New substituted C-19-andrographolide analogues with potent cytotoxic activities

Article abstract of DOI:10.1016/j.bmcl.2011.11.085

Andrographolide, the major diterpenoid lactone from Andrographis paniculata, is toxic against cancer cells. In the present study, we investigated the structure-activity relationships (SARs) of 19 andrographolide analogues which were synthesized by modific

Full text of DOI:10.1016/j.bmcl.2011.11.085

Bioorganic & Medicinal Chemistry Letters 22 (2012) 49–52
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New substituted C-19-andrographolide analogues with potent
cytotoxic activities
Uthaiwan Sirion a, Sakkasem Kasemsook a, Kanoknetr Suksen b, Pawinee Piyachaturawat b,
Apichart Suksamrarn c, Rungnapha Saeeng a,
⇑
a Department of Chemistry and Center for Innovation in Chemistry, Faculty of Science, Burapha University, Chonburi 20131, Thailand
b Department of Physiology, Mahidol University, Bangkok 10400, Thailand
c Department of Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Andrographolide, the major diterpenoid lactone from Andrographis paniculata, is toxic against cancer
cells. In the present study, we investigated the structure–activity relationships (SARs) of 19 andrographo-
lide analogues which were synthesized by modification at the three hydroxyl groups. A number of the
andrographolide analogues showed much higher cytotoxic activities than that of the parent compound
on cancer cells including P-388, KB, COL-2, MCF-7, LU-1 and ASK cells. SAR studies of the synthetic ana-
logues indicated that the introduction of silyl ether or triphenylmethyl ether group into C-19 of the par-
ent compound led to increase in toxicity against the cancer cells. The 19-O-triphenylmethyl ether
analogue 18 showed higher cytotoxic activity than the potent anticancer drug ellipticine, and this
analogue may serve as a potential structure lead for the development of new anticancer drugs.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 15 August 2011
Revised 28 October 2011
Accepted 21 November 2011
Available online 28 November 2011
Keywords:
Andrographolide
Structure modification
Cytotoxicity
19-O-Triphenylmethylandrographolide
Andrographolide (1), the major labdane diterpene was isolated
in a large quantity (2%) from the methanol extract of the aerial
parts of Andrographis paniculata.1 This natural substance has ther-
apeutic potential for a wide range of diseases.2 Its chemically mod-
In our design of the synthetic andrographolide analogues, the core
structural scaffold of 1 was kept intact to maintain the key biolog-
ical activities.2k Indeed, the double bond between C-12 and C-13
was reported to be crucial for cytotoxicity,6,7 possibly is associated
with their ability to promote alkylation of biological nucleophiles
such as enzyme through Michael addition to the exo-alkene of
ified analogues are also reported to be effective as TNF-
a
, IL-63 and
a
-glucosidase inhibitor4 as well as antibacterial2a,5 and cytotoxic6,7
agents. However, among the modified andrographolide analogues
that have been synthesized so far, only a few cases of the possible
analogues that could hold therapeutic potential over the parent
compound. Therefore, simple synthetic structures with strong bio-
activity as potential drug by convenient modification method will
be much more attractive in pharmaceutical industry.
Herein, we report the synthesis of a series of andrographolide
analogues derived from the natural substrate 1. Since compound
1 bears cytotoxic activity2k,m and is available from nature, the syn-
thesis of analogues from this compound could be of a great value in
discovering potential anticancer semisynthetic drug.
a-alkylidene-v
-butyrolactone moiety.6 Our strategy was to keep
this functional group and conversions of the three hydroxyl groups
in 1 to a series of acetyl, silyl and triphenylmethyl ether groups at
the C-19 position, and acetyl groups at the C-3 and C-14 positions,
have been undertaken.8
The first series of silyl-andrographolide analogues were prepared
as shown in Scheme 1. Compounds 2, 6 and 8 were synthesized by
silylation at C-19 position in basic condition using pyridine to obtain
tert-butyldimethylsilyl-(TBS), triisopropylsilyl-(TIPS) and tert-
butyldiphenylsilyl-(TBDPS) ether derivatives, respectively, in mod-
erate to high yields. Acetylation of the resulting silyl ether at C-3
and C-14 in the presence of acetic anhydride at 70 °C afforded the
mono- and di-acetylsilane products 3–5, 7 and 9–11.
Compounds 13–16 were obtained from the reaction of 1 in 22%,
46%, 7% and 18% yields (total 93%), respectively, after heating in the
excess acetic anhydride at 70 °C for 5 h (Scheme 2). In order to af-
ford the mono-acetyl derivative 12, compound 3 was employed as
a starting material and tert-butyldimethylsilyl ether group at C-19
was de-protected using formic acid/water in THF. Compound 17
was obtained in moderate yield under the same reaction condition
from 5.
O
14
O
HO
13
12
3
HO
HO
1
19
⇑
Corresponding author.
E-mail address: rungnaph@buu.ac.th (R. Saeeng).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.085

50
U. Sirion et al. / Bioorg. Med. Chem. Lett. 22 (2012) 49–52
O
O
O
O
O
O
O
O
R1O
HO
HO
HO
b
a
a
b
R2O
TBSO
HO
TBSO
AcO
TBSO
AcO
HO
1
: R = H, R2 = Ac
2
3
4
5
3
12
1
: R = Ac, R2 = H
O
O
1
: R = Ac, R2 = Ac
O
O
O
R1O
HO
O
O
O
c
: R = H, R2 = H
1
HO
13
O
O
: R = H, R2 = Ac
1
HO
14
15
16
c
AcO
d
: R = Ac, R2 = H
1
: R = Ac, R2 = Ac
1
R2O
AcO
HO
HO
HO
HO
HO
TIPSO
1
O
AcO
TIPSO
O
1
6
7
O
O
AcO
AcO
e
O
d
O
O
O
R1O
HO
AcO
HO
AcO
TBSO
e
f
5
17
R2O
TBDPSO
HO
TBDPSO
Scheme 2. Reagents and conditions: (a) (i) TBSCl, pyridine, rt, 1 h, 88%, (ii) Ac2O,
70 °C, 18 h, 33%; (b) HCOOH/H2O (9:1), THF, 0 °C, 30 min, 51%; (c) Ac2O, 80 °C, 1 h,
total yields of 14, 15 and 16 = 93%; (d) (i) TBSCl, pyridine, rt, 1 h, 88%, (ii) Ac2O,
145 °C, 1 h, 94%; (e) HCOOH/H2O (9:1), THF, 0 °C, 30 min, 48%.
1
: R = H, R2 = Ac
9
8
1
2
10
11
: R = Ac, R = H
1
: R = Ac, R2 = Ac
Scheme 1. Reagents and conditions: (a) TBSCl, pyridine, rt, 1 h, 92%; (b) Ac2O, 70 °C,
18 h, total yields of 3, 4 and 5 = 93%; (c) TIPSCl, pyridine, rt, 4 h, 65%; (d) Ac2O,
140 °C, 3 h, 80%; (e) TBDPSCl, pyridine, rt, 1 h, >99%; (b) Ac2O, 70 °C, 6 h, total yields
of 9, 10 and 11 = 76%.
O
O
O
O
HO
AcO
a
b
To increase structural diversities, the third series of ether ana-
logues were prepared as shown in Scheme 3. An excellent yield
of 19-O-triphenylmethylandrographolide 18 was obtained by heat-
ing the parent compound 1 in the presence of trityl chloride and
pyridine. Subsequent acetylation of the remaining two hydroxyl
groups led to the analogue 19. Finally, the protection of C-3 and
C-19 hydroxyl groups as acetonide yielded compound 20.
The cytotoxic activity of 1 and its synthetic analogues were
evaluated using in vitro sulforhodamine B (SRB) assay against six
cancer cell lines including P-388 (murine leukaemia cell line), KB
(human epidermoid carcinoma of the mouth), COL-2 (human colon
cancer), MCF-7 (human breast cancer), LU-1 (human lung cancer),
and ASK (rat glioma). All tested samples were dissolved in DMSO
(0.1%). Ellipticine was used as a positive control. It is a potent anti-
cancer agent which exhibits several modes of mechanisms against
cancer cells.9 The results were expressed as ED50 values (drug con-
O
O
HO
Ph3CO
AcO
Ph3CO
HO
19
18
O
O
HO
HO
HO
c
1
O
O
20
Scheme 3. Reagents and conditions: (a) TrCl, pyridine, 70 °C, 2.5 h, 99%; (b) Ac2O,
140 °C, 1 h, 33%; (c) 2,2-dimethoxypropane, PPTS, acetone, 97%.
centration causing 50% growth inhibition) in
Table 1.
lM, are shown in
However, changing of the silyl-TBS group at C-19 to other silyl
derivatives such as the more bulky triisopropylsilyl (TIPS) 6 led to
6- to 15-fold increase in cytotoxic activity. Conversion to the
TBDPS 8 resulted in a moderate increase in activity, and this com-
pound showed selectivity against P-388.
These bulky functional groups containing silicon might provide
lipophilicity to the compound, allowing it to pass the cell mem-
brane by passive diffusion. This strategy has been successfully ap-
plied in the antitumor drug analogues silatecans11 (silicon-
containing camptothecins) and silaplatins12 (cisplatin analogues).
Compound 13, containing acetyl group at C-19 position, exhib-
ited similar activity to the parent 1. However, this analogue was
also highly toxic against P-388. Our novel synthetic analogue 18
bearing triphenylmethoxyl group at C-19 was found to be the most
Ten out of 19 synthetic analogues (compounds 3–6, 8–10 and
16–18) displayed cytotoxic activities stronger than that of the
parent compound 1 in all cancer cell lines. Comparison on the cyto-
toxic activities of C-19 mono-substituted analogues 2, 6, 8, 13, and
18 revealed the importance of the protecting group of the hydroxyl
function at C-19 on the andrographolide core.
The introduction of a tert-butyldimethylsilyl (TBS) group on the
tetrahydropyran ring10 has previously been reported to result in a
significant enhancing cytotoxic activity against HL60 (human
leukaemia cells) and MCF7. Evaluation of the cytotoxic activity of
1 and TBS-derivative 2 against a series of cancer cell lines did not
show any enhancement in the present work. The differences in
sensitivity of cell lines may account for the discrepancy of results.
potent analogue against the tested cancer cells (ED50 0.45–2.86 lM)

U. Sirion et al. / Bioorg. Med. Chem. Lett. 22 (2012) 49–52
51
Table 1
Cytotoxic activity against six human cancer cells
O
O
R1O
R2O
R3O
Entry
Compound
ED50
(l
M)a (SRB assay)
KB
R1
R2
R3
P-388
COL-2
MCF-7
LU-1
ASK
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
1
2
3
4
5
6
7
8
H
H
H
Ac
Ac
H
Ac
H
H
H
Ac
H
Ac
H
Ac
H
Ac
H
Ac
Ac
H
Ac
H
H
2.25
8.39
1.71
1.03
0.34
0.88
1.41
0.68
0.50
0.33
1.67
4.78
2.04
2.65
1.62
1.40
0.69
0.45
1.92
27.37
40.71
11.15
10.74
3.62
2.73
7.39
11.13
9.86
10.54
29.04
35.65
15.26
35.22
26.38
11.62
11.66
2.42
13.60
17.10
11.31
8.94
2.23
2.73
13.59
9.65
3.80
10.70
12.28
27.21
12.94
15.26
15.23
10.82
10.16
2.73
15.40
46.96
9.79
10.44
2.84
2.88
13.95
8.15
12.98
48.81
9.62
10.44
2.92
16.18
15.51
8.17
3.41
1.22
2.85
7.56
5.54
10.47
11.96
32.81
58.13
16.71
22.39
16.31
11.41
11.48
2.86
TBS
TBS
TBS
TBS
TIPS
TIPS
TBDPS
TBDPS
TBDPS
TBDPS
H
3.01
18.79
11.47
10.93
11.86
52.92
30.59
13.99
34.72
19.77
12.41
8.75
9
H
7.53
9.63
10
11
12
13
14
15
16
17
18
19
Ac
Ac
H
H
H
Ac
Ac
Ac
H
45.70
26.43
18.25
19.69
18.47
10.02
10.95
2.72
Ac
Ac
Ac
Ac
Ac
Ac
H
H
CPh3
CPh3
O
0.88
8.37
Ac
Ac
9.77
22.79
22.68
15.29
O
20
21
20
H
3.78
2.44
66.45
2.46
39.80
2.72
42.80
2.71
52.98
1.62
52.22
3.56
O
O
Ellipticine
a
Cell lines used are P-388 (murine leukaemia cell line); KB (human epidermoid carcinoma of the mouth); COL-2 (human colon cancer); MCF-7 (human breast cancer); LU-1
(human lung cancer); and ASK (rat glioma). Ellipticine (Ellipt) was used as a positive control. The results were expressed as ED50 values (drug concentration causing 50%
growth inhibition) in M. See Supplementary Table 1 for the detail of each value which represents mean SE from three different experiments performed in triplicate.
l
and, most importantly, it was more potent than the reference drug
ellipticine. A variety of compounds containing the trityl motif have
been report to possess anticancer properties because of the size and
hydrophobic nature of the trityl pharmacophore and function
through different mechanisms of action.13 It might be suspected
that anticancer compounds possessing this functional group share
a common mode of cell death induction.
With the success of the modified silyl and triphenylmethyl
analogues (6, 8 and 18), addition of the acetyl groups to C-3 and
C-14 of the resulting silyl and triphenylmethyl analogues were
investigated, including the variations of hydroxyl groups either at
C-3 or C-14 of other compounds.
and 20 did not alter the cytotoxic activity, except that the ana-
logues 16 and 17 exhibited better activities than the parent 1.
In conclusion, we have successfully modified the hydroxyl
groups at C-3, C-4 and C-19 of andrographolide (1) to 19 analogues.
To our knowledge, this is the first report on the silyl and triphen-
ylmethyl ethers at C-19 of 1 as the essential structural features
for cytotoxic activities against a series of cancer cell lines. The most
potent analogue 18 was simply prepared in one step with excellent
yield (99%) by using the starting material 1 which was isolated in a
large quantity from A. paniculata. Owing to the less synthetic step
involved and the commercially available inexpensive reagents, this
new synthetic compound may serve as a potential lead compound
for the development of new anticancer drugs.
Interestingly, introduction of acetyl groups to C-3 and C-14 of
the silyl analogue 2 to give the analogue 5 resulted in 7- to 24-fold
increase in cytotoxicity; it was potent against P-388, COL-2 and
Acknowledgements
ASK with ED50 values of 0.34, 2.23 and 1.22 lM, respectively, and
was more potent than ellipticine. On the other hand, the diacetyl
analogues 7, 11, and 19 showed sharp decreases in cytotoxic activ-
ities in comparison with the analogues 6, 8, and 18, respectively,
which did not bear the acetyl groups at C-3 and C-14.
The activities of compounds bearing acetyl group at C-3 and
C-14 positions were compared among those of the 3-acetyl ana-
logues 3 with the C-14 acetyl analogues 4, and the analogue 9 with
10. The presence of one acetyl group at C-3 or C-14 of 4 and 9 led to
a dramatic increase in the cytotoxic activity as compared with
compound 2. However, the introduction of acetyl group at either
C-3 or C-14 gave similar activities.
This work was supported by Burapha University annual grant
(to R.S.) and the Center of Excellence for Innovation in Chemistry
(PERCH-CIC). Partial support from the Strategic Basic Research
Grant of The Thailand Research Fund (to A.S.) and Mahidol Univer-
sity (to P.P.) are gratefully acknowledged. Special thanks to Miss
Suthiporn Chaichana (Mahidol University) for high resolution mass
analysis.
Supplementary data
Supplementary data (General procedures and spectral data.)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2011.11.085.
In addition, the introduction of mono-, di-, tri-acetyl, and aceto-
nide groups to the parent compound 1 to give the analogues 12–17

52
U. Sirion et al. / Bioorg. Med. Chem. Lett. 22 (2012) 49–52
5. (a) Wang, Z.; Yu, P.; Zhang, G.; Xu, L.; Wang, D.; Wang, L.; Zeng, X. Bioorg. Med.
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