Inhibition of topoisomerase II α activity and induction of apoptosis in mammalian cells by semi-synthetic andrographolide analogues

Article abstract of DOI:10.1007/s10637-012-9868-9

Summary: Topoisomerase II α enzyme plays a critical role in DNA replication process. It controls the topologic states of DNA during transcription and is essential for cell proliferation. Human DNA topoisomerase II α (hTopo II α) is a promising chemotherap

Full text of DOI:10.1007/s10637-012-9868-9

Invest New Drugs (2013) 31:320–332
DOI 10.1007/s10637-012-9868-9
PRECLINICAL STUDIES
Inhibition of topoisomerase II α activity and induction
of apoptosis in mammalian cells by semi-synthetic
andrographolide analogues
Jintapat Nateewattana & Rungnapha Saeeng &
Sakkasem Kasemsook & Kanoknetr Suksen &
Suman Dutta & Surawat Jariyawat &
Arthit Chairoungdua & Apichart Suksamrarn &
Pawinee Piyachaturawat
Received: 21 May 2012 /Accepted: 2 August 2012 /Published online: 17 August 2012
#
Springer Science+Business Media, LLC 2012
Summary Topoisomerase II α enzyme plays a critical role
in DNA replication process. It controls the topologic states
of DNA during transcription and is essential for cell prolif-
eration. Human DNA topoisomerase II α (hTopo II α) is a
promising chemotherapeutic target for anticancer agents
against a variety of cancer types. In the present study,
andrographolide and its structurally modified analogues
were investigated for their inhibitory activities on hTopo II
α enzyme. Five out of nine andrographolide analogues
potently reduced hTopo II α activity and inhibited cell
proliferation in four mammalian cell lines (Hela, CHO,
BCA-1 and HepG2 cells). IC50 values for cytotoxicity of
analogues 3A.1, 3A.2, 3A.3, 1B and 2C were 4 to 7 μM.
Structure-activity relationship studies revealed that both
core structure of andrographolide and silicon based mole-
cule of functional group were important for the inhibition of
hTopo II α activity whereas position C-19 of analogues was
required for anti-proliferation. In addition, the analogue 2C
at 10 μM concentration inhibited hTopo II α, and induced
apoptosis with nuclear fragmentation and formation of apo-
ptotic bodies in HepG2 cells. The analogue 2C may, there-
fore, have a therapeutic potential as effective anticancer
agent targeting the hTopo II α functions.
.
.
Keywords Andrographolide analogues Apoptosis
.
Cancer DNA topoisomerase II α inhibitor
Introduction
:
J. Nateewattana P. Piyachaturawat
DNA topoisomerases are one of the promising molecular
targets for developing anticancer drugs [1]. Inhibitors of
DNA topoisomerase have been proposed as the most effec-
tive anticancer agents such as campthothecin, etoposide and
doxorubicin [2]. They are chosen for the first treatment of
many cancers including cervical adenocarcinoma and ovar-
ian cancers [3, 4]. DNA topoisomerases are ubiquitous and
highly conserved enzymes involved in mainly genomic
integrity of cells. These enzymes are responsible for the
relaxation of DNA during critical cellular processes includ-
ing replication, recombination, transcription and repair sys-
tem by transiently breaking one or two strands of DNA.
Recently, a number of semi-synthetic compounds derived
from natural products have been demonstrated to greatly
improve anticancer activities [5]. Modified natural com-
pounds could, therefore, render greater potency and efficacy
for the inhibition of cancer growth and proliferation [5–10].
For example, etoposide, a synthetically modified compound
Toxicology Graduate Program, Faculty of Science,
Mahidol University,
Bangkok 10400, Thailand
:
R. Saeeng S. Kasemsook
Department of Chemistry, Faculty of Science, Burapha University,
Chonburi 20131, Thailand
:
:
:
:
K. Suksen S. Dutta S. Jariyawat A. Chairoungdua
P. Piyachaturawat (*)
Department of Physiology, Faculty of Science,
Mahidol University,
Bangkok 10400, Thailand
e-mail: pawinee.pia@mahidol.ac.th
P. Piyachaturawat
e-mail: pawinee@gmail.com
A. Suksamrarn
Department of Chemistry, Faculty of Science,
Ramkhamhaeng University,
Bangkok 10240, Thailand

Invest New Drugs (2013) 31:320–332
321
derived from Podophyllum peltatum, is used for treatment of
lung and testicular cancers with much higher potency and
efficacy than its parent form [5].
several times. The combined ethyl acetate extracts were
concentrated in vacuo. The crude sample was re-
crystallized in methanol to give pure andrographolide-1 as
a white solid compound (9.4484 g, 0.94 % yield). The
structure of andrographolide obtained from the plant extract
Andrographis paniculata (Burm.f.) Nees (Acanthaceae)
is a medicinal plant widely used in many Asian countries
including China, India, Taiwan, and Thailand. Its major
biological active compound is Andrographolide, a diterpe-
noid lactone which has been reported to have various bio-
logical activities including anti-cancer activity [5]. It inhibits
HepG2 (hepatocellular carcinoma) cell proliferation and
induces caspase independent cell death [11]. Recently, a
number of andrographolide analogues have been developed
and demonstrated to have much greater therapeutic potency
for cancer treatments [5–10]. They also induced apoptosis in
human cervical, breast and liver cancer cell lines [12, 13].
Apoptosis is a highly regulated and a crucial process found
in all multi-cellular organisms. Apoptotic cell death is not
only implicated in regulatory mechanisms of cells but also
an important way of anti-cancer treatments as the process
eliminates cancer cells without inducing inflammation [14].
In the present study we semi-synthesized andrographo-
lide analogues and investigated their inhibitory activity on
human topoisomerase II α (hTopo II α) enzyme in vitro,
anti-proliferation effects on different types of cell lines, and
induction of apoptosis in HepG2 cells. This is the first report
showing direct inhibition of recombinant hTopo II α in vitro
by andrographolide analogues.
1
was identified based on H- and 13C-NMR spectral data
comparing with commercial andrographolide (purchased
from Sigma-Aldrich, CAS No. 5588-58-7).
Andrographolide analogues derived from the natural sub-
stituents were semi-synthesized by changing the core struc-
ture and functional groups at C-3, C-12, C-17, and C-19.
The methods for modifying the structure of andrographolide
analogues in structure 1 have been described previously [9].
Modifications of andrographolide analogues in structure 2
and 3 were shown in Fig. 1. Structures of andrographolide
analogues were identified by using IR, NMR and HRMS
(ESI).
Andrographolide (1)
White solid, yield 0.94 %; IR (Neat): 3196, 3097, 1646,
1
1403, 1178, 1023 cm-1; H NMR (400 MHz, CD3OD): δ
6.75 (1H, td, J06.0, 1.0 Hz, H-12), 4.92 (1H, d, J06.0 Hz,
H-14), 4.79 (1H, brs, H-17b), 4.57 (1H, brs, H-17a), 4.37
(1H, dd, J010.0, 6.0 Hz, H-15b), 4.07 (1H, dd, J010.0,
2.0 Hz, H-15a), 4.02 (1H, d, J011.0 Hz, H-19b), 3.27 (1H,
d, J011.0 Hz, H-19a), 3.23−3.17 (1H, m), 2.58−2.43 (2H,
m), 2.33 (1H, dt, J07.0, 2.0 Hz), 1.98−1.64 (5H, m), 1.35−
1.15 (4H, m), 1.12 (3H, s, H-18), 0.65 (3H, s, H-20); 13C
NMR (100 MHz, CD3OD): δ 172.72 (C-16), 149.48 (C-12),
148.76 (C-8), 129.74 (C-13), 109.24 (C-17), 80.92 (C-3),
76.17 (C-15), 66.64 (C-14), 64.96 (C-19), 57.38 (C-9),
56.32 (C-5), 43.68 (C-4), 39.96 (C-10), 38.96 (C-7), 38.13
(C-1), 29.00 (C-2), 25.73 (C-6), 25.20 (C-11), 23.37 (C-18),
15.54 (C-20).
Materials and methods
Reagents
MEM medium, antibiotic-antimicotic and trypsin were pur-
chased from Gibco Invitrogen Co (Scotland, UK). Protein-
ase K and APO-BrdU™ TUNEL Assay Kit were purchased
from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum
was purchased from Thermoscientific (CramLington, UK).
Etoposide, sulforhodamine B (SRB) dye were purchased
from Sigma Aldrich (St. Louis, MO, USA). All other chem-
icals were of analytical grade and commercially obtained.
19-TBDPS-andrographolide (1A)
Andrographolide (1) (10.0 mg, 0.0285 mmol) in pyridine
(200 μL) was stirred and tert-butyldiphenylsilyl chloride
(TBDPSCl) (100 μL, 0.391 mmol) was added at room
temperature with continuous stirring for 1 h. Then, the
reaction mixture was diluted with EtOAc (20 mL) and
quenched with H2O (20 mL), followed by extraction with
EtOAc (3×10 mL). The combined organic layer was
washed with brine (10 mL), dried over sodium sulfate, and
then concentrated under reduced pressure. The residue was
purified by column chromatography (50 % EtOAc/n-hex-
ane) to give 19-TBDPS-Andrographolide (1A) in quantita-
tive yield (16.8 mg) as a white solid, Rf 0.40 (50 % EtOAc/
n-hexane). Mp: 139–143 °C; IR (Neat): 3412, 2933, 1759,
1674, 1428, 1050, 703 cm−1;1H NMR (400 MHz, CDCl3): δ
7.68−7.62 (4H, m, PhH), 7.48−7.38 (6H, m, PhH), 6.91
Isolation of andrographolide and semi-synthesis
of andrographolide analogues
Andrographolide (1) was isolated from dried aerial part and
root of Andrographis paniculata plant, which were har-
vested from Pak Tho district, Ratchaburi province, Thailand
(September, 2008). The dried raw material was stored at
room temperature for 110 days and was extracted with
methanol at room temperature for 4 days. The concentrated
extract was first fractionated between hexane and water.
Then, the water layer was re-extracted with ethyl acetate

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Invest New Drugs (2013) 31:320–332
Fig. 1 Three groups of andrographolide analogues. Reagents and condi-
tions: a R3SiCl, pyridine for 1A and 1B; b i) TBSCl, pyridine, ii) Ac2O for
1C; c i) 2,2-dimethoxy propane, PPTS, acetone, ii) PDC, CH2Cl2, iii)
AcOH/H2O (7:3); d R3SiCl, pyridine for 2A and 2B; e i) TBSCl, pyridine,
ii) Ac2O for 2C; f mCPBA, CH2Cl2/MeOH; g TBDPSCl, pyridine for
3A.1; h i) TBDPSCI, pyridine, ii) Ac2O, reflux for 3A.2 and 3A.3
(1H, td, J06.0, 1.0 Hz, H-12), 4.98 (1H, brd, J06.0 Hz, H-
14), 4.78 (1H, brs, H-17b), 4.51 (1H, brs, H-17a), 4.42 (1H,
dd, J010.5, 6.0 Hz, H-15b), 4.22 (1H, dd, J010.5, 2.0 Hz,
H-15a), 4.17 (1H, d, J010.0 Hz, H-19b), 3.37 (1H, d, J0
10.0 Hz, H-19a), 3.35 (1H, dd, J012.0, 4.0 Hz, H-3), 2.52
(1H, ddd, J016.0, 7.5, 3.0 Hz, H-11b), 2.43 (1H, ddd, J0
16.0, 11.0, 6.5 Hz, H-11a), 2.29 (1H, ddd, J012.0, 3.5,
2.0 Hz, H-9), 1.94−1.57 (5 H, m), 1.31 (3H, s, H-18),
1.28−1.12 (4H, m), 1.04 (9H, s, SiC(CH3)3), 0.45 (3H, s,
H-20); 13C NMR (100 MHz, CDCl3): δ 169.97 (C-16),
148.62 (C-12), 146.47 (C-8), 135.68 (Ph), 135.53 (Ph),
132.38 (Ph), 131.92 (Ph), 130.03 (Ph), 130.00 (Ph),
128.03 (C-13), 127.87 (Ph), 127.84 (Ph), 108.60 (C-17),
80.30 (C-3), 74.30 (C-15), 66.08 (C-14), 65.91 (C-19),
56.02 (C-9), 55.27 (C-5), 42.78 (C-4), 38.86 (C-10), 37.65
(C-7), 37.09 (C-1), 28.49 (C-2), 26.79 (SiC(CH3)3), 24.55
(C-11), 23.62 (C-6), 23.11 (C-18), 19.05 (SiC(CH3)3), 15.22
(C-20); HRMS (ESI) m/z calcd for C36H48O5SiNa [M+Na]+
611.3169, found 611.3162.
19-TIPS-andrographolide (1B)
Andrographolide (1) (81.1 mg, 0.231 mmol) in pyridine
(500 μL) was stirred and triisopropyl chloride (TIPSCl)
(250 μL, 1.48 mmol) was added at room temperature with
continuous stirring for 4 h. Then, the reaction mixture was
diluted with EtOAc (20 mL), quenched with H2O (20 mL),
and extracted with EtOAc (3×20 mL). The combined or-
ganic layer was washed with brine (10 mL), dried over
sodium sulfate, and then concentrated under reduced pres-
sure. The residue was purified by column chromatography
(40 % EtOAc/n-hexane) to give 19-TIPS-Andrographolide
(1B) in 65 % yield (75.9 mg) as a white solid, Rf 0.49 (40 %
EtOAc/n-hexane). Mp: 62–65 °C; IR (Neat): 3377, 2941,

Invest New Drugs (2013) 31:320–332
323
1745, 1674, 1460, 1054, 755 cm−1; H NMR (400 MHz,
CDCl3): δ 6.97 (1H, td, J07.0, 2.0 Hz, H-12), 5.04 (1H, brd,
J06.0 Hz, H-14), 4.89 (1H, brs, H-17b), 4.59 (1H, brs, H-
17a), 4.52 (1H, d, J07.0 Hz, OH), 4.46 (1H, dd, J010.5,
6.0 Hz, H-15b), 4.31 (1H, d, J010.0 Hz, H-19b), 4.26 (1H,
dd, J010.5, 2.0 Hz, H-15a), 3.49 (1H, d, J010.0 Hz, H-
19a), 3.32−3.29 (1H, m, H-3), 2.62−2.38 (3H, m), 2.07
(1H, d, J07.0 Hz, OH), 2.02−1.65 (5H, m), 1.29 (3H, s, H-
18), 1.30−1.20 (4H, m), 1.06 (18H, s, 3 x (SiCH(CH3)2)),
1.06 (3H, s, 3 x (SiCH(CH3)2)), 0.69 (3H, s, H-20); 13C
NMR (100 MHz, CDCl3): δ 169.97 (C-16), 148.67 (C-8),
146.58 (C-12), 128.10 (C-13), 108.73 (C-17), 80.26 (C-3),
74.34 (C-15), 66.15 (C-14), 65.72 (C-19), 56.14 (C-9),
55.23 (C-5), 42.73 (C-4), 38.97 (C-10), 37.78 (C-7), 37.15
(C-1), 28.64 (C-2), 24.70 (C-11), 23.85 (C-6), 23.01 (C-18),
17.96 (3 x (SiCH(CH3)2)), 15.53 (C-20), 11.66 (3 x (SiCH
(CH3)2)); HRMS (ESI) m/z calcd for C29H50O5SiNa [M+
Na]+ 529.3325, found 529.3325.
1.24 (4H, m), 0.94 (3H, s, H-18), 0.89 (9H, s, SiC(CH3)3),
0.81 (3H, s, H-20), 0.05 (6H, s, Si(CH3)2); 13C-NMR
(100 MHz, CDCl3): δ 170.76 (COCH3), 170.51 (COCH3),
169.08 (C-16), 150.65 (C-12), 147.31 (C-8), 123.83 (C-13),
108.36 (C-17), 80.12 (C-3), 71.59 (C-15), 67.85 (C-14),
63.73 (C-19), 56.11 (C-9), 55.57 (C-5), 42.47 (C-4), 39.05
(C-10), 38.27 (C-7), 37.40 (C-1), 29.70 (C-2), 25.89 (SiC
(CH3)3), 25.32 (C-11), 24.40 (C-6), 23.30 (C-18), 21.27
(COCH3), 20.73 (COCH3), 18.25 (SiC(CH3)3), 14.37 (C-
20), -5.90 (Si(CH3)2); HRMS (ESI) m/z calcd for
C30H48O7SiNa [M+Na]+ 571.3067, found 571.3065.
1
12-hydroxy-14-dehydroandrographolide (2)
Andrographolide (1) (400 mg, 1.14 mmol) in acetone
(45.0 mL) was stirred and 2,2-dimethoxypropane
(1.26 mL, 10.3 mmol) was added, followed by addition
of pyridinium p-toluenesulfonate (PPTS) (14.3 mg,
0.0571 mmol). The reaction mixture was stirred at room
temperature for 2 h. Then, the mixture was diluted with
EtOAc (50 mL), quenched with saturated NaHCO3,
washed with H2O, and extracted with EtOAc (3×
50 mL). The combined organic layer was washed with
brine (10 mL), dried over sodium sulfate, and then
concentrated under reduced pressure. The residue was
purified by column chromatography (30 % EtOAc/n-
hexane) to give 3,19-isopropylideneandrographolide in
97 % yield as a white solid.
To a stirred solution of 3,19-isopropylideneandrogra-
pholide (71.8 mg, 0.20 mmol) in CH2Cl2 (3.0 mL),
pyridinium dichromate (PDC) (7.5 mg, 0.02 mmol)
was added. The reaction mixture was continuously
stirred at room temperature for 6 h, then was diluted
with EtOAc (20 mL) and quenched with saturated
NaHCO3, washed with H2O, and extracted with EtOAc
(3×50 mL). The combined organic layer was washed
with brine (10 mL), dried over sodium sulfate, and then
concentrated under reduced pressure. The residue was
purified by column chromatography (100 % EtOAc) to
give 12-hydroxy-14-hydro-3,19-isopropylidene- androg-
rapholide in 84 % yield as a white solid.
12-hydroxy-14-dehydro-3,19-isopropylideneandrogra-
pholide (409 mg, 1.05 mmol) was added into a mixture of
CH3COOH/H2O (7:3) (10 mL). The reaction was stirred at
room temperature for 30 min. After the reaction was com-
pleted, the reaction mixture was diluted with EtOAc
(50 mL) and quenched with saturated NaHCO3, washed
with H2O, and extracted with EtOAc (3×50 mL). The
combined organic layer was washed with brine (20 mL),
dried over sodium sulfate, and then concentrated under
reduced pressure. The residue was purified by column chro-
matography (100 % EtOAc) to give 12-hydroxy-14-dehy-
droandrographolide (2) (313 mg) (?% yield) as a white
19-TBS-3,14-Ac-andrographolide (1C)
Andrographolide (1) (10.0 mg, 0.0285 mmol) in pyridine
(200 μL) was stirred and tert-butyldimethylsilyl chloride
(TBDMSCl) (50.0 mg, 0.332 mmol) was added at room
temperature with continuous stirring for 1 h. Then, the
reaction mixture was diluted with EtOAc (20 mL),
quenched with H2O (20 mL), and extracted with EtOAc
(3×20 mL). The combined organic layer was washed with
brine (10 mL), dried over sodium sulfate, and then concen-
trated under reduced pressure. The residue was purified by
column chromatography (50 % EtOAc/n-hexane) to give
19-TBS-Andrographolide in 92 % yield as a white solid.
To a stirred solution of 19-TBS-Andrographolide (78.9 mg,
0.170 mmol) in acetic anhydride (1.0 mL) was heated to
70 °C. After the stirring was continued at 70 °C for 18 h, the
reaction mixture was diluted with EtOAc (20 mL) and
quenched with saturated NaHCO3, washed with H2O, and
extracted with EtOAc (3×20 mL). The combined organic
layer was washed with brine (10 mL), dried over sodium
sulfate, and then concentrated under reduced pressure. The
residue was purified by column chromatography (30 %
EtOAc/n-hexane) to give 19-TBS-3,14-Ac-Andrographo-
lide (1C) in 44 % yield (40.6 mg) as a white solid, Rf 0.78
(50 % EtOAc/n-hexane). Mp: 97–103 °C; IR (Neat): 3237,
2953, 1760, 1737, 1432, 1248, 751 cm−1 1H NMR
;
(400 MHz, CDCl3): δ 7.02 (1H, td, J07.0, 1.5 Hz, H-12),
5.92 (1H, d, J06.0 Hz, H-14), 4.88 (1H, brs, H-17b), 4.58
(1H, dd, J011.0, 5.5 Hz, H-3), 4.54 (1H, dd, J011.0,
6.0 Hz, H-15b), 4.49 (1H, brs, H-17a), 4.24 (1H, dd, J0
11.0, 1.5 Hz, H-15a), 3.82 (1H, d, J010.5 Hz, H-19b), 3.60
(1H, d, J010.5 Hz, H-19a), 2.48 (1H, ddd, J016.5, 6.5,
3.0 Hz, H-9), 2.44−2.32 (2H, m, H-11), 2.12 (3H, s,
COCH3), 2.05 (3H, s, COCH3), 1.96−1.60 (5H, m), 1.36−

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Invest New Drugs (2013) 31:320–332
solid, Rf 0.49 (100 % EtOAc). Mp: 136–140 °C; IR (Neat):
19-TIPS-12-hydroxy-14-dehydroandrographolide (2B)
1
3351, 2937, 1746, 1448, 1034 cm−1; H NMR (400 MHz,
CDCl3): δ 7.28 (1H, brd, J01.0 Hz, H-14), 4.91 (1H, brs, H-
17b), 4.83 (2H, brs, H-15), 4.73 (1H, brs, H-17a), 4.52 (1H,
brt, J07.0 Hz, H-12), 4.15 (1H, d, J011.0 Hz, H-19b), 3.45
(1H, dd, J010.0, 6.0 Hz, H-3), 3.29 (1H, d, J011.0 Hz, H-
19a), 2.66 (1H, brs, OH), 2.42 (1H, ddd, J014.0, 3.5,
2.5 Hz, H-7b), 2.03 (1H, ddd, J014.0, 9.0, 1.5 Hz), 1.97−
1.75 (6H, m), 1.56 (1H, d, J011.0 Hz), 1.34−1.05 (3H, m),
1.22 (3H, s, H-18), 0.63 (3H, s, H-20); 13C NMR (100 MHz,
CDCl3): δ 172.94 (C-16), 148.22 (C-8), 145.27 (C-14),
136.09 (C-13), 107.86 (C-17), 80.53 (C-3), 70.46 (C-15),
67.61 (C-12), 64.09 (C-19), 55.33 (C-5), 53.25 (C-9), 42.92
(C-4), 39.21 (C-10), 38.21 (C-7), 36.73 (C-1), 30.29 (C-11),
28.20 (C-2), 23.98 (C-6), 22.67 (C-18), 15.42 (C-20);
HRMS (ESI) m/z calcd for C20H30O5Na [M+Na]+
487.2856, found 487.2858.
12-hydroxy-14-dehydroandrographolide (2) (51.0 mg,
0.14 mmol) in pyridine (500 μL) was stirred and triiso-
propyl chloride (TIPSCl) (300 μL, 1.40 mmol) was added
at room temperature with continuous stirring for 8 h. The
reaction mixture was diluted with EtOAc (20 mL) and
quenched with H2O (20 mL), and extracted with EtOAc
(3×20 mL). The combined organic layer was washed with
brine (10 mL), dried over sodium sulfate, and then concen-
trated under reduced pressure. The residue was purified by
column chromatography (40 % EtOAc/n-hexane) to give
19-TIPS-12-hydroxy-14-dehydro andrographolide (2B) in
67 % yield (73.8 mg) as a white solid, Rf 0.47 (60 %
EtOAc/n-hexane). Mp: 95–98 °C; IR (Neat): 3380, 2942,
1
2867, 1750, 1460, 1056, 883 cm-1; H NMR (400 MHz,
CDCl3): δ 7.27 (1H, brs, H-14), 4.90 (1H, brs, H-17b), 4.84
(2H, brs, H-15), 4.73 (1H, brs, H-17a), 4.56–4.49 (1H, m,
H-12), 4.47 (1H, d, J07.0 Hz, OH), 4.29 (1H, d, J010.0 Hz,
H-19b), 3.47 (1H, d, J010.0 Hz, H-19a), 3.30 (1H, ddd, J0
13.0, 7.0, 5.0 Hz, H-3), 2.62 (1H, brd, J05.0 Hz, OH), 2.41
(1H, dm, J013.0 Hz, H-7b), 2.03 (1H, ddd, J014.0, 8.0,
2.0 Hz), 1.96−1.76 (5H, m), 1.74−1.62 (1H, m), 1.57 (1H,
d, J010.0 Hz, H-9), 1.27 (3H, s, H-18), 1.26−1.00 (6H, m),
1.07 (18H, d, J05.0 Hz, 3xSiCH(CH3)), 0.64 (3H, s, H-20);
13C NMR (100 MHz, CDCl3): δ 172.98 (C-16), 148.11 (C-
8), 145.11 (C-14), 136.07 (C-13), 107.72 (C-17), 80.20 (C-
3), 70.44 (C-15), 67.31 (C-12), 65.65 (C-19), 55.32 (C-5),
53.22 (C-9), 42.74 (C-4), 39.23 (C-10), 38.21 (C-7), 36.91
(C-1), 30.27 (C-11), 28.58 (C-2), 24.05 (C-6), 22.95 (C-18),
17.91 (3xSiCH(CH3)2), 15.64 (C-20), 11.63 (3xSiCH
(CH3)2),; HRMS (ESI) m/z calcd for C26H44O5SiNa [M+
Na]+ 487.2856, found 487.2858.
19-Tr-12-hydroxy-14-dehydroandrographolide (2A)
12-hydroxy-14-dehydroandrographolide (2) (41.9 mg,
0.01 mmol) in pyridine (500 μL) was stirred and tert-
butyldiphenylsilyl chloride (TBDPSCl) (150 μL,
0.57 mmol) was added at room temperature with continu-
ous stirring for 1 h. The reaction mixture was diluted with
EtOAc (20 mL) and quenched with H2O (20 mL), and
extracted with EtOAc (3×10 mL). The combined organic
layer was washed with brine (10 mL), dried over sodium
sulfate, and then concentrated under reduced pressure. The
residue was purified by column chromatography (60 %
EtOAc/n-hexane) to give 19-TBDPS-12-hydroxy-14-dehy-
droandrographolide (2A) in 82 % yield (70.4 mg) as a white
solid, Rf 0.51 (50 % EtOAc/n-hexane). Mp: 71–73 °C; IR
(Neat): 2933, 2859, 1753, 1054, 750 cm−1 1H NMR
;
(400 MHz, CDCl3): δ 7.68−7.61 (4H, m, PhH), 7.48−
7.37 (6H, m, PhH), 7.24 (1H, brs, H-14), 4.83 (2H, brs,
H-15), 4.81 (1H, brs, H-17b), 4.65 (1H, brs, H-17a), 4.58
(1H, dd, J012.5, 6.5 Hz, H-12), 4.40 (1H, d, J07.5 Hz,
OH), 4.15 (1H, d, J010.0 Hz, H-19b), 3.33 (1H, d, J0
10.0 Hz, H-19a), 3.36−3.29 (1H, m, H-3), 2.54 (1H, d,
J06.5 Hz, OH), 2.29 (1H, ddd, J013.0, 3.5, 2.5 Hz, H-7b),
2.02−1.71 (5H, m), 1.68−1.59 (2H, m), 1.51 (1H, d, J0
10.0 Hz), 1.30 (3H, s, H-18), 1.18−0.94 (3H, m), 1.04 (9H,
s, SiC(CH3)3 ), 0.39 (3H, s, H-20); 13C NMR (100 MHz,
CDCl3): δ 172.94 (C-16), 148.05 (C-8), 145.07 (C-14),
136.05 (C-13), 135.70 (2xPh), 135.54 (2xPh), 132.38
(Ph), 132.05 (Ph), 129.99 (2xPh), 127.88 (2xPh), 127.81
(2xPh), 107.64 (C-17), 80.29 (C-3), 70.42 (C-15), 67.38
(C-12), 65.89 (C-19), 55.40 (C-5), 53.19 (C-9), 42.83 (C-
4), 39.17 (C-10), 38.12 (C-7), 36.88 (C-1), 30.21 (C-11),
28.49 (C-2), 26.80 (SiC(CH3)3 ), 23.81 (C-6), 23.11 (C-18),
19.07 (SiC(CH3)3), 15.38 (C-20); HRMS (ESI) m/z calcd
for C26H44O5SiNa [M+Na]+ 487.2856, found 487.2858.
19-TBS-3,12-Ac-14-dehydroandrographolide (2C)
12-hydroxy-14-dehydroandrographolide (2) (40.8 mg,
0.12 mmol) in pyridine (500 μL) was stirred and tert-butyl-
dimethylsilyl chloride (TBDMSCl) (91.7 mg, 0.58 mmol)
was added at room temperature with continuous stirring for
1 h. The reaction mixture was diluted with EtOAc (20 mL)
and quenched with H2O (20 mL) and extracted with EtOAc
(3×20 mL). The combined organic layer was washed with
brine (10 mL), dried over sodium sulfate, and then concen-
trated under reduced pressure. The residue was purified by
column chromatography (50 % EtOAc/n-hexane) to give
19-TBS-12-hydroxy-14-dehydroandrographolide in 77 %
yield (43.0 mg) as a white solid.
To a stirred solution of 19-TBS-12-hydroxy-14-dehy-
droandrographolide (20.1 mg, 0.04 mmol) in acetic anhy-
dride (1.0 mL) was heated to 145 °C. Thereafter the stirring
was continued at 145 °C for 1.5 h, the reaction mixture was
diluted with EtOAc (20 mL) and quenched with saturated

Invest New Drugs (2013) 31:320–332
325
NaHCO3, washed with H2O and extracted with EtOAc (3×
20 mL). The combined organic layer was washed with brine
(10 mL), dried over sodium sulfate, and then concentrated
under reduced pressure. The residue was purified by column
chromatography (40 % EtOAc/n-hexane) to give 19-TBS-
3,12-Ac-14-dehydro andrographolide (2C) in 83 %
(22.9 mg) as a white solid, Rf 0.42 (40 % EtOAc/n-hexane).
Mp: 104–107 °C; IR (Neat): 2934, 1756, 1738, 1254, 1028,
752 cm-1; 1H NMR (400 MHz, CDCl3): δ 7.35 (1H, brs, H-
14), 5.66 (1H, dd, J09.5, 4.5 Hz, H-12), 4.90 (1H, brs, H-
17b), 4.83 (2H, brs, H-15), 4.75 (1H, brs, H-17a), 4.56−
4.53 (1H, m, H-3), 3.79 (1H, d, J010.5 Hz, H-19b), 3.57
(1H, d, J010.5 Hz, H-19a), 2.36 (1H, dm, J012.0 Hz, H-
7b), 2.20 (1H, ddd, J013.5, 9.0, 1.0 Hz), 2.06 (3H, s,
COCH3), 2.04 (3H, s, COCH3), 1.94 (1H, J013.5, 11.0,
5.0 Hz), 1.85−1.74 (3H, m), 1.73−1.63 (3H, m), 1.47 (1H,
d, J010.0 Hz, H-9), 1.21−1.11 (2H, m, H-5, H-11a), 0.91
(3H, s, H-18), 0.87 (9H, s, SiC(CH3)3), 0.76 (3H, s, H-20),
0.01 (6H, s, Si(CH3)2); 13C NMR (100 MHz, CDCl3): δ
171.44 (C-16), 170.77 (COCH3), 170.00 (COCH3), 148.26
(C-8), 147.05 (C-14), 132.63 (C-13), 107.56 (C-17), 80.17
(C-3), 70.04 (C-15), 68.31 (C-12), 63.59 (C-19), 55.61 (C-
5), 52.31 (C-9), 42.51 (C-4), 39.25 (C-10), 38.59 (C-7),
37.06 (C-1), 27.11 (C-11), 25.85 (SiC(CH3)3) 25.53 (C-2),
24.34 (C-6), 23.06 (C-18), 21.27 (COCH3), 21.12 (COCH3),
18.20 (SiC(CH3)3), 14.43 (C-20); HRMS (ESI) m/z calcd for
C26H44O5SiNa [M+Na]+ 487.2856, found 487.2858.
s, H-20); 13C NMR (100 MHz, CDCl3): δ 170.20 (C-16),
145.87 (C-12), 129.20 (C-13), 80.13 (C-3), 73.67 (C-15),
65.12 (C-19), 63.83 (C-14), 60.24 (C-8), 54.54 (C-5), 53.36
(C-9), 50.94 (C-17), 42.67 (C-4), 39.73 (C-10), 36.78 (C-1),
35.85 (C-7), 27.09 (C-2), 22.78 (C-11), 22.70 (C-6), 21.26
(C-18), 15.10 (C-20); HRMS (ESI) m/z calcd for
C26H44O5SiNa [M+Na]+ 487.2856, found 487.2858.
19-TBDPS-8,17-epoxy andrographolide (3A.1)
8,17-epoxy andrographolide (3A) (10.0 mg, 0.03 mmol) in
pyridine (500 μL) was stirred and tert-butyldiphenylsilyl
chloride (TBDPSCl) (200 μL, 0.76 mmol) was added at
room temperature. Thereafter, the stirring was continued at
room temperature for 1 h. The reaction mixture was diluted
with EtOAc (20 mL) and quenched with H2O (20 mL), and
extracted with EtOAc (3×10 mL). The combined organic
layer was washed with brine (10 mL), dried over sodium
sulfate, and then concentrated under reduced pressure. The
residue was purified by column chromatography (50 %
EtOAc/n-hexane) to give 19-TBDPS-8,17-epoxy androgra-
pholide (3A.1) in 81 % yield (67.5 mg) as a white solid, Rf
0.36 (50 % EtOAc/n-hexane).Mp: 135–137 °C; IR (Neat):
1
2931, 1750, 1671, 1472, 1275, 1048, 749 cm−1; H NMR
(400 MHz, CDCl3): δ 7.70−7.63 (4H, m, PhH), 7.49−7.38
(6H, m, PhH), 6.76 (1H, ddd, J010.5, 5.5, 1.5 Hz, H-12),
4.94 (1H, brd, J05.5 Hz, H-14), 4.34 (1H, dd, J010.0,
5.5 Hz, H-15b), 4.23 (1H, dd, J010.0, 1.5 Hz, H-15a),
4.15 (1H, d, J010.0 Hz, H-19b), 3.40 (1H, d, J010.0 Hz,
H-19a), 3.40−3.34 (1H, m, H-3), 2.76 (1H, dd, J03.5,
1.5 Hz, H-17b), 2.53 (1H, d, J03.5 Hz, H-17a), 2.02−
1.87 (2H, m), 1.82−1.60 (6H, m), 1.34 (3H, s, H-18),
1.31−1.12 (4H, m), 1.06 (9H, s, SiC(CH3)3), 0.56 (3H, s,
H-20); 13C NMR (100 MHz, CDCl3): δ170.01 (C-16),
145.78 (C-12), 135.69 (2xPh), 135.56 (2xPh), 132.35 (Ph),
131.95 (Ph), 130.05 (Ph), 130.03 (Ph), 129.11 (C-13),
127.90 (2xPh), 127.86 (2xPh), 79.93 (C-3), 73.59 (C-15),
65.57 (C-14), 65.14 (C-19), 60.20 (C-8), 54.65 (C-5), 53.39
(C-9), 50.74 (C-17), 42.66 (C-4), 39.77 (C-10), 36.69 (C-1),
35.86 (C-7), 27.70 (C-2), 26.77 (SiC(CH3)3), 23.08 (C-11),
22.69 (C-6), 21.22 (C-18), 19.05 (SiC(CH3)3), 15.23 (C-
20); HRMS (ESI) m/z calcd for C26H44O5SiNa [M+Na]+
487.2856, found 487.2858.
8,17-epoxy andrographolide (3A)
Andrographolide 1 (301.5 mg, 0.86 mmol) in a mixture of
CH2Cl2/methanol (5:1) (12.0 mL) was stirred and m-chlor-
operoxybenzoic acid (m-CPBA) (297.7 mg, 1.72 mmol) was
added. Thereafter, the stirring was continued at room tem-
perature for 19 h, the solvent was then removed by evapo-
rator. The reaction mixture was diluted with EtOAc (50 mL)
and quenched with saturated NaHCO3, washed with H2O,
and extracted with EtOAc (3×50 mL). The combined or-
ganic layer was washed with brine (10 mL), dried over
sodium sulfate, and then concentrated under reduced pres-
sure. The residue was purified by column chromatography
(100 % EtOAc) to give 8,17-epoxy andrographolide (3A) in
72 % yield (225.5 mg) as a white solid, Rf 0.42 (100 %
EtOAc). Mp: 147–149 °C; IR (Neat): 3282, 2927, 1739,
1673, 1456, 1035 cm-1;1H NMR (400 MHz, CDCl3): δ 6.79
(1H, ddd, J011.0, 5.5, 1.5 Hz, H-12), 5.01−4.96 (1H, m, H-
14), 4.36 (1H, dd, J09.5, 6.0 Hz, H-15b), 4.26 (1H, dd, J0
9.5, 1.5 Hz, H-15a), 4.17 (1H, d, J011.0 Hz, H-19b), 3.52
(1H, dd, J011.0, 4.0 Hz, H-19a), 3.35 (1H, t, J09.0 Hz, H-
3), 2.88 (1H, dd, J03.5, 1.5 Hz, H-17b), 2.80 (1H, brd, J0
8.5 Hz, OH), 2.68 (1H, d, J03.5 Hz, H-17a), 2.42 (1H, brs,
OH), 2.06−1.72 (8H, m), 1.50−1.41 (2H, m), 1.35−1.15
(2H, m), 1.27 (3H, s, H-18), 1.26−1.16 (2H, m), 0.82 (3H,
19-TBDPS-3-Ac-8,17-epoxy andrographolide (3A.2) and 19-
TBDPS-3,12-AC-8,17-epoxy andrographolide (3A.3)
19-TBDPS-8,17-epoxy andrographolide (3A.1) (80.0 mg,
0.13 mmol) in acetic anhydride (1.0 mL) was stirred and
heated to 70 °C. Thereafter, the stirring was continued at
70 °C for 12 h. The reaction mixture was diluted with
EtOAc (20 mL) and quenched with saturated NaHCO3,
washed with H2O and extracted with EtOAc (3×20 mL).

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Invest New Drugs (2013) 31:320–332
The combined organic layer was washed with brine
(10 mL), dried over sodium sulfate, and then concentrated
under reduced pressure. The residue was purified by column
chromatography (30 % EtOAc/n-hexane) to give 19-
TBDPS-3-Ac-8,17-epoxy andrographolide (3A.2) in 11 %
yield (9.70 mg) as a white solid, Rf 0.20 (30 % EtOAc/n-
hexane) and 19-TBDPS-3,14-Ac-8,17-epoxy andrographo-
lide (3A.3) in 77 % yield (70.0 mg) as a white solid, Rf 0.49
(30 % EtOAc/n-hexane).
19-TBDPS-3-Ac-8,17-epoxy andrographolide (3A.2)
Mp: 81–84 °C; IR (Neat): 2935, 1763, 1675, 1466, 1245,
1083, 704 cm−1; 1H NMR (400 MHz, CDCl3): δ 7.70−7.64
(4H, m, PhH), 7.47−7.35 (6H, m, PhH), 6.77 (1H, ddd, J0
10.5, 5.0, 1.5 Hz, H-12), 4.94 (1H, brd, J05.5 Hz, H-14),
4.56 (1H, dd, J012.0, 4.5 Hz, H-3), 4.35 (1H, dd, J010.5,
5.5 Hz, H-15b), 4.26 (1H, dd, J010.5, 1.5 Hz, H-15a), 3.13
(1H, brs, OH), 3.85 (1H, d, J010.0 Hz, H-19b), 3.73 (1H, d,
J010.0 Hz, H-19a), 2.88 (1H, d, J03.0 Hz, H-17b), 2.65
(1H, d, J03.0 Hz, H-17a), 2.05−1.97 (1H, m), 1.91 (3H, s,
COCH3), 1.87−1.48 (7H, m), 1.45−1.19 (4H, m), 1.07 (9H,
s, SiC(CH3)3), 1.05 (3H, s, H-18), 0.79 (3H, s, H-20); 13C
NMR (100 MHz, CDCl3): δ 170.52 (C-16), 169.99
(COCH3), 145.72 (C-12), 135.86 (2xPh), 135.77 (2xPh),
133.47 (Ph), 133.28 (Ph), 129.70 (2xPh), 129.16 (C-13),
127.64 (2xPh), 127.58 (2xPh), 79.62 (C-3), 73.61 (C-15),
65.57 (C-14), 63.85 (C-19), 60.65 (C-8), 54.86 (C-5), 53.38
(C-9), 50.80 (C-17), 42.85 (C-4), 39.98 (C-10), 36.95 (C-1),
36.38 (C-7), 26.95 (SiC(CH3)3), 23.53 (C-2), 22.86 (C-11),
22.79 (C-6, COCH3), 21.09 (C-18), 19.27 (SiC(CH3)3),
14.53 (C-20); HRMS (ESI) m/z calcd for C26H44O5SiNa
[M+Na]+ 487.2856, found 487.2858.
Cell culture and in vitro anti-proliferation assay
Four mammalian cell lines were used; HepG2 (Hepatocel-
lular carcinoma), Hela (Cervical carcinoma), CHO (Chinese
hamster ovary) and UISO-BCA1 (Human breast carcino-
ma). HepG2, Hela, and CHO cells were obtained from the
American Type Culture Collection (ATCC, Manassas,
USA). UISO-BCA1 was a kind gift from Dr. John M
Pezzuto (Department of Phamacognosy and Pharmacy, Col-
lege of Pharmacy, University of Illinois at the Chicago, Illi-
nois, USA). All cell lines were grown in MEM with 10 % fetal
bovine serum (FBS) at 37 °C in a humidified atmosphere
containing 5 % CO2. The final concentration of DMSO for
dissolving the test compounds was less than 0.1 %.
The sulphorhodamine (SRB) assay which is a colorimetric
assay was used for evaluation of cell growth. Total cell num-
ber is indirectly estimated by staining total cellular protein
with the dye SRB [15]. Cells were plated onto 96-well plates
at densities of 4×103 cells/well and were incubated at 37 °C
for 24 h before experimentation. Stock solutions of analogues
were prepared in DMSO and subsequently diluted to the
indicated final concentrations containing DMSO less than
0.1 %. Cells were treated with andrographolide analogues at
different concentrations for 72 h. Each treatment was per-
formed in triplicates. DMSO 0.1 % was used as a vehicle
control. After incubation, cells were fixed in trichloroacetic
acid (TCA), washed and stained with SRB. The bound stain
was solubilized with Tris -base, pH 10.5. The absorbance was
measured at 515 nm. The IC50 values (concentration of the
tested compound that caused 50 % inhibition of cell growth)
were estimated using GraphPad Prism version 5.01(GraphPad
Software Inc, CA, USA).
19-TBDPS-3,12-Ac-8,17-epoxy andrographolide (3A.3)
Mp: 92–94 °C; IR (Neat): 2939, 1734, 1241, 1079, 749 cm-1;
1H NMR (400 MHz, CDCl3): δ 7.71−7.63 (4H, m, PhH),
7.47−7.34 (6H, m, PhH), 7.08 (1H, td, J07.0, 1.0 Hz, H-12),
5.86 (1H, brd, J05.5 Hz, H-14), 4.53 (1H, dd, J011.0, 4.0 Hz,
H-3), 4.50 (1H, dd, J011.5, 5.5 Hz, H-15b), 4.22 (1H, dd, J0
11.5, 1.5 Hz, H-15a), 3.84 (1H, d, J011.0 Hz, H-19b), 3.37
(1H, d, J011.0 Hz, H-19a), 2.60 (1H, d, J04.0 Hz, H-17b),
2.53 (1H, d, J04.0 Hz, H-17a), 2.11 (3H, s, COCH3), 2.15−
1.92 (5H, m), 1.90 (3H, s, COCH3), 1.84−1.39 (5H, m), 1.31
−1.17 (2H, m), 1.06 (9H, s, SiC(CH3)3), 1.04 (3H, s, H-18),
0.80 (3H, s, H-20); 13C NMR (100 MHz, CDCl3): δ 170.61
(COCH3), 170.51 (COCH3), 169.01 (C-16), 150.92 (C-12),
135.82 (2xPh), 135.74 (2xPh), 133.44 (Ph), 133.39 (Ph),
129.67 (Ph), 129.64 (Ph), 127.62 (2xPh), 127.59 (2xPh),
123.19 (C-13), 79.63 (C-3), 71.57 (C-15), 67.57 (C-14),
63.83 (C-19), 58.20 (C-8), 54.99 (C-5), 54.13 (C-5), 49.55
(C-17), 42.82 (C-4), 39.98 (C-10), 37.57 (C-1), 36.18 (C-7),
26.92 (SiC(CH3)3), 23.58 (C-2), 23.01 (C-11), 22.81 (C-6,
COCH3), 21.06 (COCH3), 20.86 (C-18), 19.25 (SiC(CH3)3),
14.66 (C-20); HRMS (ESI) m/z calcd for C26H44O5SiNa [M+
Na]+ 487.2856, found 487.2858.
Topo II α mediated supercoiled pBR322 DNA relaxation
assay
The activity of human DNA topoisomerase II α (p170 form)
(hTopoII α) (TopoGEN, Florida, USA) on the relaxation of
supercoiled plasmid DNA pBR322 (Fermentas, Maryland,
USA) was determined by measuring the conversion of super-
coiled pBR322 plasmid to relaxed form [16]. The reaction
mixture contained 4 μL of Buffer A (0.5 M Tris-HCl (pH 8.0);
1.5 M NaCl; 0.1 M MgCl2; 5 mM dithiothreitol (DTT) and
300 μg/mL BSA), 4 μL of Buffer B (20 mM ATP), 0.5 μg
plasmid pBR322, and 1U topoisomerase II α enzyme. To
evaluate the inhibitory action of the andrographolide ana-
logues, the compounds were dissolved in DMSO at different
concentrations in a total volume 20 μL (final DMSO concen-
tration was less than 0.1 %). The reaction mixture was incu-
bated for 30 min at 37 °C, and terminated by adding 0.1 %
SDS and 1 μL of 1 mg/mL proteinase K. The mixture was
further incubated at 50 °C for 30 min. The products were
subjected to electrophoresis using 1 % agarose gel

Invest New Drugs (2013) 31:320–332
327
electrophoresis at 1.5 V/cm in TBE (Tris Borate EDTA)
buffer. The gel was stained with 0.5 μg/mL ethidium bromide
for 30 min and destained in distilled water for 30 min. The gel
was visualized and quantified by Gel documentation (Chem-
Genius, Syngene, UK). Inhibition of hTopo II α was estimated
from the density of supercoiled plasmid in agarose gel.
incubation, the slides were gently washed with phosphate
buffer saline (PBS) and stained with DAPI for 20 min at
37 °C. Cell morphologies were examined under a fluorescent
microscope (HB-10101AF, Nikon, Japan) at magnification of
400X, and photographed. The apoptotic cells were character-
ized by nuclear condensation and fragmentation.
DNA fragmentation, a hallmark of apoptosis, was detected
by using APO-BrdU™ TUNEL assay kit (Invitrogen, Carls-
bad, CA, USA) following the manufacturer’s instruction.
Briefly, the HepG2 cells, plated in a 6-well plate (5.0×105
cells/well), were incubated at 37 °C for 24 h. Cells were then
treated with andrographolide analogues for 24 and 48 h. After
incubation, cells were trypsinized, re-suspended and incubated
Detection of apoptosis
The apoptotic cell death in HepG2 cells was first investigated
by examining their morphological alterations. HepG2 cells
(3×104 cells/mL) were seeded on sterile slides and exposed
to andrographolide analogues for the indicated period. After
Fig. 2 a. Inhibitory effect of andrographolide analogue 2C on hTopo
II α activity by the pBR322 DNA cleavage assay in cell free system. In
lane C, DMSO (1 %) was used as a vehicle control. In lane D, Etopo-
side, a hTopo II inhibitor, at 100 μM was used as a positive control.
Lanes E, F, G were andrographolide analogue 2C at various concen-
trations (0.1, 1, and 25 μM). b. Inhibitory effects of andrographolide
analogues 3A.1, 3A.2, 3A.3 and 1B at 0.1, 1 and 25 μM on hTopo II α
activity in cell free system

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Invest New Drugs (2013) 31:320–332
Table 1 Inhibition of human topoisomerase II α activity by Androg-
rapholide Analogues
in 1 % paraformaldehyde on ice. The incubated cells were
fixed in 70 % cold ethanol overnight. The fixed cells were
incubated with DNA labeling solution (0.75 μL of Terminal-
deoxynucleotidyl Transferase (TdT) enzyme in 10 μL TdT
reaction buffer, 8 μL of BrdUTP, and 31.25 μl of H2O) for
120 min at 37 °C. At the end of incubation, cells were re-
suspended in antibody solution (5 μl of the Alexa Fluor® 488
dye-labeled anti-BrdU antibody and 95 μl of rinse buffer) and
incubated for 30 min at room temperature. Propidium iodide/
RNaseA mixture was added into cell suspension and further
incubated for 30 min at room temperature. Solution was mixed
gently every 5 min in dark. Twenty thousand events in all
treatments sets were analyzed using BD FACS Canto TM flow
cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Compounds
R1
H
R2
H
R3
H
Topoisomerase II
α Inhibition (%)
Androrapholide
None
Analogues
1A
H
H
TBDPS
TBDPS
TBDPS
TBDPS
TBDPS
TIPS
None
2A
H
H
18.2 0.10
100.0 0.00
100.0 0.00
100.0 0.00
100.0 0.00
67.5 0.10
None
3A.1
3A.2
3A.3
1B
H
H
H
Ac
Ac
H
Ac
H
2B
H
H
TIPS
1C
Ac
Ac
Ac
Ac
TBS
2C
TBS
100.0 0.00
100.0 0.00
Statistical analysis
Etoposide
Data were presented as means+SEM. Each value was
obtained from at least three independent experiments.
Data are means SEM percent inhibition by andrographolide analogues at
100 μM on the activity of recombinant human topoisomerase II α. Etopo-
side was used as positive control and andrographolide for a negative
control. TBDPS: tert-butyldiphenylsilyl, TIPS: triisopropylsilyl, TBS:
tert-butyldimethylsilyl. Percent inhibition presented in μM was obtained
from 3 independent experiments
Results
Synthesis of semi-synthetic andrographolide analogues
To determine the potency of the active andrographolide
analogues, percent inhibition of hTopo II α was tested at
0.1, 1 and 25 μM of various analogues. As shown in Table 2,
analogues 3A.1, 3A.2, 3A.3 and 2C strongly inhibited the
topoisomerase II α activity at concentration of 1 μM.
Figure 1 shows the semi-synthetic andrographolide ana-
logues used in the experiment. The analogues were obtained
after the modifications of both the core structure and func-
tional groups of andrographolide. The analogue structure-1
contains the core structure of andrographolide. In the
structure-2, the core structure was modified to contain dou-
ble bond at C-14 and hydroxyl group (-OH) at C-12 whereas
the structure 3 was modified to contain epoxide at C-17
position. At C-19 position, all analogues contained one of
the following silicon based molecules including triisopro-
pylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), or tert-
butyldimethylsilyl (TBS).
Anti-proliferative effect of andrographolide analogues
The analogues with potent topoisomerase II α inhibitory
activity including analogues 3A.1, 3A.2, 3A.3, 1B, and 2C
were further investigated for their anti-proliferative activities
in four mammalian cell lines (HepG2, Hela, CHO and UISO-
BCA-1) using SRB assay. IC50 values of these analogues in
four cell lines were estimated to be 4 to 7 μM (Table 3).
Inhibition of recombinant human topoisomerase II α
activity by andrographolide analogues
Table 2 Inhibition of human topoisomerase II α activity by androg-
rapholide analogues
The ability of andrographolide analogues to inhibit recom-
binant human topoisomerase II α activity was evaluated by
measuring the extent of conversions of supercoiled pBR322
DNA plasmid to relaxed form (Fig. 2, Table 1). Androgra-
pholide, the parent compound did not affect topoisomerase
II α enzyme activity. Etoposide, a topoisomerase poison,
was used as a positive control since, at 100 μM, it stabilizes
the DNA cleavable complex as well as completely inhibits
recombinant human topoisomerase II α activity. As shown
in Fig. 2 and Table 1, andrographolide analogues 3A.1,
3A.2, 3A.3, 1B and 2C at concentration of 100 μM fully
inhibited activities of topoisomerase II α enzyme.
Compounds
Percent Inhibition
0.1 μM
1 μM
25 μM
3A.1
3A.2
3A.3
1B
40.11 0.02
37.11 0.02
37.08 0.02
15.4 0.03
17.11 0.01
100 0.00
100 0.00
100 0.00
15.6 0.02
100 0.00
100 0.00
100 0.00
100 0.00
100 0.00
100 0.00
2C
Data are means SEM percent inhibition by andrographolide analogues
at 0.1 μM, 1 μM and 25 μM. Percent inhibition presented in μM
obtained from 3 independent experiments

Invest New Drugs (2013) 31:320–332
Table 3 Cytotoxicity of
Andrographolide analogues
(IC50 values) in various mam-
malian cancer cell lines
329
Compounds
IC50 (μM)
HepG2
Hela
0.92 0.06
CHO
UISO-BCA1
Etoposide
2.61 0.28
45.65 1.30
5.95 0.19
5.53 0.05
5.50 0.04
5.79 0.01
5.57 0.04
46.77 1.46
100 0.36
6.30 0.06
6.14 0.01
6.00 0.03
5.86 0.08
6.03 0.01
6.17 0.46
27.46 0.25
4.91 0.04
4.49 0.05
4.44 0.01
5.38 0.08
5.24 0.03
Andrographolide
24.62 0.03
4.74 0.06
4.94 0.04
4.85 0.04
5.15 0.10
5.06 0.13
3A.1
3A.2
3A.3
1B
IC50 value presented in μM was
obtained from 3 independent
experiments which were con-
ducted in triplicates. Data show
means SEM
2C
Interestingly, IC50 values of all these five andrographolide
analogues on CHO cells were approximately 6 μM, which
was much lower than that of etoposide (46 μM).
induced apoptosis in HepG2 cells. This is the first report that
andrographolide analogues inhibit recombinant human top-
oisomerase II α enzymes.
Topoisomerase inhibitors have been suggested as inter-
esting anti-cancer drugs [1, 4]. In this study, we showed that
andrographolide analogues 3A.1, 3A.2, 3A.3, 1B and 2C at
100 μM concentration effectively inhibited the activity of
topoisomerase II α. Of note, these analogues are relatively
more potent than etoposide, a specific topoisomerase II α
inhibitor [3]. To our knowledge, there has been no report on
the inhibitory action of andrographolide (diterpenoid lac-
tone) as well as andrographolide analogues on topoisomerse
IIα enzymes. In 2006, Miyata and coworkers [17] reported
inhibitory action of twelve diterpene compounds derived
from the roots of Euphorbia kansui on topoisomerase II
enzymes. However, the structure of diterpene is different
from diterpenoid lactone of andrographolide. In the present
study, our analogues were obtained from the modifications
on both the core structure and functional groups of androg-
rapholide. The analogue structure-1 contains the core struc-
ture of andrographolide whereas the analogue structure-2
possesses double bond at C-14 and hydroxyl group (-OH)
at C-12. On the other hand, the double bond at C-17 of the
core structure was changed to epoxide in the analogue
structure-3. At C-19 position, all analogues contained one
of the following silicon based molecules including triiso-
propylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and
Apoptosis induced by andrographolide analogues
Andrographolide analogue 2C, which was one of the highest
inhibitor on hTopo II α, was further investigated for its ability
to induce cell death in HepG2 cells by using DAPI staining for
DNA. As illustrated in Fig. 3, the analogue 2C effectively
induced DNA fragmentation and condensation at 24 h. Induc-
tion of apoptosis in HepG2 cells was further confirmed by
using flow-cytometric APO-BrdU™ TUNEL assay, which
detected the fragmentation of DNA. Analog 2C (10 μM)
markedly induced apoptosis of HepG2 cells by 24 h (Fig. 4).
Percent cell death was approximately 78+0.02 %, which was
much higher (about 15 folds) than those induced by the parent
andrographolide and the positive control etoposide.
Discussion
The present study demonstrated that five out of nine androg-
rapholide analogues exhibited potent inhibitory activities on
topoisomerase II α in vitro. The active analogues were also
cytotoxic to a number of mammalian cell lines including Hela,
CHO, BCA1 and HepG2. In addition, these compounds
Fig. 3 Effect of andrographolide
analogue 2C on condensation
and fragmentation of DNA in
HepG2 cells which were treated
with a vehicle (0.1 % DMSO)
and b andrographolide analogue
2C (10 μM) for 24 h. Apoptotic
cells were stained with DAPI and
seen under fluorescence micros-
copy at magnification 400X. The
arrow (→) represents apoptosis
cells with nuclear condensation
as well as fragmentation of DNA

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Invest New Drugs (2013) 31:320–332
Fig. 4 FACS analysis showing
apoptosis in HepG2 cells
induced by andrographolide
analogues. Andrographolide
analogue 2C at 10 μM induced
apoptotic cell death after
treatment for 24 h, detected by
TUNEL assay. Detection of
apoptotic nuclei was done by
using Flow-cytometric APO-
BrdU™ TUNEL assay kit in
20,000 events. a: 0.1 % DMSO,
b: 10 μM etoposide, c: 10 μM
andrographolide and d: 10 μM
andrographolide analogue 2C
tert-butyldimethylsilyl (TBS). The silicon base compounds
have been widely used in synthetic organic chemistry to
protect hydroxyl group. The inhibition of hTopo II α activ-
ity by our andrographolide analogues depends on both core
structure and functional group at C-19 position. The pres-
ence of TBDPS group at C-19 on the compounds having
different core structures (1A, 2A, 3A.1) caused different
inhibitory activity on hTopo II α. The effect of core struc-
ture was also observed in the analogues 1C and 2C with the
same TBS group at C-19. Thus, analogue 1C at 100 μM had
no inhibitory activity whereas analogue 2C completely
inhibited hTopo II α activity. Among the three core struc-
tures, the core structure 3 seems to be the most active
moiety. Addition of acetyl group on the core structure
(3A.2 and 3A.3) did not alter the inhibitory activity. These
results indicate that the core structure of andrographolide in
the analogues is essential for the inhibitory activity on
hTopo II α. However, when the functional group of androg-
rapholide at C-19 was changed to TIPS, the compounds
with core structure 1 (1B) and core structure 2 (2B) showed
increases in the inhibitory activities. The non-active ana-
logue 1A became fully active when its functional group at
C-19 was replaced by TIPS, i.e. analogue 1B. Likewise, the
activity of 2A was substantially increased in 2B. When the
activities among the compounds with core structure 1 were
compared, only that containing TIPS (1B) inhibited the
activity of hTopo II α. For the compounds with structure 2
(2A, 2B and 2C), all analogues inhibited hTopo II α enzyme
at varying degree. Of these, analogue 2C containing TBS
gave the highest inhibitory potency compared to 2A and 2B.
These results indicated the importance of functional group at
C-19 position of andrographolide analogues in providing
the inhibitory activity against hTopo II α.
Topoisomerase II inhibitors have been used for treatment of
a variety of cancers including lung cancer, leukemia, sarcoma,
breast cancer and ovarian cancer [3]. In the present study, the
anti-cancer potential of the andrographolide analogues were
also observed. These compounds effectively inhibited prolifer-
ation of HepG2, Hela and CHO cell lines (Table 3), which have
previously been reported to response to topoisomerase II inhib-
itors [12, 13, 19, 20]. Andrographolide analogues containing
one of silicon based molecules (TIPS, TBDPS and TBS) at C-
19 position in all 3 structures possess an anti-proliferative
activity. The inhibition concentration 50 (IC50) of the active
andrographolide analogues ranged from 4–7 μM, which were
much lower than that of the parent andrographolide, suggesting
that the functional group at C-19 position is essential for the
anti-proliferative properties on four mammalian cell lines. Re-
cently, our groups have reported the importance of functional
group containing silicon at C-19 position of the andrographolide
analogue structure 1 in the anti-proliferation effect on a panel of
cancer cells [9]. By using silicon, the increases in lipophilicity
and stability of the compounds have been reported [21].
Etoposide is widely used for treatment of cancers by
inhibiting hTopo II α activity [3]. However, there are a
number of cancers that resist to etoposide treatment. In the
present study, CHO was used since it is not sensitive to
etoposide [18] and the parent form of andrographolide.

Invest New Drugs (2013) 31:320–332
331
under the National Research universities (NRU). We are grateful to Prof.
Chumpol Pholpramool for his critical reading on the manuscript.
Interestingly, this cell line is sensitive to our andrographo-
lide analogues. The mechanism involved in the resistance to
anticancer drugs may, at least, be related to drug transport,
drug-target interaction and drug detoxification [22]. It is pos-
sible that our analogues may have different inhibitory mech-
anism from that of etoposide. Currently, several etoposide
analogues have been developed by modifying its structure to
make a prodrug to improve its inhibitory activity in the resis-
tant cells [23]. For the cells that are resistant to etoposide, our
modified analogues may be useful as a prodrug.
Conflict of interest None.
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induction has been observed in HepG2 cells treated with
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In conclusion, we have semi-synthesized a new family of
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activity toward recombinant human topoisomerase II α,
caused cytotoxicity in various types of mammalian cell lines,
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Acknowledgements This research project is supported by Mahidol
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ogy, Science & Technology Postgraduate Education and Research De-
velopment Office (PERDO), Ministry of Education, Thailand, and the
Office of the Higher Education Commission and Mahidol University

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