Synthesis and in vitro cytotoxicity of andrographolide-19-oic acid analogues as anti-cancer agents

Article abstract of DOI:10.1016/j.bmcl.2013.04.010

The synthesis of a series of andrographolide-19-oic acid derivatives was described and their in vitro anti-tumor activity against two human cell lines was evaluated. Most compounds were found to exhibit significant cytotoxicity, better than andrographolide, and compounds 9d and 9b were identified as the most potent with IC₅₀ values of 1.18 and 6.28 μm against HCT-116 and MCF-7 cell lines, respectively. The preliminary results indicated that the oxidation of C-19-hydroxyl group of andrographolide to corresponding carboxyl group and the subsequent esterification of the formed carboxylic acid led to considerable improvement in cytotoxicity against the cancer cells.

Full text of DOI:10.1016/j.bmcl.2013.04.010

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3166–3169
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro cytotoxicity of andrographolide-19-oic acid
analogues as anti-cancer agents
⇑
Dongsheng Chen, Yaping Song, Yunlong Lu, Xiaowen Xue
Department of Medicinal Chemistry, China Pharmaceutical University, 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 synthesis of a series of andrographolide-19-oic acid derivatives was described and their in vitro anti-
tumor activity against two human cell lines was evaluated. Most compounds were found to exhibit sig-
nificant cytotoxicity, better than andrographolide, and compounds 9d and 9b were identified as the most
Received 2 January 2013
Revised 24 March 2013
Accepted 3 April 2013
Available online 10 April 2013
potent with IC50 values of 1.18 and 6.28 lm against HCT-116 and MCF-7 cell lines, respectively. The pre-
liminary results indicated that the oxidation of C-19-hydroxyl group of andrographolide to corresponding
carboxyl group and the subsequent esterification of the formed carboxylic acid led to considerable
improvement in cytotoxicity against the cancer cells.
Keywords:
Andrographolide
Structure modification
Andrographolide-19-oic acid derivatives
Cytotoxicity
Ó 2013 Elsevier Ltd. All rights reserved.
Andrographolide 1, chemically named as 2(3H)-furanone,
3-[2-[decahydro-6-hydroxy-5- (hydroxymethyl)-5,8a-dimethyl-2-
methylene-1-napthalenyl]ethylidene]dihydro-4-hydroxy (Fig. 1),
is the major labdane diterpenoidal constituent isolated from the
plant Andrographis paniculata (family Acanthaceae),1,2 which is
used extensively as the traditional Chinese medicine. Androgra-
pholide exhibits a wide spectrum of biological activities including
antibacterial,3 antiinflammatory,4 antimalarial,5 immunomodula-
tion,6 antithrombotic,7 and hepatoprotective effect.8 In recent past,
the compound is reported for its anti-tumor activity.9,10 Several
mechanisms have been proposed for its anti-cancer activity, such
as cytotoxicity against cancer cells,11 induction of cell-cycle ar-
rest,12 and induction of apoptosis.13
The promising anti-cancer activity of andrographolide makes it
a good lead. Various semi-synthetic analogues have been synthe-
sized and evaluated in order to find out a better candidate holding
therapeutic potential over the parent compound.14–16 Most struc-
ture modifications were focused on the functionallization of
3,14,19-hydroxy groups of andrographolide. Stanslas reported that
14-acetylandrographolide is more potent from 60 NCI human
cancer cell lines in vitro screen compared to andrographolide.17
C-3-hydroxyl group of parent compound has been oxidized to
the keto group,19 while increased significantly after the C-19-hy-
droxyl group has been oxidized to the carboxylic group.20
Unfortunately, the
a-alkylidene-c-butyrolactone core moiety of
andrographolide was destroyed during the bioconversion of
C-19-hydroxyl group into carboxyl group. Therefore, it is natural
for us to envisage that the andrographolide-19-oic acid analogues
with the a-alkylidene-c-butyrolactone core moiety unbroken may
have much better anti-tumor activity than andrographolide
(Fig. 2). To our surprise, no attempt was made to design the
andrographolide analogues bearing these structural characters. In
this Letter, we report the synthesis and biological evaluation of
new andrographolide-19-oic acid analogues as potent cytotoxic
agents.
The first series of andrographolide-19-oic acid analogues were
prepared as shown in Scheme 1. Andrographolide was used as
starting material since it is readily available from nature. The
O
14
HO
O
In another report, the
a-alkylidene-c-butyrolactone moiety of
1
andrographolide was believed to play a major role in the activity
profile.18 He et al. recently studied the biotransformed products
from andrographolide by Rhizopus stolonifer ATCC 12939 and
Aspergillus ochraceus, and found that the antiproliferative activities
against MCF-7 and HCT-116 cell lines were decreased after the
1
17
8
H
3
4
HO
19
OH
1
⇑
Corresponding author. Tel./fax: +86 25 83199600.
E-mail address: xwenxue@cpu.edu.cn (X. Xue).
Figure 1. Andrographolide.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.010

D. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3166–3169
3167
O
AcO
O
favorable to the activity 17,18
H
HO
OH
O
O
O
14
AcO
O
HO
O
HO
unfavorable to
the activity19
O
12
1
17
8
H
H
H
3
4
HO
HO
19
OH
O
CO2H
1
OH
O
O
O
14
R1O
O
12
1
H
HO
H
3
CO2H
4
R3O
19
favorable to the activity 20
CO2R2
Figure 2. Design strategy for the new andrographolide-19-oic acid analogues.
O
O
O
HO
O
HO
O
R1O
O
c
a
b
H
H
H
HO
O
O
OH
O
O
1
2
3a-3c
O
O
O
R1O
O
R1O
O
R1O
O
d
e
H
H
H
HO
HO
HO
CHO
5a-5c
CO2H
OH
4a-4c
6a-6c
a R1 = Ac; b R1 = Bz; c R1 = p-Methoxycinnamoyl
Scheme 1. Reagents and conditions: (a) 2,2-dimethoxypropane, p-TsOH (cat.), Tol/DMSO, 80 °C, 1.5 h, 93%; (b) for 3a: Ac2O, reflux 1.5 h; for 3b: BzCl, TEA, DMAP, CH2Cl2, rt,
4 h; for 3c: p-methoxycinnamic acid/Tf2O, TEA, CH2Cl2, 0 °C; then 2, 0 °C to rt; (c) HOAc–H2O (7:3), rt, 42–85% for two-steps; (d) TEMPO, TBAI, NCS, CH2Cl2, K2CO3–KHCO3
buffer, 0 °C, 9 h, 83-91%; (e) NaClO2–NaH2PO4, isopantene, t-BuOH, DMSO, 0 °C to rt,48 h, 42–59%.
hydroxyl groups at C-3 and C-19 of androgropholide were pro-
tected by reacting with 2,2-dimethoxypropane at the presence of
catalytic p-TsOH according to a modified literature approach,21 to
furnish 3,19-isopropylideneandrographolide 2. The hydroxyl group
at C-14 was then acylated with corresponding acylating agents
(acetic anhydride, benzoyl chloride, and p-methoxycinnamic

3168
D. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3166–3169
Table 1
trifluoromethanesulfonic anhydride respectively) to provide
3a–3c. Here, aliphatic acetyl, aromatic benzoyl and conjugated p-
methoxycinnamoyl were chosen to investigate the influence of
different types of acyl groups on the anti-tumor activity. The iso-
propylidene protecting group of 3a–3c was removed by stirring
with aqueous acetic acid at room temperature, followed by selec-
tive oxidation of primary hydroxyl group at C-19 position with
TEMPO–NCS system22 to yield corresponding aldehydes 5a–5c.
The formed aldehyde group of 5a–5c was further oxidized to car-
boxyl group with NaClO2–NaH2PO4 and isopentene,23 affording
the first series target compounds 6a–6c.
The second and third series of andrographolide analogues
8a–8c and 9a–9f were synthesized as shown in Scheme 2. Com-
pounds 5a–5c were acetylated with acetic anhydride under reflux
to furnish 3-acetylated products 7a–7c. The aldehyde group of
7a–7c was then oxidized to carboxyl group with the same method
described in Scheme 1, to afford the second series targets 8a–8c.
The carboxyl group of 8a–8c was esterificated with MeI or BnBr
at the presence of K2CO3 in DMF at room temperature to provide
the third series targets 9a–9f.
Evaluation of in vitro cell growth inhibitory effects of andrographolide analogues in
against two cell lines
O
R1O
O
H
R3O
CO2R2
Entry
Compound
Cell growth inhibition in terms of IC50 (l
M)a
R1
R2
R3
HCT-116
MCF-7
1
2
3
4
5
6
7
8
9
10
11
12
13
14
6a
6b
6c
8a
8b
8c
9a
9b
9c
9d
9e
9f
Ac
Bz
H
H
H
H
H
H
Me
Bn
Me
Bn
Me
Bn
H
H
H
29.24
36.85
32.39
20.90
17.81
9.73
2.29
2.48
3.01
1.18
35.66
40.20
95.00
>100
44.57
43.31
11.80
6.28
10.62
8.37
19.73
8.92
p-MCb
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Bz
p-MC
Ac
Ac
Bz
Bz
p-MC
p-MC
The structure of the target compounds was identified using
spectroscopic techniques (IR, 1H NMR, 13C NMR, and mass spectro-
scopic analyses).24
3.01
3.00
24.91
13.36
The cytotoxic activity of 1 and its synthetic analogues were
evaluated by MTS method,25 using in vitro assay against two can-
cer cell lines: HCT-116 (human colon cancer) and MCF-7 (human
breast cancer). The reason why these two cell lines were selected
is that andrographolide was found most effective against colon
cancer cell lines, followed by breast cancer cell lines.12,17 All tested
samples were dissolved in DMSO (0.1%). cis-Platin was selected as
positive control. The concentrations that cause 50% inhibition of
cancer cell growth against various cell lines are expressed as IC50
values and are summarized in Table 1.
1
>100
89.83
cis-Platin
a
IC50 was expressed as an average value of two experiments.
p-MC = p-methoxycinnamoyl.
b
better anti-tumor activities against these two cell lines than posi-
tive control cis-platin, with 9d and 9b displaying the strongest
activities with IC50 values of 1.18 and 6.28 lm against HCT-116
and MCF-7, respectively. All test compounds were found more
effective against HCT-116 than MCF-7, which could be accounted
for by the difference in sensitivity between these two cell lines.
The esterification of andrographolide-19-oic acid derivatives con-
tributes significantly to the potent cytotoxicity (compounds 8a vs
9a–9b, 8b vs 9c–9d, and 8c vs 9e–9f), and more interestingly,
Almost all 12 synthesized analogues exhibited cytotoxic activi-
ties stronger than that of parent compound 1 in both cancer lines
(except for the compound 8a, which displayed almost no activity
against MCF-7). The cytotoxicity of these compounds might be
attributed to their ability to inhibit proliferation and induce apop-
tosis in cancer cells. Seven compounds 8c and 9a–9f even showed
O
O
O
R1O
O
R1O
O
R1O
O
g
f
H
H
H
AcO
AcO
HO
CO2H
CHO
CHO
5a R1 = Ac
5b R1 = Bz
5c R1 = p-MC
7a R1 = Ac
8a R1 = Ac
7b R1 = Bz
8b R1 = Bz
7c R1 = p-MC
8c R1 = p-MC
9a R1 = Ac, R2 = Me
9b R1 = Ac. R2 = Bn
9c R1 = Bz, R2 = Me
9d R1 = Bz, R2 = Bn
9e R1 = p-MC, R2 = Me
9f R1 = p-MC, R2 = Bn
O
R1O
O
h
H
CO2R2
AcO
p-MC = p-Methoxycinnamoy
Scheme 2. Reagents and conditions: (f) Ac2O, reflux 1.5 h, 60–78%; (g) NaClO2–NaH2PO4, isopantene, t-BuOH, DMSO, 0 °C to rt,48 h, 50–76%; (h) for 9a, 9c and 9e: MeI, K2CO3,
DMF, rt, 1 h, 60–62%; for 9b, 9d and 9f: BnBr, K2CO3, DMF, rt, 1 h, 49–55%.

D. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3166–3169
3169
the size of ester group has an important influence on the activity
References and notes
against MCF-7: the compounds with benzyl ester group (9b, 9d
and 9f) express a better activity than those with methyl ester
group (9a, 9c and 9e). However, acetylation of C-3-hydroxyl group
seems unfavorable to the activity against MCF-7, while favorable to
the activity against HCT-116 (compounds 6a vs 8a, 6b vs 8d, and 6c
vs 8c). Moreover, for the structure modification of C14-hydroxyl
group of andrographolide-19-oic acid analogues with acyl group,
what should be noted is that the type of the acyl group has no obvi-
ous effect on the activity: whatever the acyl group is, aliphatic
acetyl, aromatic benzoyl or conjugated p-methoxycinnamoyl, the
activity keeps almost unchangeable.
In summary, we have successfully synthesized twelve androg-
rapholide-19-oic acid analogues and biologically evaluated their
in vitro anti-tumor activities against human cancer cell lines
HCT-116 and MCF-7. To our knowledge, this is the first attempt
to oxidize the C-19-hydroxyl group of andrographolide to corre-
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core moiety intact meanwhile. The preliminary results indicated
that the oxidation of C-19-hydroxyl of the parent compound to car-
boxylic acid group, especially the subsequent esterification of the
formed carboxylic acid, indeed led to a big increase in activity
against the tested cancer cells. The most potent analogue 9d,
whose preparation was relatively simply achieved in seven-steps
from andrographolide, showed 11-fold higher cytotoxic activity
against HCT-116 than the anticancer drug cis-platin, which made
it a promising structure lead for the development of new antican-
cer drugs. Studies in this direction are in progress.
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Acknowledgments
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24. The structure of the compounds 6a–c, 8a–c, and 9a–f was confirmed by
spectroscopic techniques. For example, methyl 3-acetyl-14-acetyl-
andrographolide-19-oate (9a): IR (KBr): 3449, 3127, 1763, 1735, 1724, 1676,
We thank China Pharmaceutical University and the Fundamen-
tal Research Funds for the Central Universities (Program No.
JKY2011082) for the financial support. Mr. Ying Zhang is appreci-
ated for his help in HRMS measurement. We also thank Drs.
Xiangyang Xu and Yong Zhao for their guidance in biological test.
1647, 1396, 1251, 1234, 1218, 1155, 1025 cmÀ1 1H NMR (300 MHz, CDCl3) d:
;
7.01 (1H, t, J = 6.5 Hz, H-12), 5.92 (1H, d, J = 5.8 Hz, H-14), 4.96 (1H, s, H-17b),
4.61–4.52 (3H, m, H-3, H-15b and H-17a), 4.27–4.23 (1H, d, J = 11.2 Hz, H-15a),
3.66 (3H, s, CO2CH3), 2.52–2.29 (4H, m), 2.12 (3H, s, CH3CO), 2.07 (3H, s,
CH3CO), 2.01–1.94 (1H, m), 1.86–1.80 (3H, m), 1.60–1.30 (4H, m), 1.25 (3H, s,
H-18), 0.65 (3H, s, H-20); 13C NMR (75 MHz, CDCl3) d: 173.8, 170.9, 170.4,
169.0, 150.1, 146.4, 124.0, 109.0, 79.0, 71.5, 67.8, 55.6, 55.3, 51.1, 48.4, 39.3,
37.6, 37.1, 25.2, 25.2, 24.5, 24.0, 21.2, 20.6, 12.4; HRMS (ESI) m/z calcd for
Supplementary data
C
25H34O8Na [M+Na]+ 485.2151, found 485.2152.
25. Riss, T. L.; Moravec, R. A. Mol. Biol. Cell 1992, 3, 184a.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.04.
010.