Synthesis, cytotoxicity, and structure-activity relationship (SAR) studies of andrographolide analogues as anti-cancer agent

Article abstract of DOI:10.1016/j.bmcl.2010.09.126

A series of analogues of andrographolide, prepared through chemo-selective functionalization at C14 hydroxy, have been evaluated for in vitro cytotoxicities against human leukemic cell lines. Two of the analogues (6a, 9b) exhibited significant potency. Preliminary studies on structure-activity relationship (SAR) revealed that the α-alkylidene-γ-butyrolactone moiety of andrographolide played a major role in the activity profile. The structures of the analogues were established through spectroscopic and analytical data.

Full text of DOI:10.1016/j.bmcl.2010.09.126

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6947–6950
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, cytotoxicity, and structure–activity relationship (SAR) studies
of andrographolide analogues as anti-cancer agent
Bimolendu Das a, Chinmay Chowdhury a, , Deepak Kumar a, Rupashree Sen b, Rajneeta Roy a, Padma Das a,
⇑
Mitali Chatterjee b
a Indian Institute of Chemical Biology (CSIR), 4, Raja S.C. Mullick Road, Kolkata 700 032, India
b Institute of Post Graduate Medical Education and Research, 244 B, A.J.C. Bose Road, Kolkata 700 020, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 June 2010
Revised 15 September 2010
Accepted 27 September 2010
Available online 23 October 2010
A series of analogues of andrographolide, prepared through chemo-selective functionalization at C14
hydroxy, have been evaluated for in vitro cytotoxicities against human leukemic cell lines. Two of the
analogues (6a, 9b) exhibited significant potency. Preliminary studies on structure–activity relationship
(SAR) revealed that the a-alkylidene-c-butyrolactone moiety of andrographolide played a major role in
the activity profile. The structures of the analogues were established through spectroscopic and analytical
data
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Andrographolide
C14-ester
Analogue
Anti-cancer
Apoptosis
Mankind has benefited enormously through drugs discovered
from natural resources. Roughly around 50% of the currently used
anti-cancer drugs were discovered from studies on these second-
ary metabolites.1 Therefore, the development of novel therapeutic
agents relies largely on the library of natural products and/or nat-
ural product based molecules. Primarily this involves synthetic
transformation of the lead molecule(s) allowing rapid access of
complex molecular entities with diversified structures.
The herb Andrographis paniculata Nees (Acanthaceae) is popular
in India, China and other Asian countries due to its different uses in
traditional medicine. The metabolites isolated from this herb are
mainly diterpenoids, flavonoids and sterols. The major constituent
is andrographolide, a labdane diterpene. Extracts of this herb and
its various constituents are reported to have a broad range of bio-
logical activities, such as antidiabetic,2 antimalarial,3 antibacterial,4
antiinflammatory,5 hepatoprotective,6 and others.7 Besides these,
the aerial parts of A. paniculata have been traditionally used as
medicine to treat cancer.8 In recent past, the potent anti-cancer
activities of andrographolide have indeed been established.9 How-
ever, despite its impressive biological activities, the major draw-
back of andrographolide is poor water solubility making it
difficult to prepare formulations for clinical use. So, various semi-
synthetic analogues are being developed10 and evaluated in order
to find out a better lead. Notably, Jada et al. recently reported10c
that 14-acetylandrographolide is more potent (in vitro) against
leukemia compared to andrographolide. In another report,10d DRF
3188, a novel derivative of andrographolide having
a,b-unsatu-
rated ester side chain at C14, was shown to have better anti-cancer
activity than the parent molecule.
During the course of studies on structural modifications of bio-
active natural products for value addition,11 we have chosen
andrographolide for chemo-selective functionalization at C14 hy-
droxy in order to develop the pharmacophore(s) possessing better
apoptotic index than andrographolide. Andrographolide 1 contains
(a) two double bonds (D D a-alkylidene-c-buty-
12 and 8(17)), (b) an
rolactone, and (c) three hydroxyl groups at C3, C14, and C19.
However, we opted for selective functionalization at C14 hydroxyl
group which is allylic in nature. The idea was that transformation
of andrographolide into an appropriate ester derivative (at C14)
should increase the solubility as well as activity. If esterases cleave
O
O
possible cleavage
by esterase
14
8
O
R
O
esterase
1
17
3
HO
H
19
HO
⇑
Corresponding author. Tel.: +91 33 2499 5862; fax: +91 33 2473 0284.
E-mail address: chinmay@iicb.res.in (C. Chowdhury).
Figure 1. Ester analogue of andrographolide and possible site of cleavage.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.126

6948
B. Das et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6947–6950
the ester bond under physiological conditions releasing androgra-
pholide in vivo, the ester analogue may be considered as a pro-
drug12 (Fig. 1). In pursuance of this goal, we have synthesized a
new class of andrographolide analogues having ester linkage at
C14 connected through a spacer with a terminal acid moiety and
evaluated their cytotoxicities (in vitro) in human leukemic cell
lines. The results obtained so far are reported herein.
Andrographolide was isolated in high yield (0.54%) from the
leaves of A. paniculata and used as starting material for derivatiza-
tion. The synthetic pathways used in the present study are depicted
in Scheme 1. Initially, the hydroxyl groups at C3 and C19 of
andrographolide were protected to furnish 3,19-isopropylidenean-
dro- grapholide 2, which served as the key intermediate in the
preparation of analogue library. Synthesis of the desired ester 5a
was smoothlyachieved by treatment of intermediate 2 with succinic
anhydride 4a in dry dichloromethane in the presence of catalytic
amount of 4-dimethylaminopyridine (DMAP) at room temperature.
Encouraged by the result, the higher homologue 5b was prepared
(42%) using glutaric anhydride (4b). Removal of the isopropylidene
moiety of the products 5a and 5b was carried out by exposure to
aqueous acetic acid (3:7) affording the targeted analogues 6a and
6b in yields of 72% and 86%, respectively. In the next phase, this reac-
moderate yields (41% in each case). Subsequent removal of the iso-
propylidene group from the intermediates 8a and 8b furnished
(96% and 88%) the targeted products 9a and 9b, respectively.
In order to understand the role of the
a-alkylidene part attached
to the -butyrolactone ring of andrographolide for structure–activity
c
relationship (SAR) studies, we planned to prepare the corresponding
saturated derivatives. Accordingly, chemo-selective reduction of the
double bond (D
12) of intermediate 2 was accomplished (56% yield)
using sodium borohydride in methanol instead of the costly reagent
NiCl2 as used earlier.13 Next, we adopted our earlier strategy for
attachment of ester side chain at C14 of the saturated intermediate
3 using succinic, glutaric, maleic and phthalic anhydrides (4a, 4b,
7a, and 7b), which resulted in the formation of the intermediate
products 10a, 10b, 12a, and 12b, respectively. Finally, synthesis of
the targeted saturated analogues 11a–b and 13a–b was performed
with good yields (86–89%) by deprotection of the isopropylidene
group as depicted in scheme 1. Purifications of the final analogues
were performed through usual silica gel chromatography followed
by semipreparative HPLC.14 The structures of the products15 were
established based on spectroscopic (IR, NMR and mass spectra) and
analytical data.16
In vitro screening and structure–activity relationship (SAR) studies:
In vitro screening was carried out in selected human leukemic cell
lines (U937, K562, and THP1); parallel to this, cytotoxicities against
normal cell lines (NIH3T3 and L132) were also checked. The
tion protocol was applied for the installation of
a,b-unsaturated
ester at C14 of intermediate 2 using maleic and phthalic anhydride
(7a and 7b). The expected products 8a and 8b were produced with
O
O
O
14
O
O
HO
O
HO
HO
12
1
17
ii
i
8
3
HO
O
O
O
H
H
H
1
2 (70%)
O
HO
3 (56%)
Andrographolide
O
R1
R2
O
O
O
O
R1
R2
O
O
O
( )n
iii
( )n
4
iii
iii
n=1,2
iii
7
7
O
O
n=1,2
O
O
4
7a:R1=R2=H
7a: R1= R2= H
4a ( n=1)
4b ( n=2)
4a ( n=1)
4b ( n=2)
7b:R1+R2=
7b: R1+ R2
=
-CH=CH-CH=CH-
-CH=CH-CH=CH-
O
O
O
O
O
O
O
O
HO2C
HO2C
O
O
O
O
( )n
( )n
O
O
HO2C
O
HO2C
O
R1
R2
R1
R2
O
O
O
O
H
H
H
10
H
O
O
12
12a: R1= R2 = H (41%)
12b: R1 = R2
O
O
10a (n=1, 50%)
10b (n=2, 52%)
5
8a:R1=R2=H (41%)
8b:R1+R2=
5a (n=1, 36%),
5b (n=2, 42%)
8
=
-CH=CH-CH=CH- (42%)
-CH=CH-CH=CH- (41%)
iv
iv
iv
iv
O
O
O
O
O
O
O
O
HO2C
O
O
O
HO2C
R1 R2
( )n
O
O
H
( )n
O
HO2C
O
HO2C
O
H
R1 R2
HO
HO
HO
HO
H
HO
H
9
11
6
HO
HO
13
HO
13a: R1= R2 = H (86%)
13b: R1 + R2
-CH=CH-CH=CH- (89%)
9a: R1=R2=H (96%)
11a (n=1, 86%)
11b (n=2, 88%)
6a (n=1, 72%)
6b (n=2, 86%)
9b: R1+R2= -CH=CH-CH=CH- (88%)
=
Scheme 1. Synthesis of C14-ester derivatives of andrographolide. Reagents and conditions: (i) 2,2-dimethoxypropane, p-TsOH (cat.), acetone, reflux, 2 h ; (ii) sodium
borohydride, MeOH, 0 °C to rt, 1 h ; (iii) succinic or glutaric or maleic or phthalic anhydride (4a or 4b or 7a or 7b), 4-dimethylaminopyridine (cat.), DCM, rt, 4–6 h ; (iv) acetic
acid/water (7:3), rt, 30 min.

B. Das et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6947–6950
6949
Table 1
Evaluation of in vitro cell growth inhibitory effects of andrographolide analogues in
various cell linesa,b,c
Entry
Compound
Cell growth inhibition in terms of IC50 (lM)
U937
THP1
K562
L132
NIH3T3
1
2
3
4
5
6
7
8
9
5a
5b
6a
6b
8a
8b
9a
9b
19.53
15.63
5.47
9.76
ND
14.55
9.72
6.27
NA
10.01
26.15
5.84
7.53
14.84
12.16
7.77
5.68
ND
31.39
36.46
25.30
19.55
ND
26.92
41.95
41.12
ND
21.72
15.13
8.50
19.50
31.11
18.84
12.78
10.91
ND
36.69
ND
10.20
22.06
ND
16.03
11.33
8.01
ND
Figure 3. Morphological and nuclear changes of U937 cells upon treatment with
analogue 6a; untreated U937 cells (C) following treatment with 6a (5.47
l
M) for
11a
24 h (D) stained with Hoechst 33258 and observed under
microscope (100Â).
a Leica confocal
10
11
12
13
11b
13a
13b
NA
NA
NA
12.87
NA
NA
NA
6.69
NA
NA
NA
41.85
NA
NA
NA
53.8
NA
NA
NA
44.5
Andrographolide
pholide but have lower specificity (toward normal cell lines) than
andrographolide. Further optimization of these structures (6a and
9b) is being carried out to remove this lacuna.
In addition, evaluation of the induction of apoptosis by ana-
logue 6a was carried out using flow cytometry20 and confocal
microscopy.21 Cells (U937) treated with an IC50 concentration
a
U937, THP1, and K562 are human leukemic cell lines; whereas L132 and
NIH3T3 are normal cell lines.
b
NA means not active and IC50 >50 lM.
ND means not detected.
c
standard MTS-PMS cell viability assay17 was carried out18 for
determining the antiproliferative activities of the compounds syn-
thesized. The results of the studies are summarized in Table 1.
Interestingly, two of the synthesized analogues (6a and 9b) inhib-
ited the proliferation of U937 and THP1 cells by 50% or more at
(5.47 lM) of 6a for 24 h showed Annexin-V positivity that was
ten fold higher than untreated cells (46.02% vs 4.6%, Fig. 2). The
confocal images of cells similarly treated with 6a and stained with
Hoechst 33258, showed formation of apoptotic bodies along with
membrane blebbing, whereas untreated cells had intact nuclei
(Fig. 3).
concentration less than 6.5 lM (Table 1, entries 3 and 8), while
others displayed the cytotoxicity at higher concentrations. Among
the analogues synthesized, the most active agent was 6a, while 9b
was slightly weaker (Table 1, entry 3 vs 8). Thus, analogue 6a
emerged as the best lead candidate.
In conclusion, in our endeavor to develop promising anti-cancer
agent(s) based on andrographolide, we have synthesized a new
family of andrographolide analogues and evaluated their in vitro
activities against different leukemic cell lines. The structure–activity
relationship (SAR) studies indicated that the major role is played by
It is interesting to note that activities of the unsaturated (D12
)
compounds (6a–b and 9a–b) are lost when the double bond be-
tween C12 and C13 ( -alkylidene) is reduced leading to the forma-
tion of analogues 11a–b and 13a–b (Table 1, entries 9–12). This
observation clearly indicates that the presence of the -alkylidene
part attached to the -butyrolactone ring is crucial for exhibiting
the a-alkylidene-c-butyrolactone moiety present in andrographolide.
a
Two of the analogues (6a and 9b) were found to be promising for fur-
ther structural modifications guided by the information as obtained
in the SAR studies. Studies in this direction are in progress.
a
c
cytotoxicity. Possibly, the activity of andrographolide and their
unsaturated analogues (6a–b and 9a–b) is associated with their
ability to promote alkylation of biological nucleophiles (e.g., enzymes)
Acknowledgments
This work is supported by the CSIR net-work projects (NWP
0009 and IAP 0001). Partial financial support from DST (project
no: SR/S1/OC-42/2009), India is also gratefully acknowledged.
B.D. and D.K. thank CSIR, New Delhi, for the award of fellowships.
through Michael addition19 to the
a-alkylidene-c-butyrolactone
moiety. Furthermore, the results of SAR studies show that increase
in lipophilicity by protection of 3,19-hydroxyl groups of androgra-
pholide has detrimental effects on the activity profile (Table 1, en-
tries 1 vs 3, 2 vs 4, 5 vs 7, 6 vs 8). Andrographolide itself was found
to be significantly active against THP1 cells but only moderately so
against U937 cells (Table 1, entry 13). The IC50 values of analogues
6a and 9b appear to be lower compared to andrographolide against
both cancer and normal cell lines. Thus, the screening studies indi-
cate that analogues 6a and 9b are more cytotoxic than androgra-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.09.126.
References and notes
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Andrographolide-14
a-O-succinate 6a: gum, 72% yield; IR (neat): 3425, 2938,
1739 cmÀ1 1H NMR (CDCl3, 300 MHz): d 7.06 (1H, t, J = 6.4 Hz), 5.94 (1H, d,
;
J = 5.1 Hz), 4.89 (1H, s), 4.56 (1H, dd, J = 11.5, 6.4 Hz), 4.53 (1H, s), 4.25–4.17
(2H, m), 3.93 (2H, br), 3.52–3.49 (1H, m), 3.33 (1H, d, J = 10.8 Hz), 2.74–2.64
(4H, m), 2.50–2.39 (3H, m), 2.00–1.96 (1H, m), 1.82–1.73 (5H, m), 1.30–1.13
(6H, m), 0.67 (3H, s); 13C NMR (CDCl3, 75 MHz): d 175.7, 172.1, 169.2, 150.9,
146.5, 123.6, 108.7, 80.2, 71.5, 68.0, 64.0, 55.7, 54.9, 42.4, 38.7, 37.5, 36.7, 28.7,
28.6, 27.8, 25.1, 23.6, 22.6, 14.9; ESI-MS: m/z 473.14 [M+Na]+. Anal. Calcd for
C
24H34O8: C, 63.98; H, 7.61. Found: C, 64.02; H, 7.56.
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NIH3T3 and L132 cells using MTS-PMS assay (see Ref. 17 for details). Briefly,
cells (2.5–5.0 Â 104/200
l
L) were seeded in 96-well tissue culture plates and
incubated with compounds (0–50
lM) for 48 h at 37 °C, 5% CO2. Following
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treatment, MTS [3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-
(4-sulphonyl)-2H-tetrazolium, inner salt] (2.0 mg/ml) and PMS (phenazine
14. HPLC was performed on RP C18 column (300 mm  7.8 mm, 7.0
l
m) using
methosulphate) (0.92 mg/ml) were added in a ratio of 10:1 (20 lL per well).
methanol/acetonitrile/water/acetic acid (49:10:40:1 or 34:10:55:1) as mobile
phase; flow rate = 3 ml/min; tR = 6.8, 8.9, 16.9, 17.2, 17.8 and 18.4 min (for
compounds 6a, 6b, 9a, 9b, 13a and 13b, respectively).
After incubation for 3 h at 37 °C, the resulting absorbances at 490 nm were
measured in an ELISA reader. The specific absorbance that represented
formazan production was calculated by subtraction of background
absorbance from total absorbance. The mean % viability was calculated as
follows: Mean specific absorbance of treated cells  100/Mean specific absorbance
of untreated cells
The results were expressed as IC50 values, that is, the concentration that
inhibited 50% of cell growth, enumerated by graphic extrapolation using Graph
pad prism software (version 5).
15. All the synthesized final analogues were generally preserved at 0 to À4 °C and
remained stable for months. Even allowing them to stand at rt for several
weeks did not lead to any decomposition (HPLC analysis). Besides, few of the
final analogues (6a, 9a, 11a–b, 13a) were found to be soluble in chloroform but
all of them were fairly soluble in methanol and dimethyl sulfoxide (DMSO) as
well.
16. General procedure for the synthesis of acid 5/8/10/12 (step iii): To a well stirred
solution of compound 2/3 (0.512 mmol) in dry dichloromethane (8 mL) were
added acid anhydride 4/7 (0.615 mmol) and a catalytic amount (4 mg) of 4-
dimethylamino-pyridine (DMAP). The whole mixture was stirred under argon
atmosphere at room temperature for 4–6 h. The solvent was evaporated under
reduced pressure and the residue was purified through silica gel (100–200
mesh) column chromatography (ethyl acetate–petroleum ether) to afford the
desired product 5/8/10/12.
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(c) Hardy, L. W.; Finer-Moore, J. S.; Montfort, W. R.; Jones, M. O.; Santi, D. V.;
Stroud, R. M. Science 1987, 235, 448.
20. Flow cytometric analysis: Double staining for Annexin V-FITC and propidium
iodide (PI) was performed. Briefly, U937 cells were incubated without or with
6a (IC50 = 5.47 lM) for 24 h at 37 °C, 5% CO2. Cells were then washed twice in
phosphate buffered saline (PBS, 0.02 M, pH 7.2) and resuspended in Annexin-V
binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). Annexin
V-FITC and propidium iodide were then added according to the manufacturer’s
instructions and incubated for 15 min in the dark at 25 °C. Data was acquired
3,19-Isopropylideneandrographolide-14-
a-O-succinate 5a: gum, 36% yield; IR
(neat): 3421, 2936, 1738, 1676 cmÀ1 1H NMR (CD3OD, 300 MHz): d 6.98 (1H, t,
;
J = 6.3 Hz), 6.04 (1H, d, J = 5.1 Hz), 4.93 (1H, s), 4.63–4.57 (2H, m), 4.30 (1H, d,
J = 11.1 Hz), 4.04 (1H, d, J = 11.7 Hz), 3.54–3.51 (1H, m), 3.20 (1H, d,
J = 11.7 Hz), 2.64 (4H, s), 2.57–2.52 (2H, m), 2.47–2.43 (1H, m), 2.07–1.97
(3H, m), 1.83–1.78 (3H, m), 1.41 (3H, s), 1.36–1.28 (6H, m), 1.22 (3H, s), 0.98
(3H, s); 13C NMR (CD3OD, 75 MHz): d 175.5, 173.6, 171.4, 151.7, 149.0, 125.6,
109.3, 100.3, 78.0, 73.1, 69.5, 64.9, 57.1, 53.4, 39.5, 38.9, 38.7, 35.5, 29.9, 29.6,
27.5, 26.5, 26.2, 25.7, 24.3, 16.5; ESI-MS: m/z 513.01 [M+Na]+. Anal. Calcd for
using
a FACS Calibur flow cytometer and analyzed with Cell Quest Pro
software.
21. Confocal microscopy: Apoptotic cells were characterized by nuclear
condensation of chromatin and/or nuclear fragmentation. Briefly, U937 cells
were incubated with an IC50 concentration (5.47
with ice cold PBS and stained with Hoechst 33258 (10
were mounted on poly -lysine coated slides and analyzed in a laser scanning
l
M) of 6a (for 24 h), washed
lg/ml, 30 min). The cells
C27H38O8: C, 66.10; H, 7.81. Found: C, 66.04; H, 7.86.
L
General procedure for the synthesis of acid 6/9/11/13 (step iv): To a well stirred
solution of ester intermediate 5/8/10/12 (0.50 mmol) in 1,4-dioxane (3 mL)
confocal microscope (Leica TCS SP2 system, Leica microsystem, Heidelberg,
Germany; 100Â). At least 20 microscopic fields were observed for each sample.