Design, synthesis and antibacterial activity of novel andrographolide derivatives

Article abstract of DOI:10.1016/j.bmc.2010.04.094

A series of andrographolide derivatives were synthesized through a facile condensation reaction with different carboxylic acids. The new compounds were characterized and screened for their antibacterial activities. A number of the new compounds significantly reduced bacterial quorum sensing virulence factors production in Pseudomonas aeruginosa, essential for pathogenesis. Compound 11b showed the best activity among all the new compounds.

Full text of DOI:10.1016/j.bmc.2010.04.094

Bioorganic & Medicinal Chemistry 18 (2010) 4269–4274
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and antibacterial activity of novel andrographolide derivatives
*
Zhongli Wang, Pei Yu , Gaoxiao Zhang, Lipeng Xu, Dingyuan Wang, Liang Wang, Xiangping Zeng,
*
Yuqiang Wang
Institute of New Drug Research and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine & New Drug Research,
Jinan University College of Pharmacy, Guangzhou 510632, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of andrographolide derivatives were synthesized through a facile condensation reaction with dif-
ferent carboxylic acids. The new compounds were characterized and screened for their antibacterial
activities. A number of the new compounds significantly reduced bacterial quorum sensing virulence fac-
tors production in Pseudomonas aeruginosa, essential for pathogenesis. Compound 11b showed the best
activity among all the new compounds.
Received 23 March 2010
Revised 27 April 2010
Accepted 28 April 2010
Available online 20 May 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Andrographolide derivatives
Synthesis
Antibacterial
Quorum sensing
1. Introduction
that attenuate bacterial virulence rather than growth is a novel
way for antibacterial drug research.
Bacteria communicate with each other by means of different
classes of signal molecules. In general, each bacterial cell produces
a small amount of one or more signal molecules, which are subse-
quently released into the environment. When the signal mole-
cule(s) reach a certain threshold, they promote the transcription
of genes necessary for bacterial group functions.1–4 These group
phenomena are largely under the control of a cell–cell signaling
pathway called quorum sensing (QS), which plays a considerable
role in the establishment of both symbiotic and pathogenic rela-
tionships with eukaryotic hosts.5 As quorum sensing represents a
strategy to control bacterial growth, there is significant interest
in the development of QS-targeting agents.6,7
The Gram-negative Pseudomonas aeruginosa (P. aeruginosa) is a
model bacterium for studying QS and biofilm formation. P. aerugin-
osa produces two different acyl homoserine lactones (AHLs) as QS
signal molecules, that is, N-3-oxododecanoylhomoserine lactone
(3-oxo-C12-HSL) and N-butanoylhomoserine lactone (C4-HSL).
These molecules are sensed by three receptors: LasR, QscR, and RhlR.
LasRand OscRcommonlyreceive3-oxo-C12-HSL butoperatedistinct
regulons. RhlR also regulate its own regulon by sensing C4-HSL.8,9 QS
controls secondary metabolism, bioluminescence, protein secretion,
motility, virulence factor production, plasmid transfer and biofilm
formation in P. aeruginosa.10–12 Therefore, finding QS antagonists
Andrographolide (Andro, 1, Fig. 1), a natural bicyclic diterpeniod
lactone, is extracted as the main phytoconstituent from Androgra-
phis paniculata Nees, a herb commonly used in India, China and
southeast Asia for the treatment of a large variety of illness, such
as gastric disorders, cold, influenza and other infectious dis-
eases.13,14 Andro exhibits several pharmacological activities, includ-
ing antiinflammatory, immunomodulatory, antibacterial and
antiviral infections.15–22 Although the herb, Andro and its deriva-
tives have been used to treat bacterial infections for many years,
these drugs have been found to have minimal or no direct inhibition
on bacterial growth, especially on P. aeruginosa.18–20
Our laboratory recently developed Andro derivatives as autoin-
ducer mimics that can inhibit the growth of both Gram-positive
and the Gram-negative bacteria.19 We have demonstrated that
O
O
O
O
HO
O
O
S
S
CH2
CH2
HO
HO
HO
HO
1
* Corresponding authors. Tel.: +86 20 8522 8481; fax: +86 20 8522 4766.
E-mail addresses: pennypeiyu@yahoo.com.cn (P. Yu), yuqiangwang2001@
yahoo.com (Y. Wang).
(Andro)
AL-1
Figure 1. Structures of Andro and AL-1.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.094

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Z. Wang et al. / Bioorg. Med. Chem. 18 (2010) 4269–4274
some of these compounds, such as AL-1 (Fig. 1), a conjugate of
R-( )-lipoic acid and Andro, had significant effects on the QS sys-
osa growth at a concentration of up to 2 mM (data not show), and
the result is consist with our previous findings.19
a
tem in P. aeruginosa, leading to strong inhibition of biofilm forma-
tion.19 In search for more effective new compounds and uncover
the mechanism of drug action, we herein report a new series of An-
dro derivatives and their preliminary evaluation for activity in
inhibiting the QS.
Production of pyocyanin by P. aeruginosa is positively regulated
by quorum sensing signals including AHLs.24–26 AL-1 and Andro
have been shown to inhibit the P. aeruginosa QS system.19,20 We
therefore examined the effect of the new Andro derivatives on pyo-
cyanin production. P. aeruginosa were grown for overnight to mid
log phase (OD600 0.5) and were then diluted to an OD600 of 0.05, ali-
quoted to test tubes containing the new compounds. Following an-
other 10 h of growth, pyocyanin was extracted, and was then
quantified. The results are shown in Figure 2. We found that all
of the tested compounds, 3b–11b, Andro and AL-1, inhibited pyo-
cyanin production. Among them, compound 11b and AL-1 were
the most potent. Compounds 3b–10b also significantly inhibited
pyocyanin production, and all were more potent than the natural
Andro.
Protease is another virulence factor produced and excreted by
P. aeruginosa under QS control. We then tested the new compounds
for their anti-protease activity. A colorimetric method employing
azocasein as a substrate was used to measure protease activity,
and the results are shown in Figure 3. Compounds AL-1, 5b, 8b
and 11b almost completely suppressed protease production at
0.5 mM. Compounds 7b, 9b, 10b and Andro had similar activity.
Analysis of the SAR of the new compounds reveals that: (1) a
side chain, aliphatic straight or cyclic, at the C14 of the natural An-
dro increases activity inhibiting the production of virulence fac-
tors; (2) Compounds such as 5b and 8b significantly inhibit the
production of protease, but are much less potent inhibiting pro-
duction of pyocyanin, suggesting that the compounds act through
different pathways on inhibition of the productions of these two
virulence factors; (3) In contrast to compounds 5b and 8b which
inhibits pyocyanin and proteinase productions with different
potencies, AL-1 and compound 11b potently inhibit the produc-
tions of both pyocyanin and protease, the discrepancies between
these results remain to be elucidated; (4) The fact that AL-1, with
a disulfide bond, and compound 11b, without a disulfide bond, are
equally effective inhibiting the productions of pyocyanin and pro-
tease suggests that the disulfide bond plays insignificant role for
antibacterial activity. It is important to point out that AL-1, in
addition to its antibacterial and antiviral activity,19,20 also exhibits
2. Results and discussion
2.1. Chemistry
In this study, we designed new Andro derivatives (Scheme 1),
structurally similar to natural AHLs to develop QS inhibitors of
P. aeruginosa and biofilm formation. The LasI of P. aeruginosa pri-
marily catalyzes synthesis of 3-oxo-C12-HSL, and RhlI directs the
synthesis of C4-HSL. The length of acyl side chain and its substitu-
tions provide signal specificity.23 Thus, we coupled acyl side chains
with different length to Andro’s C14 position (3b–7b) to find out
whether substitution on the side chain making any difference to
their biological activity.
(E)-3-(Furan-2-yl) acrylic acid, adamantaneacetic acid and
(E)-3-cyclohexylacrylic acid were conjugated to Andro making
compounds 8b, 9b and 10b. This is to understand whether differ-
ent cyclic-substitution is important in biological activity. Our
previously synthesized AL-1 is superior to the natural Andro in
inhibiting QS of P. aeruginosa and biofilm formation,19,20 and is
recently found to significantly reduces the expression of two regu-
lators, LasI and RhlI in P. aeruginosa.20 In this report, we design and
synthesized compound 11b, similar to AL-1, but without the disul-
fide bond in the ring to investigate the biological functions of the
disulfide bond to its anti-QS activity.
2.2. Biological activity evaluations
We have previously found that Andro compounds did not di-
rectly inhibit bacterial growth.19,20 To find if the new compounds
directly inhibit bacterial growth or not, we used the zone of inhibi-
tion method to measure the inhibition of P. aeruginosa growth. We
found that the new compounds were not active against P. aerugin-
O
O
O
O
O
O
O
HO
HO
R
O
c
a
b
CH2
CH2
CH2
O
HO
HO
O
O
O
3a-11a
1 (Andro)
2
O
O
O
R=
R=
8a, 8b
3a, 3b R= CH2(CH2)9CH3
4a, 4b
5a, 5b R= CH2(CH2)3CH3
O
R
O
R= CH2(CH2)5CH3
9a, 9b
CH2
10a, 10b
11a, 11b
R=
R=
HO
HO
R= CH2(CH2)2CH3
R= CH=CHCH3
6a, 6b
7a, 7b
3b-11b
Scheme 1. Reagents and conditions: (a) 2,2-dimethoxypropane/benzene/DMSO/reflux/1 h; (b) RCOOH/DCM/ClCOOEt/NEt3/15–24 h; (c) AcOH/H2O/30 min.

Z. Wang et al. / Bioorg. Med. Chem. 18 (2010) 4269–4274
4271
0.5
0.45
0.4
*
*
0.35
0.3
*
*
*
*
*
*
*
0.25
0.2
*
*
0.15
0.1
0.05
0
Con Andro AL-1
3b
4b
5b
6b
7b
8b
9b
10b
11b
*
Figure 2. Inhibition of pyocyanin production in P. aeruginosa PAO1. Con, control; the concentration of all compounds was 0.5 mM. P < 0.05 compared to control group.
1.4
1.2
1
0.8
0.6
0.4
0.2
0
*
*
*
*
*
*
*
*
*
*
Con Andro AL-1
3b
4b
5b
6b
7b
8b
9b
10b
11b
*
Figure 3. Inhibition of protease production in P. aeruginosa PAO1. Con, control; the concentration of all compounds was 0.5 mM. P < 0.05 compared to control group.
significant anti-diabetic activity both in vitro and in experimental
animal model.27 In contrast, we found that compound 11b had
minimal activity in experimental diabetic animal model (unpub-
lished data). These results indicate that the disulfide bond of AL-
1 plays a critical role in its anti-diabetic activity, and AL-1 acts
through different mechanisms for its antimicrobial and anti-dia-
betic activity. We have recently found that AL-1 significantly sup-
pressed the gene expressions of LasI and RhlI in P. aeruginosa.20
were purchased from Alfa Aesar Company (Beijing). Silica gel
plates (Merck F254) were used for thin layer chromatography.
Dichloromethane was distilled over calcium hydride prior to use.
Chromatographic purification was performed with silica gel
(200–400 mesh).
4.1. 14-Lauroyl andrographolide (3b)
A stirred solution of the lauric acid (400 mg, 2.0 mmol) and tri-
ethylamine (0.63 mL, 4.58 mmol) in dry dichloromethane (16 mL)
was maintained at 0 °C under a nitrogen atmosphere. Ethyl chloro-
formate (0.33 mL, 3.36 mmol) was added, and the reaction mixture
was stirred for 1 h. Compound 228 (200 mg, 0.51 mmol) was added
dropwise to the reaction mixture, which was then stirred at room
temperature for 18 h. After quenching with water (30 mL), the or-
ganic layer was separated and the aqueous layer was extracted with
dichloromethane (3Â 10 mL). The combined organic extracts were
dried with Na2SO4 and were concentrated in vacuo. The product
was purified by column chromatography using ethyl acetate–petro-
leum ether (1:2), affording compound 3a as a colorless liquid
(150 mg, 51.2% yield). 1H NMR (CDCl3): 7.01 (t, J = 6.08 Hz, 1H),
5.92 (d, J = 6.0 Hz, 1H), 4.86 (s, 1H), 4.54 (m, J = 10 Hz, 1H), 4.48 (s,
1H), 4.18 (m, J = 3.6 Hz, 2H), 3.42 (t, J = 7.6 Hz, 2H), 3.28 (d,
J = 10.8 Hz, 1H), 2.48–2.30 (m, 5H), 2.0–1.95 (m, 1H), 1.88–1.74 (m,
4H), 1.74–1.54 (m, 3H), 1.38 (s, 3H), 1.34 (s, 3H), 1.28 (s, 3H), 0.92
(s, 3H), 0.86 (t, J = 6.6 Hz, 3H). MS (EI) [M+H]+ m/z 573.8. Withoutfur-
ther purification, compound 3a was added to a diluted acetic acid
solution (AcOH/H2O = 7:3, 10 mL), and the solution was stirred at
room temperature for 30 min. Water was added, and the solution
was neutralized with NaHCO3. The product was extracted with
dichloromethane, and the organic phase was dried over Na2SO4
and was then filtered. The product was purified by column chroma-
tography using ethyl acetate–petroleum ether (2:1), affording com-
pound 3b as a colorless liquid (54 mg, 38.7% yield). MS (EI) [M+H]+
m/z 533.9. 1H NMR (CDCl3): 7.01 (t, J = 6.08 Hz, 1H), 5.92 (d,
3. Conclusions
We have designed and synthesized a series of new Andro deriv-
atives as the AHL mimics, and tested them for inhibition of viru-
lence factors production. None of the compounds were active
against P. aeruginosa growth up to 2 mM concentration, but all of
them inhibited pyocyanin production and protease expression.
Compounds 5b, 8b, 11b and AL-1 almost completely suppressed
protease expression. The fact that compound 11b and AL-1 are al-
most equally potent inhibiting productions of both pyocyanin and
protease suggests that antioxidative activity of AL-1 is not impor-
tant for inhibition of virulence factors production in P. aeruginosa.
To explore the mechanism of action for this novel class of antibac-
terial agents, we are conducting further experiments, and will re-
port the results in due course.
4. Experimental
1H NMR spectra were recorded on a Bruker AV 400 spectrome-
ter at 400 MHz. Electrospray ionization mass spectra (ESI-MS)
were obtained on a Finnigan LCQ Advantage MAX mass spectrom-
eter (ABI company, 4000 Q TRAP). High-resolution mass spectra
(HRMS) were obtained on a JEOL JMS-700 mass spectrometer.
Lauric acid, octanoic acid, hexanoic acid, pentanoic acid, E-2-bute-
noic acid, (E)-3-(furan-2-yl) acrylic acid and adamantaneacetic acid

4272
Z. Wang et al. / Bioorg. Med. Chem. 18 (2010) 4269–4274
J = 6.0 Hz, 1H), 4.86 (s, 1H), 4.54 (m, J = 10 Hz, 1H), 4.48 (s, 1H), 4.18
(m, 2H), 3.42(t, J = 7.6 Hz, 2H), 3.28(d, J = 10.8 Hz, 1H), 2.48–2.30(m,
5H), 2.0–1.95 (m, 1H), 1.88–1.74 (m, 4H), 1.74–1.54 (m, 3H), 0.86 (t,
J = 6.6 Hz, 3H), 0.64 (s, 3H). HRMS (ESI, m/z) calcd for C32H52O6Na
555.3662, found 555.3659.
romethane, and the organic phase was dried over Na2SO4 and was
then filtered. The product was purified by column chromatography
using ethyl acetate–petroleum ether (2:1), affording compound 5b
(57.4 mg, 41.7% yield) as a colorless liquid. MS (EI) [M+Na]+ m/z
471.7. 1H NMR (CDCl3): 7.0 (t, J = 6.8 Hz, 1H), 5.93 (d, J = 6.0 Hz,
1H), 4.86 (s, 1H), 4.56 (m, 1H), 4.50 (s, 1H), 4.22–4.16 (m, 2H),
3.47 (m, 1H), 3.32 (d, J = 10.8 Hz, 1H), 2.48–2.34 (m, 5H), 1.35–
1.28 (m, 5H), 1.25 (s, 3H), 0.90 (t, J = 6.6 Hz, 3H), 0.67 (s, 3H). HRMS
(ESI, m/z) calcd for C26H40O6Na 471.2723, found 471.2717.
4.2. 14-Octanoyl andrographolide (4b)
A stirred solution of the octanoic acid (300 mg, 2.0 mmol) and
triethylamine (0.63 mL, 4.58 mmol) in dry dichloromethane
(16 mL) was maintained at 0 °C under a nitrogen atmosphere, ethyl
chloroformate (0.33 mL, 3.36 mmol) was added, and the reaction
mixture was stirred for 1 h. Compound 2 (200 mg, 0.51 mmol)
was added dropwise to the reaction mixture, which was stirred
at room temperature for 18 h. After quenching with water
(30 mL), the organic layer was separated, and the aqueous layer
was extracted with dichloromethane (3Â 10 mL). The combined
organic extracts were dried with Na2SO4 and concentrated in va-
cuo. The product was purified by column chromatography using
ethyl acetate–petroleum ether (1:2), affording compound 4a
(153.5 mg, 58% yield) as a colorless liquid. 1H NMR (CDCl3): 6.99
(t, J = 6.8 Hz, 1H), 5.92 (d, J = 6.0 Hz, 1H), 4.88 (s, 1H), 4.56–4.52
(m, 2H), 4.18 (dd, J = 2.0, 9.2 Hz, 2H), 3.93 (d, J = 11.6 Hz, 1H),
3.48 (m, 1H), 3.16 (d, J = 11.6 Hz, 1H), 2.45–2.37 (m, 2H), 2.34 (t,
J = 7.6 Hz, 2H), 2.02–1.93 (m, 2H), 1.80–1.58 (m, 6H), 1.38 (s, 3H),
1.34 (s, 3H), 1.18 (s, 3H), 0.92 (s, 3H), 0.87 (t, J = 6.8 Hz, 3H). MS
(EI) [M+H]+ m/z 517.8. Without further purification, compound
4a was added to a diluted acetic acid solution (AcOH/H2O = 7:3,
10 mL), and the solution was stirred at room temperature for
30 min. Water was added, and the solution was neutralized with
NaHCO3. The product was extracted with dichloromethane, and
the organic phase was dried over Na2SO4 and was then filtered.
The product was purified by column chromatography using ethyl
acetate–petroleum ether (2:1), affording compound 4b (53.5 mg,
37.9% yield) as a colorless liquid. MS (EI) [M+Na]+ m/z 499.7. 1H
NMR (CDCl3): 6.97 (t, J = 6.0 Hz, 1H), 5.90 (d, J = 6.0 Hz, 1H), 4.85
(s, 1H), 4.53 (m, 1H), 4.47 (s, 1H), 4.20–4.13 (m, 2H), 3.43 (t,
J = 8.0 Hz, 2H), 3.28 (d, J = 10.8 Hz, 1H), 2.41–2.31 (m, 5H), 1.21
(s, 3H), 0.86 (t, J = 6.8 Hz, 3H), 0.64 (s, 3H). HRMS (ESI, m/z) calcd
for C28H44O6Na 499.3036, found 499.3033.
4.4. 14-Valeryl andrographolide (6b)
A stirred solution of the pentanoic acid (0.23 mL, 2.0 mmol) and
triethylamine (0.63 mL, 4.58 mmol) in dry dichloromethane
(16 mL) was maintained at 0 °C under a nitrogen atmosphere. Ethyl
chloroformate (0.33 mL, 3.36 mmol) was added and the reaction
mixture was stirred for 1 h. Compound 2 (200 mg, 0.51 mmol)
was added dropwise to the reaction mixture, which was stirred
at room temperature for 20 h. After quenching with water
(30 mL), the organic layer was separated and the aqueous layer
was extracted with dichloromethane (3Â 10 mL). The combined
organic extracts were dried with Na2SO4 and were then concen-
trated in vacuo. The product was purified by column chromatogra-
phy using ethyl acetate–petroleum ether (1:2), affording
compound 6a (126.4 mg, 51.8% yield) as a colorless liquid. 1H
NMR (CDCl3): 6.98 (t, J = 6.8 Hz, 1H), 5.91 (d, J = 6.0 Hz, 1H), 4.87
(s, 1H), 4.55–4.51 (m, 2H), 4.18 (dd, J = 2.0, 9.2 Hz, 1H), 3.92 (d,
J = 11.6 Hz, 1H), 3.46 (m, 1H), 3.15 (d, J = 11.6 Hz, 1H), 3.28 (d,
J = 10.8 Hz, 1H), 2.49–2.28 (m, 5H), 1.37 (s, 3H), 1.33 (s, 3H), 1.17
(s, 3H), 1.27–1.21 (m, 3H), 0.92–0.88 (m, 6H). MS (EI) [M+H]+ m/z
475.7. Without further purification, compound 6a was added to a
diluted acetic acid solution (AcOH/H2O = 7:3, 10 mL), and the solu-
tion was stirred at room temperature for 30 min. Water was added,
and the solution was neutralized with NaHCO3. The product was
extracted with dichloromethane, and the organic phase was dried
over Na2SO4 and was then filtered. The product was purified by
column chromatography using ethyl acetate–petroleum ether
(2:1), affording compound 6b (48.96 mg, 42.57% yield) as a color-
less liquid. MS (EI) [M+Na]+ m/z 457.7. 1H NMR (CDCl3): 7.0 (t,
J = 6.8 Hz, 1H), 5.92 (d, J = 6.0 Hz, 1H), 4.88 (s, 1H), 4.55 (m, 1H),
4.50 (s, 1H), 4.22–4.16 (m, 2H), 3.48 (m, 1H), 3.33 (d, J = 11.2 Hz,
1H), 2.44–2.34 (m, 5H), 2.04–1.95 (m, 9H), 1.38–1.21 (m, 8H),
0.92 (t, J = 7.2 Hz, 3H), 0.66 (s, 3H). HRMS (ESI, m/z) calcd for
C25H38O6Na 457.2561, found 457.2566.
4.3. 14-Caproyl andrographolide (5b)
A stirred solution of the hexanoic acid (0.26 mL, 2.0 mmol) and
triethylamine (0.63 mL, 4.58 mmol) in dry dichloromethane
(16 mL) was maintained at 0 °C under a nitrogen atmosphere, ethyl
chloroformate (0.33 mL, 3.36 mmol) was added, and the reaction
mixture was stirred for 1 h. Compound 2 (200 mg, 0.51 mmol)
was added dropwise to the reaction mixture, which was stirred
at room temperature for 20 h. After quenching with water
(30 mL), the organic layer was separated and the aqueous layer
was extracted with dichloromethane (3Â10 mL). The combined or-
ganic extracts were dried with Na2SO4 and were then concentrated
in vacuo. The product was purified by column chromatography
using ethyl acetate–petroleum ether (1:2), affording compound
5a (150.1 mg, 60% yield) as a colorless liquid. 1H NMR (CDCl3):
7.03 (t, J = 6.08 Hz, 1H), 5.94 (d, J = 6.0 Hz, 1H), 4.91 (s, 1H), 4.59–
4.55 (m, 2H), 4.22 (dd, J = 2.0, 9.2 Hz, 1H), 4.13 (dd, J = 7.2, 7.2 Hz,
2H), 3.96 (d, J = 11.6 Hz, 1H) 3.51 (m, 1H), 3.19 (d, J = 11.6 Hz,
1H), 2.47–2.35 (m, 5H), 1.88–1.74 (m, 4H), 1.74–1.54 (m, 3H),
1.42 (s, 3H), 1.37 (s, 3H), 1.21 (s, 3H), 0.95 (s, 3H), 0.92 (t,
J = 7.2 Hz, 3H). MS (EI) [M+H]+ m/z 489.7. Without further purifica-
tion, compound 5a was added to a diluted acetic acid solution
(AcOH/H2O = 7:3, 10 mL), and the solution was stirred at room
temperature for 30 min. Water was added, and the solution was
neutralized with NaHCO3. The product was extracted with dichlo-
4.5. 14-(E-2-Crotonoyl) andrographolide (7b)
A stirred solution of the E-2-butenoic acid (180 mg, 2.0 mmol)
and triethylamine (0.63 mL, 4.58 mmol) in dry dichloromethane
(16 mL) was maintained at 0 °C under a nitrogen atmosphere. Ethyl
chloroformate (0.33 mL, 3.36 mmol) was added and the reaction
mixture was stirred for 1 h. Compound 2 (200 mg, 0.51 mmol) was
added dropwise to the reaction mixture, which was stirred at room
temperature for 20 h. After quenching with water (30 mL), the or-
ganic layer was separated and the aqueous layer was extracted with
dichloromethane (3Â 10 mL). The combined organic extracts were
driedwithNa2SO4 andwere thenconcentratedinvacuo. Theproduct
was purified by column chromatography using ethyl acetate–petro-
leum ether (1:2), affording compound 7a (100 mg, 42.59% yield) as a
colorless liquid. 1H NMR (CDCl3): 7.05 (t, J = 6.8 Hz, 1H), 6.0 (d,
J = 6.0 Hz, 1H), 5.8 (dd, J = 1.6, 14 Hz, 1H), 4.9 (s, 1H), 4.58 (dd,
J = 4.8, 6.4 Hz, 1H), 4.55 (s, 1H), 4.26 (dd, J = 2.0, 9.2 Hz, 1H), 3.97
(d, J = 3.6 Hz, 1H), 3.5 (dd, J = 4.0, 4.8 Hz, 2H), 3.18 (d, J = 11.6 Hz,
1H), 2.5–2.40 (m, 5H), 2.04–1.95 (m, 2H), 1.88–1.6 (m, 6H), 1.74–
1.54 (m, 3H), 1.42 (s, 3H), 1.38 (s, 3H), 1.21 (s, 3H), 0.94 (s, 3H). MS
(EI) [M+Na]+ m/z 481.7. Without further purification, compound 7a

Z. Wang et al. / Bioorg. Med. Chem. 18 (2010) 4269–4274
4273
was added to a diluted acetic acid solution (AcOH/H2O = 7:3, 10 mL),
and the solution was stirred at room temperature for 30 min. Water
was added, and the solution was neutralized with NaHCO3. The
product was extracted with dichloromethane, and the organic phase
was dried over Na2SO4 and was then filtered. The product was puri-
fied by column chromatography using ethyl acetate–petroleum
ether (2:1), affording compound 7b (37 mg, 40.54% yield) as a color-
less liquid. MS (EI) [M+Na]+ m/z 441.6. 1H NMR (CDCl3): 7.05 (t,
J = 6.08 Hz, 1H), 5.99 (d, J = 6.0 Hz, 1H), 5.87 (dd, J = 1.6, 14 Hz, 1H),
4.88 (s, 1H), 4.58 (dd, J = 5.2, 6.0 Hz, 1H), 4.52 (s, 1H), 4.26 (dd,
J = 2.0, 9.2 Hz, 1H), 4.18 (d, J = 11.2 Hz, 1H), 3.49 (m, 1H), 3.33 (d,
J = 11.2 Hz, 1H), 2.51–2.34 (m, 3H), 1.25–1.10 (s, 7H), 0.9–0.8 (m,
5H), 0.67 (s, 3H). HRMS (ESI, m/z) calcd for C24H34O6Na 441.2253,
found 441.2248.
matography using ethyl acetate–petroleum ether (1:2), affording
compound 9a (110 mg, 38% yield) as a colorless liquid. 1H NMR
(CDCl3): 7.05 (t, J = 6.08 Hz, 1H), 5.90 (d, J = 6.0 Hz, 1H), 4.91 (s,
1H), 4.55 (m, J = 10 Hz, 1H), 4.45 (s, 1H), 4.19 (m, J = 3.6 Hz, 2H),
3.40 (t, J = 7.6 Hz, 2H), 3.25 (d, J = 10.8 Hz, 1H), 2.40–2.30 (m, 5H),
1.9–1.70 (m, 5H), 1.70–1.55 (m, 3H), 1.38 (s, 3H), 1.34 (s, 3H), 1.28
(s, 3H), 0.92 (s, 3H). MS (EI) [M+H]+ m/z 567.4. Without further puri-
fication, compound 9a was added to a diluted acetic acid solution
(AcOH/H2O = 7:3, 10 mL), and the solution was stirred at room tem-
perature for 30 min. Water was added, and the solution was neutral-
ized with NaHCO3. The product was extracted with dichloro
methane, and the organic phase was dried over Na2SO4 and was then
filtered. The product was purified by column chromatography using
ethyl acetate–petroleum ether (2:1), affording compound 9b
(27 mg, 26.5% yield) as a colorless liquid. MS (EI) [M+Na]+ m/z
549.6. 1H NMR (CDCl3): 7.03 (t, J = 6.11 Hz, 1H), 6.01 (d, J = 5.7 Hz,
1H), 4.85 (s, 1H), 4.54 (m, J = 9.8 Hz, 1H), 4.48 (s, 1H), 4.18 (m,
J = 4.0 Hz, 2H), 3.42 (t, J = 7.3 Hz, 2H), 3.28 (d, J = 9.8 Hz, 1H), 2.48–
2.30 (m, 5H), 2.0–1.95 (m, 1H), 1.88–1.74 (m, 4H), 1.74–1.54 (m,
3H), 0.64 (s, 3H). HRMS (ESI, m/z) calcd for C32H46O6Na 549.3192,
found 549.3186.
4.6. 14-(E-3-Furan-2-yl-acryloyl) andrographolide (8b)
A stirred solution of the (E)-3-(furan-2-yl) acrylic acid (283 mg,
2.0 mmol) and triethylamine (0.63 mL, 4.58 mmol) in dry dichloro-
methane (16 mL) was maintained at 0 °C under a nitrogen atmo-
sphere. Ethyl chloroformate (0.33 mL, 3.36 mmol) was added and
the reaction mixture was stirred for 1 h. Compound 2 (200 mg,
0.51 mmol) was added dropwise to the reaction mixture, which
was stirred at room temperature for 20 h. After quenching with
water (30 mL), the organic layer was separated and the aqueous
layer extracted with dichloromethane (3Â 10 mL). The combined
organic extracts were dried with Na2SO4 and concentrated in va-
cuo. The product was purified by column chromatography using
ethyl acetate–petroleum ether (1:2), affording compound 8a
(112.3 mg, 43% yield) as a yellow liquid. 1H NMR (CDCl3): 7.53 (s,
1H), 7.46 (d, J = 12.4 Hz, 1H), 7.06 (t, J = 5.6 Hz, 1H), 6.69 (d,
J = 2.8 Hz, 1H), 6.51 (m, 1H), 6.3 (d, J = 12.8 Hz, 1H), 6.05 (d,
J = 3.2 Hz, 1H), 4.89 (s, 1H), 4.66 (dd, J = 4.0, 4.8 Hz, 1H), 4.57 (s,
1H), 4.30 (dd, J = 1.2, 7.6 Hz, 1H), 3.95 (d, J = 9.2 Hz, 1H), 3.49 (m,
1H), 3.46 (s, 1H), 3.16 (d, J = 9.6 Hz, 1H), 2.52–2.39 (m, 3H), 2.04–
1.68 (m, 8H), 1.39 (s, 3H), 1.36 (s, 3H), 1.20 (s, 3H), 1.31–1.23 (m,
5H), 0.92 (s, 3H). MS (EI) [M+Na]+ m/z 533.6. Without further puri-
fication, compound 8a was added to a diluted acetic acid solution
(AcOH/H2O = 7:3, 10 mL), and the solution was stirred at room
temperature for 30 min. Water was added, and the solution was
neutralized with NaHCO3. The product was extracted with dichlo-
romethane, and the organic phase was dried over Na2SO4 and was
then filtered. The product was purified by column chromatography
using ethyl acetate–petroleum ether (2:1), affording compound 8b
(47.6 mg, 46% yield) as a yellow power. MS (EI) [M+H]+ m/z 471.6.
1H NMR (CDCl3): 7.50 (s, 1H), 7.43 (d, J = 12.1 Hz, 1H), 7.01 (t,
J = 5.8 Hz, 1H), 6.69 (d, J = 2.8 Hz, 1H), 6.49 (m, 1H), 6.25 (d,
J = 12.3 Hz, 1H), 6.15 (d, J = 3.5 Hz, 1H), 4.92 (s, 1H), 4.61 (dd,
J = 4.0, 4.8 Hz, 1H), 4.59 (s, 1H), 4.30 (dd, J = 1.2, 7.6 Hz, 1H), 3.98
(d, J = 9.2 Hz, 1H), 3.45 (s, 1H), 3.18 (d, J = 10 Hz, 1H), 2.07–1.65
(m, 6H), 1.26 (s, 3H), 1.35–1.20 (m, 7H), 0.89 (s, 3H). HRMS (ESI,
m/z) calcd for C23H33O7 469.2206, found 469.2263.
4.8. 14-(E-3-Cyclohexylacryloyl) andrographolide (10b)
A stirred solution of the (E)-3-cyclohexylacrylic acid29 (315 mg,
2.0 mmol) and triethylamine (0.63 mL, 4.58 mmol) in dry dichloro-
methane (16 mL) was maintained at 0 °C under a nitrogen atmo-
sphere. Ethyl chloroformate (0.33 mL, 3.36 mmol) was added and
the reaction mixture was stirred for 1 h. Compound 2 (200 mg,
0.51 mmol) was added dropwise to the reaction mixture, which
was stirred at room temperature for 24 h. After quenching with
water (30 mL), the organic layer was separated and the aqueous
layer was extracted with dichloromethane (3Â 10 mL). The com-
bined organic extracts were dried with Na2SO4 and were then con-
centrated in vacuo. The product was purified by column
chromatography using ethyl acetate–petroleum ether (1:2), afford-
ing compound 10a (113 mg, 43% yield) as a colorless liquid. 1H
NMR (CDCl3): 7.04 (t, J = 6.12 Hz, 1H), 6.0 (d, J = 4.8 Hz, 1H), 5.78
(dd, J = 1.2, 11.6 Hz, 1H), 4.89 (s, 1H), 4.60 (dd, J = 4.0, 4.8 Hz,
1H), 4.52 (s, 1H), 4.28 (dd, J = 1.6, 7.2 Hz, 1H), 4.0 (d, J = 9.6 Hz,
1H), 3.50 (m, 1H), 3.15 (d, J = 9.2 Hz, 1H), 2.48–2.42 (m, 4H), 1.43
(s, 3H), 1.38 (s, 3H), 1.32–1.27 (m, 9H), 1.22 (s, 3H), 0.93 (s, 3H).
MS (EI) [M+H]+ m/z 527.7. Without further purification, compound
10a was added to a diluted acetic acid solution (AcOH/H2O = 7:3,
10 mL), and the solution was stirred at room temperature for
30 min. Water was added, and the solution was neutralized with
NaHCO3. The product was extracted with dichloromethane, and
the organic phase was dried over Na2SO4 and was then filtered.
The product was purified by column chromatography using ethyl
acetate–petroleum ether (2:1), affording compound 10b
(51.2 mg, 49% yield) as a colorless liquid. MS (EI) [M+H]+ m/z
487.6. 1H NMR (CDCl3): 7.01 (t, J = 6.10 Hz, 1H), 5.96 (d,
J = 5.2 Hz, 1H), 5.77 (dd, J = 1.2, 11.6 Hz, 1H), 4.86 (s, 1H), 4.58
(dd, J = 4.0, 4.8 Hz, 1H), 4.49 (s, 1H), 4.26 (dd, J = 1.6, 7.2 Hz, 1H),
4.17 (d, J = 8.8 Hz, 1H), 3.47 (m, 1H), 3.31 (d, J = 8.8 Hz, 1H),
2.44–2.37 (m, 4H), 2.2–2.15 (m, 1H), 2.0–1.95 (m, 1H), 1.35–1.27
(m, 4H), 1.25 (s, 3H), 0.91–0.79 (m, 2H), 0.64 (s, 3H). HRMS (ESI,
m/z) calcd for C29H41O6 485.2881, found 485.2903.
4.7. 14-Adamantaneacetyl andrographolide (9b)
A
stirred solution of the adamantaneacetic acid (400 mg,
2.0 mmol) and triethylamine (0.63 mL, 4.58 mmol) in dry dichloro-
methane (16 mL) was maintained at 0 °C under a nitrogen atmo-
sphere. Ethyl chloroformate (0.33 mL, 3.36 mmol) was added and
the reaction mixture was stirred for 1 h. Compound 2 (200 mg,
0.51 mmol) was added dropwise to the reaction mixture, which
was stirred at room temperature for 20 h. After quenching with
water (30 mL), the organic layer was separated and the aqueous
layer was extracted with dichloromethane (3Â 10 mL). The com-
bined organic extracts were dried with Na2SO4 and were then con-
centrated in vacuo. The product was purified by column chro
4.9. 14-(5-Cyclopentylvaleryl) andrographolide (11b)
A stirred solution of the 5-cyclopentylpentanoic acid30 (350 mg,
2.0 mmol) and triethylamine (0.63 mL, 4.58 mmol) in dry dichloro-
methane (16 mL) was maintained at 0 °C under a nitrogen atmo-
sphere. Ethyl chloroformate (0.33 mL, 3.36 mmol) was added and
the reaction mixture was stirred for 1 h. Compound 2 (200 mg,

4274
Z. Wang et al. / Bioorg. Med. Chem. 18 (2010) 4269–4274
0.51 mmol) was added dropwise to the reaction mixture, which
was stirred at room temperature for 24 h. After quenching with
water (30 mL), the organic layer was separated and the aqueous
layer was extracted with dichloromethane (3Â 10 mL). The com-
bined organic extracts were dried with Na2SO4 and concentrated
in vacuo. The product was purified by column chromatography
using ethyl acetate–petroleum ether (1:2), affording compound
11a (132 mg, 47.6% yield) as a colorless liquid. MS (EI) [M+H]+
m/z 543.7. 1H NMR (CDCl3): 7.02 (t, J = 6.10 Hz, 1H), 5.96 (d,
J = 5.2 Hz, 1H), 4.91 (s, 1H), 4.59–4.55 (m, 2H), 4.22 (dd, J = 1.6,
7.2 Hz, 1H), 3.97 (d, J = 9.2 Hz, 1H), 3.50 (m, 1H), 3.18 (d,
J = 9.2 Hz, 1H), 2.45–2.36 (m, 4H), 2.25–2.14 (m, 3H), 2.07–1.97
(m, 3H), 1.87–1.82 (m, 4H), 1.71–1.60 (m, 7H), 1.32–1.22 (m,
7H), 1.38 (s, 3H), 1.34 (s, 3H), 1.28 (s, 3H), 0.92 (s, 3H). Without fur-
ther purification, compound 11a was added to a diluted acetic acid
solution (AcOH/H2O = 7:3, 10 mL), and the solution was stirred at
room temperature for 30 min. Water was added, and the solution
was neutralized with NaHCO3. The product was extracted with
dichloromethane, and the organic phase was dried over Na2SO4
and was then filtered. The product was purified by column chro-
matography using ethyl acetate–petroleum ether (2:1), affording
compound 11b (61 mg, 50% yield) as a colorless liquid. MS (EI)
[M+H]+ m/z 503.6. 1H NMR (CDCl3): 7.03 (t, J = 5.20 Hz, 1H), 5.94
(d, J = 5.2 Hz, 1H), 4.90 (s, 1H), 4.58 (dd, J = 4.4, 4.8 Hz, 1H), 4.52
(s, 1H), 4.26 (dd, J = 1.6, 7.2 Hz, 1H), 4.20 (d, J = 8.8 Hz, 1H), 3.50
(m, 1H), 3.2 (d, J = 8.8 Hz, 1H), 2.45–2.36 (m, 4H), 2.25–2.14 (m,
3H), 2.07–1.97 (m, 3H), 1.87–1.82 (m, 4H), 1.71–1.60 (m, 7H),
1.27 (s, 3H), 0.68 (s, 3H). HRMS (ESI, m/z) calcd for C30H45O6
501.3199, found 501.3216.
Acknowledgements
This work was supported in part by grants from the China
Natural Science Fund (30772642 and 30973618 to YW), The
Guangdong Province (2008A030101007 to YW) and Guangzhou
City (2008Z1-E691 to PY) Science and Technology Funds as well
as the 211 Project of Jinan University. The authors thank Professor
Lixin Zhang of the Institute of Microbiology, Chinese Academy of
Sciences for his critical and valuable comments and Ms. Linda
Wang of Yale University for her proofreading the manuscript.
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