Synthesis and preliminary antihyperlipidaemic activities evaluation of andrographolide derivatives

Article abstract of DOI:10.2174/157340612800493629

Recent studies indicated that andrographolide was a potential antihyperlipidaemic therapeutic agent. In the paper, the synthesis of a series of andrographolide derivatives was described and their antihyperlipidaemic activities were evaluated in vivo. As compared with TG, TC, HDL-C and LDL-C concentrations, some of the derivatives exhibited better antihyperlipidaemic effects than positive control atromide. Therein, compound 6i, which was the most potent compound, could serve as a new lead for further development of antihyperlipidaemic agents.

Full text of DOI:10.2174/157340612800493629

Medicinal Chemistry, 2012, 8, 293-298
293
Synthesis and Preliminary Antihyperlipidaemic Activities Evaluation of
Andrographolide Derivatives
Bin Wanga,b,#, Chunlei Tanga,#, Yaodan Hana, Ruzhou Guoa, Hai Qiana,* and Wenlong Huanga,*
a
Center of Drug Discovery, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
bTechnology Center, Jiangsu Yabang Pharmaceutical Group, Changzhou 213163, China
Abstracts: Recent studies indicated that andrographolide was a potential antihyperlipidaemic therapeutic agent. In the pa-
per, the synthesis of a series of andrographolide derivatives was described and their antihyperlipidaemic activities were
evaluated in vivo. As compared with TG, TC, HDL-C and LDL-C concentrations, some of the derivatives exhibited
better antihyperlipidaemic effects than positive control atromide. Therein, compound 6i, which was the most potent com-
pound, could serve as a new lead for further development of antihyperlipidaemic agents.
Keywords: Andrographolide, antihyperlipidaemic activities, derivatives, synthesis.
INTRODUCTION
Swiss albino mice were 42% (100 mg/kg·d) and 62% (500
mg/kg·d), respectively. And for 14-cinnamoyl-3,19-dihy-
droxy-8,17- epoxy andrographolide, percentage reduction of
the plasma triglyceride (TG) of male Swiss albino mice were
42% (100 mg/kg·d), 52% (250 mg/kg·d) and 62% (500
mg/kg·d), respectively [10, 11]. It indicated that Andro de-
rivatives benefit the antihyperlipidaemic treatment and could
be regarded as an excellent lead compound.
Cholesterol plays a major role in the assembly of mem-
branes and performs other important biological functions in
human heart health. However, excess plasma cholesterol
leads to the development of hyperlipidemia and atheroscle-
rotic cardiovascular disease such as coronary heart disease
and stroke, which are among the leading causes of death in
many countries.The adverse effects on treating hyperlipide-
mia urged the search of new antihyperlipidaemic agents and
targets [1-3]. Natural products are important sources of new
drugs and leads with antihyperlipidaemic effects such as
Artichoke, Green tea, Guggul, Korean ginseng and Fenu-
greek, inspiring and motivating researchers to look for new
drugs from plants and natural sources [4, 5].
In our previous studies, isoandrographolide (Isoandro, 2),
which is much more stable than Andro while has similar
biological activities to Andro, such as anti-inflammatory,
anticancer and anti-HIV properties [12-14], was chosen as
the lead generation and the derivatives were synthesized.
Isoandro and the derivatives have showed the inhibitory ac-
tivities on LPS-induced TNF-ꢁ, IL-6 and COX-2 expression
in mouse macrophages, therein, 19-O-triphenylmethyl iso-
andrographolide displayed the best activity, suggesting that it
could be taken as an optimized lead generation for further
biological activities studies [13]. Clofibric acid (4-chloro-
phenyloxyisobutyric acid) is the active metabolite of the
blood lipid regulators clofibrate, etofibrate, and etofyllin-
clofibrate, which can lower elevated serum lipids by reduc-
ing the very lowdensity lipoprotein fraction [15]. In this
study, we disclose the procedures for the synthesis of a series
of derivatives by the combination of 19-O-triphenylmethyl
isoandrographolide and substituted phenyloxyisobutyric acid
with the antihyperlipidaemic activities evaluated in vivo.
Andrographolide (Andro, 1) is a bicyclic diterpenoid lac-
tone isolated as the major active constituent from leaves of
Andrographis paniculata, a traditional herbal medicine used
for centuries in China, India for the treatment of various dis-
eases such as respiratory infection, acute gastroenteritis, lar-
yngitis, bacterial dysentery and diarrhea [6]. Andro was
known to possess a wide spectrum of bioactivities such as
anti-inflammatory, antibacterial, immunomodulatory and etc
[7].
Andro exerts its anti-apoptotic potential via activation of
the Akt-BAD pathway in HUVECs and thus may represent a
candidate of therapeutic agent for atherosclerosis, in which
the NF-ꢀB/IꢀB regulatory system plays a key role [8, 9]. In
vivo study showed that after being administered orally at 30
to 500 mg/kg·d dose of 3,19-diacetyl-14-(2'-acetyl-3'-
acetamido-3'-phenyl)propionyl andrographolide for 6 days,
percentage reduction of the plasma triglyceride (TG) of male
RESULTS AND DISCUSSION
Synthesis
The synthesis of these derivatives is summarized in
Scheme 1. Substituted phenol 3 was treated with sodium
hydroxide in acetone at room temperature and then chloro-
form was added to afford substituted phenyloxyisobutyric
acid 4 in 67% yield. As our previous studies [13], Andro 1
was treated with cons. hydrochloric acid at room temperature
to afford Isoandro 2 in 70% yield. Treatment of 2 with
#These authors contributed equally to this work.
ꢀAddress correspondence to these authors at the School of Pharmacy, China
Pharmaceutical University, Nanjing 210009, People’s Republic of China;
Tel: +86-(0) 25-83271302; Fax: +86-(0) 25-83271297; E-mail: qian-
hai24@163.com; Tel: +86-(0)25-83271480; Fax: +86-(0)25-83271480;
E-mail: ydhuangwenlong@126.com.
1875-6638/12 $58.00+.00
© 2012 Bentham Science Publishers

294 Medicinal Chemistry, 2012, Vol. 8, No. 2
Wang et al.
O
OH
O
a
OH
R
R
3
4
O
O
O
O
O
HO
O
O
O
d
c
b
O
O
O
O
4
H
H
H
H
O
O
HO
HO
HO
R
O
HO
HO
O
5
1
2
6a-k
6h: R=4-Br
6i: R=4-NO2
6j: R=4-tBu
6k:
6a; R=H
6b: R=2-CH3
6c: R=3-CH3
6d: R=4-CH3
6e: R=2-OCH3
6f: R=4-F
R
=
6g; R=4-Cl
Scheme 1. a) 1) NaOH/acetone; 2) 1 mol/L CHCl3; 3) dilute HCl; b) Cons. HCl; c) TrCl, N-Methylmorpholine, CH2Cl2, rt; d) EDC·HCl,
DMAP, CH2Cl2, rt.
triphenylchloromethane (TrCl) in the presence of N-
methylmorpholine in CH2Cl2 at room temperature afforded
trityl ether 5 in 80% yield. Compound 5 was reacted with
different substituted phenyloxyisobutyric acid 4 and 1-(3-
its increased penetration of arterial walls [16]. For conven-
ience, the percentage changes in serum TG, TC, HDL-C and
LDC-C of andro and its derivatives compared with the fat
control group were obtained and summarized in Table 1. By
comparison of 6a, 6b, 6c and 6d, which significantly low-
ered the serum TC by 7.85%, 5.24%, -3.14% and 17.28%,
and serum LDL-C by 5.36%, 12.50%, 8.93% and 16.07%,
respectively, it was very obvious that 6d exhibited best lipid-
lowering activities. It indicates that substitution at position 4
of the benzene ring was better than position 2 and 3. By con-
trast with 6d, 6g, 6h, 6i and 6j, which significantly lowered
the serum TC by 17.28%, 2.62%, 6.81%, 14.66% and 5.24%,
and serum LDL-C by 16.07%, 0.00%, 8.93%, 28.57% and
10.71%, respectively, It is quite clear that 6i exhibited best
lipid-lowering activities and the preliminary activity se-
quence decressed in the following order: 6i>6d>6j>6h>6g.
It indicates that electron withdrawing substituents (such as
nitro group) of position 4 of the benzene ring were beneficial
to increasing lipid-lowering activities. As to the halogen sub-
stituents of position 4 of the benzene ring, it decressed in the
following order: 4-Br>4-Cl>4-F.
dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
(EDC·HCl) in the presence of DMAP in CH2Cl2 at room
temperature afforded 6a-k.
In vivo Activities
Andro and its derivatives were tested for the antihyper-
lipidaemic activities with Male SD rats which were intragas-
trically given fat emulsion to afford hyperlipidemia and then
given the corresponding drug (160 mg/kg·d) for 7 days.
Blood samples were collected via carotid artery, centrifuged
and analyzed for serum total triglyceride (TG), cholesterol
(TC), high-density lipoprotein cholesterol (HDL-C), and
low-density lipoprotein cholesterol (LDL-C), respectively,
on an Olympus AU 400 automatic biochemistry analyzer.
The results of TG, TC, HDL-C and LDL-C of Andro and its
derivatives were summarized in Table 1. Serum TC and
LDL-C were significantly lowered by Andro and most of its
derivatives compared with the fat control group. The positive
control atromide also lowered serum TC and LDL-C signifi-
cantly. Serum HDL-C was increased by some Andro deriva-
tives.
It was very obvious that compounds 1, 6d and 6i signifi-
cantly lowered the serum TC by 19.90%, 17.28%, and
14.66% and serum LDL-C by 28.57%, 16.07% and 28.57%,
respectively, which were already close to normal control
group and considered a beneficiary effect in the treatment of
dyslipidemia condition. These groups were comparable with
standard drug atromide at the same dose of 160 mg/kg body
weight decreased the levels of serum TC by 17.28% and
serum LDL-C by 14.29%. The lipid-lowering effect of other
As it is known, elevated levels of serum TG and LDL-C,
accompanied by reduced HDL-C levels, are often associated
with an increased risk of coronary heart disease. In particu-
lar, research shows that LDL to be the most dangerous
among the serum lipids, and the oxidation of LDL leads to

Synthesis and Preliminary Antihyperlipidaemic Activities Evaluation
Medicinal Chemistry, 2012, Vol. 8, No. 2 295
Table 1. The Assay Data of Antihyperlipidaemic Activities for Andro and its Derivatives
Compd.a
TGe (%f)
TCe(%f)
HDL-Ce(%f)
LDL-Ce (%f)
1
0.47±0.09(+6.82%)
0.47±0.04*(+6.82%)
0.49±0.10(+11.36%)
0.44±0.08(0)
1.53±0.24**(-19.90%)
1.74±0.27**(-8.90%)
1.67±0.26**(-12.57%)
1.76±0.23**(-7.85%)
1.81±0.10(-5.24%)
1.97±0.09(+3.14%)
1.58±0.22*(-17.28%)
1.78±0.25**(-6.81%)
2.13±0.32**(+11.52%)
1.86±0.15(-2.62%)
1.78±0.14(-6.81%)
1.63±0.17(-14.66%)
1.81±0.26**(-5.24%)
1.87±0.27**(-2.09%)
1.39±0.09* (-27.23%)
1.58±0.20*(-17.28%)
1.91±0.19
1.03±0.14(-13.45%)
1.20±0.18(+0.84%)
1.08±0.20(-9.24%)
1.14±0.14(-4.20%)
1.19±0.09(0)
0.40±0.12*(-28.57%)
0.42±0.10(-25.00%)
0.50±0.10**(-10.71%)
0.53±0.12(-5.36%)
0.49±0.09(-12.50%)
0.51±0.09(-8.93%)
0.47±0.12*(-16.07%)
0.47±0.10(-16.07%)
0.68±0.09(+21.43%)
0.56±0.08(0)
2
5
6a
6b
0.39±0.05**(-11.36%)
0.39±0.07(-11.36%)
0.45±0.07(+2.27%)
0.43±0.05*(-2.27%)
0.47±0.04**(+6.82%)
0.52±0.11(+18.18%)
0.42±0.04**(-4.55%)
0.42±0.05*(-4.55%)
0.48±0.06(+9.09%)
0.44±0.05*(0)
6c
1.34±0.10(+12.61%)
0.97±0.17(-18.49%)
1.20±0.15(+0.84%)
1.29±0.21*(+8.40%)
1.19±0.09(0)
6d
6e
6f
6g
6h
1.17±0.08(-1.68%)
1.08±0.10(-9.24%)
1.18±0.17(-0.84%)
1.28±0.19(+7.56%)
1.01±0.10(-15.13%)
1.05±0.13(-11.76%)
1.19±0.16
0.51±0.08(-8.93%)
0.40±0.07*(-28.57%)
0.50±0.10**(-10.71%)
0.45±0.08(-19.64%)
0.37±0.06* (-33.93%)
0.48±0.09(-14.29%)
0.56±0.11
6i
6j
6k
NCb
Atromidec
FCd
0.50±0.13* (+13.64%)
0.54±0.08(+22.73%)
0.44±0.06
aEach value represents means ± SD from 8 rats. bNormal control group. cPositive control group.dFat control group.
emmol/L serum. fThe percentage changes in serum TG, TC, HDL-C and LDL-C of andro and its derivatives compared with the fat control group. Calculation formula: (Vn-
*
V
FC)/VFCꢀ100% (Vn stands for TG, TC, HDL-C or LDL-C value of one of the compounds,). P<0.05, **P<0.01 vs FC (assessed by one-way ANOVA with Bonferroni’s Multiple
Comparison Test).
Andro derivatives was modest when compared to 1, 6d and
6i derivatives.
F254 plates with separated compounds visualized by iodine
vapour. Melting points (uncorrected) were determined on a
RY-1 MP apparatus (Tianjin Analytical Apparatus Corp,
Tianjin, China). Mass spectra were obtained on
a
CONCLUSION
HP1100LC/MSD mass spectrometer. 1H-NMR and 13C-
NMR spectra were recorded in CDCl3 or DMSO-d6 on a
Brucker ACF-300 MHz spectrometer with TMS as internal
standard. IR spectra were detected on a Nicolet Impact 410
IR spectrometer.
In summary, a series of Andro derivatives have been syn-
thesized and evaluated for antihyperlipidaemic activities in
vivo. Some of the derivatives exhibited better antihyperlipi-
daemic effects than or equal to positive control atromide
with the aid of analyzing the concentrations of TG, TC,
HDL-C and LDL-C, respectively; especially compound 6i
was the most potent compound, which raised a challenge for
lead optimization based on the structure of 19-O-
triphenylmethyl isoandrographolide and could serve as a
new lead for further development of antihyperlipidaemic
agents. A closure look into the structure activity relationship
indicates that the substitution at position 4 and with electron
withdrawing is benefit to the antihyperlipidaemic activities.
Further studies on biological evaluation, structure activity
relationship and mechanism of this new class of compounds
are currently in progress and the results will be reported in
due course.
Chemistry
Andro (1 >98 %) was purchased from Zelang Medical
Technology Co., Ltd (Nanjing, China) and was further puri-
fied by recrystallization from ethanol before use. All other
reagents were of AR grade and used without any further pu-
rification.
Preparation of Substituted Phenyloxyisobutyric Acid (4)
To a solution of sodium hydroxide (4.0 g, 100.0 mmol) in
acetone (20 ml) at room temperature was added different
substituted phenol 3 (20.0 mmol, dissolved in 20 ml acetone)
dropwise. The reaction mixture was stirred under refluxing
for 0.5 h, added chloroform (2.0 ml, 24.0 mmol, dissolved in
8 ml acetone) dropwise and stirred under refluxing for 3.5 h.
Then, the whole solution was evaporated to obtain a residue,
diluted with water (50 ml) and washed with dichloromethane
EXPERIMENTAL SECTION
General
Reactions and the resulted products were monitored by
thin-layer chromatography (TLC) on pre-coated silica gel

296 Medicinal Chemistry, 2012, Vol. 8, No. 2
Wang et al.
1H-NMR (CDCl3, 300 MHz, ppm) ꢂ: 7.43-7.18 (m, 18H, Ph-
H, H-14, H-3’, 5’), 6.98 (t, 1H, J=7.2 Hz, H-4’), 6.70 (d, 2H,
J=7.5 Hz, H-2’, 6’), 4.77 (s, 2H, H-15), 4.57 (m, 1H, H-12),
4.50 (d, 1H, J=8.2 Hz, H-3), 3.34 (d, 1H, J=9.9 Hz, H-19a),
3.18 (d, 1H, J=9.9 Hz, H-19b), 2.30 (dd, 1H, J=8.1 Hz, H-
11a), 2.08 (dd, 1H, J=8.1 Hz, H-11b), 1.98-1.61 (several
protons), 1.53 (s, 3H, -OCOCH(CH3)a), 1.50 (s, 3H, -
OCOCH(CH3)b), 1.46-0.88 (several protons), 1.14 (s, 3H, H-
17), 1.04 (s, 3H, H-18), 0.42 (s, 3H, H-20).
(25 mlꢀ3). The water layer was neutralized with 5 mol/L
HCl and then filtered. The filter cake was washed with wa-
ter, dried over infrared lamp and purified by recrystallization
with ethyl acetate/petroleum ether (EtOAc/PE).
Preparation of Isoandrographolide (2)
To Andro 1 (5.0 g, 14.3 mmol) was added concentrated
hydrochloric acid (81 ml, 810 mmol), and the reaction mix-
ture was stirred at room temperature for 24 h. Then, the
whole solution was diluted with ice water (200 ml) and then
filtered. The filter cake was washed with ice water, dissolved
in dichloromethane(200 ml), washed with saturated sodium
bicarbonate (75 mlꢀ3) and saturated brine (75 mlꢀ3), dried
over Na2SO4 and evaporated to obtain a residue purified by
recrystallization with ethyl acetate. White powder; 70 %
yield; mp: 201-202 °C (literature values mp: 198-200 °C)
[17].
3-O-(2-methylphenyloxyisobutyryl)-19-O-tritylisoandrogra-
pholide (6b)
White powder; 77.0 % yield; mp 176-177 °C. MS (ESI)
m/z: 791.4 (M+Na)+; IR (KBr, cm-1) ꢁ: 3496.11, 2922.82,
2868.41, 1757.87, 1739.07, 1637.56, 1599.55, 1491.92,
1449.09, 1227.2,1147.69, 1049.73, 748.99; 1H-NMR
(CDCl3, 300 MHz, ppm) ꢂ: 7.40-7.11 (m, 16H, Ph-H, H-14),
7.01 (t, 1H, J=7.6 Hz, H-3’), 6.88 (t, 2H, J=7.6 Hz, 7.9 Hz,
H-4’, 6’), 6.61 (d, 1H, J=7.9 Hz, H-5’), 4.77 (s, 2H, H-15),
4.58 (s, 1H, H-12), 4.55 (s, 1H, , H-3), 3.38 (d, 1H, J=9.7
Hz, H-19a), 3.20 (d, 1H, J=9.7 Hz, H-19b), 2.30 (dd, 1H,
J=8.1 Hz, H-11a), 2.05 (dd, 1H, J=8.1 Hz, H-11b), 2.02 (s,
3H, Ph-CH3), 1.95-1.60 (several protons), 1.52 (s, 3H, -
OCOCH(CH3)a), 1.49 (s, 3H, -OCOCH(CH3)b), 1.20-0.89
(several protons), 1.25 (s, 3H, H-17), 1.08 (s, 3H, H-18),
0.43 (s, 3H, H-20).
Preparation of 19-O-tritylisoandrographolide (5)
To a solution of 2 (2.1 g, 6.0 mmol) and N-
Methylmorpholine in anhydrous dichloromethane (25 ml) at
room temperature was added triphenylmethyl chloride (1.70
g, 7.8 mmol, dissolved in 10 ml dichloromethane) dropwise,
and the reaction mixture was stirred for 12 h. Then, the
whole solution was diluted with dichloromethane (50 ml),
washed with saturated brine (30 mlꢀ3), dried over Na2SO4
and evaporated to obtain a residue purified by recrystalliza-
tion with EtOAc/PE. White powder; 79.5 % yield; mp 208
°C (dec.). MS (ESI) m/z: 615.2 (M+Na)+; IR (KBr, cm-1) ꢁ:
3546, 3061, 2927, 2844, 1754, 1741, 1734, 1448, 1207, 933,
3-O-(3-methylphenyloxyisobutyryl)-19-O-tritylisoandrogra-
pholide (6c)
White powder; 90.4 % yield; mp 177-178°C. MS (ESI)
m/z: 791.4 (M+Na)+; IR (KBr, cm-1) ꢁ: 3473.08, 3055.57,
3030.02, 1748.91, 1724.51, 1637.99, 1606.92, 1585.87,
1488.73, 1448.44, 1369.64, 1174.67, 1092.83, 1074.21,
1
705; H-NMR (CDCl3, 300 MHz, ppm) ꢂ: 7.51-7.22 (m,
16H, ArH, H-14), 4.78 (s, 2H, H-15), 4.57 (t, 1H, J=7.2 Hz,
H-12), 4.10 (d, 1H, J=8.2 Hz, H-3), 3.46 (d, 1H, J=9.6 Hz,
H-19a), 3.21 (m, 2H, H-19b, 3-OH), 2.30 (dd, 1H, J=7.2 Hz,
H-11a), 2.05 (m, 1H, H-1), 1.92 (m, 1H, H-11b), 1.65-1.27
(several protons), 1.14 (s, 3H, H-17), 1.03 (t, 3H, H-18), 0.93
(s, 3H, H-20); 13C-NMR (CDCl3, 75 MHz, ppm) ꢂ: 172.5,
143.4, 143.1, 138.2, 128.4, 128.0, 127.2, 87.7, 82.6, 80.6,
77.5, 77.0, 76.6, 73.0, 70.5, 64.8, 58.4, 52.7, 42.4, 39.3, 35.8,
35.6, 33.0, 31.5, 27.4, 23.3, 18.2, 16.1.
1
831.10, 768.99, 711.98; H-NMR (CDCl3, 300 MHz, ppm)
ꢂ: 7.40-7.16 (m, 16H, Ph-H, H-14), 7.07 (t, 1H, J=7.7 Hz,
7.9 Hz, H-3’), 6.80 (d, 2H, J=7.4 Hz, H-2’, 4’), 6.49 (d, 1H,
J=7.4 Hz, H-6’), 4.77 (s, 2H, H-15), 4.56 (m, 1H, H-12),
4.50 (s, 1H, H-3), 3.36 (d, 1H, J=9.7 Hz, H-19a), 3.19 (d,
1H, J=9.7 Hz, H-19b), 2.26 (s, 3H, Ph-CH3), 2.08 (dd, 1H,
J=7.8 Hz, H-11a), 1.90 (dd, 1H, J=7.8 Hz, H-11b), 1.68-1.49
(several protons), 1.51 (s, 3H, -OCOCH(CH3)a), 1.49 (s, 3H,
-OCOCH(CH3)b), 1.25 (s, 3H, H-17), 1.08 (s, 3H, H-18),
0.42 (s, 3H, H-20).
General Procedure for the Preparation of 6a-k
To a solution of 5 (1.19 g, 2.0 mmol), different substi-
tuted phenyloxyisobutyric acid 4 (2.4 mmol), DMAP (cata-
lytic amount) in anhydrous dichloromethane (25 ml) at room
temperature was added EDC·HCl (0.58 g, 3.0 mmol, dis-
solved in 15 ml anhydrous dichloromethane) dropwise, and
the reaction mixture was stirred until the end of the reaction.
Then, the whole solution was diluted with dichloromethane
(20 ml), washed with water (30 mlꢀ3) and saturated brine
(30 mlꢀ3), dried over Na2SO4 and evaporated to obtain a
residue purified by column chromatography (EtOAc/PE).
3-O-(4-methylphenyloxyisobutyryl)-19-O-tritylisoandrogra-
pholide (6d)
White powder; 62.4 % yield; mp 180-181 °C. MS (ESI)
m/z: 791.4 (M+Na)+; IR (KBr, cm-1) ꢁ: 3473.69, 3087.09,
3055.44, 3030.02, 2991.28, 2956.75, 2923.83, 2898.05,
1748.68, 1724.47, 1606.01, 1585.61, 1488.66, 1448.41,
1
1145.79, 1135.13, 1092.85, 831.13; H-NMR (CDCl3, 300
MHz, ppm) ꢂ: 7.42-7.15 (m, 16H, Ph-H, H-14), 6.81 (d, 2H,
J=7.2 Hz, H-3’, 5’), 6.63 (d, 2H, J=7.2 Hz, H-2’, 6’), 4.80
(s, 2H, H-15), 4.66 (m, 1H, H-12), 4.60 (s, 1H, H-3), 3.35 (d,
1H, J=9.6 Hz, H-19a), 3.21 (d, 1H, J=9.6 Hz, H-19b), 2.45
(dd, 1H, J=7.8 Hz, H-11a), 2.29 (s, 3H, Ph-CH3), 2.21 (dd,
1H, J=7.8 Hz, H-11b), 2.11-1.03 (several protons), 1.62 (s,
3H, -OCOCH(CH3)a), 1.60 (s, 3H, -OCOCH(CH3)b), 1.10
(s, 3H, H-17), 0.96 (s, 3H, H-18), 0.89 (s, 3H, H-20).
3-O-phenyloxyisobutyryl-19-O-tritylisoandrographolide
(6a)
White powder; 58.3 % yield; mp 172-173 °C. MS (ESI)
m/z: 777.4 (M+Na)+; IR (KBr, cm-1) ꢁ: 3433.56, 3057.61,
2975.31, 2950.98, 2918.40, 1759.97,1728.13, 1598.64,
1491.35, 1448.8, 1156.66, 1074.65, 1001.04, 759.16, 707.09;

Synthesis and Preliminary Antihyperlipidaemic Activities Evaluation
Medicinal Chemistry, 2012, Vol. 8, No. 2 297
3-O-(2-methoxylphenyloxyisobutyryl)-19-O-tritylisoandro-
grapholide (6e)
82.6, 79.4, 77.4, 77.0, 76.6, 73.2, 70.5, 63.0, 58.2, 52.7, 42.1,
39.1, 36.3, 35.8, 32.9, 31.6, 26.4, 24.5, 23.3, 23.0, 19.6, 15.2.
White powder; 81.3 % yield; mp 186-187 °C. MS (ESI)
m/z: 807.4 (M+Na)+; IR (KBr, cm-1) ꢀ: 3462.39, 2982.06,
2929.89, 1757.25, 1496.79, 1449.22, 1257.75, 1144.95,
1125.72, 1044.31, 1024.24, 749.74, 709.71; 1H-NMR
(CDCl3, 300 MHz, ppm) ꢁ: 7.42-7.17 (m, 16H, Ph-H, H-14),
7.00 (m, 2H, H-4’, 5’), 6.88 (m, 2H, H-3’, 6’), 4.80 (s, 2H,
H-15), 4.60 (m, 1H, H-12), 4.58 (s, 1H, H-3), 3.80 (s, 3H,
Ph-OCH3), 3.39 (d, 1H, J=9.6 Hz, H-19a), 3.28 (d, 1H, J=9.6
Hz, H-19b), 2.40 (m, 1H, H-11a), 2.20 (m, 1H, H-11b), 2.05-
0.85 (several protons), 1.54 (s, 6H, -OCOCH(CH3)2), 1.23 (s,
3H, H-17), 0.85 (s, 3H, H-18), 0.49 (s, 3H, H-20).
3-O-(4-nitrylphenyloxyisobutyryl)-19-O-tritylisoandrogra-
pholide (6i)
White powder; 77.5 % yield; mp 200-201 °C. MS (ESI)
m/z: 822.4 (M+Na)+; IR (KBr, cm-1) ꢀ: 3454.16, 3089.40,
2933.15, 2852.22, 1750.38, 1736.36, 1719.53, 1630.01,
1589.73, 1549.13, 1458.17, 1285.36, 1164.11, 1091.37,
1
1052.48, 811.40, 745.36, 720.14; H-NMR (DMSO-d6, 300
MHz, ppm) ꢁ: 8.09 (d, 2H, J=9.1 Hz, H-3’, 5’), 7.39-7.23
(m, 16H, Ph-H, H-14), 7.07 (t, 1H, J=7.7 Hz, 7.9 Hz, H-3’),
6.65 (d, 2H, J=9.1 Hz, H-2’, 6’), 4.78 (s, 2H, H-15), 4.55
(m, 1H, H-12), 4.48 (s, 1H, H-3), 3.27 (d, 1H, J=9.6 Hz, H-
19a), 3.14 (d, 1H, J=9.6 Hz, H-19b), 2.30 (dd, 1H, J=8.0 Hz,
H-11a), 2.05 (dd, 1H, J=8.0 Hz, H-11b), 1.99-0.97 (several
protons), 1.62 (s, 3H, -OCOCH(CH3)a), 1.61 (s, 3H, -
OCOCH(CH3)b), 1.18 (s, 3H, H-17), 1.08 (s, 3H, H-18),
0.42 (s, 3H, H-20); 13C-NMR (CDCl3, 75 MHz, ppm) ꢁ:
173.2, 172.6, 143.9, 143.1, 138.3, 128.9, 127.9, 127.6, 126.9,
125.4, 119.4, 117.2, 87.4, 83.1, 82.7, 80.1, 77.4, 77.0, 76.6,
73.1, 70.5, 62.9, 58.2, 52.7, 42.0, 39.1, 36.2, 35.7, 32.8, 31.5,
26.5, 24.4, 23.2, 23.0, 19.5, 15.2.
3-O-(4-fluorophenyloxyisobutyryl)-19-O-tritylisoandrogra-
pholide (6f)
White powder; 85.6% yield; mp 198-199 °C. MS (ESI)
m/z: 795.4 (M+Na)+; IR (KBr, cm-1) ꢀ: 3455.09, 3078.77,
2995.06, 2983.44, 1755.35, 1741.88, 1637.53, 1504.04,
1449.04, 1205.94, 1126.60, 772.05, 751.17, 710.17; 1H-
NMR (DMSO-d6, 300 MHz, ppm) ꢁ: 7.42-7.22 (m, 16H, Ph-
H, H-14), 7.07 (m, 2H, H-3’, 5’), 6.71 (m, 2H, H-2’, 6’),
4.83 (s, 2H, H-15), 4.48 (d, 1H, J=8.7 Hz, H-12), 4.36 (s,
1H, H-3), 3.26 (d, 1H, J=9.6 Hz, H-19a), 3.08 (d, 1H, J=9.6
Hz, H-19b), 2.09 (dd, 1H, J=7.8 Hz, H-11a), 1.98 (dd, 1H,
J=7.8 Hz, H-11b), 1.90-1.06 (several protons), 1.43 (s, 6H, -
OCOCH(CH3)2), 1.14 (s, 3H, H-17), 1.01 (s, 3H, H-18), 0.36
(s, 3H, H-20).
3-O-(4-tert-butylphenyloxyisobutyryl)-19-O-tritylisoandro-
grapholide (6j)
White powder; 95.6 % yield; mp 96-97 °C. MS (ESI)
m/z: 833.4 (M+Na)+; IR (KBr, cm-1) ꢀ: 3448.46, 3058.02,
2961.38, 2869.97, 1758.86, 1608.35, 1510.25, 1490.80,
1448.83, 1383.87, 1365.26, 1281.04, 1129.97, 1060.33,
3-O-(4-chlorophenyloxyisobutyryl)-19-O-tritylisoandrogra-
pholide (6g)
1
832.36746.58, 706.39; H-NMR (CDCl3, 300 MHz, ppm) ꢁ:
7.45-7.17 (m, 18H, Ph-H, H-14, H-3’, 5’), 6.84 (d, 1H, J=8.7
Hz, H-2’, 6’), 4.80 (s, 2H, H-15), 4.68 (m, 1H, H-12), 4.56
(s, 1H, H-3), 3.40 (d, 1H, J=9.6 Hz, H-19a), 3.29 (d, 1H,
J=9.6 Hz, H-19b), 2.40 (dd, 1H, J=8.0 Hz, H-11a), 1.93 (dd,
1H, J=8.0 Hz, H-11b), 2.10-0.85 (several protons), 1.51 (s,
3H, -OCOCH(CH3)a), 1.48 (s, 3H, -OCOCH(CH3)b), 1.27
(s, 9H, Ph-C(CH3)3), 1.17 (s, 3H, H-17), 1.08 (s, 3H, H-18),
0.46 (s, 3H, H-20).
White powder; 76.0 % yield; mp 163-164 °C. MS (ESI)
m/z: 811.3 (M+Na)+; IR (KBr, cm-1) ꢀ: 3453.96, 2993.70,
1754.22, 1742.05, 1636.64, 1490.89, 1449.14, 1271.41,
1246.05, 1127.69, 771.64, 751.28, 709.66; 1H-NMR (CDCl3,
300 MHz, ppm) ꢁ: 7.41-7.26 (m, 16H, Ph-H, H-14), 7.20 (m,
1H, H-3’, 5’), 6.77 (d, 1H, J=9.0 Hz, H-2’, 6’), 4.81 (s, 2H,
H-15), 4.66 (m, 1H, H-12), 4.61 (s, 1H, H-3), 3.48 (d, 1H,
J=9.6 Hz, H-19a), 3.28 (d, 1H, J=9.6 Hz, H-19b), 2.44 (dd,
1H, J=7.8 Hz, H-11a), 2.20 (dd, 1H, J=7.8 Hz, H-11b), 2.46-
1.04 (several protons), 1.61 (s, 6H, -OCOCH(CH3)2), 1.24 (s,
3H, H-17), 0.96 (s, 3H, H-18), 0.91 (s, 3H, H-20).
3-O-(2-naphthoxyisobutyryl)-19-O-tritylisoandrographolide
(6k)
White powder; 93.0 % yield; mp 104-105 °C. MS (ESI)
m/z: 827.4 (M+Na)+; IR (KBr, cm-1) ꢀ: 3453.83, 3057.48,
1757.81, 1630.07, 1599.02, 1508.58, 1448.50, 1384.57,
1368.83, 1282.04, 1128.88, 764.78, 746.61, 706.60; 1H-
NMR (DMSO-d6, 300 MHz, ppm) ꢁ: 7.87 (d, 1H, J=8.1 Hz,
H-5’), 7.79 (d, 1H, J=9.0 Hz, H-4’), 7.68 (d, 1H, J=7.8 Hz,
H-8’), 7.49-7.23 (m, 18H, Ph-H, H-14, H-6’, H-7’), 6.30 (s,
1H, H-1’), 6.86 (d, 1H, J=9.0 Hz, H-3’), 5.10 (s, 2H, H-15),
4.50 (s, 1H, H-12), 4.31 (s, 1H, H-3), 3.27 (d, 1H, J=9.9 Hz,
H-19a), 3.01 (d, 1H, J=9.9 Hz, H-19b), 2.04 (dd, 1H, J=8.0
Hz, H-11a), 2.90 (dd, 1H, J=8.0 Hz, H-11b), 1.56 (s, 6H, -
OCOCH(CH3)2), 1.51-0.88 (several protons), 1.23 (s, 3H, H-
17), 0.85 (s, 3H, H-18), 0.27 (s, 3H, H-20); 13C-NMR
(CDCl3, 75 MHz, ppm) ꢁ: 173.9, 172.5, 153.3, 143.9, 143.0,
138.4, 134.0, 129.1, 128.9, 127.6, 127.3, 126.8, 126.3, 124.1,
121.0, 112.4, 111.0, 110.6, 87.1, 82.7, 82.4, 79.2, 77.5, 77.1,
76.7, 73.2, 70.5, 63.4, 58.3, 52.7, 42.1, 39.2, 36.3, 35.7, 32.9,
31.6, 26.6, 24.5, 23.3, 22.9, 19.6, 15.1.
3-O-(4-bromophenyloxyisobutyryl)-19-O-tritylisoandrogra-
pholide (6h)
White powder; 79.2 % yield; mp 106-107 °C. MS (ESI)
m/z: 855.3 (90%), 857.3 (100%) (M+Na)+; IR (KBr, cm-1) ꢀ:
3454.02, 2925.95, 1758.16, 1486.99, 1448.47, 1384.41,
1369.18, 1238.07, 1129.88, 1072.17, 823.92, 764.75, 706.69;
1H-NMR (DMSO-d6, 300 MHz, ppm) ꢁ: 7.41-7.24 (m, 18H,
Ph-H, H-14, H-3’, 5’), 6.58 (d, 2H, J=8.7 Hz, H-2’, 6’), 4.83
(s, 2H, H-15), 4.46 (d, 1H, J=7.7 Hz, H-12), 4.35 (s, 1H, H-
3), 3.26 (d, 1H, J=9.6 Hz, H-19a), 3.05 (d, 1H, J=9.6 Hz, H-
19b), 2.09 (dd, 1H, J=7.8 Hz, H-11a), 1.92 (dd, 1H, J=7.8
Hz, H-11b), 1.92-1.04 (several protons), 1.47 (s, 6H, -
OCOCH(CH3)2), 1.14 (s, 3H, H-17), 1.01 (s, 3H, H-18), 0.38
(s, 3H, H-20); 13C-NMR (CDCl3, 75 MHz, ppm) ꢁ: 173.6,
172.5, 154.6, 143.9, 143.0, 138.4, 131.9, 130.8, 130.6, 130.6,
129.9, 129.8, 129.0, 127.9, 127.9, 126.1, 114.1, 87.2, 82.7,

298 Medicinal Chemistry, 2012, Vol. 8, No. 2
Wang et al.
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IN VIVO ACTIVITIES
Animals
Male Sprague-Dawley rats weighing between 150 g and
180 g at the start of treatment and housed individually in a
room with a controlled temperature (23±1°C) and a 12 h/12
h light/dark cycle and a relative humidity of 55 ± 5%, with
free access to commercial pellet food and water.
[3]
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[5]
Chemicals and Reagents
Atromide (Xiyue pharm, Shanxi, China), triglyceride
reagent kit (BioSino, Beijing, China), cholesterrol reagent kit
(BioSino, Beijing, China), high-density lipoprotein choles-
terol reagent kit (BioSino, Beijing, China), low-density lipo-
protein cholesterol reagent kit (BioSino, Beijing, China).
[6]
[7]
Standard substances: TG, TC (Randox, Antrim, UK),
HDL-C, LDL-C (BioSino, Beijing, China).
[8]
Methods
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Nanduri, S.; Rajagopal, S.; Pothukuchi, S.; Pillaj, S.B.K.;
Chakrabarti, R. Compounds having antitumor actnity: process for
their preparation and pharmaceutical compositions containing
them. India. Patent WO2001057026A1, Jan 11, 2011.
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antitumor activity of isoandrographolide derivatives. J. China
Pharmaceut Univ., 2005, 36(6), 504-510.
Li, J.; Huang, W.L.; Zhang, H.B.; Wang, X.Y.; Zhou, H.P.
Synthesis of andrographolide derivatives and their TNF-ꢁ and IL-6
expression inhibitory activities. Bioorg. Med. Chem. Lett., 2007,
17(24), 6891-6894.
After one-week adaptive breeding, 136 male SD rats
were divided into 17 groups randomly: normal control group
(1 group), fat group (16 group, including a fat control
group), with 8 in each group. All but normal control group
was intragastrically given fat emulsion (10 ml/kg·d) in addi-
tion to normal feeding for 21 days. Afterward, except the
normal black control group and the fat control group, each of
the other groups was given the corresponding drug. After
given for 7 days, the rats were fasted for 12 h and then anes-
thetized by intraperitoneal injection of 1.0 g/kg Urethane.
Blood was obtained from the arteria cervicalis, the viscera
were excised, weighed and tested for tissue section. Blood
placed in sterile tubes, serum samples were isolated from the
blood by centrifugation (4000 rpm for 30 min) and analyzed
for serum total triglyceride (GPO-POD UV method), choles-
terol (cholesterol oxidase method), high-density lipoprotein
cholesterol (one-step method), and low-density lipoprotein
cholesterol (one-step method), respectively, on an Olympus
AU 400 automatic biochemistry analyzer (Olympus, Tokyo,
Japan) [18-20].
[10]
[11]
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[13]
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Wang, B.; Ge, L.; Huang, W.L.; Zhang, H.B.; Qian, H.; Li, J.;
Zheng, Y.T. Synthesis and Preliminary Anti-HIV Activities of
Andrographolide Derivatives. Med. Chem., 2010, 6(4), 252-258.
Emblidge, J.P.; DeLorenzo, M.E. Preliminary risk assessment of
the lipid-regulating pharmaceutical clofibric acid, for three
estuarine species. Environ. Res., 2006, 100(2), 216-226.
Vaidya, H.; Rajani, M.; Sudarsanam, V.; Padh, H.; Goyal, R.
Antihyperlipidaemic activity of swertiamarin,
a secoiridoid
glycoside in poloxamer-407-induced hyperlipidaemic rats. J. Nat.
Med., 2009, 63(4), 437-442.
Consisting of fat emulsion: 10% cholesterol, 25% lard
oil, 2% sodium cholate, 1% propylthiouracil, 10% Tween-
80, 20% propylene glyco and 32% water.
[17]
Pramanick, S.; Banerjee, S.; Achari, B.; Das, B.; Sen, A.K.;
Mukhopadhyay, S.; Neuman, A.; Prange, T. Andropanolide and
Isoandrographolide, Minor Diterpenoids from Andrographis
paniculata: Structure and X-ray Crystallographic Analysis. J. Nat.
Prod., 2006, 69(3), 403-405.
ACKNOWLEDGEMENTS
[18]
[19]
Pan, D.D.; Zhang, D.Z. Function and safety assessment of
Lactococus lactis subsp. lactis LB12 as potential probiotic strain.
Afr. J. Biotechnol., 2008, 7(22), 4037-4044.
The work was supported by the National Natural Science
Foundation of China (No. 30772647) and the special major
science and technology project of "Creation of major new
drugs" (No. 2009ZX09102-033).
Shen, L.; Zhang, Y.; Wang, A.; Sieber-McMaster, E.; Chen, X.;
Pelton, P.; Xu, J.Z.; Yang, M.; Zhu, P.; Zhou, L. Synthesis and
structure-activity relationships of thiadiazole-derivatives as potent
and orally active peroxisome proliferator-activated receptors ꢁ/ꢂ
dual agonists. Bioorg. Med. Chem., 2008, 16(6), 3321-3341.
Khanna, A.K.; Rizvi, F.; Chander, R. Lipid lowering activity of
Phyllanthus niruri in hyperlipemic rats. J. Ethnopharmacol., 2002,
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Received: January 12, 2011
Revised: August 05, 2011
Accepted: August 13, 2011