Synthesis and biological evaluation of novel exo-methylene cyclopentanone tetracyclic diterpenoids as antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2010.11.055

The structure of exo-methylene cyclopentanone, which exists in nature tetracyclic diterpenoids products, has been proved to be an innate group for the treatment of cancer and inflammation. In this letter, four different scaffolds of tetracyclic diterpenoi

Full text of DOI:10.1016/j.bmcl.2010.11.055

Bioorganic & Medicinal Chemistry Letters 21 (2011) 130–132
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel exo-methylene cyclopentanone
tetracyclic diterpenoids as antitumor agents
⇑
Jing Li, Dayong Zhang , Xiaoming Wu
Center of Discovery Drug, School of Pharmacy, China Pharmaceutical University, 24 Road 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:
The structure of exo-methylene cyclopentanone, which exists in nature tetracyclic diterpenoids products,
has been proved to be an innate group for the treatment of cancer and inflammation. In this letter, four
different scaffolds of tetracyclic diterpenoids including the structure exo-methylene cyclopentanone
were synthesized from steviol and isosteviol and evaluated in vitro for their antitumor activity against
three human cancer lines. Compounds 1a, 1b, 2b and 3b showed significant cytotoxicity, particularly, tet-
racyclic diterpenoids 2b, 3b were identified as the most potent and selective anticancer agents superior
Received 13 August 2010
Revised 22 October 2010
Accepted 10 November 2010
Available online 16 November 2010
Keywords:
ent-Kaurane
Steviol
Isosteviol
to adriamycin with IC50 values of 0.9
respectively.
l
M and 1.5
l
M, against Hep-G2 and MDA-MB-231 cell lines,
Ó 2010 Elsevier Ltd. All rights reserved.
exo-Methylene cyclopentanone
Antitumor agents
Tetracyclic diterpenoids, especially ent-kaurane diterpenoids
with an enone system in ring D, constitute an important class of
natural products which exhibit interesting pharmacological activ-
eties.1,2 Natural and synthetic ent-kaurane diterpenoids show
broad spectrum of biological activities such as anti-inflammatory,
anti-tuberculosis, and anticancer.3,4 Different mechanisms have
been proposed such as the inhibition of the neutrophile response
due to the inhibition of cytosolic Ca2+, as well as the inhibition of
prostaglandin E2 production through the suppression of nuclear
cell lines in vitro (Fig. 1). Zhang et al.11 discovered that ent-kaurane
diterpenoids semipinnated brake isolated from Pteris semipinnata
L had significant cytotoxicity superior to 5-FU. Inflexusin and
rabdoserrin D, isolated from Isodon phyllostachys by Li et al.12
,
were demonstrated good cytotoxicity against K569 cell. These
natural ent-kaurane diterpenoids all contain the structure of
exo-methylene cyclopentanone in ring D.
For the reason above we try to build up a crucial structure frag-
ment of exo-methylene cyclopentanone in the ring D of steviol (the
aglycone of stevioside, which shares the same mother ring with
oridonin but has little antitumor or anti-inflammatory activity)
factor-
more relevant to the anti-inflammatory effect of ent-kaurane, and
it make these compounds good candidates for NF- B activity
modulation through interference with the steps leading up to the
release of I B kinase (IKK).
The inhibitory activity of ent-kaurane toward NF-
jB (NF-j
B) activation.5 The latter mechanism seems to be
j
j
HO
OH
H
jB has been
accounted for by invoking the presence of reactive centers and
various studies have focused on the exo-methylene cyclopenta-
none group. This reactive group interacted with biological nucleo-
philes such as the mercapto group of the cysteine residue in the
OH
O
OH
O
H
O
OH
H
OH
H
OH
oridonin
inflexusin
DNA-binding domain of the NF-jB subunit through an irreversible
Micheal-type addition.6,7 A large number of ent-kaurane diterpe-
noids isolated from different natural plants have been reported
to display interesting biological activities. Fujita et al. and Xu
et al.8–10 reported that the natural ent-kaurane diterpenoids
oridonin and its derivatives with an enone system in ring D had
significant cytotoxicity against BGC-7901, BEL-7402 and SW-480
OH
Y
X
X
H
H
COOR
H
H
Y
COOR
1 X=CH2,Y=O
3
X=O,Y=CH2
4 X=CH2,Y=O
2
X=O,Y=CH2
⇑
Corresponding author. Tel./fax: +86 25 83271307.
E-mail address: cpuzdy@yahoo.com.cn (D. Zhang).
Figure 1. Typical natural tetracyclic diterpenoids and target compounds.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.055

J. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 130–132
131
OH
OAc
OH
OH
OH
OH
OR
O
OH
a
b
b
c
H
H
H
H
H
H
OH
O
H
H
H
H
H
H
COOH
COOR
COOH
13
d,e
COOH
COOH
COOH
12
5, R=H
11, R=Ac
1a, R=H
1b, R=Bn
5
steviol
a
c
OH
OH
OH
Scheme 1. Reagents and conditions: (a) SeO2, t-BuOOH, THF (59.5%); (b) PDC, DMF
(71.5% for 1a); (c) BnBr, K2CO3, DMF, KI (78.7%).
OH
O
O
f
h
H
H
H
OTs
OTs
and isosteviol (steviol’s rearrangement isomer) to obtain a series of
novel anticancer compounds.13,15 Previous work proved that the
modification of steviol and isosteviol was effective for increasing
its cytotoxicity. Tao et al.14 reported that hydroxyl groups in ring
D of isosteviol may play an important role in binding to some
receptors. Terai et al. demonstrated that steviol and its derivatives
show a positive response in forward mutation assay using Salmo-
nella typhimurium TM677 in the presence of metabolic activation
system.15 Herein, we report the facile synthesis and biological
activities of four different scaffolds of tetracyclic diterpenoids, in
which the compound 1 and 4 have a structure 16-exo-methy-
lene-15-cyclopentanone while the compound 2 and 3 have a struc-
ture 15-exo-methylene-16-cyclopentanone. (Fig. 1)
H
H
H
COOR1
COOBn
COOR1
2a, R1=H
14
15, R1=Bn
g
2b, R1=Bn
16, R1=H
Scheme 3. Reagents and conditions: (a) Ac2O, DMAP, TEA, THF; (b) O3/O2, CH2Cl2,
Ph3P (85.6% for two-steps); (c) HCHO (aq), NaOH, C2H5OH–H2O, 75 °C, 3 h (78.8%);
(d) BnBr, K2CO3, DMF, KI; (e) TsCl, pyridine, DMAP, rt (53.7% for two-steps); (f) PDC,
DMF (63.6%); (g) 10% Pd-C, H2, C2H5OH (85.7%); (h) pyridine, DMAP, reflux (53.6%).
OH
OH
OH
O
c
a
H
H
OTs
H
Our approaches to synthesize the title compounds are described
below. The synthesis of 1a and 1b is shown in Scheme 1. Starting
from steviol, treatment with SeO2 and t-BuOOH led to 16-hydroxy
steviol 5. Oxidation of 16-hydroxy steviol 5 with PDC gave the de-
sired compound 1a.16,17 Esterification of 1a with benzyl bromide
provided 1b successfully. Preparation of the corresponding com-
pound of isosteviol with 15-carbonyl-16-exo-methylene structure
is illustrated in Scheme 2. Benzyl bromide was added to isosteviol
6 followed by Wittig reaction and SeO2 oxidation to give 9.18,19
However, the synthesis of compounds 4a and 4b was unsuccessful
under the same conditions as above. Furthermore, Swern oxidation
of 9 and 10 resulted in the formation of the target compound 4 in
good yield.20–22
COOBn
COOR
COOH
17, R=H
18, R=Bn
19
b
O
O
d
f
H
H
OTs
COOR1
3a, R1=H
3b, R1=Bn
COOR1
20, R1=Bn
21, R1=H
e
Scheme 4. Reagents and conditions: (a) HCHO (aq), NaOH, C2H5OH–H2O, 75 °C, 3 h
(56.1%); (b) BnBr, K2CO3, DMF, KI (70.4%); (c) TsCl, pyridine, DMAP, rt (68.8%);
(d) PDC, DMF (74.0%); (e) 10% Pd–C, H2, C2H5OH, (84.1%); (f) pyridine, DMAP, reflux
(73.6%).
The synthetic approaches employed to prepare another two
skeletons are outlined in Scheme 3 and 4. Normal way was to con-
struct a hydroxymethyl in the a-position of 15-ketone, and then to
access 16-ketone-15-ene structure by eliminating monomolecular
H2O.23–26 Any attempt to obtain 16-ketone-15-hydroxymethyl
failed to meet our expectation. Therefore, one abnormal Cannizz-
Scheme 3 illustrates the synthesis of compounds 2a and 2b.
Before hydroxymethylation of the
a-position in 15-ketone, we
aro-type reaction was adopted, the
a-position of 16-ketone was
needed to synthesize ent-13-acetoxyl-16-oxo-17-demethylkaura-
ne-19-acid 12 by protecting 13-hydroxy and oxidizing the double
bond with Ac2O and O3, respectively.28,29 After the diol 13 had been
successfully achieved, we attempted to selectively oxidize 15-posi-
tion secondary alcohol with sodium hypochlorite and acetic anhy-
dride as reference,23,24 but the anticipated compound had not been
obtained. So we had to protect the primary alcohol of 13 selectively
before oxidation of 15-hydroxy group. We used TsCl instead of tri-
tylchloride to protect the primary alcohol of 13, on account of a
large steric of tritylchloride.30–32 Oxidation of 14 with PDC gave
15-oxo product 15. Finally, elimination of the TsOH provided the
target compound 2b. Compound 2a was obtained through elimina-
tion of 16, which was accessed by removing the benzyl of 15 with
10% Pd-C. Using the same methodology of the conversion of 12 to
2a and 2b, target compounds 3a and 3b were synthesized starting
from isosteviol 7 as shown in Scheme 4.
hydroxymethylated and 16-carbonyl was reduced in one-step.14,27
O
b
H
H
H
H
COOR
COOR
6, R=H
7, R=Bn
a
8
c
e
H
H
O
OH
d
H
H
COOR
The structure of the target compounds was elucidated using
COOR
spectroscopic techniques (IR, 1H and 13C NMR, ESI/MS).
4a, R=H
4b, R=Bn
9, R=Bn
10, R=H
Steviol, isosteviol and novel tetracyclic diterpenoids 1–4 were
assayed for their in vitro cytotoxicity against three human cancer
cell lines: breast carcinoma (MDA-MB-231), hepatocellular carci-
Scheme 2. Reagents and conditions: (a) BnBr, K2CO3, DMF, KI; (b) CH3P+Ph3IÀ,
n-BuLi, THF, reflux (88.1% for two-steps); (c) SeO2, t-BuOOH, THF (85.9%); (d) 10%
Pd-C, 85% HCOOH (84.6%); (e) (COCl)2, DMSO, TEA (72.2%).

132
J. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 130–132
Table 1
Acknowledgments
In vitro cytotoxicity data of tetracyclic diterpenoids 1–4
Compound
Antitumor activity in 48 h (IC50
,
l
M)a
MGC-803
We thank the National Natural Science Foundation of China(No.
30973607) and the ‘Six Talent Peak’ Foundation of Jangsu Province
of China for financial support (No. Y092003).
MDA-MB-231
Hep-G2
Steviol
ND
ND
ND
1a
1b
2a
ND
2.51
ND
3.69
2.70
ND
3.38
1.80
ND
Supplementary data
2b
Isosteviol
3a
3b
1.24
ND
ND
1.58
ND
0.95
ND
ND
ND
ND
2.41
ND
ND
2.22
ND
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.11.055.
4a
4b
ND
2.26
2.38
ND
2.08
3.66
ND
2.53
3.57
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Adriamycin
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a
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In summary, we prepared four different scaffolds of tetracyclic
diterpenoids that inhibited the growth of MDA-MB-231, Hep-G2,
and MGC-803 cancer cell lines at micromolar concentration.
Compounds 1a, 1b, 2b, and 3b have significant cytotoxicity against
part of cancer cell lines. In isosteviol series, compounds 3a, 4a, and
4b exhibit poor cytotoxicity. The preliminary anticancer activity
study of both steviol and isosteviol series reveals that the exo-
methylene cyclopentanone moiety is beneficial for anticancer
activity. Further, initial antitumor activity results of tetracyclic
diterpenoids 1–4 have generated further interest to optimize their
activity and investigate specific biological targets of these com-
pounds at molecular level.