lu, W. Y.; Hafner, M.; MacDonald, J. F.; Tymianski, M.
Science 1999, 284, 1845.
C41H46N3O15S: 852.2644, found 852.2629. Spectrum data of compounds 10a–f,
15a–c, 15e and 15f, and experiment procedures of MTT assay, cell cycle study,
Annexin-V and PI double staining cellular apoptosis analysis and
mitochondrial membrane potential effects could be found in Supplementary
material.
5. Cho, D. H.; Nakamura, T.; Fang, J.; Cieplak, P.; Godzik, A.; Gu, Z.; Lipton, S. A.
Science 2009, 324, 102.
29. Sánchez-Martínez, C.; Gelbert, L. M.; Lallena, M. J.; de Dios, A. Bioorg. Med.
Chem. Lett. 2015, 25, 3420.
30. Blank, M.; Shiloh, Y. Cell Cycle 2007, 6, 686.
6. Wei, X. Q.; Charles, I. G.; Smith, A.; Ure, J.; Feng, G. J.; Huang, F. P.; Xu, D.;
Muller, W.; Moncada, S.; Liew, F. Y. Nature 1995, 375, 408.
7. Carpenter, A. W.; Schoenfisch, M. H. Chem. Soc. Rev. 2012, 41, 3742.
8. Wang, P. G.; Xian, M.; Tang, X.; Wu, X.; Wen, Z.; Cai, T.; Janczuk, A. J. Chem. Rev.
2002, 102, 1091.
9. Duan, W.; Li, J.; Inks, E. S.; Chou, C. J.; Jia, Y.; Chu, X.; Li, X.; Xu, W.; Zhang, Y. J.
Med. Chem. 2015, 58, 4325.
10. Han, C.; Huang, Z. J.; Zheng, C.; Wan, L. D.; Zhang, L. W.; Peng, S. X.; Ding, K.; Ji,
H. B.; Tian, J. D.; Zhang, Y. H. J. Med. Chem. 2013, 56, 4738.
11. Liu, M. M.; Chen, X. Y.; Huang, Y. Q.; Feng, P.; Guo, Y. L.; Yang, G.; Chen, Y. J.
Med. Chem. 2014, 57, 9343.
31. Wong, R. S. J. Exp. Clin. Cancer Res. 2011, 30, 87.
32. Raffa, D.; Maggio, B.; Plescia, F.; Cascioferro, S.; Raimondi, M. V.; Cancemi, G.;
D’Anneo, A.; Lauricella, M.; Cusimano, M. G.; Bai, R.; Hamel, E.; Daidone, G.
Bioorg. Med. Chem. 2015, 23, 6305.
33. Yang, F. R.; Li, Y.; Zhang, X. H.; Wang, M.; Guo, H. R.; Ruan, W. J. Bioorg. Med.
Chem. Lett. 2015, 25, 3592.
Please cite this article in press as: Li, D.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.07.059