Synthesis of new andrographolide derivatives and evaluation of their antidyslipidemic, LDL-oxidation and antioxidant activity

Article abstract of DOI:10.1016/j.ejmech.2013.09.002

Andrographis paniculata, native to Taiwan, Mainland China and India, is a medicinal herb, which possesses various biological activities including anti-atherosclerosis. Andrographolide (1) has been identified as one of the active constituents against atherosclerosis. In continuation of our drug discovery program we synthesized few novel derivatives of 1 to improve their antidyslipidemic, LDL-oxidation and antioxidant activity. The tosylated derivative 7 has been turned out to be more potent than the parent compound and comparable activity with marketed antidyslipidemic drugs.

Full text of DOI:10.1016/j.ejmech.2013.09.002

European Journal of Medicinal Chemistry 69 (2013) 439e448
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of new andrographolide derivatives and evaluation of their
antidyslipidemic, LDL-oxidation and antioxidant activity
,
,
Sukanya Pandeti a 1, Ravi Sonkar b 1, Astha Shukla a, Gitika Bhatia b,
Narender Tadigoppula a
,
*
a Medicinal and Process Chemistry Division, CSIR e Central Drug Research Institute, Lucknow 226 001, U.P., India
b Biochemistry Division, CSIR e Central Drug Research Institute, Lucknow 226 001, U.P., India
a r t i c l e i n f o
a b s t r a c t
Article history:
Andrographis paniculata, native to Taiwan, Mainland China and India, is a medicinal herb, which pos-
sesses various biological activities including anti-atherosclerosis. Andrographolide (1) has been identi-
fied as one of the active constituents against atherosclerosis. In continuation of our drug discovery
program we synthesized few novel derivatives of 1 to improve their antidyslipidemic, LDL-oxidation and
antioxidant activity. The tosylated derivative 7 has been turned out to be more potent than the parent
compound and comparable activity with marketed antidyslipidemic drugs.
Received 22 May 2013
Received in revised form
29 August 2013
Accepted 2 September 2013
Available online 12 September 2013
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Andrographolide derivatives
Andographis paniculata
LPL activity
Antidyslipidemic activity
Anti oxidant activity
LDL oxidation
1. Introduction
drug having multifold properties such as lipid lowering and anti-
oxidant activities mutually is in great demand.
Hypercholesterolemia and hypertriglyceridemia are either,
alone or together major risk factors contributing to prevalence and
severity of coronary artery disease and the progression of stroke,
atherosclerosis, hyperlipidemia [1e3], which are leading cause of
death in the developed and developing countries. High levels of
low-density lipoprotein (LDL) accumulate in the extracellular sub
endothelial space of arteries and are highly atherogenic and toxic to
vascular cells thereby leading to atherosclerosis, hypertension,
obesity, diabetes, functional depression in some organs, etc [4,5].
Oxidative stress is also a major contributing factor for peroxidative
damage to lipoproteins, which is dependable for initiation and
progression of atherosclerosis in hyperlipidemic subjects [6].
Hyperlipidemia may also provoke abnormalities with oxidation of
fatty acids leading to the development of ketone bodies as well as
making liver and muscle resistance to insulin which start and
progress diabetes in patients [7]. To rise above these ailments, a
The discovery of new drugs from traditional medicine is not a
new phenomenon. It has been reported that traditional systems
have immense potential to cure various diseases [8,9]. Plants have
always been an ideal source of drugs and many of the currently
available drugs have been derived directly or developed from plants
(Fig. 1). For example, metformin is currently used as antidiabetic
agent in the treatment of type-2 diabetes. Metformin and its ana-
logs [10] were synthesized on the basis of a natural product lead viz,
galegine [11], which was isolated from the seeds of Galega officinali
(Fig. 1). The synthetic cholesterol lowering statins such as fluvas-
tatin [12], cerivastatin [13] were developed on the basis of natural
product leads, that is, mevastatin [14]. The fish oils, which contain
fatty acids such as eicosapentaenoic acid and docosahexenoic acids
(Fig. 1), have been reported for their lowering effect on triglycerides
and cholesterol [15].
As a part of our drug discovery program we identified few
antidyslipidemic agents such as aegeline from Aegle marmelos [16]
and 4-hydroxyisoluecine from Trigonella foenum graecum seeds
(Fig. 1) [17]. In continuation of this program we explored Androg-
raphis paniculata. A. paniculata (Burm. f.) Nees (Acanthaceae)
(A. paniculata, Chuanxinlian), native to Taiwan, Mainland China and
India, is a medicinal herb with an extremely bitter in taste [18],
* Corresponding author. Tel.: þ91 522 2612411; fax: þ91 522 2623405.
E-mail
addresses:
t_narendra@cdri.res.in,
tnarender@rediffmail.com
(N. Tadigoppula).
1
These authors contributed equally.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.002

440
S. Pandeti et al. / European Journal of Medicinal Chemistry 69 (2013) 439e448
Fig. 1. Naturally occurring and synthetic antidiabetic and antidyslipidemic agents.
which possesses anti inflammation, anti cancer, immunomodula-
tion, anti infection, anti hepatotoxicity, anti atherosclerosis, anti
oxidation and anti hyperglycemic effect [19e29]. According to
Chinese medicinal system, A. paniculata ’cools’ and relieves internal
heat, inflammation and pain and is used for detoxification [30e32].
Antihyperlipidemic and antioxidant activities of the extracts and
the constituents of A. paniculata have been explored by several
researchers in recent years. Furthermore, an alcoholic extract of the
plant and its major constituent 1 have been shown to anti-
hyperlipidemic agent through bioactivity-guided chromatographic
fractionation. However, no systematic attempt has been made to
improve the antihyperlipidemic and antioxidant activity of 1 and to
synthesize novel and potent agents. Towards this goal, we have
synthesized a series of new derivatives from 1 and evaluated lipid
lowering activity in triton induced hyperlipidemic rat model.
Further we have studied the dose dependent lipid lowering activity,
lipoprotein cholesterol level, LPL activity, antioxidant and LDL
oxidation.
endocyclic double bond D
13(14), with the simultaneous removal of
C-14 hydroxyl and compound 11 was synthesized from 1 with
NaBH4 and Amberlyst-15 (Scheme 2).
8(17)
Selective epoxidation of the exocyclic double bond
D
of 1
with m-CPBA led to 12. Our efforts to oxidize the C-3, 19 hydroxyl
groups of 1 with DMP yielded 13. To understand the role of the two
8(17)
12(13)
olefin bonds
D
and
D
in 1, 14 and 15 were prepared by
selectively reducing the double bond (Scheme 3).
2.2. Biological evaluation
2.2.1. Lipid lowering activity of triton induced hyperlipidemic rat
Administration of triton WR-1339 in rats induced marked
hyperlipidemia as evidenced by increase in the plasma levels of TC
(þ2.11 folds), PL (þ2.37 folds), TG (þ2.44 folds) followed by
decrease in PHLA level (ꢀ24%) as compared to control rats. Treat-
ment of hyperlipidemic rats with all the andographolide derivatives
(2e16) at the dose of 100 mg/kg p.o. reversed the plasma levels of
lipid but with varying extents. The parent compound, andogra-
pholide (1) is causing a decrease in plasma levels of TC, PL and TG by
27%, 26% and 28% respectively followed 16% increase in PHLA level
as compared to triton induced rats. The synthesized derivatives
inhibited cholesterol biosynthesis and potentiated the activity of
lipolytic enzymes to early clearance of lipids from circulation in
triton induced hyperlipidemia. Among these derivatives (2e16), the
sulfonyl derivative (7) turned out to be most potent lipid lowering
agent, causing a decrease in plasma levels of TC, PL and TG by 33%,
37% and 35% respectively followed 18% increase in PHLA level as
compared to triton induced rats at the dose of 100 mg/kg, where as
the marketed lipid lowering drugs gemfibrozil and fenofibrate
decreased the levels of TC, PL and TG in plasma by 30%, 30%, 28%
and 32%, 33%, 33% respectively followed 16% and 17% increase in
PHLA level as compared to triton induced rats at the same dose.
Derivatives 5 and 8 showed moderate activity, caused a decrease in
plasma levels of TC, PL and TG levels by 20%, 22%, 22% and 22%, 20%,
20% respectively followed 13% and 12% increase in PHLA level as
compared to triton induced rats (Table 1). All other modifications
such as hydrogenation of double bonds (9, 10, 14, 15), epoxidation
(12), oxidation (13), acetylation (16) lead to decreased activity to its
parent compound (1).
2. Results and discussion
2.1. Chemistry
The structure of andrographolide contains a
a
-alkylidene
D
g-
8(17)
butyrolactone moiety, two olefin bonds
D
and
12(13), and
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 struc-
tural features.
In order to determine the importance of hydroxyl groups of
andrographolide 1 for antihyperlipidemic and antioxidant activity.
Initially, the hydroxyl groups at C-3 and C-19 of 1 were protected as
an isopropylidene to give 2 and then allylic hydroxyl at C-14 was
acetylated with acetic anhydride to provide 3. The compound 3’s
isopropylidine ring was hydrolyzed to get 14-Acetyl andrographo-
lide (4) with 1N HCl. Sulfonylation reaction was carried out on 14-
acetyl andrographolie (4) to generate derivatives 5 and 6 and
subsequently hydrolyzed with NaOMe to give deacetylated com-
pound 7 and 8 respectively (Scheme 1).
To understand the role of the exocyclic double bond
D
12(13) in 1,
9 was prepared by selectively reducing the conjugated double bond
with NaBH4 and NiCl2 6H2O. To further study, the role of allylic
2.2.2. Effect of compounds 1 and 7 on lipid lowering at different
doses in triton induced hyperlipidemic rats
hydroxyl at C-14 and the conjugated double bond, 10 was prepared,
Since the tosylated derivative 7 has shown better activity profile
than its parent compound 1 and comparable profile to marketed
12(13)
in which the exocyclic double bond
D
isomerized to the

S. Pandeti et al. / European Journal of Medicinal Chemistry 69 (2013) 439e448
441
Scheme 1. Synthesis of sulfonyl derivatives 5e8 of andrographolide (1).
drugs (gemfibrozil and fenofibrate) a dose dependent activity study
was carried out. As shown in Table 2, the administration of triton to
rats increased their plasma levels of TC (þ3.45 folds), PL (þ2.86
folds), TG (þ3.15 folds) followed by decrease in PHLA level (ꢀ41%)
as compared to control rats. The efficacy of compounds 1 and 7
were studied at different doses in 25e200 mg/kg body weight.
Compound 7 lowered the TC by 16%, PL by 15% and TG by 14%
followed by increase in PHLA level by 10% (Table 2) at the dose of
25 mg/kg and at a dose of 50 mg/kg it lowered the TC by 22%, PL by
22%, TG by 20% and increased the PHLA level by 13%. At a higher
dose of 100 mg/kg it lowered the TC by 33%, PL by 37%, TG by 35%
and increased the PHLA level by 18%. A marginal improvement in
the activity profile has been observed at further higher doses such
as 150 and 200 mg/kg b.w. (Table 2).
Scheme 2. Hydrogenation of andrographolide (1) by using NaBH4/NiCl2$6H2O and NaBH4, amberlyst-15.

442
S. Pandeti et al. / European Journal of Medicinal Chemistry 69 (2013) 439e448
Scheme 3. Acetylation, hydrogenation, oxidation and epoxidation of andrographolide (1).
Table 1
Percentage (%) change of plasma lipids with the treatment of andrographolide (1) and its derivatives (2-16) in Triton-induced hyperlipidemic rats at dose of 100 mg/kg body
weight.
Animal groups
TC (mg/dl)
PL (mg/dl)
TG (mg/dl)
PHLA (nmol of free fatty
acids formed/h/ml of plasma)
Control
Triton
86.49 ꢁ 6.34
85.70 ꢁ 6.41
88.42 ꢁ 7.63
16.02 ꢁ 1.72
182.89 ꢁ 17.34C (þ2.11 Fold)
133.50 ꢁ 10.06*** (ꢀ27)
153.62 ꢁ 10.63* (ꢀ16)
159.50 ꢁ 11.63* (ꢀ16)
165.20 ꢁ 10.88* (ꢀ13)
151.91 ꢁ 12.39** (ꢀ20)
167.10 ꢁ 14.73 (ꢀ12)
122.53 ꢁ 6.81*** (ꢀ33)
142.65 ꢁ 9.56** (ꢀ22)
160.94 ꢁ 10.26* (ꢀ12)
151.79 ꢁ 11.38* (ꢀ17)
148.14 ꢁ 9.88* (ꢀ19)
155.45 ꢁ 11.33* (ꢀ15)
159.11 ꢁ 9.93* (ꢀ13)
157.28 ꢁ 8.73* (ꢀ14)
162.72 ꢁ 9.61* (ꢀ11)
148.14 ꢁ 9.29* (ꢀ19)
125.86 ꢁ 11.72*** (ꢀ30)
129.12 ꢁ 10.36*** (ꢀ32)
203.72 ꢁ 17.11C (þ2.37 Fold)
150.75 ꢁ 11.08*** (ꢀ26)
179.27 ꢁ 12.22* (ꢀ12)
175.19 ꢁ 13.82* (ꢀ14)
177.23 ꢁ 12.32* (ꢀ13)
158.90 ꢁ 9.73** (ꢀ22)
175.19 ꢁ 14.86 (ꢀ14)
128.34 ꢁ 7.53*** (ꢀ37)
162.97 ꢁ 9.38** (ꢀ20)
183.34 ꢁ 11.37* (ꢀ10)
175.19 ꢁ 12.89* (ꢀ14)
169.08 ꢁ 14.84* (ꢀ17)
169.08 ꢁ 9.86* (ꢀ17)
183.34 ꢁ 13.19* (ꢀ10)
169.08 ꢁ 9.83* (ꢀ17)
177.23 ꢁ 11.82* (ꢀ13)
171.12 ꢁ 10.84* (ꢀ16)
138.00 ꢁ 12.34*** (ꢀ30)
136.49 ꢁ 9.59*** (ꢀ33)
215.78 ꢁ 15.12C (þ2.44 Fold)
155.36 ꢁ 11.05*** (ꢀ28)
183.41 ꢁ 11.73* (ꢀ15)
187.72 ꢁ 15.81* (ꢀ13)
183.41 ꢁ 13.79* (ꢀ15)
168.30 ꢁ 10.46** (ꢀ22)
192.04 ꢁ 14.68 (ꢀ11)
140.25 ꢁ 9.53*** (ꢀ35)
172.62 ꢁ 13.48** (ꢀ20)
192.04 ꢁ 13.72* (ꢀ11)
189.88 ꢁ 9.81* (ꢀ12)
176.93 ꢁ 13.09* (ꢀ18)
185.57 ꢁ 12.89* (ꢀ14)
194.20 ꢁ 10.13* (ꢀ10)
183.41 ꢁ 11.46* (ꢀ15)
183.41 ꢁ 13.32* (ꢀ15)
176.93 ꢁ 11.18* (ꢀ18)
150.22 ꢁ 13.08*** (ꢀ28)
144.57 ꢁ 10.28*** (ꢀ33)
12.04 ꢁ 0.59C (ꢀ24)
Triton þ Compound 1
Triton þ Compound 2
Triton þ Compound 3
Triton þ Compound 4
Triton þ Compound 5
Triton þ Compound 6
Triton þ Compound 7
Triton þ Compound 8
Triton þ Compound 9
Triton þ Compound 10
Triton þ Compound 11
Triton þ Compound 12
Triton þ Compound 13
Triton þ Compound 14
Triton þ Compound 15
Triton þ Compound 16
Triton þ Gemfibrozil
Triton þ Fenofibrate
13.96 ꢁ 1.22* (þ16)
NS
12.76 ꢁ 0.93
12.64 ꢁ 0.83
12.88 ꢁ 1.03
(þ6)
(þ5)
(þ7)
NS
NS
13.60 ꢁ 1.16* (þ13)
12.76 ꢁ 0.92 (þ6)
14.20 ꢁ 1.18* (þ18)
13.48 ꢁ 1.10* (þ12)
NS
12.64 ꢁ 0.96
12.76 ꢁ 0.81
(þ5)
(þ6)
NS
13.24 ꢁ 0.86* (þ10)
NS
12.76 ꢁ 1.16
12.52 ꢁ 0.63
12.64 ꢁ 1.03
12.52 ꢁ 0.81
(þ6)
(þ4)
(þ5)
(þ4)
NS
NS
NS
13.36 ꢁ 0.88* (þ11)
14.89 ꢁ 1.41* (þ16)
14.08 ꢁ 1.26* (þ17)
Each parameter represents pooled data from 6 rats/group and values are expressed as mean ꢁ s.d. cp < 0.001, triton treated group compared with control group and *p < 0.05;
**p < 0.01; ***p < 0.001 triton plus compounds groups compared with triton treated group only. Note: ns (non significant) and f (fold change over control group). Units are amg/
dl; nmol of free fatty acids formed/h/ml of plasma.

S. Pandeti et al. / European Journal of Medicinal Chemistry 69 (2013) 439e448
443
Table 2
Percentage (%) change of plasma lipids with the treatment of andrographolide (1) and 7 in triton-induced hyperlipidemic rats at different doses.
Animal groups
TC (mg/dl)
PL (mg/dl)
TG (mg/dl)
PHLA (nmol of free fatty
acids formed/h/ml of plasma)
Control
Triton
81.32 ꢁ 6.92
83.42 ꢁ 6.12
85.26 ꢁ 8.00
18.62 ꢁ 1.66
280.70 ꢁ 25.16c (þ3.45 Fold) 238.60 ꢁ 18.63c (þ2.86 Fold) 269.15 ꢁ 15.36c (þ3.15 Fold) 10.84 ꢁ 0.74c (ꢀ41)
Triton þ Compound 1
25 mg/kg b.w
50 mg/kg b.w
100 mg/kg b.w
150 mg/kg b.w
200 mg/kg b.w
Triton þ Compound 7
25 mg/kg b.w
50 mg/kg b.w
100 mg/kg b.w
150 mg/kg b.w
200 mg/kg b.w
241.40 ꢁ 19.88* (ꢀ14)
221.75 ꢁ 20.06** (ꢀ21)
204.91 ꢁ 18.22*** (ꢀ27)
199.29 ꢁ 16.24*** (ꢀ29)
199.25 ꢁ 15.48*** (ꢀ29)
209.96 ꢁ 18.82* (ꢀ12)
190.88 ꢁ 14.06** (ꢀ20)
176.56 ꢁ 16.74*** (ꢀ26)
169.40 ꢁ 17.00*** (ꢀ29)
167.02 ꢁ 13.39*** (ꢀ30)
228.77 ꢁ 20.00* (ꢀ15)
212.62 ꢁ 18.56** (ꢀ21)
193.78 ꢁ 12.02*** (ꢀ28)
188.40 ꢁ 16.21*** (ꢀ30)
188.38 ꢁ 17.72*** (ꢀ30)
11.92 ꢁ 0.86* (þ10)
12.24 ꢁ 1.02* (þ13)
12.57 ꢁ 0.76* (þ16)
12.79 ꢁ 1.24* (þ18)
12.75 ꢁ 0.86* (þ18)
235.78 ꢁ 12.63* (ꢀ16)
218.94 ꢁ 15.81** (ꢀ22)
188.06 ꢁ 13.33*** (ꢀ33)
185.26 ꢁ 12.89*** (ꢀ34)
185.26 ꢁ 11.06*** (ꢀ34)
202.81 ꢁ 10.42* (ꢀ15)
186.10 ꢁ 12.26** (ꢀ22)
150.31 ꢁ 8.43*** (ꢀ37)
145.54 ꢁ 9.28*** (ꢀ39)
143.16 ꢁ 7.93*** (ꢀ40)
155.09 ꢁ 12.86*** (ꢀ35)
157.47 ꢁ 11.48*** (ꢀ34)
231.46 ꢁ 17.36* (ꢀ14)
215.32 ꢁ 13.81** (ꢀ20)
174.94 ꢁ 12.53*** (ꢀ35)
169.56 ꢁ 8.82*** (ꢀ37)
169.56 ꢁ 11.32*** (ꢀ37)
180.33 ꢁ 16.00*** (ꢀ33)
177.63 ꢁ 17.05*** (ꢀ34)
11.92 ꢁ 0.73* (þ10)
12.24 ꢁ 0.091* (þ13)
12.79 ꢁ 1.13* (þ18)
12.79 ꢁ 0.89* (þ18)
12.79 ꢁ 0.77* (þ18)
13.00 ꢁ 1.22** (þ20)
13.00 ꢁ 1.28*** (þ20)
Triton þ Gemfibrozil (100 mg/kg b.w) 188.06 ꢁ 16.28*** (ꢀ33)
Triton þ Fenofibrate (100 mg/kg b.w)
182.45 ꢁ 13.36*** (ꢀ35)
Each parameter represents pooled data from 6 rats/group and values are expressed as mean ꢁ s.d. cp < 0.001, triton treated group compared with control group and *p < 0.05;
**p < 0.01; ***p < 0.001 triton plus compounds groups compared with triton treated group only. Note: ns (non significant) and f (fold change over control group). Units are amg/
dl; bmol of free fatty acids formed/h/ml of plasma.
2.2.3. Effect of compounds 1 and 7 on plasma lipoprotein lipids in
triton induced hyperlipidemic rats
Compounds 1 and 7 (100 mg/kg b.w) improve the serum lipo-
proteins cholesterol level in triton induced hyperlipidemic rats. The
analysis of hyperlipidemic serum of triton administered rats
showed significantly increase in the level of VLDL-C and LDL-C
followed by decrease in HDL-C as compared to control rats. Treat-
ment with compounds 1 and 7 significantly reversed the increased
levels of VLDL-TC and LDL-TC and decreased HDL-TC in triton
induced hyperlipidemic rats (Fig. 2). Here also the tosylated de-
rivative 7 showed better activity profile than its parent compound 1
and the activity is comparable to marketed drugs.
Fig. 2. Effect of compounds 1 and 7 on lipoprotein metabolism in triton induced hyperlipidemic rats. Blood was drawn after 18 h of hyperlipidemia was developed by administration
of triton WR-1339 with and without compounds 1 and 7. Gemfibrozil and fenofibrate were taken as reference drug (100 mg/kg). (A) Very low density lipoprotein (VLDL). (B) Low
density lipoprotein. (C) High density lipoprotein. Values are expressed as the mean ꢁ SD (n ¼ 6). *P < 0.05; **P < 0.01; ***P < 0.001; NS (non significant).

444
S. Pandeti et al. / European Journal of Medicinal Chemistry 69 (2013) 439e448
Fig. 4. Compounds 1 and 7 reduced the LDL-oxidation at 200 mg/ml concentration in
normal donor blood sample. Data expressed as mean ꢁ S.D. ***P < 0.001; compared to
treated values with untreated (control).
A significant decrease in superoxide anions (33% and 41%) and hy-
droxyl radicals (36%e46%) were observed by compounds 1 and 7.
Fig. 3. Compounds 1 and 7 (100 mg/kg) re-activates LPL activity significantly in Triton
induced hyperlipidemic rats. Each parameter represents pooled data from 6 rats/group
and values are expressed as mean ꢁ S.D. *P < 0.05; **P < 0.01; ***P < 0.001; NS (non
significant). Gemfibrozil and Fenofibrate are taken as reference drug.
Furthermore, compounds 1 and 7 at 200
somal lipid per-oxidation (38%e40%).
mg/ml reduced the micro-
2.2.6. Effect of compounds 1 and 7 on LDL oxidation
Aerobic oxidation of LDL even in the absence of metal ions
caused formation of TBARS (n mol MDA/mg protein), which were
greatly increased by 10e15 folds in presence of Cuþ2. Addition of
compounds 1 and 7 at 200 mg/ml concentrations in above reaction
mixture protected LDL against oxidative changes (Fig. 4).
2.2.4. Effect of compounds 1 and 7 LPL activities in triton induced
hyperlipidemic rats
Further the compounds 1 and 7 were studied for LPL activity in
triton induced hyperlipidemic rats. Administration of triton in rats
markedly decreases in LPL activity in liver. After treatment with
compounds 1 and 7 LPL activity was significantly increased similar
to standard drug gemfibrozil and fenofibrate (Fig. 3).
3. Conclusion
In conclusion we have prepared few new derivatives of Androg-
rapholide (1) and evaluated their antidyslipidemic, LDL oxidation and
antioxidant activity. The tosylated derivative 7 has turned out to be
more potent than the parent compound 1 and the activity profile is
comparable to the marketed antidyslipidemic drugs.
2.2.5. Antioxidant activity
Recent studies have demonstrated that the generation of large
quantities of reactive oxygen species can cause activation of lipid
peroxidation, protein modification, which leads to cardiovascular
diseases(CVD)[33e35] thereforewehavescreenedalcoholicfortheir
antioxidant activity using earlier reported method [36]. The scav-
4. Experimental section
enging potential of compounds 1 and 7 at 200 mg/ml against forma-
tionofO2ꢀ andOHC innonenzymatic systems werestudied(Table 3).
4.1. General chemistry
Table 3
IR spectra were recorded on PerkineElmer RX-1 spectrometer
using either KBr pellets (or) in neat. 1H NMR, 13C NMR, DEPT-90 and
DEPT-135 spectra were run on Bruker Advance DPX 300 MHz and
200 MHz in CDCl3.Chemical shifts are reported as values in ppm
relative to CHCl3/DMSO with TMS as internal standard. ESI mass
spectra were recorded on JEOL SX 102/DA-6000. Thin layer chro-
matography (TLC) plates of silica gel 60 GF254 of Merck Company
were used for identification and purity check of compounds.
Chromatography was executed with silica gel (60e120 mesh) using
mixtures of chloroform, methanol and hexane as eluants. Visuali-
zation was obtained under UV light and spraying with 10% sulfur-
icsulfuric acid in methanol.
Effect of andographolide (1) and its derivatives (2-16) on superoxide anions, hy-
droxyl radicals and lipid-peroxidation at 200 mg/ml dose.
Dose
(200
Superoxide
anions (%)
(n mol formazone
formed/minute)
Hydroxyl
Lipid-peroxidation (%)
(n mol MDA
formed/h/mg protein)
m
g/ml)
radicals (%)
(n mol MDA
formed/h/mg
protein)
1
2
3
4
5
6
7
8
ꢀ33***
ꢀ15*
ꢀ36***
ꢀ11*
ꢀ38***
ꢀ17*
ꢀ23***
ꢀ19**
ꢀ27***
ꢀ31***
ꢀ41***
ꢀ23***
ꢀ14*
ꢀ25***
ꢀ17**
ꢀ30***
ꢀ28***
ꢀ46***
ꢀ25***
ꢀ11*
ꢀ26***
ꢀ19**
ꢀ29***
ꢀ26***
ꢀ40***
ꢀ26***
ꢀ10*
4.2. Collection of medicinal plant
9
10
11
12
13
14
15
16
ꢀ11*
ꢀ16*
ꢀ13*
A. paniculata .Nees (Whole plant) was collected from India and
the authentification was done by the Botany Division of Central
Drug Research Institute, Lucknow.
ꢀ16*
ꢀ18*
ꢀ13*
ꢀ17*
ꢀ15*
ꢀ14*
ꢀ16*
ꢀ12*
ꢀ14*
ꢀ19*
ꢀ15*
ꢀ17*
ꢀ13*
ꢀ17*
ꢀ14*
4.3. Extraction
ꢀ12*
ꢀ16*
ꢀ11*
Standards
(200 g/ml)
ꢀ54***
(Allopurinol)
ꢀ50***
(Manitol)
ꢀ55***
m
(a-tocopherol)
Powdered A. paniculata .Nees (Whole plant) (4 kg) were placed
in glass percolator with 95% ethanol (10 L) and allowed to stand for
24 h at room temperature. The percolate was collected and these
Each value is mean ꢁ sd of six values, *p < 0.05; **p < 0.01; ***p < 0.001; ns (non
significant) experimental values compared with control values.

S. Pandeti et al. / European Journal of Medicinal Chemistry 69 (2013) 439e448
445
processes were repeated for four times. The combined percolate of
40 L was evaporated under reduced pressure at 50 ꢂC to afford
ethanol extract. The weight of extract was found to be 300 g.
4.05 (d, J ¼ 9.73 Hz, 1H), 3.86 (d, J ¼ 10.33 Hz, 1H), 2.18 (s, 3H), 1.31
(s, 6H), 1.08 & 0.65 (each s, 3H); ESI - MS: 433 [M þ H].þ
4.6.3. Synthesis of (S, E)-4-(2-((1R, 4aS, 5R, 6R, 8aS)-6-hydroxy-5-
(hydroxymethyl)-5, 8a-dimethyl-2-methylene decahydronaphthalen-
1-yl)ethylidene)-5-oxotetrahydrofuran-3-yl acetate (4)
4.4. Fractionation
The ethanol extract was macerated with hexane. The hexane
soluble fraction was separated and evaporated under reduced
pressure to afford hexane fraction (F001, 80 g). Distilled water was
added to hexane insoluble portion, which was fractionated with
chloroform and the resultant solution was evaporated under
reduced pressure to afford chloroform fraction (F002, 120 g). The
water soluble fraction was evaporated under reduced pressure at
60 ꢂC to afford the aqueous fraction (F003, 70 g).
To a solution of compound 3 (100 mg, 0.00023 mol) in THF (5 mL)
was added 1 N HCl (5 mL). The mixture was stirred at 20 ꢂC for 2 h,
and then extracted with ethyl acetate (3 ꢃ 25 mL), the organic layer
was washed with water, dried over anhyd Na2SO4 and evaporated
under reduced pressure. Then the crude product was chromato-
graphed on silica gel to afford the desired compound 4.Yield: 95%; IR
(KBr) 3683, 3620, 3436, 3019, 2975, 2400, 1602, 1522, 1475, 1423,
1216, 1045, 928, 876, 849, 768, 758, 669, 627 cmꢀ1; 1H NMR (DMSO,
300 MHz)
d
6.60 (t, J ¼ 6.83 Hz, 1H), 5.68 (d, J ¼ 6.23 Hz, 1H), 4.91 (d,
4.5. Isolation of diterpenoid
J ¼ 4.93 Hz,1H), 4.79 (s,1H), 4.60 (s,1H), 4.52 (t, J ¼ 6.53 Hz,1H), 3.81
(d, J ¼ 9.83 Hz, 1H), 3.62 (d, J ¼ 10.13 Hz, 1H), 2.30 (s, 3H), 1.06 & 0.63
(each s, 3H); ESI - MS: 393 [M þ H].þ
Chloroform fraction (120 g) was chromatographed on a column
of silica gel (60e120 mesh) and eluted with chloroform e methanol
(96:4) to afford compound 1 (2 g) as colorless needles. The com-
pound’s visualization was done under UV light and by spraying
with 10% sulfuric acid in methanol, which also gives positive Lie-
bermanneBurchard test for terpenoids.
4.6.4. General procedure for synthesis of sulfonyl compounds (5, 6)
Compound 4 (100 mg, 0.00025 mol) magnetically stirred in a
solution of pyridine (2 mL) and tosyl chloride/2-Mesitylene sulfonyl
chloride at 60-70 ꢂC for 4 h. Solvents were removed by vacuum, and
then extracted with ethyl acetate (3 ꢃ 25 mL), the organic layer was
washed with water, dried over anhyd Na2SO4 and evaporated under
reduced pressure. Then the crude product was chromatographed
on silica gel to afford the desired compounds 5, 6.
Andrographolide (1): IR (KBr) 3435, 2956, 2917, 2849, 2143,
1639, 1465, 1377, 1218, 1073, 1018, 771, 655 cmꢀ1; 1H NMR (DMSO,
300 MHz)
d
6.62 (t, J ¼ 6.73 Hz, 1H), 5.72 (d, J ¼ 6.14 Hz, 1H), 5.04 (d,
J ¼ 4.92 Hz, 1H), 4.81 (s, 1H), 4.63 (s, 1H), 4.37 (t, J ¼ 6.37 Hz, 1H),
4.02 (d, J ¼ 9.41 Hz, 1H), 3.82 (d, J ¼ 10.24 Hz, 1H) 1.09 & 0.66 (each
s, 3H); 13C NMR (DMSO, 75 MHz) 170.41, 148.04, 146.76, 129.43,
108.71, 78.94, 74.78, 64.99, 63.13, 55.95, 54.84, 42.73, 40.79, 37.96,
36.97, 28.35, 24.43, 24.40, 23.52, 15.21; ESI - MS 350 [M þ H].þ
4.6.4.1. (S, E)-4-(2-((1R, 4aS, 5R, 6R, 8aS)-6-hydroxy-5, 8a-dimethyl-
2-methylene-5-tosyloxymethyl) decahydronaphthalen-1-yl) ethyl-
idene)-5-oxotetrahydrofuran-3-yl acetate 5. Yield: 70%; IR (KBr)
3664, 3425, 3073, 2986, 2471, 2252, 2125, 1751, 1639, 1508, 1418,
1242,1221,1031, 881, 819, 758, 665, 621, 568 cmꢀ1; 1H NMR (DMSO,
4.6. Preparation of andrographolide derivatives
300 MHz)
d
7.51 (d, J ¼ 8.02 Hz, 2H), 7.18 (d, J ¼ 7.93 Hz, 2H), 6.64 (t,
4.6.1. Synthesis of (S, E)-4-hydroxy-3-(2-((4aR, 6aS, 7R, 10aS, 10bR)-
3, 3, 6a, 10b-tetramethyl-8-methylenedecahydro-1H-naphtho[2,1-d]
[1,3]dioxin-7-yl)ethylidene)dihydrofuran-2(3H)-one (2)
J ¼ 6.53 Hz, 1H), 4.94 (d, J ¼ 6.73 Hz, 2H), 4.83 (s,1H), 4.64 (s, 1H),
4.50 (t, J ¼ 6.42 Hz, 1H), 4.04 (d, J ¼ 9.59 Hz, 1H), 3.85 (d,
J ¼ 10.13 Hz, 1H), 2.32 (s, 3H), 2.18 (s, 3H), 1.10 & 0.67 (each s, 3H);
ESI - MS: 547 [M þ H].þ
Iodine (30 mg) was dissolved in acetone (5 mL) and compound 1
(100 mg, 0.00028 mol) was added and stirred at the ambient tem-
perature (28 ꢂC). The reaction was monitored by TLC and was found
to be completewhen the compound 1 went into solution. Iodine was
destroyed by the addition of dilute aqueous sodium hydroxide so-
lution. The resulting solutionwould be colorless. The isopropylidene
derivative was extracted with ethyl acetate (3 ꢃ 25 mL), the organic
layer was washed with water, dried over anhyd Na2SO4 and evap-
orated under reduced pressure. Then the crude product was chro-
matographed on silica gel to afford the desired compound 2. Yield:
90%; IR (KBr) 3854, 3400, 3016, 2946, 2834, 2515, 2402, 2219, 2039,
1642, 1449, 1415, 1216, 1111, 1023, 928, 878, 769, 668 cmꢀ1; 1H NMR
4.6.4.2. (S, E)-4-(2-((1R, 4aS, 5R, 6R, 8aS)-6-hydroxyl-5-((mesi-
tylsulfonyloxy) methyl)-5, 8a-dimethyl-2-methylene decahydronaph-
thalen-1-yl) ethylidene)-5-oxotetrahydrofuran-3-yl acetate 6.
Yield: 70%; IR (KBr) 3424, 3019, 2979, 2400, 1743, 1637, 1452, 1383,
1215, 1088, 1013, 851, 758, 683, 669, 582, 548 cmꢀ1
;
1H NMR
(DMSO, 300 MHz)
d
6.79 (s, 2H), 6.62 (t, J ¼ 6.63 Hz, 1H), 5.81 (d,
J ¼ 6.47 Hz, 1H), 4.92 (d, J ¼ 4.83 Hz, 1H), 4.81 (s,1H), 4.62 (s, 1H),
4.52 (t, J ¼ 6.42 Hz, 1H), 4.03 (d, J ¼ 9.83 Hz, 1H), 3.83 (d,
J ¼ 10.33 Hz, 1H), 2.65 (s, 6H), 2.33 (s, 3H), 2.18 (s, 3H), 1.08 & 0.65
(each s, 3H); ESI - MS: 575 [M þ H].þ
(DMSO, 300 MHz)
d
6.62 (t, J ¼ 6.75 Hz, 1H), 5.14 (d, J ¼ 6.21 Hz, 1H),
4.91 (d, J ¼ 4.87 Hz, 1H), 4.81 (s,1H), 4.63 (s, 1H), 4.35 (t, J ¼ 6.42 Hz,
1H), 4.02 (d, J ¼ 9.40 Hz, 1H), 3.83 (d, J ¼ 10.13 Hz, 1H), 1.36 (s, 6H),
1.08 & 0.66 (each s, 3H); ESI - MS: 391 [M þ H]þ.
4.6.5. General procedure for synthesis of deacetylated sulfonyl
compounds (7, 8)
To a solution of compound 5 (100 mg, 0.00018 mol) in methanol
(5 mL) was added sodium methoxide (0.00025 mol). The mixture
was stirred at room temperature for 1 h, and then extracted with
ethyl acetate (3 ꢃ 25 mL), the organic layer was washed with water,
dried over anhyd Na2SO4 and evaporated under reduced pressure.
Then the crude product was chromatographed on silica gel to afford
the desired compound.
4.6.2. Synthesis of (S, E)-5-oxo-4-(2-((4aR, 6aS, 7R, 10aS, 10bR)-3,
3,6a, 10b-tetramethyl-8-methylenedecahydro-1H-naphtho[2,1-d]
[1,3]dioxin-7-yl)ethylidene)tetrahydrofuran-3-yl acetate (3)
Compound 2 (100 mg, 0.00025 mol) magnetically stirred in a
solution of pyridine (2 mL) and acetic anhydride (0.00128 mol) at
60-70 ꢂC for 4 h. The reaction mixture was put into cold water for
crystallization, then filtered and dried to get desired compound
3.Yield: 98%; IR (KBr) 3413, 3018, 2948, 2837, 2400, 2129, 1638,
4.6.5.1. ((1R, 2R, 4aS, 5R, 8aS)-2-hydroxyl-5-((E)-2-((S)-4-hydroxyl-2-
oxodihydrofuran-3(2H)-ylidene) ethyl)-1, 4a-dimethyl-6-
methylenedecahydronaphthalen-1-yl) methyl 4-methylbenzenesulfonate
7. Yield: 85%; IR (KBr) 3375, 3020, 2980, 2941, 2402, 1747, 1605,
1449, 1412, 1216, 1019, 927, 770, 669 cmꢀ1
300 MHz)
J ¼ 4.73 Hz, 1H), 4.81 (s,1H), 4.62 (s, 1H), 4.58 (t, J ¼ 6.46 Hz, 1H),
;
1H NMR (DMSO,
d
6.61 (t, J ¼ 6.69 Hz, 1H), 5.67 (d, J ¼ 6.24 Hz, 1H), 4.92 (d,
1452, 1216, 1084, 1013, 927, 854, 761, 674, 582, 546 cmꢀ1 1H NMR
;

446
S. Pandeti et al. / European Journal of Medicinal Chemistry 69 (2013) 439e448
(DMSO, 300 MHz)
d
7.51 (d, J ¼ 8.10 Hz, 2H), 7.15 (d, J ¼ 7.83 Hz, 2H),
in THF (5 mL) and the reaction was stirred at room temperature for
2 h, and then extracted with ethyl acetate (3 ꢃ 25 mL), the organic
layer was washed with water, dried over anhyd Na2SO4 and evapo-
rated under reduced pressure. Then the crude product was chro-
matographed on silica gel to afford the desired compound 11 and 9.
Yield: 75%; IR (KBr) 3424, 2925, 2855, 1746, 1643, 1454, 1378, 1218,
6.60 (t, J ¼ 6.68 Hz, 1H), 4.92 (d, J ¼ 6.54 Hz, 2H), 4.80 (s, 1H), 4.61 (s,
1H), 4.37 (t, J ¼ 6.37 Hz, 1H), 4.04 (d, J ¼ 9.54 Hz, 1H), 3.85 (d,
J ¼ 10.14 Hz,1H), 2.29 (s, 3H),1.07 & 0.64 (each s, 3H); 13C NMR (DMSO,
75 MHz) 170.46, 148.03, 146.76, 144.80, 138.96, 129.40, 128.80, 125.95,
108.72, 78.90, 74.81, 72.94, 63.09, 55.93, 54.81, 42.70, 38.91, 37.94,
36.94, 28.33, 24.43, 24.37, 23.52, 21.26, 15.21; ESI - MS 505 [M þ H].þ
1037, 893, 767, 668 cmꢀ1 1H NMR (CDCl3, 300 MHz)
; d 7.16 (s, 1H),
6.84 (t, J ¼ 6.56 Hz, 1H), 6.13 (d, J ¼ 15.79 Hz,1H), 4.81 (d, J ¼ 6.12 Hz,
2H), 4.18 (d, J ¼ 9.79 Hz, 1H), 3.47 (d, J ¼ 10.36 Hz, 1H), 1.25 & 0.80
(each s, 3H); 13C NMR(CDCl3, 75 MHz) 170.43, 148.12, 143.09, 129.21,
113.98, 111.07, 109.15, 80.72, 61.19, 55.64, 54.63, 42.87, 38.55, 38.23,
36.56, 28.04, 22.95, 22.68, 15.91; ESI-MS: 333 [M þ H].þ
4.6.5.2. ((1R, 2R, 4aS, 5R, 8aS)-2-hydroxyl-5-((E)-2-((S)-4-hydroxyl-2-
oxodihydrofuran-3(2H)-ylidene) ethyl)-1, 4a-dimethyl-6-
methylenedecahydronaphthalen-1-yl) methyl 2, 4, 6-trimethyl benzene
sulfonate 8. Yield: 85%; IR (KBr) 3411, 2940, 1743, 1668, 1452, 1213,
1086, 1013, 852, 763, 681, 582, 547 cmꢀ1; 1H NMR (DMSO, 300 MHz)
d
6.92 (s, 2H), 6.75 (t, J ¼ 6.86 Hz,1H), 5.58 (d, J ¼ 6.28 Hz,1H), 5.04 (d,
4.6.9. Synthesis of (S, E)-4-hydroxy-3-(2-((1S, 2S, 4aS, 5R, 6R, 8aR)-
6-hydroxy-5-(hydroxymethyl)-5, 8a-dimethyloctahydro-1H-spiro
[naphthalene-2, 20-oxirane]-1-yl) ethylidene) dihydrofuran-2(3H)-
one (12)
To a solution of compound 1 (100 mg, 0.00028 mol) in methanol
(5 mL) was added m-CPBA (0.00033 mol). The mixture was stirred
at room temperature for 12 h, and then extracted with ethyl acetate
(3 ꢃ 25 mL), the organic layer was washed with water, dried over
anhyd Na2SO4 and evaporated under reduced pressure. Then the
crude product was chromatographed on silica gel to afford the
desired compound 12. Yield: 80%; IR (KBr) 3427, 2987, 2471, 2252,
2126, 1647, 1508, 1416, 1242, 1223, 1154, 1028, 820, 758, 664,
J ¼ 4.88 Hz, 1H), 4.94 (s, 1H), 4.75 (s, 1H), 4.51 (t, J ¼ 6.46 Hz,1H), 4.16
(d, J ¼ 9.73 Hz,1H), 3.99 (d, J ¼ 10.52 Hz,1H), 2.64 (s, 6H), 2.31 (s, 3H),
1.21 & 0.78 (each s, 3H); 13C NMR (DMSO, 75 MHz) 170.44, 148.05,
146.78, 141.93, 137.56, 136.53, 130.54, 129.42, 108.71, 78.91, 74.81,
73.05, 63.11, 55.94, 54.83, 42.72, 37.95, 36.96, 28.34, 24.43, 23.52,
23.09, 20.75, 15.21; ESI - MS 533 [M þ H].þ
4.6.6. Synthesis of (4S)-4-hydroxyl-3-(2-((1R, 4aS, 5R, 6R, 8aS)-6-
hydroxyl-5-(hydroxymethyl)-5, 8a-dimethyl-2 -methylenedecahy
dronaphthalen-1-yl) ethyl) dihydrofuran ꢀ2(3H)-one (9)
To a magnetically stirred solution of compound 1 (100 mg,
0.00028 mol) in methanol (10 mL) was added gradually NiCl2.6H2O
(0.00042 mol) at rt. When the clear solution acquired a greenish color,
the whole reaction mixture was brought to 0 ꢂC and NaBH4
(0.00036 mol) was added portion wise. After addition of NaBH4, the
whole solution was stirred for 30 min at 0 ꢂC to rt. Methanol was
removed by vacuum, and then extracted with ethyl acetate
(3 ꢃ 25 mL), the organic layer was washed with water, dried over
anhyd Na2SO4 and evaporated under reduced pressure. Then the
crude product was chromatographed on silica gel to afford the desired
compound 9. Yield: 90%; IR (KBr) 3684, 3614, 3405, 3021, 2401, 1640,
622 cmꢀ1; 1H NMR (DMSO, 300 MHz)
d
6.71 (t, J ¼ 6.68 Hz,1H), 4.83
(s, 1H), 4.36 (t, J ¼ 6.38 Hz, 1H), 4.13 (s, 1H), 3.99 (d, J ¼ 9.56 Hz, 1H),
3.87 (d, J ¼ 10.16 Hz, 1H), 2.76 (d, J ¼ 2.68 Hz, 1H), 2.70 (d,
J ¼ 2.56 Hz, 1H), 1.09 & 0.80 (each s, 3H); 13C NMR(DMSO, 75 MHz)
170.89, 148.74, 128.23, 80.33, 79.90, 74.86, 65.44, 63.50, 59.40,
54.76, 53.94, 50.19, 42.93, 37.52, 36.64, 29.83, 28.10, 23.82, 23.15,
15.67; ESI-MS: 367 [M þ H].þ
4.6.10. Synthesis of (1R, 4aS, 5R, 8aS)-5-((E)-2-((S)-4 -hydroxy-2-
oxodihydrofuran-3(2H)-ylidene) ethyl)-1, 4a -dimethyl-6-
1522, 1424, 1215, 1039, 928, 761, 671, 627 cmꢀ1
300 MHz)
1H), 4.63(s,1H),4.39(t,J¼ 6.56Hz,1H), 4.13(d, J¼ 9.73 Hz,1H), 4.03 (d,
J ¼ 10.49 Hz, 1H), 1.09 (s, 3H), 0.66 (s, 3H); 13C NMR (DMSO, 75 MHz)
170.42, 146.77, 108.71, 78.92, 74.79, 68.99, 63.12, 55.96, 54.84, 48.74,
42.80, 37.97, 36.98, 28.35, 24.43, 23.53,15.22; ESI e MS: 353 [M þ H].þ
;
1H NMR (DMSO,
methylene-2-oxodecahydronaphthalene-1-carbaldehyde (13)
To a magnetically stirred solution of compound 1 (100 mg,
0.00028 mol) in DCM (5 mL), the whole reaction mixture was
brought to 0 ꢂC and DMP (0.00084 mol) was added. After addition
of DMP, the whole solution was stirred for 30 min at 0 ꢂC to rt.
DCM was removed by vacuum, and neutralized with saturated
NaHCO3 then extracted with ethyl acetate (3 ꢃ 25 mL), the organic
layer was washed with water, dried over anhyd Na2SO4 and
evaporated under reduced pressure. Then the crude product was
chromatographed on silica gel to afford the desired compound 13.
Yield: 70%; IR (KBr) 3624, 3405, 3015, 2947, 2835, 2403, 2037,
d
5.71 (d, J ¼ 6.14 Hz, 1H), 5.05 (d, J ¼ 4.87 Hz, 1H), 4.82 (s,
4.6.7. Synthesis of 3-(2-((1R, 4aS, 5R, 6R, 8aS)-6-hydroxyl-5-(hydroxy
methyl)-5, 8a-dimethyl-2 -methylenedecahydronaphthalen-1-yl)
ethyl) furan-2(5H)-one (10)
To a solution of compound 9 (100 mg, 0.00028 mol) in methanol
(5 mL) was added sodium carbonate (0.00025 mol). The mixture
was stirred at 50 ꢂC for 2 h, and then extracted with ethyl acetate
(3 ꢃ 25 mL), the organic layer was washed with water, dried over
anhyd Na2SO4 and evaporated under reduced pressure. Then the
crude product was chromatographed on silica gel to afford the
desired compound 10. Yield: 80%; IR (KBr) 3435, 3018, 2990, 2937,
1794, 1751, 1644, 1452, 1383, 1345, 1217, 1148, 1078, 1028, 894, 845,
1638, 1450, 1415, 1217, 1022, 927, 767, 669 cmꢀ1
;
1H NMR (CDCl3,
9.76 (s, 1H), 6.91 (t, J ¼ 6.68 Hz, 1H), 5.01 (d,
300 MHz)
d
J ¼ 6.23 Hz, 2H), 4.95 (s, 1H), 4.64 (s, 1H), 4.46 (t, J ¼ 6.38 Hz, 1H),
0.87 & 0.68 (each s, 3H); 13C NMR (CDCl3, 75 MHz) 209.08, 201.04,
173.27, 148.98, 143.87, 130.08, 121.93, 110.04, 70.56, 65.01, 56.52,
55.50, 43.71, 39.46, 39.09, 37.43, 28.89, 23.82, 23.54, 16.75; ESI-MS:
347 [M þ H].þ
756, 668 cmꢀ1; 1H NMR (CDCl3, 300 MHz)
d 7.08 (s, 1H), 4.86 (s, 1H),
4.76 (s, 2H), 4.58 (s, 1H), 3.45 (d, J ¼ 9.51 Hz, 1H), 3.31
(d,J ¼ 10.26 Hz, 1H), 1.22 & 0.62 (each s, 3H); 13C NMR(CDCl3,
75 MHz) 174.77, 147.20, 144.49, 135.00, 107.68, 80.83, 70.51, 64.54,
56.42, 55.66, 43.17, 39.39, 38.54, 37.24, 28.55, 24.88, 24.35, 23.10,
22.30, 15.58; ESI-MS: 335 [M þ H].þ
4.6.11. Synthesis of (4S, E)-4-hydroxy-3-(2-((1R, 4aS, 5R, 6R, 8aS)-6-
hydroxy-5-(hydroxymethyl)-2, 5, 8a -trimethyldecahydronaphthalen-
1-yl) ethylidene) dihydrofuran-2(3H)-one (14)
Compound 1 (100 mg, 0.00028 mol) was dissolved in MeOH and
added Pd (10% on carbon) in a bottle under atmosphere of nitrogen.
Then nitrogen was completely replaced by hydrogen in parr as-
sembly. The reaction was allowed to run for 1 h under pressure of
50 psi of H2. After completion of reaction (1 h, TLC monitoring), the
catalyst was removed by filtration through celite, solvent was
removed under vacuum, to afford the desired compound 14. Yield:
90%; IR (KBr) 3435, 3019, 1639, 1427, 1216, 1026, 758, 669 cmꢀ1; 1H
4.6.8. Synthesis of (E)-3-(2-((1R, 4aS, 5R, 6R, 8aS)-6-hydroxy-5-
(hydroxymethyl)-5, 8a-dimethyl-2 -methylenedecahydronaphthalen-
1-yl) ethylidene) furan-2(3H)-one (11)
NaBH4 (0.00033 mol) was added over 5 min to a stirred mixture of
compound 1 (100 mg, 0.00028 mol) and Amberlyst-15 (0.00142 mol)

S. Pandeti et al. / European Journal of Medicinal Chemistry 69 (2013) 439e448
447
NMR (DMSO, 300 MHz)
d
6.61 (t, J ¼ 6.71 Hz, 1H), 4.81 (s, 1H), 4.62
compound 1 and compound 7 in same model at different doses
(50, 100 & 150 mg/kg b.w).
(s, 1H), 4.38 (t,J ¼ 6.32 Hz, 1H), 4.01 (d, J ¼ 9.56 Hz, 1H), 3.82 (d,
J ¼ 10.23 Hz, 1H), 1.17 (s, 3H), 0.87 (d, J ¼ 6.80 Hz, 3H), 0.65 (s, 3H);
13C NMR(DMSO, 75 MHz) 170.37, 148.02, 129.41, 78.90, 74.75, 64.96,
63.09, 55.93, 54.82, 42.71, 40.78, 37.94, 36.95, 28.32, 24.40, 23.49,
15.82, 15.18; ESI-MS: 353 [M þ H].þ
4.7.3. Lipoprotein measurement
Plasma was fractionated into very low density lipoprotein
(VLDL), low density lipoprotein (LDL) and high density lipoprotein
(HDL) by poly anionic precipitation methods. Plasma and lipo-
proteins were analyzed for their total cholesterol (TC), phospho-
lipid (PL), and triglyceride (TG) by standard procedures reported
earlier [38].
4.6.12. Synthesis of (4S)-4-hydroxy-3-(2-((1R, 4aS, 5R, 6R, 8aS)-6-
hydroxy-5-(hydroxymethyl)-2, 5, 8a -trimethyldecahydronaphthalen-
1-yl) ethyl) dihydrofuran-2(3H)-one 15
Yield: 90%; IR (KBr) 3436, 3019, 1637, 1215, 1035, 928, 757, 669,
484 cmꢀ1;; 1H NMR (DMSO, 300 MHz)
d
4.86 (s, 1H), 4.67 (s, 1H),
4.7.4. Lipoprotein lipase activity in liver of triton induced
hyperlipidemic rats
Liver was homogenized (10%, w/v) in cold 100 mM phosphate
buffer pH 7.2 and used for the assay of total lipolytic activity of li-
poprotein lipase (LPL) [39].
4.44 (t, J ¼ 6.28 Hz, 1H), 4.06 (d, J ¼ 9.76 Hz, 1H), 3.86 (d,
J ¼ 10.34 Hz, 1H), 1.22 (s, 3H), 0.80 (d, J ¼ 6.86 Hz, 3H), 0.70 (s, 3H);
ESI-MS: 355 [M þ H]. þ
4.6.13. Synthesis of (1R, 2R, 4aS, 5R, 8aS)-5-((E)-2-((S)-4- acetoxy-
2-oxodihydrofuran e 3 (2H)-ylidene) ethyl)-1-(acetoxymethyl)-1,
4a-dimethyl-6-methylenedecahydronaphthalen-2-yl acetate 16
Yield: 98%; IR (KBr) 3684, 3618, 3436, 3019, 2976, 2400, 1602,
1521, 1476, 1422, 1215, 1045, 928, 877, 849, 770, 669, 627,
4.7.5. Antioxidant activity
Superoxide anions were generated enzymatically by xanthine
(160 mM), xanthine oxidase (0.04 U), and nitroblue tetrazolium
(320
m
M) in absence or presence of compound 1 and 7 at con-
g/mL in 100 mM phosphate buffer (pH 8.2).
497 cmꢀ1
;
1H NMR (DMSO, 300 MHz)
d
6.90 (t, J ¼ 6.71 Hz, 1H),
centrations 200 m
5.70 (d,J ¼ 6.16 Hz, 1H), 5.20 (d, J ¼ 4.98 Hz, 1H), 5.09(s,1H), 4.90
(s, 1H), 4.67 (t, J ¼ 6.42 Hz, 1H), 4.32 (d, J ¼ 9.76 Hz, 1H), 4.11 (d,
J ¼ 10.56 Hz, 1H), 2.23 (s, 9H), 1.36 & 0.94 (each s, 3H); ESI-MS:
477 [M þ H].þ
Compounds were sonicated well in phosphate buffer before use.
The reaction mixtures were incubated at 37 ꢂC and after 30 min
the reaction was stopped by adding 0.5 mL glacial acetic acid. The
amount of formazone formed was calculated spectrophotomet-
rically. In another set of experiment effect of compounds on the
generation of hydroxyl radical was also studied by non-
enzymatic reactants. Briefly, ꢄOH were generated in a non-
enzymatic system comprising deoxy ribose (2.8 mM), FeS-
O4.7H2O (2 mM), sodium ascorbate (2.0 mM) and H2O2 (2.8 mM)
in 50 mM KH2PO4 buffer (pH 7.4) to a final volume of 2.5 mL. The
above reaction mixtures in the absence or presence of test
compounds were incubated at 37 ꢂC for 90 min. The test com-
pounds were also studied for their inhibitory action against
microsomal lipid peroxidation in vitro by nonenzymatic inducer.
Reference tubes and reagents blanks were also run simulta-
neously. Malondialdehyde (MDA) contents in both experimental
and reference tubes were estimated spectrophotometrically by
4.7. Biological experiments
4.7.1. Animals
Rats (Charles Foster strain, male, adult, body wt 200e225 g)
were kept in a room with controlled temperature at 25e26 ꢂC,
humidity 60e80% and 12/12 h light/dark cycle (light on from 8.00
AM to 8 PM) under hygienic conditions. Animals were acclimatized
for one week before starting the experiment. The animals had free
access to the normal diet and water.
4.7.2. Antidyslipidemic activity in triton induced hyperlipidemic rat
model
Rats were divided into different groups- control, triton
treated and triton plus extraction, fractionation and derivatives
of compound 1 treated containing six animals in each group. In
the acute experiment of 18 h, hyperlipidemia was developed by
administration of triton WR-1339 (Sigma Chemical Company, St.
Louis, MO, USA) at a dose of 400 mg/kg, b.w. intraperitoneally
(i.p) to animals of all the treated groups with extraction, frac-
tionation and derivatives of compound1 were macerated with
gum acacia, suspended in water and fed simultaneously with
triton at a dose of 100 mg/kg p.o. to the animals of treated
groups, diet was withdrawn. Animals of control and triton group
without treatment with extraction, fractionation and derivatives
of compound 1 were given same amount of gum acacia sus-
pension (vehicle). After 18 h of treatment the animals were
anaesthetized with thiopentone solution (50 mg/kg b.w.) pre-
pared in normal saline and 1 mL blood was withdrawn from
retro-orbital sinus using glass capillary in EDTA coated tubes
(3.0 mg/ml blood). The blood was centrifuged at 2500 ꢃ g for
10 min at 4 ꢂC and plasma was separated. Plasma was diluted
with normal saline (ratio of 1:3) and used for analysis of total
cholesterol (TC), Phospholipid (PL) and triglyceride (TG) by
standard enzymatic methods. Using Beckmann auto-analyzer
and standard kit purchase from Merck Company and post-
heparin lipolytic activity (PHLA) was assayed using spectropho-
tometer [37]. After the confirmation of most active compounds
in primary screening we further, evaluate the activity of
thiobarbituric acid [40]. Alloprinol, Mannitol and a-tocopherol
were used as standard drugs for superoxide, hydroxylations and
microsomal lipid peroxidation.
4.7.6. LDL oxidation
Serum was separated from the blood of normolipemic donors
who were fasted overnight and fractionated into very low-
density lipoprotein (VLDL), low density lipoprotein (LDL) and
high density lipoprotein (HDL) by ultracentrifugation [41]. The
lipoproteins preparations were dialyzed against 150 mM NaCl
containing EDTA (0.02% w/v) in presence of N2 gas in cold. The
purity of LDL was checked on polyacrylamide gel electrophoresis.
LDL (0.71 mg) and CuCl2$2H2O (10 mM) in the absence or pres-
ence of compound 2 in 50 mM phosphate buffer saline (pH 7.4)
to a final volume of 1.5 mL, was incubated at 37 ꢂC for 16 h. The
level of lipid peroxides in unoxidized LDL, oxidized LDL with
Cuþþ in the absence or presence of compounds 1 and 7 at dose of
200
mg/mL were assayed as thiobarbuteric acid reactive sub-
stances (TBARS). Briefly the reaction mixture contained 0.5 mL
SDS (8% w/v), 0.5 ml glacial acetic acid, 1.5 ml TBA (0.8% w/v) was
heated in a boiling water bath for 1 h. After cooling up to room
temperature optical density of reaction mixture was read at
532 nm with respective reagent blank. The level of lipid peroxide
as nmol of Malondialdehyde formed was calculated by taking
absorption Coefficient of MDA as 1.78 ꢃ 105 cmꢀ1 Mꢀ1 mg
protein.

448
S. Pandeti et al. / European Journal of Medicinal Chemistry 69 (2013) 439e448
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Acknowledgments
Authors are thankful to Dr. T.K. Chakraborty, Director, CSIR-CDRI
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