Indole-based fibrates as potential hypolipidemic and antiobesity agents

Article abstract of DOI:10.1021/jm201697v

Hypolipidemic and antiobesity effects of the newly synthesized indole-based fibrates were evaluated in Triton WR-1339 and high fat diet (HFD)-induced hyperlipidemic rats. Preliminary screening of all the synthesized compounds was done by using an acute model (Triton model), in which compounds 3f and 3l showed significant antidyslipidemic activity. Furthermore, these compounds 3f and 3l were found to induce significant weight loss in the visceral fat mass of HFD-fed hyperlipidemic rats without affecting the normal feeding behavior. Histological examination of the liver of rats supplemented with 3f and 3l revealed a significant decrease in steatosis when compared to the effect of the standard drug fenofibrate. Additional effects such as an increase in lecithin cholesterol acyl-transferase (LCAT) enzyme level and increased receptor mediated catabolism of I¹³¹-low density lipoproteins (LDL) confirm and reinforce the efficacy of both of these compounds as a new class of dual-acting hypolipidemic and antiobesity agents.

Full text of DOI:10.1021/jm201697v

Article
pubs.acs.org/jmc
Indole-Based Fibrates as Potential Hypolipidemic and Antiobesity
Agents
Koneni V. Sashidhara,*,† Manoj Kumar,† Ravi Sonkar,‡ Bhanu Shankar Singh,§ A. K. Khanna,‡
and Gitika Bhatia‡
†Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute (CSIR-CDRI), Lucknow 226 001, India
‡Biochemistry Division, CSIR-Central Drug Research Institute (CSIR-CDRI), Lucknow 226 001, India
§Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Raebareli 229 010, India
S
* Supporting Information
ABSTRACT: Hypolipidemic and antiobesity effects of the newly synthesized indole-based fibrates were evaluated in Triton
WR-1339 and high fat diet (HFD)-induced hyperlipidemic rats. Preliminary screening of all the synthesized compounds was
done by using an acute model (Triton model), in which compounds 3f and 3l showed significant antidyslipidemic activity.
Furthermore, these compounds 3f and 3l were found to induce significant weight loss in the visceral fat mass of HFD-fed
hyperlipidemic rats without affecting the normal feeding behavior. Histological examination of the liver of rats supplemented with
3f and 3l revealed a significant decrease in steatosis when compared to the effect of the standard drug fenofibrate. Additional
effects such as an increase in lecithin cholesterol acyl-transferase (LCAT) enzyme level and increased receptor mediated
catabolism of I131-low density lipoproteins (LDL) confirm and reinforce the efficacy of both of these compounds as a new class of
dual-acting hypolipidemic and antiobesity agents.
is characterized by elevated triglycerides, decreased high density
lipoproteins (HDL) levels, and irregular LDL composition.8
Furthermore, an increase in plasma lipids may lead to ischemic
heart disease, myocardial infarction, and cerebrovascular
incidents. These conditions are responsible for one-third of
deaths in industrialized nations. Fibrates have been extensively
used for the past several decades in the management of
combined dyslipidaemia,9 but they require high doses to show
significant efficacy10 and are also associated with primary
muscle injury, especially when used in combination with a
statin.11,12 A more effective drug having both antiobesity
properties and hypolipidemic activity is in great demand.
In the design of new drugs, the development of hybrid
molecules through the combination of different pharmaco-
phores may lead to compounds with interesting biological
profiles. We have recently reported some coumarin-based
hybrids, which have shown diverse biological properties such as
anticancer, anti-inflammatory, and antithrombotic activities.13 A
series of coumarin-bis-indole hybrids has also been reported to
INTRODUCTION
■
Obesity has become a serious health problem in the
industrialized world.1 Analysis of worldwide epidemiological
data reveals that the frequency of this disorder has gone up
steeply, probably because of unhealthy living habits.2 In
addition, it has a huge impact on lifestyle-related disorders
such as coronary heart disease, atherosclerosis, and diabetes.3
Obesity involves an increased visceral fat mass and plasma lipid
profile, as well as an increase in body weight. Most marketed
antiobesity drugs are appetite suppressants (anorectics), which
act directly on the central nervous system (CNS) and decrease
food consumption by altering the central adrenergic or the
serotonergic system.4 Despite a rising worldwide epidemic of
obesity, there are currently only a very small number of
antiobesity drugs available to manage the problem. Orlistat5
(lipase inhibitor; reduces dietary fat absorption) and sibutr-
amine6 (appetite suppressant; inhibits reuptake of serotonin
and norepinephrine) are the only two Food and Drug
Administration (FDA)-approved drugs that are currently
available for the long-term treatment of obesity. Both of
these drugs are of limited efficacy and are associated with severe
side effects.7 The primary dyslipidemia associated with obesity
Received: December 16, 2011
Published: February 17, 2012
© 2012 American Chemical Society
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Figure 1. Designing of bis-indole−fibrate hybrids based on indole and fibrate scaffolds showing lipid lowering and antiobesity activity.
have significant antihyperlipidemic activity in HFD-fed
hamsters.14 The indole group is an essential part of a number
of compounds with significant biological activity. Indole
derivatives are known to exert antitubercular,15 anticancer,16,17
antiviral,18 and antioxidant activities.19 Fluvastatin [3-hydroxy-
3-methyl-glutaryl (HMG)-CoA reductase inhibitor], which
includes an indole moiety, is a synthetic member of the statin
class of drug, used to lower cholesterol and prevent
cardiovascular disease. The indole pharmacophore is also
found in a variety of antiobesity agents.20 Figure 1 shows
chemical structures of some bioactive indoles with antiobesity
and hypolipidemic activity. Herein, we wish to describe the
synthesis of novel indole−fibrate hybrids and their utility as
potential antiobesity and antidyslipidemic agents.
Table 1. Percentage (%) Decrease of Plasma Lipids with the
Treatment of Compound (3a−l) in Triton-Induced
Hyperlipidemic Rats at the Dose of 100 mg/kg Body
a
Weight
compd
3a
TC
PL
TG
−12*
−10*
−12*
−10*
−25***
−12*
−10*
NS
3b
−9
NS
3c
−6
−10*
NS
3d
−6NS
−8
−8
NS
NS
NS
3e
−9
−5
3f
−22**
−10*
−9
−10*
−11*
−23***
−10*
−25***
NS
3g
−6
−6
−5
NS
NS
NS
3h
−9
−9
NS
NS
3i
NS
NS
3j
−9
−9
RESULTS AND DISCUSSION
3k
−12*
−15*
−12*
■
3l
−20**
−33***
−14*
−35***
−27***
−35***
The antidyslipidemic activities of bis-indole derivatives 3a−l
were evaluated in an in vivo Triton model.21 Administration of
Triton WR-1339 in rats induced marked hyperlipidemia as
evidenced by an increase in the plasma level of total cholesterol
(TC, 2.66-fold), phospholipids (PL, 2.36-fold), and triglyceride
(TG, 3.02-fold) as compared to the control. Treatment of
hyperlipidemic rats with bis-indole derivatives at a dose of 100
mg/kg po reversed the plasma levels of lipid with varying
extents (Table 1). Compounds 3f and 3l significantly lowered
the TC, PL, and TG by 22%, 23%, and 25%, and 20%, 14%, and
27% respectively, while compounds 3a−d, 3g, and 3i−k
showed mild activity. By comparison, the standard drug
gemfibrozil at the same dose decreased the levels of TC, PL,
and TG in plasma by 33%, 35%, and 35%, respectively.
gemfibrozil
a
Units are mean
SD of six rats. The Triton-treated group was
compared with the control group, and Triton plus compound-treated
group was compared with the triton group only. ***P < 0.001. **P <
0.01. *P < 0.05 and NS = not significant.
With these promising activities in hand, dose-dependent (at
50, 100, 150 mg/kg body weight) studies on compounds 3f and
3l were performed on a different set of experimental animals,
which exhibited concentration-dependent effects. Both com-
pounds showed similar activity patterns, and at a dose of 150
mg/kg body weight, 3f and 3l showed activities comparable to
those of the standard drug gemfibrozil (Table 2).
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Table 2. Dose−Response Effects of Compounds 3f and 3l in Triton-Induced Hyperlipidemic Rats on Different Lipid
a
Parameters and Post-Heparin Lipolytic and Lecithin Cholesterol Acyl-Transferase Activities
b
b
b
c
d
compd
Triton
dose
TC
Pl
TG
PHLA
LCAT
−29***
+11*
−37***
+8*
3f
50 mg/kg
100 mg/kg
150 mg/kg
50 mg/kg
100 mg/kg
150 mg/kg
100 mg/kg
−16*
−15*
−12*
−22**
−33***
−15*
−20**
−35***
−35***
−23***
−29***
−16*
−14**
−29***
−31***
−25***
−26***
−13*
−27***
−27***
−31***
+12*
+15*
+10*
+15*
+12*
+18*
+11*
+15*
3l
+20*
+21*
gemfibrozil
+27***
+27***
a
Units are the mean SD of six rats. Triton-treated group compared with the control group and triton plus compound-treated group compared with
b
c
d
the Triton group only. ***P < 0.001. **P < 0.01. *P < 0.05. Unit: mg/dL. Unit: nmol cholesterol released/h/L plasma. Unit: nmol free fatty acid
formed/h/mL plasma.
Figure 2. Effects of compounds 3f and 3l on body weight gain, food intake, and food efficiency ratio of HFD-fed hyperlipidemic rats Animals were
sacrificed after 8 weeks of ND and HFD consumption with or without 3f, 3l, and fenofibrate (100 mg/kg/d). (A) Change in body weight gain (g),
(B) food intake, and (C) FER. Values are expressed as the mean SD (n = 6). **P < 0.01; ***P < 0.001. FER = (body weight gain for the
experimental period (g)/food intake for the experimental period (g)).
It is known that the LCAT enzyme converts free cholesterol
into cholesteryl ester (a more hydrophobic form of
cholesterol), which is subsequently converted into HDL.
Compounds 3f and 3l showed this beneficiary effect as each
significantly increased the level of LCAT enzyme in Triton-
treated hyperlipidemic rats in a concentration-dependent
manner. Furthermore, both compounds also increased post
heparin lipolytic activities as shown in Table 2. Since
compounds 3f and 3l showed significant activity in the
Triton-induced hyperlipidemic model, these two compounds
were further tested in the HFD-fed hyperlipidemic model,
which is similar in feeding behavior to humans. Several
biological experiments were carried out such as the study of
their effects on lipolytic enzymes, hepatic biochemical
parameters, specific binding of I125-LDL, and fecal bile acid
excretions in HFD-induced hyperlipidemic rats, and the results
of various studies are discussed next.
Effects of 3f and 3l on Body Weight and Visceral Fat
Mass of HFD-Induced Hyperlipidemic Rats. Adult Charles
Foster rats were given HFD (to induce hyperlipidemia) or
normal diet (ND) for four weeks. Subsequently, the ND rats
were given ND + vehicle, and HFD-fed rats were divided into
four groups and given HFD + vehicle, HFD + 3f, and HFD +
3l, and HFD + fenofibrate were given treatment for another
four weeks. HFD increased the body weight in rats, but
supplementing the HFD rats with either 3f or 3l (100 mg/kg/
d) suppressed the gain in body weight without affecting the
food intake, comparable in effect to the standard drug
fenofibrate (Figure 2). The food efficiency ratio in the HFD
+ 3f and HFD + 3l groups was significantly lower than that in
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Figure 3. Effects of 3f and 3l on the visceral fat-pad weights of HFD-fed hyperlipidemic rats. Animals were sacrificed after 8 weeks of ND and HFD
consumption with or without 3f, 3l, and fenofibrate (100 mg/kg/d). Visceral fat-pad weights in each group are shown. (A) Mesenteric, (B) gonadal,
(C) inguinal, and (D) perirenal fat mass. Values are expressed as the mean SD (n = 6). **P < 0.01; ***P < 0.001.
a
Table 3. Effects of Fenofibrate Derivatives and Fenofibrates on Serum Lipids in HFD Induced Hyperlipidemic Rats
parameters
control
HFD
HFD + 3f
HFD + 3l
HFD + fenofibrate
TC (mg/dL)
PL (mg/dL)
TG (mg/dL)
85.59 7.66
240.39 13.69***
180.49 13.26***
(−25% over HFD)
168.35 10.25***
(−24% over HFD)
262.30 10.75***
(−25% over HFD)
173.46 12.26***
(−28% over HFD)
166.66 9.17***
(−25% over HFD)
256.65 10.79***
(−27% over HFD)
51.95 4.40
153.05 10.21***
(−36% over HFD)
146.35 6.38***
(−34% over HFD)
233.89 7.14***
(−33% over HFD)
(+2.80-fold over control)
81.85 6.43
88.53 9.67
66.82 3.04
221.65 6.38***
(+2.70-fold over control)
350.37 17.35***
(+3.95-fold)
LCAT (nmol cholesterol released/h/L plasma)
37.06 1.82***
(−41% over control)
54.57 3.94***
(+32% over HFD)***
56.44 3.70***
(+34% over HFD)
(+29% over HFD)
a
Each parameter represents pooled data from 6 rats/group, and values are expressed as the mean SD. ***P < 0.001, HFD group compared with
the control group and HFD plus compound/fenofibrate groups compared with HFD.
the HFD group (Figure 2). The relative weights of the
mesenteric, gonadal, perirenal, and inguinal fat-pads in HFD +
3f and HFD + 3l rats were significantly lower than those in the
HFD-fed rats (Figure 3).
Effects of 3f and 3l on the Lipid Profile and LCAT
Activity in HFD-Induced Hyperlipidemic Rats. The
administration of HFD to rats increased their plasma levels of
TC (2.8-fold), PL (2.7-fold), and TG (3.95-fold) followed by a
decrease in LCAT activity (41%) as compared to control rats.
As shown in Table 3, feeding of 3f and 3l significantly reversed
the increased levels of TC, PL, and TG. Furthermore,
compound 3f increased the level of LCAT by 32%, whereas
fenofibrate increased the level by 34% (Table 3).
Effects of 3f and 3l on the Lipid Composition in
Serum Lipoproteins of HFD-Induced Hyperlipidemic
Rats. As shown in Table 4, the analysis of hyperlipidemic
serum of HFD administered rats showed marked increase in
the level of lipids and apoproteins constituting β-lipoproteins,
and these effects were pronounced for very low density
lipoprotein (VLDL-TG, 2.18-fold) and low-density lipoprotein
total cholesterol (LDL-TC, 4.08-fold). Treatment with 3f and
3l significantly reduced the increased levels of VLDL-TG (24%
and 17%, respectively) as well as LDL-TC (24% and 22%), PL
(25% and 20%), TG (25% and 21%), and apo-LDL (25% and
21%), respectively, in hyperlipidemic rats. Concurrently, the
decreased levels of HDL lipid and apo-HDL in these animals
were partially recovered (Table 4).
Effects of 3f and 3l on Hepatic Biochemical
Parameters in HFD-Induced Hyperlipidemic Rats. As
shown in Table 5, HFD administration to rats also caused
marked accumulation of TC (2.16-fold), PL (1.92-fold), and
TG (1.89-fold), followed by a decrease in LPL activity (39%) in
liver tissue. However, treatment with 3f and 3l caused a
decrease in the levels of TC (23% and 26%, respectively), PL
(28% and 23%), and TG (27% and 24%), followed by
reactivation of LPL activity (20% and 14%), respectively, in
hyperlipidemic animals.
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Table 4. Effects of Bis-Indole Derivatives and Fenofibrate on Blood Lipids and Lipolytic Enzymes in HFD Induced
a
Hyperlipidemic Rats
parameters
(mg/dL plasma)
control
HFD
HFD + 3f
HFD + 3l
HFD + fenofibrate
VLDL-TC
7.99 1.20
25.77 0.83***
(3.68-fold over control)
20.22 1.34**
(−21% over HFD)
21.99 1.20*
(−15% over HFD)
18.57 0.68***
(−28% over HFD)
VLDL-PL
VLDL-TG
apoprotein
LDL-TC
LDL-PL
14.67 1.24
38.00 1.83
6.12 0.87
0.56 1.00
12.30 0.72
14.47 1.49
16.92 2.08
44.32 3.22
36.47 2.35
15.48 1.72
166.71 5.56
35.01 2.48***
29.33 1.98*
31.10 2.50*
26.61 3.53***
(2.38-fold over control)
(−16% over HFD)
(−11% over HFD)
(−24% over HFD)
83.2 2.88***
(2.18-fold over control)
62.93 2.57***
(−24% over HFD)
9.68 1.06***
(−26% over HFD)
39.72 0.86***
(−24% over HFD)
33.09 2.35***
(−25% over HFD)
28.31 0.98**
(−21% over HFD)
22.36 1.49***
(−25% over HFD
39.71 3.28*
(+14% over HFD)
69.06 4.40*
(−17% over HFD)
8.85 0.90***
(−33% over HFD)
40.76 1.17**
(−22% over HFD)
35.04 2.40*
(−20% over HFD)
26.89 1.42***
(−25% over HFD)
23.55 1.49**
(−21% over HFD)
58.13 2.01***
(−30% over HFD)
8.80 0.56***
(−35% over HFD)
37.73 0.48***
(−28% over HFD)
31.52 0.85***
(−28% over HFD)
26.22 0.55***
(−27% over HFD)
21.37 1.18***
(−28% over HFD)
40.60 3.26*
(+16% over HFD)
13.18 0.88***
(2.15-fold over control)
52.59 3.00***
(4.08-fold over control)
43.92 2.05***
(3.57-fold over control)
LDL-TG
apoprotein
HDL-TC
HDL-PL
36.03 1.33***
(3.57-fold over control)
29.88 2.08***
(1.76-fold over control)
34.08 4.46**
(−19% over control)
29.55 2.01***
(−23% over control)
11.60 1.43***
(−25% over control)
28.35 11.96***
(−23% over control)
38.57 3.67*
(+12% over HFD)
33.98 1.56*
(+13% over HFD
32.28 1.95NS
34.95 1.56*
(+15% over HFD)
(+8% over HFD)
HDL-TG
apoprotein
13.10 1.72*
(+11% over HFD)
13.46 1.60*
(+14% over HFD)
13.82 1.71*
(+16% over HFD)
152.48 6.03*
(+16% over HFD)
149.42 5.75*
(+14% over HFD)
155.64 5.04*
(+17% over HFD)
a
Each parameter represents pooled data from 6 rats/group, and values are expressed as the mean SD. *P < 0.05. **P < 0.01. ***P < 0.001, HFD
group compared with the control group and HFD plus compound/fenofibrate groups compared with HFD.
Effects of 3f and 3l on Liver Histopathology of HFD-
Induced Rats. Figure 4 shows the effects of 3f and 3l on the
hepatic morphology of rats fed with a high-fat diet. The control
group showed normal hepatic histology. HFD administration
caused macrovesicular steatosis in the centrilobular area, but
liver fibrosis was not observed. The extent of steatosis was
considerably reduced in rats fed with HFD plus either 3f or 3l
(Figure 4).
Effects of Bis-Indole Derivatives and Fenofibrate on
I125-LDL Catabolism in Liver Plasma Membrane of HFD-
Induced Hyperlipidemic Rats. Administration of HFD
suppressed the specific binding of I125-LDL in the liver plasma
membrane by 54%. Compounds 3f and 3l, and fenofibrate
reversed the receptor-mediated catabolism of LDL (24%, 18%,
and 30%, respectively) in treated animals (Figure 5).
Effects of 3f and 3l on Fecal Excretion of Bile Acids.
HFD administration to rats caused a significant decrease in the
fecal excretion of cholic acid (41%) and deoxycholic acid (46%)
over control, and these levels were shown to be recovered by
the treatment with 3f (26% and 21%) and 3l (28% and 31%) in
HFD-treated rats (Table 6).
Preliminary screening of bis-indole hybrids on Triton WR-
1339-treated hyperlipidemic rats resulted in two promising
compounds, 3f and 3l, in comparison to gemfibrozil by
improving the lipid profile and enzymatic activity of plasma
LPL and LCAT. Further studies on these two compounds were
carried out using HFD-induced hyperlipidemic rats. The HFD-
induced hyperlipidemic rat model is more suitable for study
because the rats consume food and water ad libitum, similar to
human eating patterns, rather than being force-fed. The assayed
period of high-energy feeding induces a marked weight increase
in rats. In this study, we have found that HFD feeding for four
weeks resulted in hyperlipidemia, which was associated with
increased body weight. Moreover, administration of 3f and 3l
for the next four weeks significantly reduced body weight and
visceral fat mass, which indicates the degradation of adipose
tissue or inhibition of its formation. These beneficial effects of
3f and 3l were not because of decreased food intake, as the
amount of food consumed per animal was unchanged.
Additionally, the in vivo examined compounds did not show
any toxic effects at the doses studied, and almost all the animals
survived and looked normal both macroscopically and by
autopsy, demonstrating low general toxicity of the synthesized
compounds.
Dyslipidemia in obesity, characterized by increased trigly-
cerides and LDL, and decreased HDL, is the most important
factor that leads to coronary artery diseases.22 In our study, 3f
and 3l supplementation decreased the plasma TC, PL, and TG
levels in HFD-fed rats, possibly by inhibition in of their
exogenous absorption and formation. Compound 3f and 3l also
increased the LCAT activity, which plays a key role in
lipoprotein metabolism contributing to an increased level of
HDL.
Increased endogenous production of TG-enriched hepatic
VLDL, and decreased TG uptake in the peripheral tissues is the
secondary factor contributing to hyperlipidemia in HFD rats.23
TG enhances the ectopic accumulation of lipid stores in the
liver and is associated with a number of diseases such as
metabolic syndrome and type 2 diabetes.24 High TC levels
increase the risk of developing coronary heart disease (CHD),
and high levels of LDL and VLDL are also a risk factor for
CHD, while high HDL is helpful in transporting excess
cholesterol to the liver for excretion in bile.25
The endogenous effects of 3f and 3l on lipid metabolism
include increased receptor-mediated catabolism of LDL (Figure
6)26 as well as in lipolytic activity of the liver and in the level of
plasma HDL-TC, with a decrease of β-lipoprotein-lipid and
improved hepatic lipid profile.
Compounds 3f and 3l enhanced the synthesis of LDL-
apoprotein (Apo-B) as well as receptor protein to accelerate the
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turnover of cholesterol.27 It also decreased the VLDL levels in
plasma, which is mainly synthesized in the liver and is a carrier
for various lipids. In addition, compounds 3f and 3l increased
LPL levels in plasma, which catalyzes the hydrolysis of various
lipids on lipoprotein particles such as chylomicrons and VLDL
and turns them into smaller remnants that are rapidly cleared
from the bloodstream. The decreased lipid levels in the liver
were also complemented by increased bile acid secretion in
feces.
In the liver, HFD increases fatty acid synthesis and facilitates
the movement of free fatty acids from plasma.28 It also
decreases β-oxidation of free fatty acids, which may, in turn,
cause TG accumulation in the liver.29,30 The higher level of TG
in hepatocytes causes cellular dysfunction and may damage the
liver parenchyma.31 On giving HFD, macrovasicular dysfunc-
tion (fatty degeneration) occurs in the liver. The risk factors
from steatosis in the liver are diverse and are frequently
associated with obesity since the liver is the primary organ for
lipid metabolism. From our study, we have found that in HFD
groups liver macrovasicular steatosis arises, but administration
of compounds 3f and 3l to HFD-fed animals noticeably
lowered the extent of steatosis (Figure 4). These results may
indicate that compounds 3f and 3l could be beneficial for
patients with hepatic steatosis.
CONCLUSIONS
■
In conclusion, a novel series of bis-indole based fibrates was
synthesized and evaluated for their antihyperlipidemic and
antiobesity potentials in two animal models. Compounds 3f
and 3l emerged as promising agents for both dyslipidemia and
obesity as these compounds significantly modulated blood
plasma lipids and reduced visceral body masses without
affecting the feeding pattern of the animals. Additional studies
such as an increase in LCAT enzyme level and increased
receptor mediated catabolism of I131-LDL confirm and
reinforce the efficacy of both these compounds as a new class
of dual acting hypolipidemic and antiobesity agents. Fur-
thermore, the compound supplement did not apparently
adversely affect the animals. Compounds 3f and 3l seem to
be good candidates for the development of a new type of dual-
acting drug.
EXPERIMENTAL SECTION
■
General. All reagents were commercial and were used without
further purification. Chromatography was carried on silica gel (60−
120 and 100−200 mesh). All reactions were monitored by thin-layer
chromatography (TLC), and silica gel plates with fluorescence F254
were used. Melting points were taken in open capillaries on a Complab
melting point apparatus and are presented uncorrected. Infrared
spectra were recorded on a Perkin-Elmer FT-IR RXI spectropho-
1
tometer. H NMR and 13C NMR spectra were recorded using Bruker
Supercon Magnet DPX-200 and DRX-300 spectrometers (operating at
1
200 and 300 MHz for H and 75 MHz for 13C) using CDCl3 as
solvent and tetramethylsilane (TMS) as internal standard. Chemical
shifts are reported in parts per million. Electrospray ionization mass
spectra (ESIMS) were recorded on Thermo Lcq Advantage Max-IT.
High resolution mass spectra (HRMS) were recorded on 6520 Agilent
Q Tof LC MS/MS (Accurate mass). Elemental analyses were
performed on a Vario EL-III C, H, N, S analyzer (Germany), and
values were within 0.5% of the calculated values; therefore, these
compounds meet the criteria of >95% purity. Additionally, purity of
one of the key compounds was measured by a reverse phase HPLC
with the following conditions, and purity of the compound was >95%.
Analytical HPLC was performed using a C18 reverse phase column
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Figure 4. Histological findings of the livers of rats supplemented with or without 3f and 3l. In the HFD group, macrovesicular steatosis is present in
the centrilobular area (H&E, 200×), whereas steatosis (H&E, 200×) decreases in the 3f, 3l, and fenofibrate supplemented HFD-fed rat group.
active compounds (3l) was determined by HPLC (see Supporting
Information).
Ethyl 2-(4-Formylphenoxy)acetate (2a). To a mixture of 4-
hydroxybenzaldehyde (12.3 mmol, 1.5 g), ethylbromoacetate (12.2
mmol, 2.05 g), and K2CO3 (24.6 mmol, 3.4 g) was added acetonitrile
(50 mL). The reaction mixture was refluxed for 3 h. After completion
of the reaction, K2CO3 was removed in a sintered funnel, and the
filtrate was concentrated under vacuum and subjected to column
chromatography (silica gel 100−200 mesh, EtOAc/hexane) to give
pure 2a as a colorless liquid.
Following the above procedure, other derivatives of 4-hydrox-
ybenzaldehyde were synthesized. The structures of known inter-
mediates were ascertained by spectroscopic methods and by
comparison with the reported literature (1a−e and 2a−d).32,33
Ethyl 4-(4-(Bis(1-methyl-1H-indol-3-yl)methyl)phenoxy)-
butanoate (3l). A mixture of ethyl 4-(4-formylphenoxy)butanoate
(2d) (1.1 mmol, 0.24 g), N-methylindole (1c) (2.2 mmol, 0.3 g), and
Figure 5. Effects of bis-indole derivatives and fenofibrate I125-LDL
I2 (53.83 mg, 0.21 mmol) in acetonitrile (20 mL) was stirred at room
catabolism in the liver plasma membrane of HFD-induced hyper-
temperature for 30 min. After completion of the reaction, the mixture
lipidemic rats. Animals were sacrificed after 8 weeks of ND and HFD
treated with aq. Na2S2O3 solution (5%, 10 mL) and the product was
consumption with or without 3f, 3l, and fenofibrate (100 mg/kg/d).
extracted with CHCl3 (3 × 25 mL). The combined organic layers were
Compounds 3f and 3l, and fenofibrate increase the I125-LDL
dried with anhydrous sodium sulfate, concentrated in vacuo, and
catabolism in the plasma membrane of the liver in HFD-inducd
purified by column chromatography to afford 3l.
hyperlipidemic rat. Values are expressed as the mean SD (n = 6).
The compounds (3a−l) were prepared in a manner similar to the
**P < 0.01; ***P < 0.001.
procedure described above.
Ethyl 2-(4-(Di(1H-indol-3-yl)methyl)phenoxy)acetate (3a). Light
(4.6 × 259 mm; Waters 5 μM) on a Merck Hitachi D-7000
brown gum; yield, 85%. IR (KBr): 3403, 3016, 1691, 1592, 1067 cm−1.
instrument.
1H NMR (CDCl3, 300 MHz) δ: 7.74 (brs, 2H), 7.32 (d, 2H, J = 7.9
HPLC Conditions. Method: Isocratic solution of acetonitrile
Hz), 7.22−7.07 (m, 6H), 6.97−6.92 (m, 2H), 6.70 (d, 2H, J = 8.6 Hz),
(80%) and 1% acetic acid−water; flow rate of 0.5 mL/min for 25
6.39 (s, 1H), 6.38 (s, 1H), 5.75 (s, 1H), 4.50 (s, 2H), 4.21 (q, 2H, J =
min; and was detected at UV 254 nm.
7.1 Hz), 1.24 (t, 3H, J = 7.1 Hz). 13C NMR (CDCl3, 75 MHz): 169.5,
The route followed for the preparation of bis-indole derivatives
156.2, 137.5, 136.7, 129.8, 127.1, 123.8, 121.8, 119.9, 119.6, 119.1,
from 1a−e and 2a−d is outlined in Scheme 1. Reaction of 4-
114.4, 111.3, 65.6, 61.5, 39.3, 14.2. ESI-MS: (m/z) 423 (M − H)+.
hydroxybenzaldehyde with appropriate bromo esters in the presence
Anal. Calcd for C27H24N2O3: C, 76.39; H, 5.70; N, 6.60. Found: C,
of K2CO3 in acetonitrile under reflux conditions furnished substituted
benzaldehydes (2a−d). An efficient electrophilic substitution of
76.31; H, 5.81; N, 6.53.
Ethyl 2-(4-(Bis(1-methyl-1H-indol-3-yl)methyl)phenoxy)acetate
suitable indoles (1a−e) with these substituted benzaldehydes
derivatives (2a−d) using catalytic iodine in acetonitrile at room
temperature furnished final bis-indole-fibrate hybrids (3a−l) in good
to excellent yields. The structures of the compounds were
substantiated by 1H NMR, 13C NMR, mass spectrometry, and IR
spectroscopy. The purity of these compounds was ascertained by TLC
and spectral analysis, and furthermore, the purity of one of the most
(3b). Light brown gum; yield, 87%. IR (KBr): 3011, 1695, 1598,
1069 cm−1. 1H NMR (CDCl3, 300 MHz) δ: 7.36 (d, 2H, J = 9.0 Hz),
7.28−7.15 (m, 6H), 7.00−6.95 (m, 2H), 6.80 (d, 2H, J = 8.6 Hz), 6.50
(s, 2H), 5.82 (s, 1H), 4.57 (s, 2H), 4.25 (q, 2H, J = 7.1 Hz), 3.65 (s,
6H), 1.27 (t, 3H, J = 7.1 Hz). 13C NMR (CDCl3, 75 MHz): 169.2,
156.3, 137.9, 137.5, 129.8, 128.3, 127.5, 121.5, 120.1, 118.7, 118.5,
114.5, 109.2, 65.7, 61.4, 39.3, 32.7, 14.3. ESI-MS: (m/z) 451 (M −
Table 6. Effects of Fenofibrate Derivatives and Fenofibrates on Fecal Bile Acid Excretion in HFD Induced Hyperlipidemic
a
Rats
parameters (μg/g)
control
HFD
HFD+3f
HFD+3l
HFD+fenofibrate
cholic acid
81.90 8.53
48.25 6.69***
(−41%over control)
65.04 3.93 **
(+26% over HFD)
60.82 4.86 **
(+21%over HFD)
69.74 3.71***
(+31% over HFD)
deoxycholic acid
51.82 3.06
27.82 3.11***
(−46%over control)
38.91 4.07***
(+28%over HFD)
40.25 3.38 ***
(+31%over HFD)
1.76 3.47***
(+33% over HFD)
a
Each parameter represents pooled data from 6 rats/group, and values are expressed as the mean SD. **P < 0.01. ***P < 0.001, HFD group
compared with the control group and HFD plus compound/fenofibrate groups compared with HFD.
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Figure 6. Effects of compounds 3f and 3l on lipoprotein metabolism, LCAT, LPL, and LDR receptor binding in HFD-induced rats.
H)+. Anal. Calcd for C29H28N2O3: C, 76.97; H, 6.24; N, 6.19. Found:
C, 76.82; H, 6.15; N, 6.24.
(d, 2H, J = 8.7 Hz), 6.55 (brs, 2H), 5.80 (s, 1H), 5.80 (s,1H), 4.70 (q,
1H, J = 6.7 Hz), 4.25−4.14 (m, 2H), 4.04 (q, 4H, J = 7.2 Hz), 1.59 (d,
4H, J = 6.8 Hz), 1.35 (t, 6H, J = 7.2 Hz), 1.22 (t, 3H, J = 7.1 Hz). 13C
NMR (CDCl3, 75 MHz): 172.6, 156.0, 137.8, 136.5, 129.8, 127.8,
126.7, 121.3, 120.4, 118.6, 115.0, 109.2, 61.3, 40.9, 39.5, 18.7, 15.6,
14.3. ESI-MS: (m/z) 493 (M − H)+. HRMS m/z calcd for
C32H34N2O3 (M − H)+ 493.2490; found, 493.2478.
Ethyl 2-(4-(Bis(1-ethyl-1H-indol-3-yl)methyl)phenoxy)acetate
(3c). Light brown solid; yield, 93%; mp 120−121 °C. IR (KBr):
1
3016, 1691, 1592, 1067 cm−1. H NMR (CDCl3, 300 MHz) δ: 7.36−
7.22 (m, 6H), 7.18−7.13 (m, 2H), 6.98−6.93 (m, 2H), 6.80 (d, 2H, J
= 8.3 Hz), 6.56 (brs, 2H), 5.82 (s, 1H), 4.56 (brs, 2H), 4.24 (q, 2H, J
= 7.1 Hz), 4.02 (q, 4H, J = 7.2 Hz), 1.34 (t, 6H, J = 7.2 Hz), 1.26 (t,
3H, J = 7.1 Hz). 13C NMR (CDCl3, 75 MHz): 169.3, 156.3, 138.0,
136.5, 129.8, 127.7, 126.7, 121.3, 120.3, 118.6, 118.6, 114.5, 109.2,
65.8, 61.4, 40.9, 39.5, 15.6, 14.3. ESI-MS: (m/z) 479 (M − H)+. Anal.
Calcd for C31H32N2O3: C, 77.47; H, 6.71; N, 5.83. Found: C, 77.53;
H, 6.75; N, 5.88.
Ethyl 2-(4-(Bis(1,2-dimethyl-1H-indol-3yl)methyl)phenoxy)-
propanoate (3g). Light brown gum; yield, 89%. IR (KBr): 2980,
1
1684, 1589, 1038 cm−1. H NMR (CDCl3, 300 MHz) δ: 7.21−7.01
(m, 6H), 6.89−6.88 (m, 2H), 6.82−6.73 (m, 4H), 5.97 (s, 1H), 4.70
(q, 1H, J = 6.9 Hz), 4.22−4.15 (m, 2H), 3.60 (brs, 6H), 2.12 (brs,
6H), 1.58 (d, 3H, J = 6.8 Hz), 1.24−1.19 (m, 3H). 13C NMR (CDCl3,
75 MHz): 172.5, 155.9, 137.3, 136.7, 133.6, 130.1, 127.9, 120.1, 119.7,
118.7, 114.9, 113.3, 108.4, 61.3, 39.1, 29.8, 29.5, 18.7, 14.3, 10.8. ESI-
MS: (m/z) 493 (M − H)+. Anal. Calcd for C32H34N2O3: C, 77.70; H,
6.93; N, 5.66. Found: C, 77.78; H, 6.86; N, 5.75.
Ethyl 2-(4-(Bis(1-methyl-1H-indol-3-yl)methyl)phenoxy)-
propanoate (3d). Light brown solid; yield, 87%; mp 115−116 °C.
IR (KBr): 3011, 1697, 1588, 1050 cm−1. 1H NMR (CDCl3, 300 MHz)
δ: 7.35 (d, 2H, J = 7.9 Hz), 7.28−7.15 (m, 6H), 6.99−6.95 (m, 2H),
6.78 (d, 2H, J = 8.6 Hz), 6.49 (brs, 2H), 5.81 (s, 1H), 4.69 (q, J = 8.6
Hz, 1H), 4.23−4.14 (m, 2H), 3.65 (brs, 6H), 1.58 (d, 3H, J = 6.8 Hz),
1.22 (t, 3H, J = 7.1 Hz). 13C NMR (CDCl3, 75 MHz): 172.5, 156.1,
137.7, 137.5, 129.7, 128.3, 127.5, 121.5, 120.2, 118.7, 118.6, 115.0,
109.1, 61.3, 39.3, 32.7, 18.7, 14.2. ESI-MS: (m/z) 465 (M − H)+. Anal.
Calcd for C30H30N2O3: C, 77.23; H, 6.48; N, 6.00. Found: C, 77.32;
H, 6.55; N, 5.91.
Ethyl 2-(4-(Bis(2-methyl-1H-indol-3-yl)methyl)phenoxy)-
propanoate (3e). Light brown solid; yield, 92%; mp 185−186 °C.
IR (KBr): 3390, 3016, 1691, 1595, 1045 cm−1. 1H NMR (CDCl3, 300
MHz) δ: 7.65 (s, 2H), 7.23−7.12 (m, 5H), 7.03−6.95 (m, 4H), 6.85−
6.80 (m, 2H), 6.75 (d, 2H, J = 8.7 Hz), 5.90 (s, 1H), 4.70 (q, 1H, J =
6.9 Hz), 4.24−4.15 (m, 2H), 1.99 (s, 6H), 1.59 (d, 3H, J = 6.8 Hz),
1.22 (t, 3H, J = 7.1 Hz). 13C NMR (CDCl3, 75 MHz): 172.6, 156.0,
137.0, 135.2, 131.9, 130.1, 129.0, 120.7, 119.5, 119.1, 115.0, 113.7,
110.1, 61.3, 38.6, 18.7, 14.3, 12.5. ESI-MS: (m/z) 465 (M − H)+. Anal.
Calcd for C30H30N2O3: C, 77.23; H, 6.48; N, 6.00. Found: C, 77.29;
H, 6.41; N, 6.05.
Ethyl 2-(4-(di(1H-indol-3-yl)methyl)phenoxy)-2-methylpropa-
noate (3h). Light brown gum; yield, 85%. IR (KBr): 3404, 3002,
1696, 1590, 1034 cm−1. 1H NMR (CDCl3, 300 MHz) δ: 7.84 (s, 2H),
7.14 (d, 2H, J = 7.9 Hz), 7.34−7.28 (m, 2H), 7.23−7.17 (m, 4H),
7.07−7.01 (m, 2H), 6.82 (d, 2H, J = 8.5 Hz), 6.52 (d, 2H, J = 1.6 Hz),
5.85 (s, 1H), 4.26 (q, 2H, J = 7.1 Hz), 1.64 (brs, 6H), 1.28 (t, 3H, J =
7.1 Hz). 13C NMR (CDCl3, 75 MHz): 174.6, 153.7, 138.1, 136.7,
129.4, 127.1, 123.8, 121.9, 120.0, 119.3, 119.1, 111.2, 61.5, 39.5, 25.5,
14.2. ESI-MS: (m/z) 451 (M − H)+. Anal. Calcd for C29H28N2O3: C,
76.97; H, 6.24; N, 6.19. Found: C, 76.86; H, 6.15; N, 6.29.
Ethyl 2-(4-(Bis(1-methyl-1H-indol-3-yl)methyl)phenoxy)-2-meth-
ylpropanoate (3i). Light brown solid; yield, 86%; mp 110−111 °C. IR
(KBr): 3012, 1696, 1599, 1040 cm−1. 1H NMR (CDCl3, 300 MHz) δ:
7.34 (d, 2H, J = 7.9 Hz), 7.26 (d, 2H, J = 8.2 Hz), 7.19−7.14 (m, 4H),
6.98−6.93 (m, 2H), 6.75 (d, 2H, J = 8.6 Hz), 6.48 (s, 2H), 5.79 (s,
1H), 4.19 (q, 2H, J = 7.1 Hz), 3.63 (brs, 6H), 1.56 (brs, 6H), 1.20 (t,
3H, J = 7.1 Hz). 13C NMR (CDCl3, 75 MHz): 174.5, 153.6, 138.2,
137.4, 129.3, 128.3, 127.4, 121.4, 120.1, 119.0, 118.6, 118.4, 109.1,
61.4, 39.3, 32.7, 25.5, 14.2. ESI-MS: (m/z) 479 (M − H)+. Anal. Calcd
for C31H32N2O3: C, 77.47; H, 6.71; N, 5.83. Found: C, 77.41; H, 6.79;
N, 5.85.
Ethyl 2-(4-(Bis(1-ethyl-1H-indol-3-yl)methyl)phenoxy)-
propanoate (3f). Light brown solid; yield, 90%; mp 112−113 °C.
IR (KBr): 3008, 1684, 1593, 1041 cm−1. 1H NMR (CDCl3, 300 MHz)
δ: 7.36−7.28 (m, 4H), 7.23−7.13 (m, 4H), 6.98−6.93 (m, 2H), 6.78
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a
Scheme 1. Synthesis of Substituted Bis-Indoles
a
Reagents and conditions: (a) NaH, DMF, 0 °C, 1 h. (b) K2CO3, CH3CN, reflux, 2 h. (c) l2, CH3CN, rt, 30 min.
Ethyl 2-(4-(Bis(1-ethyl-1H-indol-3-yl)methyl)phenoxy)-2-methyl-
157.3, 137.6, 136.9, 129.7, 128.3, 127.6, 121.5, 120.2, 118.7, 114.2,
109.1, 66.8, 60.5, 39.4, 32.7, 31.0, 24.9, 14.4. ESI-MS: (m/z); 479 (M
− H)+. HRMS m/z calcd for C31H32N2O3 (M − H)+ 479.2334; found,
479.2326. HPLC Purity >95%.
propanoate (3j). Light brown gum; yield, 91%. IR (KBr): 3008,
1
1685, 1597, 1038 cm−1. H NMR (CDCl3, 300 MHz) δ: 7.35−7.28
(m, 4H), 7.20−7.13 (m, 4H), 6.95 (t, 2H, J = 7.5 Hz), 6.76 (d, 2H, J =
8.5 Hz), 6.55 (brs, 2H), 5.80 (s, 1H), 4.19 (q, 2H, J = 7.0 Hz), 4.02 (q,
4H, J = 7.2 Hz), 1.57 (brs, 6H), 1.34 (t, 6H, J = 7.2 Hz), 1.21 (t, 3H, J
= 7.1 Hz). 13C NMR (CDCl3, 75 MHz): 174.5, 153.7, 138.4, 136.5,
129.5, 127.7, 126.7, 121.8, 121.3, 120.3, 119.2, 118.6, 109.2, 79.2, 61.4,
40.9, 39.6, 32.0, 29.8, 29.5, 25.5, 22.8, 15.6, 14.2. ESI-MS: (m/z) 507
(M − H)+. Anal. Calcd for C33H36N2O3: C, 77.92; H, 7.13; N, 5.51.
Found: C, 77.84; H, 7.21; N, 5.62.
Biological Materials and Methods. Animal and Diets. In vivo
experiments were conducted as per the guidelines provided by
the animal ethics committee of the Central Drug Research
Institute, Lucknow, India. Adult male Charles Foster rats
(200−225 g) bred in the animal house of the institute were
used. The animals were housed in polypropylene cages and
kept in uniform hygienic conditions, at a temperature of 25−26
°C, relative humidity of 50−70%, and 12/12 h light/dark cycle.
These were provided with standard rat pellet diet and water ad
libitum for one week before their division into three weight-
matched groups given ND and HFD. Hyperlipidemia was
induced in three groups by feeding them HFD for 30
consecutive days. After induction of hyperlipidemia, bis-indole
derivatives and fenofibrates were given by oral gavage to one
HFD-fed groups (HFD + 3f, HFD + 3l ,and HFD + fenofibrate
group), and the same volume of the vehicle (0.2% Gum acacia)
was given in other HFD and ND groups. After 30 days of
treatment, rats were fasted overnight. Blood was withdrawn
from the retro-orbital plexus using a glass capillary in an EDTA
coated tube (3 mg/mL blood). Thereafter, animals were
sacrificed, and the liver was excised gently, washed with cold
0.15 M KCl, and kept at 4 °C until analysis. Blood was
centrifuged to collect plasma/serum.
Ethyl 4-(4-(Di(1H-indol-3-yl)methyl)phenoxy)butanoate (3k).
Light brown gum; yield, 89%. IR (KBr): 3403, 3013, 1694, 1593,
1049 cm−1. 1H NMR (CDCl3, 300 MHz) δ: 7.82 (s, 2H), 7.35 (d, 2H,
J = 7.9 Hz), 7.29 (d, 2H, J = 7.5 Hz), 7.21−7.10 (m, 5H), 6.97 (t, 2H,
J = 7.3 Hz), 6.75 (d, 2H, J = 8.6 Hz), 6.54 (s, 1H), 6.54 (s, 2H), 5.79
(s, 1H), 4.12 (q, 2H, J = 7.1 Hz), 3.92 (t, 2H, J = 6.0 Hz), 2.48 (t, 2H,
J = 7.3 Hz); 2.05 (t, 2H, J = 6.9 Hz), 1.23 (t, 3H, J = 7.2 Hz). 13C
NMR (CDCl3, 75 MHz): 173.5, 157.3, 136.8, 136.5, 129.7, 127.2,
123.7, 122.0, 119.9, 120.1, 119.3, 114.3, 111.2, 66.8, 60.6, 39.5, 31.0,
24.9, 14.3. ESI-MS: (m/z); 451 (M − H)+. Anal. Calcd for
C29H28N2O3: C, 76.97; H, 6.24; N, 6.19. Found: C, 76.88; H, 6.15;
N, 6.27.
Ethyl 4-(4-(Bis(1-methyl-1H-indol-3-yl)methyl)phenoxy)-
butanoate (3l). Light brown solid; yield, 91%; mp 110−111 °C. IR
(KBr): 3018, 1697, 1590, 1042 cm−1. 1H NMR (CDCl3, 200 MHz) δ:
7.39−7.34 (m, 2H), 7.29−7.13 (m, 6H), 7.01−6.93 (m, 2H), 6.80−
6.76 (m, 2H), 6.50 (s, 2H), 5.81 (s, 1H), 4.13 (q, 2H, J = 7.1 Hz), 3.95
(t, 2H, J = 6.1 Hz), 3.64 (s, 6H), 2.49 (t, 2H, J = 7.4 Hz), 2.11−2.01
(m, 2H), 1.24 (t, 3H, J = 7.1 Hz). 13C NMR (CDCl3, 75 MHz): 173.4,
Biochemical Analysis of Plasma/Serum. Serum was fractionated
into VLDL, LDL, and HDL by polyanionic precipitation methods.
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Serum and lipoproteins were analyzed for their TC, PL, TG, and total
protein by the standard procedures reported earlier.34
(3) The World Health Report 2002: Reducing Risks, Promoting a
Healthy Life; World Health Organization: Geneva, Switzerland, 2002.
(4) Jong, W. Y. Possible anti-Obesity drugs from nature- a review.
Phytochemistry 2010, 71, 1625−1641.
(5) Drew, B. S.; Dixon, A. F.; Dixon, J. B. Obesity management:
update on orlistat. Vasc. Health Risk Manag. 2007, 3, 817−821.
(6) Tziomalos, K.; Krassas, G. E.; Tzotzas, T. The use of sibutramine
in the management of obesity and related disorders: an update. Vasc.
Health Risk Manag. 2009, 5, 441−452.
(7) (a) De Simone, G.; D’Addeo, G. Sibutramine: balancing weight
loss benefit and possible cardiovascular risk. Nutr. Metab. Cardiovasc.
Dis. 2008, 18, 337−341. (b) Slovacek, L.; Pavlik, V.; Slovackova, B.
The effect of sibutramine therapy on occurrence of depression
symptoms among obese patients. Nutr. Metab. Cardiovasc. Dis. 2008,
18, e43−e44. (c) Karamadoukis, L.; Shivashankar, G. H.; Ludeman, L.;
Williams, A. J. An unusual complication of treatment with orlistat. Clin.
Nephrol. 2009, 71, 430−432.
(8) Agulera, C. M.; Gil-Compos, M.; Canete, R.; Gil, A. Alterations in
plasma and tissue lipids associated with obesity and metabolic
syndrome. Clin. Sci. 2008, 114, 183−193.
(9) Lalloyer, F.; Staels, B. Fibrates, glitazones, and peroxisome
proliferator-activated receptors. Arterioscler., Thromb., Vasc. Biol. 2010,
30, 894−899.
(10) Davidson, M. H.; Armani, A.; McKenney, J. M.; Jacobson, T. A.
Safety considerations with fibrate therapy. Am. J. Cardiol. 2007, 99,
3C−18C.
(11) Barker, B. J.; Goodenough, R. R.; Falko, J. M. Fenofibrate
monotherapy induced rhabdomyolysis. Diabetes Care 2003, 26, 2482−
2483.
Biochemical Analysis of the Liver. The liver was homogenized
(10%, w/v) in cold 100 mM phosphate buffer at pH 7.2 and used for
the assay of total lipolytic activity of LPL.35 The lipid extract of each
homogenate prepared in CHCl3/CH3OH (2:1 v/v) was used for the
estimation of TC, PL, TG, and total protein. Human serum LDL was
prepared and radio-labeled with I125, and the binding of this I125LDL
with liver plasma membrane preparation36 was assayed as described
previously.37
Histopathology of the Liver. Histopathological study of the rat
liver of all groups was performed. A tissue section of 4 μm thickness
was cut and stained with Erhlich’s hematoxylin and eosin and
examined by a light microscope.
Fecal Bile Acids. Rat feces was collected from all experimental
groups from the day of dosing over 30 days and processed for the
cholic and deoxycholic acid estimation test.38
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed spectral analysis for all new compounds, structures,
and 1D and 2D NMR. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +91 9919317940. Fax: +91-522-2628493. E-mail:
sashidhar123@gmail.com; kv_sashidhara@cdri.res.in.
(12) Shek, A.; Ferrill, M. J. Statin-fibrate combination therapy. Ann.
Pharmacother. 2001, 35, 908−917.
Notes
(13) (a) Sashidhara, K. V.; Kumar, A.; Kumar, M.; Sarkar, J.; Sinha, S.
Synthesis and in vitro evaluation of novel coumarin-chalcone hybrids
as potential anticancer agents. Bioorg. Med. Chem. Lett. 2010, 20,
7205−7211. (b) Sashidhara, K. V.; Kumar, M.; Modukuri, R. K.;
Sonkar, R.; Khanna, A. K.; Bhatia, G.; Rai, S.; Shukla, R. Synthesis and
anti-inflammatory activity of novel biscoumarin-chalcone hybrids.
Bioorg. Med. Chem. Lett. 2011, 21, 4480−4484. (c) Sashidhara, K. V.;
Kumar, A.; Kumar, M.; Singh, S.; Jain, M.; Dikshit, M. Synthesis of
novel 3-carboxamide-benzocoumarin derivatives as orally active
antithrombotic agents. Bioorg. Med. Chem. Lett. 2011, 21, 7034−7040.
(14) Sashidhara, K. V.; Kumar, A.; Kumar, M.; Srivastava, A.; Puri, A.
Synthesis and antihyperlipidemic activity of novel coumarin bisindole
derivatives. Bioorg. Med. Chem. Lett. 2010, 20, 6504−6507.
(15) Velezheva, V. S.; Brennan, P. J.; Marshakov, V. Y.; Gusev, D. V.;
Lisichkina, I. N.; Peregudov, A. S.; Tchernousova, L. N.; Smirnova, T.
G.; Andreevskaya, S. N.; Medvedev, A. E. Novel pyridazino[4,3-
b]indoles with dual inhibitory activity against Mycobacterium tuber-
culosis and monoamine oxidase. J. Med. Chem. 2004, 47, 3455−3461.
(16) Andreani, A.; Burnelli, S.; Granaiola, M.; Leoni, A.; Locatelli, A.;
Morigi, R.; Rambaldi, M.; Varoli, L.; Landi, L.; Prata, C.; Berridge, M.
V.; Grasso, C.; Fiebig, H. H.; Kelter, G.; Burger, A. M.; Kunkel, M. W.
Antitumor activity of bis-indole derivatives. J. Med. Chem. 2008, 51,
4563−4570.
The authors declare no competing financial interest.
This work is part XVI in the series Advances in Drug Design
and Discovery CDRI #8210.
ACKNOWLEDGMENTS
■
Instrumentation facilities from SAIF, CDRI are gratefully
acknowledged. M.K. and R.S. are thankful to CSIR, New
Delhi, India for financial support. B.S.S. is thankful to the
Department of Pharmaceuticals, Ministry of Chemicals and
Fertilizers, Government of India. S.P.S. is acknowledged for
technical support. We are grateful to Dr. Tushar Kanti
Chakraborty (Director, CSIR-CDRI) for funding (MLP14i)
and for his constant encouragement in the drug discovery
program. This is CSIR-CDRI communication number 8210.
ABBREVIATIONS USED
■
HFD, high fat diet; LCAT, lecithin cholesterol acyl-transferase;
LDL, low density lipoprotein; CNS, central nervous system;
FDA, Food and Drug Administration; HDL, high density
lipoprotein; HMG CoA reductase, 3-hydroxy-3-methyl-glutaryl-
CoA reductase; TLC, thin layer chromatography; TMS,
tetramethylsilane; ESIMS, electrospray ionization mass spectra;
HRMS, high resolution mass spectra; HPLC, high-performance
liquid chromatography; TC, total cholesterol; TG, triglycerides;
PL, phospholipids; ND, normal diet; VLDL, very low density
lipoprotein; CHD, coronary heart disease
(17) Routier, S.; Peixoto, P.; Merour, J. Y.; Coudert, G.; Dias, N.;
́
́ ́
Pierre, C. B. A.; Leonce, S.; Caignard, D. H. Synthesis and biological
evaluation of novel naphthocarbazoles as potential anticancer agents. J.
Med. Chem. 2005, 48, 1401−1413.
(18) Regina, G. L.; Coluccia, A.; Piscitelli, F.; Bergamini, A.; Sinistro,
A.; Cavazza, A.; Maga, G.; Samuele, A.; Zanoli, S.; Novellino, E.;
Artico, M.; Silvestri, R. Indolyl aryl sulfones as HIV-1 non-nucleoside
reverse transcriptase inhibitors: role of two halogen atoms at the
indole ring in developing new analogues with improved antiviral
activity. J. Med. Chem. 2007, 50, 5034−5038.
REFERENCES
■
(1) (a) Wickelgren, I. Obesity: How Big a Problem? Science 1998,
280, 1364−1367. (b) Bray, G. A. Obesity: the disease. J. Med. Chem.
2006, 49, 4001−4007.
(19) Knott, K. E.; Auschill, S.; Jager, A.; Knolker, H. J. First total
̈
̈
synthesis of the whole series of the antiostatins A and B. Chem.
Commun. 2009, 12, 1467−1469.
(2) Flegal, K. M.; Carroll, M. D.; Ogden, C. L.; Johnson, C. L.
Prevalence and trends in obesity among US adults, 1999−2000. JAMA
2002, 288, 1723−1727.
(20) (a) Cowley, P. M.; Baker, J.; Barn, D. R.; Buchanan, K. I.;
Carlyle, I.; Clark, J. K.; Clarkson, T. R.; Deehan, M.; Edwards, D.;
2778
dx.doi.org/10.1021/jm201697v | J. Med. Chem. 2012, 55, 2769−2779

Journal of Medicinal Chemistry
Article
Goodwin, R. R.; Jaap, D.; Kiyoi, Y.; Mort, C.; Palin, R.; Prosser, A.;
Walker, G.; Ward, N.; Wishart, G.; Young, T. The discovery of novel
indole-2-carboxamides as cannabinoid CB1 receptor antagonists.
Bioorg. Med. Chem. Lett. 2011, 21, 497−501. (b) Morrison, A. J.;
Adam, J. M.; Baker, J. A.; Campbell, R. A.; Clark, J. K.; Cottney, J. E.;
Deehan, M.; Easson, A. M.; Fields, R.; Francis, S.; Jeremiah, F.;
Keddie, N.; Kiyoi, T.; McArthur, D. R.; Meyer, K.; Ratcliffe, P. D.;
Schulz, J.; Wishart, G.; Yoshiizumi, K. Design, synthesis, and structure-
activity relationships of indole-3-heterocycles as agonists of CB1
receptor. Bioorg. Med. Chem. Lett. 2011, 21, 506−509. (c) Pierson, P.
D.; Fettes, A.; Freichel, C.; Gatti-McArthur, S.; Hertel, C.; Huwyler, J.;
Mohr, P.; Nakagawa, T.; Nettekoven, M.; Plancher, J. M.; Raab, S.;
Richter, H.; Roche, O.; Sarmiento, R. M. R.; Schmitt, M.; Schuler, F.;
Takahashi, T.; Taylor, S.; Ullmer, C.; Wiegand, R. 5-Hydroxyindole-2-
carboxylic acid amides: Novel histamine-3 receptor inverse agonists for
the treatment of obesity. J. Med. Chem. 2009, 52, 3855−3868.
(21) Sashidhara, K. V.; Kumar, A.; Bhatia, G.; Khan, M. M.; Khanna,
A. K.; Saxena, J. K. Antidyslipidemic and antioxidative activities of 8-
hydroxyquinoline derived keto-enamine schiffs bases. Eur. J. Med.
Chem. 2009, 44, 1813−1818.
(22) Howard, B. V.; Ruotolo, G.; Robbins, D. C. Obesity and
dyslipidemia. Endocrinol. Metab. Clin. North Am. 2003, 32, 855−867.
(23) Colca, J. R.; Dailey, C. F.; Palazuk, B. J.; Hillman, R. M.; Dinh,
D. M.; Melchior, G. W.; Spilman, C. H. Pioglitazone hydrochloride
inhibits cholesterol absorption and lowers plasma cholesterol
concentrations in cholesterol-fed rats. Diabetes 1991, 40, 1669−1674.
(24) Rijzewijk, L. J.; Van der Meer, R. W.; Lubberink, M.; Lamb, H.
J.; Romijn, J. A.; Roos, A.; Twisk, J. W.; Heine, R. J.; Lammertsma, A.
A.; Smit, J. W.; Diamant, M. Liver fat content in type 2 diabetes:
relationship with hepatic perfusion and substrate metabolism. Diabetes
2010, 59, 2747−2754.
F.; Forest, M. C.; Fouchet, M. H.; Gillibert, F. J.; Grillot, D. A.;
Lambert, M. H.; Laroze, A.; Grumelec, C. L.; Linget, J. M.; Montana,
V. G.; Nguyen, V. L.; Nicodeme, E.; Patel, V.; Penfornis, A.; Pineau,
O.; Pohin, D.; Potvain, F.; Paulain, G.; Ruault, C. B.; Saunders, M.;
Taum, J.; Xu, H. E.; Xu, R. X.; Pianetti, P. M. Substituted 2-[(4-
Aminomethyl)phenoxy]-2-methylpropionic acid PPARα agonists. 1.
Discovery of a novel series of potent HDLc raising agents. J. Med.
Chem. 2007, 50, 685−695. (c) Fraga-Dubreuil, J.; Bazureau, J. P.
Efficient combination of task-specific ionic liquid and microwave
dielectric heating applied to one-pot three component synthesis of a
small library of 4-thiazolidinones. Tetrahedron 2003, 59, 6121−6130.
(34) Khanna, A. K.; Chander, R.; Singh, C.; Srivastava, A. K.; Kapoor,
N. K. Hypolipidemic activity of Terminalia chebula in rats. Fitoterpia
1993, 64, 351−356.
(35) Wing, D. R.; Robinson, D. S. Clearing-factor lipase in adipose
tissue. A possible role of adenosine 3′,5′-(cyclic)-monophosphate in
the regulation of its activity. Biochem. J. 1968, 109, 841−849.
(36) Kovanen, P. T.; Brown, M. S.; Goldstein, J. L. Increased binding
of low density lipoprotein to liver membranes from rats treated with
17 alpha-ethinyl estradiol. J. Biol. Chem. 1979, 254, 11367−11373.
(37) Singh, V.; Kaul, S.; Chander, R.; Kapoor, N. K. Stimulation of
low density lipoprotein receptor activity in liver membrane of
guggulsterone treated rats. Pharmacol. Res. 1990, 22, 37−44.
(38) Mosbach, E. H.; Klenisky, H. J.; Hal, P.; Kendall, F. E.
Determination of deoxycholic acid cholic acid in bile. Arch. Biochem.
Biophys. 1954, 51, 402−409.
(25) Bhuiyan, M. J.; Do, H. V.; Mun, S.; Jun, H. J.; Lee, J. H.; Kim, Y.
R.; Lee, S. J. Hypocholesterolemic and hypoglycemic effects of
enzymatically modified carbohydrates from rice in high-fat-fed
C57BL/6J mice. Mol. Nutr. Food Res. 2011, 55, S214−S226.
(26) Lees, R. Hyperlipidemia and Atherosclerosis HST.151: Principles
of Pharmacology; http://ocw.mit.edu/courses (Accessed on October
16, 2011).
(27) Windler, E. E.; Kovanen, P. T.; Chao, Y. S.; Brown, M. S.; Havel,
R. J. The estradiol-stimulated lipoprotein receptor of rat liver. A
binding site that membrane mediates the uptake of rat lipoproteins
containing apoproteins B and E. J. Biol. Chem. 1980, 255, 10464−
10471.
(28) Anstee, Q. M.; Goldin, R. D. Mouse models in non-alcoholic
fatty liver disease and steatohepatitis research. Int. J. Exp. Pathol. 2006,
87, 1−16.
(29) Rahman, S. M.; Wang, Y.; Yotsumoto, H.; Cha, J.; Han, S.;
Inoue, S.; Yanagita, T. Effects of conjugated linoleic acid on serum
leptin concentration, body-fat accumulation, and beta-oxidation of
fatty acid in OLETF rats. Nutrition 2001, 17, 385−390.
(30) Reddy, J. K.; Rao, M. S. Lipid metabolism and liver
inflammation. II. Fatty liver disease and fatty acid oxidation. Am. J.
Physiol. Gastrointest. Liver Physiol. 2006, 290, G852−G858.
(31) Jeon, J. R.; Kim, J. Y. Effects of pine needle extract on
differentiation of 3T3-L1 preadipocytes and obesity in high-fat diet fed
rats. Biol. Pharm. Bull. 2006, 29, 2111−2115.
(32) (a) Xu, X. H.; Liu, G. K.; Azuma, A.; Tokunaga, E.; Shibata, N.
Synthesis of indole and biindolyl triflones: Trifluoromethanesulfony-
lation of indoles with Tf2O/TTBP (2,4,6-tri-tert-butylpyridine)
system. Org. Lett. 2011, 13, 4854−4857. (b) Seefeld, M. A.; Miller,
W. H.; Newlander, K. A.; Burgess, W. J.; Dewolf, W. E. Jr.; Elkins, P.
A.; Head, M. S.; Jakas, D. R.; Janson, C. A.; Keller, P. M.; Manley, P. J.;
Rittenhouse, S. F.; Uzinskas, I. N.; Wallis, N. G.; Huffman, W. F.
Indole naphthyridinones as inhibitors of bacterial enoyl-ACP
reductases FabI and FabK. J. Med. Chem. 2003, 46, 1627−1635.
(33) (a) Bhaumik, J.; Yao, Z.; Borbas, K. E.; Taniguchi, M.; Lindsey,
J. S. Masked imidazoyl-dipyrromethanes in the synthesis of imidazole-
substituted porphyrins. J. Org. Chem. 2006, 71, 8807−8817. (b) Sierra,
M. L.; Beneton, V.; Boullay, A. N.; Boyer, T.; Brewster, A. G.; Donche,
2779
dx.doi.org/10.1021/jm201697v | J. Med. Chem. 2012, 55, 2769−2779