Synthesis and structure-activity relationships of andrographolide analogues as novel cytotoxic agents

Article abstract of DOI:10.1016/j.bmcl.2004.06.090

Andrographolide 1, the cytotoxic agent of the plant Andrographis paniculata was subjected to semi-synthetic studies leading to the preparation 19 and a number of related novel analogues. Synthesis and structure-activity relationships are discussed. Androg

Full text of DOI:10.1016/j.bmcl.2004.06.090

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4711–4717
Synthesis and structure–activity relationships of
andrographolide analogues as novel cytotoxic agentsq
a
a
Srinivas Nanduri,a, Vijay Kumar Nyavanandi, Siva Sanjeeva Rao Thunuguntla,
*
Sridevi Kasu,a Mahesh Kumar Pallerla,a P. Sai Ram,a Sriram Rajagopal,b
R. Ajaya Kumar,b Rajagopalan Ramanujam,b J. Moses Babu,c Krishnamurthi Vyas,c
A. Sivalakshmi Devi,c G. Om Reddyc and Venkateswarlu Akellaa
aDiscovery Chemistry, Dr. Reddyꢀs Laboratories Ltd, Discovery Research, Bollaram Road, Miyapur, Hyderabad 500 049, India
bDiscovery Biology, Dr. Reddyꢀs Laboratories Ltd, Discovery Research, Bollaram Road, Miyapur, Hyderabad 500 049, India
cAnalytical Research, Dr. Reddyꢀs Laboratories Ltd, Discovery Research, Bollaram Road, Miyapur, Hyderabad 500 049, India
Received 21 May 2004; accepted 25 June 2004
Abstract—Andrographolide 1, the cytotoxic agent of the plant Andrographis paniculata was subjected to semi-synthetic studies lead-
ing to the preparation of a number of potent and novel analogues. Of the analogues synthesized, while 8,17-epoxy andrographolide
6 retained the cytotoxic activity of 1, ester derivatives of 6 exhibited considerable improvement in activity. Lower activity was
observed when the epoxy moiety in the triacetate 9, derived from 6 was modified. Synthesis and structure–activity relationships
are discussed.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
O
14
HO
O
Andrographolide 1 (Fig. 1), is the major labdane diterp-
enoidal constituent of the plant Andrographis paniculata
(family Acanthaceae),1 which is used extensively in the
traditional systems of Indian and Chinese medicine.2,3
Extracts of the plant and their constituents are reported
to exhibit a wide spectrum of biological activities of
therapeutic importance including antibacterial,4 antiin-
flammatory,5 antimalarial,6,7 immuno stimulant,8
hepatoprotective,9 and antithrombotic10 properties.
Potent antiHIV activity of the succinoyl derivatives of
1 are described.11,12 The interesting cytotoxic and antitu-
mor activities of the extracts and the constituents of the
plant have been explored by several researchers in recent
years. Potent cell differentiation inducing activity on
mouse myeloid leukemia (M1) cells by the methanolic
extract of A. paniculata and its constituents is re-
ported.13 Furthermore, an alcoholic extract of the plant
12
1
17
8
3
4
HO
HO
H
19
Figure 1. Andrographolide (1).
and its major constituent 1 have been shown to affect the
cell cycle progression in prostate and breast cancer cell
lines.14 Identification of 1 as a cytotoxic agent against
mouse15 and human cancer cell lines16 through bioactiv-
ity-guided chromatographic fractionation are reported.
A thorough study on the anticancer and immunomodu-
latory potential of 1 is recently reported.17 However, no
systematic attempt has been made to improve the cyto-
toxic activity of 1 and to synthesize novel and potent
agents, except for a report on the antitumorigenic prop-
erties of alkanoyl derivatives18 of 1. In this paper, the re-
sults on the design, synthesis, and biological evaluation
of new analogues of 1 as potent cytotoxic agents are
described.
Keywords: Andrographolide; Andrographis paniculata; Cytotoxic
agents.
q DRL Publication No.: 247.
*
Corresponding author. Tel.: +91-40-23045439; fax: +91-40-
23045438; e-mail: nandurisrinivas@drreddys.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.090

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S. Nanduri et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4711–4717
2. Chemistry
As 6 retained the cytotoxic activity of 1 and possesses
a C-8 epoxy moiety as structural novelty, it was taken
as a lead structure for further modifications aimed at
understanding the contribution of the three hydroxyl
groups. A number of novel ester derivatives of 6 were
designed and synthesized to achieve this objective. Table
1 includes the novel ester derivatives synthesized and
their syntheses are given in Schemes 2–4.
The structure of andrographolide contains (1) an a-alky-
lidene c-butyrolactone moiety, (2) two olefin bonds
D8(17) and D12(13), and (3) three hydroxyls at C-3, C-19,
and C-14. Of the three hydroxyl groups, the one at C-
14 is allylic in nature, and the others at C-3 and C-19
are secondary and primary, respectively. The derivatives
of andrographolide were synthesized by modifying the
above structural features.
Thus, the three hydroxyls of 6 at C-14, C-3, and C-19
were acylated simultaneously or selectively. The triacetyl
derivative 9 was synthesized by heating 6 in acetic anhy-
dride. The structure of 9 was elucidated by spectral stud-
ies and finally confirmed by single crystal X-ray
diffraction studies.21 The perspective view of the mole-
cule is shown in Figure 2. Both the six-membered rings
adopt the chair conformation, whereas the five-mem-
bered ring is in an envelope conformation. Van der
Waals contacts stabilize the molecules in the lattice.
The absolute stereochemistry at C-4, C-9, and C-10
was assumed to be the same as that observed in the
ent-labdane structure.22,23 (Crystallographic data for 9
have been deposited with the Cambridge Crystallogra-
phic Data Centre as supplementary publication number
222220.) Based on this information, the stereochemistry
at C-8 and C-14 have been determined to be S. To exam-
ine the role of the carbon chain length in the ester func-
tionality of 9, the corresponding tripropionyl derivative
10 was synthesized by heating 6 in propionic anhydride.
Compounds 9 and 10 can also be synthesized by selec-
tively epoxidizing the D8(17) double bond of the corre-
sponding ester derivative of 1. The 3,19-diacetyl
derivative 14 was synthesized by this method (Scheme 2).
In order to determine the importance of lactone moiety
of andrographolide 1 for cytotoxic activity, it was
opened to yield andrographolic acid 2 by reported pro-
cedure.19 To understand the role of the exocyclic double
bond (D12(13)) in 1, 3 was prepared by selectively reduc-
ing the conjugated double bond. To further study, the
role of allylic hydroxyl at C-14 and the conjugated dou-
ble bond, 4 and 5 were prepared, in which the exocyclic
double bond (D12(13)) isomerized to the endocyclic dou-
ble bond (D13(14)), with the simultaneous removal of
C-14 hydroxyl. Compounds 4 and 5 were synthesized
from the triacetyl derivative of 1 by reported proce-
dures.20 On the other hand, selective epoxidation of
the exocyclic double bond (D8(17)) with m-CPBA led to
6 (Scheme 1).
In vitro cytotoxic activity screening of the above ana-
logues showed that while 2–5 demonstrated loss of activ-
ity, 6 did not exhibit any significant difference in its
activity compared to 1 (discussed in biological results).
O
HO
O
Selective esterification of the allylic alcohol at C-14 was
achieved by protecting the C-3, C-19 hydroxyls on the
decalin system with an isopropylidene moiety and subse-
quently modifying the C-14 alcohol (Scheme 3). Thus,
the 14-O-acetyl derivative was obtained by heating
8,17-epoxy-3,19-isopropylidene andrographolide 7 in
the corresponding anhydride. 14-N-Boc-glycinyl and
cinnamoyl esters were prepared by treating 7 with the
appropriate acyl carbonates (generated by the addition
of ethyl chloroformate to the corresponding acids).
Selective removal of C-3, C-19 isopropylidene protec-
tion in the above derivatives resulted in the formation
of compounds having ester groups at C-14 and hydrox-
yls at C-3 and C-19. Thus, derivatives 11, 16, and 19
were synthesized. Synthesis of derivatives 17, 18, and
20–23 with novel ester linkages at C-14 and alkyl esters
at C-3 and C-19 was carried out by deprotection of the
C-3, C-19-isopropylidene group followed by transfor-
mation of the C-3 and C-19 hydroxyls into the alkyl
esters by heating in the corresponding anhydrides.
OH
OH
HO
HO
HO
O
H
O
3
O
O
b
HO
HO
H
c
a
e
HO
HO
H
2
1
d
4
O
HO
O
O
O
HO
HO
5
H
HO
HO
H
6
Synthesis of derivatives 12, 13, and 15 (Scheme 4) having
either acyl or hydroxyl groups at C-14, C-3, and C-19
was achieved by routine selective acylations of the hyd-
roxy groups present in 6. Thus, the acetyl derivatives
were prepared by heating 8,17-epoxy-19-trityl androgra-
pholide 8 with acetic anhydride. The 3-mono-acetyl and
3,14-diacetyl derivatives formed were separated by chro-
matography. The 3,14-dipropionyl derivative was pre-
Scheme 1. Synthesis of compounds 1–6. Reagents and conditions: (a)
(i) Aq 1N NaOH/reflux, 1h, (ii) 6N HCl, 70%19; (b) (i) 2,2-dimethoxy
propane/benzene and DMSO, PPTS/reflux, 2h, 95%, (ii) NaBH4,
MeOH/rt, 1h, 85%, (iii) AcOH and H2O (7:3)/rt, 15min, 90%; (c) (i)
Ac2O/ZnCl2/rt, 1h, 97%, (ii) NaBH4, MeOH/rt, 1h, 85%, (iii) 10%
methanolic HCl/rt, 8h, 80%20; (d) (i) Ac2O/ZnCl2/rt, 1h, 97%, (ii) dry
pyridine/reflux, 4h, 80%, (iii) 10% methanolic HCl/rt, 8h, 80%20; (e)
m-CPBA/CHCl3/MeOH, rt, 6h, 90% (isomeric mixture).

S. Nanduri et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4711–4717
Table 1. Novel ester derivatives of epoxy andrographolide 6
4713
O
R1O
O
O
R2O
R3O
H
S. No.
R1
R2
R3
6
H
COCH3
H
H
COCH3
9
COCH3
COCH2CH3
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
COCH2CH3
COCH3
COCH2CH3
H
H
COCH3
COCH3
COCH3
COCH3
COCH2CH3
H
H
H
H
COCH3
H
COCH2CH3
COCH2NHBoc
COCH2NHBoc
COCH2NHBoc
COCH@CH–C6H5
COCH@CH–C6H5
COCH@CH–(4-OCH3)C6H4
COCH@CH-(3,4-di OCH3)C6H3
COCH@CH-(3,4-OCH2O-)C6H3
H
COCH3
COCH2CH3
H
COCH3
COCH2CH3
H
COCH2CH3
COCH2CH3
COCH2CH3
COCH2CH3
COCH2CH3
COCH2CH3
COCH2CH3
COCH2CH3
O
O
R1O
HO
O
O
O
O
b
R2O
R3O
HO
H
H
O
HO
HO
6
O
R1=R2=R3 = COCH3
9
a
R1=R2=R3 = COCH2CH3
10
O
O
R1O
R1O
O
O
HO
HO
c
H
1
d
O
R2O
R3O
R1 = H ; R2 = R3= COCH3
R2O
R3O
R1 = H ; R2 = R3 = COCH3 14
H
H
Scheme 2. Synthesis of compounds 9, 10, and 15. Reagents and conditions: (a) m-CPBA/DCM/MeOH/rt, 8h, 90% (isomeric mixture); (b) acetic or
propionic anhydride/reflux, 30min, 70% (required isomer); (c) acetic acid/70ꢁC, 6h, 40%18; (d) m-CPBA/DCM/rt, 4h, 70% (required isomer).
pared by heating 8 in propionic anhydride. The 19-trityl
group in the above derivatives was selectively deprotect-
ed with formic acid in dichloromethane, thus yielding
derivatives 12, 13, and 15.
The aldehyde was also accompanied by the formation of
a tetracyclic compound 25, presumably as a by product
derived from the aldehyde. Further, the aldehyde 24 was
converted to the corresponding alcohol 26 by NaBH4
reduction.
In addition to the above syntheses, in order to examine
the role of the C-8, C-17 epoxy moiety of the new deriv-
atives in exhibiting cytotoxic activity, modifications on
the epoxy moiety in the above series was performed.
These changes were made in 9 (Scheme 5).
3. Biological results and discussion
The in vitro cytotoxic activity of andrographolide 1 and
its analogues 2–26 were evaluated against breast (MCF-
7/ADR), CNS (U251), colon (SW620), lung (H522),
ovarian (SKOV3), prostate (DU145), and renal (A498)
Accordingly, the epoxide in 9 was transformed into the
corresponding aldehyde 24 by treatment with BF3ÆEt2O.

4714
S. Nanduri et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4711–4717
O
O
HO
HO
O
O
a
O
O
O
H
HO
HO
H
6
7
O
O
b,c,d
b,c
R1O
O
O
R1O
O
O
O
R2O
R3O
H
R2O
R3O
H
17 R1=COCH2NHBoc R2=R3=COCH3
18 R1=COCH2NHBoc; R2=R3=COCH2CH3
20 R1=COCH=CH-C6H5;R2=R3=COCH2CH3
11 R1=COCH3; R2=R3=H
16 R1=COCH2NHBoc; R2=R3=H
19 R1=COCH=CH-C6H5; R2=R3=H
21 R1=COCH=CH-(4-OCH3)C6H4;R2=R3=COCH2CH3
22 R1=COCH=CH-(3,4-di OCH3)C6H3;R2=R3=COCH2CH3
23 R1=COCH=CH-(3,4- OCH2O)C6H3;R2=R3=COCH2CH3
Scheme 3. Synthesis of compounds 11, 16–23. Reagents and conditions: (a) 2,2-dimethoxy propane/benzene and DMSO/PPTS/rt, 8h, 70% (required
isomer); (b) for 11 acetic anhydride/reflux, 1h, 80%; for 16–23 R1COOH/CICOOEt/NEt3/DCM/rt, 1h, 60–85%; (c) AcOH and H2O (7:3)/rt, 15min,
85%; (d) acetic or propionic anhydride/reflux, 30min, 65–75%.
O
O
O
HO
R1O
HO
O
O
O
a,b
O
c
O
HO
HO
R2O
R3O
HO
H
H
H
O
1
8
Ph
Ph
Ph
12 R1 = R3=H ; R2 = COCH3 (25% over two steps)
13 R1 = R2 = COCH3; R3=H (40%over two steps)
15 R1= R2 = COCH2CH3; R3=H (55%over two steps)
Scheme 4. Synthesis of compounds 12, 13, 15. Reagents and conditions: (a) tritylchloride/pyridine/60ꢁC, 6h, 80%; (b) m-CPBA/CH2Cl2/rt, 2h, 70%
(required isomer); (c) (i) acetic or propionic anhydride/reflux, 30min, (ii) formic acid/CH2Cl2/rt, 30min.
that causes 50% inhibition of cancer cell growth against
various cell lines are expressed as GI50 values and are
given in Table 2. The selected active analogues were fur-
ther evaluated for their in vivo antitumor activity using
modified hollow fiber assay.
In the above study, andrographolide 1 was found to
possess moderate activity against all the cell lines with
GI50 values ranging from 3 to 30lM. Loss of activity
was observed for 2 and 3 namely andrographolic acid
and 12,13-dihydro andrographolide, respectively, clearly
demonstrating that the intact lactone moiety of andro-
grapholide and the conjugated D12(13) double bond play
an important role in the observed cytotoxic activity of 1.
Further confirmation of the importance of the conju-
gated D12(13) double bond and the hydroxyl at C-14
was evident from the similar loss of activity observed
Figure 2. Single-crystal X-ray structure of 9.
for 4 and 5. However, 4 exhibited potent activity against
ovarian cell line in nanomolar concentrations. Com-
cancer cell lines using the NCI standard protocol for
screening anticancer molecules.24 The concentration
pound 6 exhibited moderate activity against all the cell
lines similar to 1 (GI50 values ranging from 4 to

S. Nanduri et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4711–4717
4715
O
AcO
O
O
O
AcO
O
H
O
O
O
a
O
AcO
AcO
H
9
AcO
AcO
AcO
AcO
H
24 (60 %)
+
H
25 (25%)
O
AcO
b
O
OH
AcO
AcO
H
26 (50%)
Scheme 5. Synthesis of compounds 24–26. Reagents and conditions: (a) BF3ÆEt2O/CH2Cl2/0ꢁC, 10min; (b) NaBH4/MeOH/0ꢁC, 30s.
Table 2. In vitro cytotoxic activities of andrographolides (1–26)
Compound
Cytotoxicity (GI50 lM)a
Breast
MCF-7/ADR
CNS
U251
Colon
SW 620
Lung
H 522
Ovarian
SK OV3
Prostate
DU145
Renal
A 498
1
15
30
ndb
nd
>100
20
5
3
>100
85
10
>100
>100
54
17
80
15
>100
>100
0.18
nd
4
20
>100
2
30
4
2
3
>100
>100
>100
30
nd
nd
nd
30
0.25
nd
7
4
>100
81
16
5
5
>100
30
2
6
20
15
3
9
2.5
1
15
2.5
1.36
5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
nd
20
20
20
30
30
7
3
50
45
30
40
35
40
4
2
20
3
25
25
50
6.5
nd
20
20
7
>100
55
20
60
30
50
2.5
>100
3
20
6
nd
40
20
40
5
7
40
>100
4
6
4
3
30
8
20
4
15
2
20
6
4
15
4
20
5.5
4
0.5
2.5
0.6
0.4
65
6
20
9
0.08
3
6
10
2
3
3
4.8
7.5
3
2
3
1.5
20
nd
40
nd
2
2.5
0.3
3
2.5
4
3.5
6
35
62
30
19
32
40
34
50
12
40
2
nd
nd
nd
40
3
7
19
a Cytotoxicity GI50 values are the concentrations corresponding to 50% growth inhibition.
b nd––not done.
30lM), indicating that the exocyclic D8(17) double bond
may not play any important role and can be replaced
with an epoxy moiety.
converted to esters for exhibiting potent activity, two
monoacetyl (11 and 12) and two diacetyl (13 and 14)
derivatives were evaluated and their activities compared
with 9. In general, all the mono and diacetyl derivatives
were found to be less active compared to 9, indicating
that all the three hydroxyls need to be converted into
their ester functionalities for potent activity. Interest-
ingly, 11 and 13, which contained an acetyl group
at C-14 were found to possess potent activity against
Significant improvement in the activity of 6 was ob-
served when it was converted to its triacetyl 9 and tripro-
pionyl 10 derivatives (GI50 values ranging from 0.25 to
5lM against most of the cell lines). To understand fur-
ther, the number of hydroxyls on 6, which need to be

4716
S. Nanduri et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4711–4717
Table 3. In vivo hollow fiber assay results of andrographolide
derivatives
ovarian and renal cell lines (GI50 values less than 10lM)
compared to 12 and 14, indicating that the presence of
an ester functionality at C-14, and possibly also at C-3
and C-19, is essential for improving the activity of 6.
In view of this observation, a number of compounds
having ester side chains at C-14 and hydroxyls or alkyl
esters at C-3 and C-19 were synthesized and screened.
Compound Dose (mg/kg) SC score IP score Total score
1b
50
100
300
100
150
100
200
100
200
25
6
14
6
10
14
6
16
28
12
2
6a
9a
2
0
8
8
16
14
28
2
17b
18b
19b
20b
8
6
Thus, 16–18 having a N-Boc-glycinyl group, and 19–23
with substituted or unsubstituted cinnamoyl side chains
at C-14 with either hydroxyls or alkyl ester groups at
C-3 and C-19 were screened.
14
2
14
0
8
4
12
8
2
6
50
14
8
14
12
14
28
20
28
Their cytotoxic activity results indicated that
100
200
14
1. The activity of 16 with C-14 N-Boc-glycinyl moiety
and hydroxyls at C-3 and C-19 improved when the
free hydroxyls were converted to their corresponding
alkyl esters 17 and 18. Further, 17 having acetyl moi-
eties at C-3 and C-19 showed better activity com-
pared to its corresponding propionyl derivative 18.
2. When conjugated esters were added as side chains at
C-14 (derivatives 19–23), the activity improved con-
siderably compared to the parent molecule 6 against
all the cell lines. While these compounds possessed
GI50 values less than 10lM against all the cell lines,
20 exhibited potent activity against breast cancer cell
line (GI50 0.08M) and 19, 21, and 22 showed superior
activity against colon cancer cell line (GI50 0.5, 0.6,
and 0.4lM, respectively). Compound 23 was found
to be less active compared to the above derivatives.
a Compound tested against 4 cell lines.
b Compounds tested against 7 cell lines.
was observed with the acylation of the hydroxyls as in
compounds 9, 17–20. Of these ester derivatives, com-
pound 19 was found to be the most potent compound
exhibiting superior activity compared to both the parent
compounds 1 and 6. Further in vivo studies of 19 in var-
ious xenograft models are in progress.
4. Conclusion
In conclusion, in our endeavor to develop promising
anticancer molecules based on the naturally occurring
andrographolide 1, we have designed and synthesized
19 and a number of related analogues possessing potent
in vitro and in vivo anticancer activity.27 An analysis of
the in vitro results indicated that (a) the intact a-alkylid-
ene c-butyrolactone moiety of andrographolide, (b) the
D12(13) double bond, (c) the C-14 hydroxyl or its ester
moiety, and (d) the D8(17) double bond or epoxy moiety
are responsible for the cytotoxic activity exhibited by
andrographolide and its analogues.
To further understand the role of the epoxy moiety pre-
sent in the above ester derivatives of 6 for their potent
activity, the epoxy modified analogues of 9 (24–26) were
studied. All the three compounds exhibited lower activ-
ity compared to 9 suggesting that the epoxide is neces-
sary for retaining activity.
In summary, most of the ester derivatives of 6, exhibited
good in vitro cytotoxic activity possessing GI50 values
ranging from 1 to 10lM. Compounds 9, 17–20 along
with the parent compounds 1 and 6, were further evalu-
ated for their in vivo antitumor activity using modified
hollow fiber animal assay.25,26 The cell lines needed for
this assay were selected from in vitro studies. Poly vinyli-
dine fluoride hollow fibers containing different types of
human cancer cell lines were implanted intraperitoneally
(IP) and subcutaneously (SC) into Swiss Albino Mice
(SAM). The compounds were administered by IP route
at two different doses for 4days. The doses were deter-
mined based on the MTD values of the compounds.
The in vivo efficacy of the compounds was evaluated
by determining the percentage growth of the cells using
MTT assay. Compounds inhibiting 50% or greater
growth compared to the vehicle control were considered
active and a score of 2 was assigned at the different doses
tested. The results obtained were tabulated in Table 3.
The results indicated that Andrographolide 1, possessed
moderate activity at 50mg/kg and good activity at
100mg/kg doses. However, the corresponding 8,17-
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epoxy derivative 6 showed moderate activity at
300mg/kg. Improvement in the in vivo activity of 6

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4717
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21. X-ray crystallographic analysis of 9: A colorless crystal of
9 C26H36O9 [0.50mm·0.40mm·0.15mm], was mounted
on a glass fiber in a random orientation. The data was
collected on a Rigaku AFC7S diffractometer with Cu Ka
˚
(k=1.5418A) radiation. The cell parameters and the
orientation matrix for data collection were obtained from
least squares refinement, using the setting angles of 25
reflections in the range of 25–33ꢁ (2h). The data were
collected at a temperature of 298K. A total of 2861 unique