Hybrids of coumarin-indole: Design, synthesis and biological evaluation in Triton WR-1339 and high-fat diet induced hyperlipidemic rat models

Article abstract of DOI:10.1039/c6md00283h

In this study, a series of coumarin-indole hybrids have been synthesized and evaluated for their lipid lowering activity. Preliminary biological screening of the synthesized compounds was undertaken in an in vitro model of the HMG-CoA reductase enzyme, an

Full text of DOI:10.1039/c6md00283h

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Bhatia, M. K. Suthar, J. K. saxena, V. Kumar and I. Siddiqi, Med. Chem. Commun., 2016, DOI:
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ARTICLE TYPE
Hybrids of coumarin-indole: Design, synthesis and biological evaluation
in triton WR-1339 and high-fat diet induced hyperlipidemic rat models†
Koneni V. Sashidhara,*a K. Bhaskara Rao,§a Ravi Sonkar ,§b Ram K. Modukuri,a Yashpal S. Chhonker,c
Pragati Kushwaha,a Hardik Chandasana,c A. K. Khanna,b Rabi S. Bhatta,c Gitika Bhatia,b Manish Kumar
Suthar,b Jitendra Kumar Saxena,bVikash Kumar,d Mohammad Imran Siddiqi.d
5
Received (in XXX, XXX) XthXXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
In this study, a series of coumarinꢀindole hybrids have been synthesized and evaluated for their lipid
10 lowering activity. Preliminary biological screening of synthesized compounds was undertaken in an in
vitro model of HMGꢀCoA reductase enzyme and the activity was confirmed in Triton WRꢀ1339 induced
hyperlipidemic rats. Among the hybrids, compound 26 was found to be the most persuasive as it
significantly reduced the serum and hepatic lipid profile in an HFDꢀfed hyperlipidemic rat model. The
mechanism of action seems to be associated with the regulation of HMGꢀCoA reductase activity in liver,
15 which is in good agreement with its binding mode studies. Compound 26 exhibited favorable
pharmacokinetic behavior for its oral administration, which underscores the potential of this template as a
new class of hypolipidemic agents.
Introduction
50
.Statins, which are HMGꢀCoA reductase inhibitors, are widely
prescribed for lowering low density lipoprotein cholesterol (LDLꢀ
C) levels and reducing cardiovascular event rates.6However, they
are associated with serious drawbacks like myalgia, muscle
cramp, and rhabdomyolysis.7 The fibrate drugs are used clinically
Hyperlipidemia is a group of disorders that involve elevated
20 levels of lipids in the blood, including serum cholesterol and
triglycerides. Epidemiological
evidence indicates that
55 to reduce the level of triglycerides (TG), free fatty acid (FFA)
levels and they enhance highꢀdensity lipoprotein cholesterol
(HDLꢀC).8 However, fibrates are known to cause myopathy and
increase creatine kinase activity.9 Statins and fibrate class of
drugs have played a major role in the treatment of dyslipidemia
60 for the past two decades. However, a combination of fibrate and
statins has met with the potential increased risk for myopathy,
which may occasionally be accompanied by rhabdomyolysis and
hyperlipidemia is the most important risk factors for the
progression of cardiovascular diseases, including coronary heart
disease.1 Hydroxyꢀ3ꢀmethylglutaryl coenzyme A (HMGꢀCoA)
25 reductase is a key enzyme, which plays central role in the
cholesterol biosynthesis. Inhibitors of HMGꢀCoA reductase
induce the expression of low density lipoprotein (LDL) receptors
in the liver and the subsequent clearance of LDL particles from
the bloodstream.2 Current therapies for the treatment of
30 hyperlipidemia and mixed dyslipidemia include statins,fibrates,
niacin, bile acid resins, and ezetimibe.3,5
renal failure.10 Therefore, there is
a urgent need of the
development of new class of lipid lowering agents which could
65 lower the elevated serum cholesterol levels without causing side
effects.
aMedicinal and Process Chemistry Division, CSIRꢀCentral Drug
Research Institute, BSꢀ10/1,Sector 10, Jankipuram Extension, Sitapur
35 Road, Lucknow 226031, India. Fax: +91ꢀ522ꢀ2628493; Tel: +91
9919317940; Eꢀmail: sashidhar123@gmail.com,
kv_sashidhara@cdri.res.in.
b Biochemistry Division, CSIRꢀCentral Drug Research Institute, BSꢀ10/1,
Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India.
40 cPharmacokinetics and Metabolism Division, CSIRꢀCentral Drug
Research Institute, BSꢀ10/1, Sector 10, Jankipuram Extension, Sitapur
Road, Lucknow 226031, India
dDivision of Molecular and Structural Biology, CSIRꢀCentral Drug
Research Institute, BSꢀ10/1, Sector 10, Jankipuram Extension, Sitapur
45 Road, Lucknow 226031, India
§ These authors contributed equally.
†
The authors declare no competing interests.
Electronic Supplementary Information (ESI) available: 1H NMR, 13C
Fig. 1 Designing of coumarinꢀindole based hybrids by pharmacophore
NMR, LCꢀMS/MS, Biological details. See DOI: 10.1039/b000000x/
hybridization.
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The concept of hybridization is an attractive approach in 50 case of remaining compounds, inhibition profile was modest. To
medicinal chemistry as it generates new scaffolds with improved
bioactivity.11 Here two distinct pharmacophores are covalently
linked into a single chemical entity so as to produce new hybrid
compound with improved efficacy and safety. In the present
context, we have combined coumarin and indole subunits to
produce novel hybrid derivatives. The indole structure is a key
component of various pharmacological agents and its derivatives
play a prominent role in medicinal chemistry due to its high
have more insight into the inhibition activity of most active
compounds, we carried out a doseꢀresponse study compared to
the atorvastatin.
5
10 receptor binding affinity.12ꢀ14 We had previously developed some
coumarinꢀbased hybrids, which showed diverse biological
properties.15aꢀi Our previous encouraging laboratory experience on
these scaffolds and the literature survey revealed that coumarin
and indole pharmacophores occur in a variety of potential
15 antidyslipidemic agents (Fig. 1).16,17
Thus keeping in view the lipid lowering potential of coumarin
and indoles, it was envisaged that the synthesis of their hybrids is
worth the attempt. Herein, we describe the design, synthesis and
biological evaluation of novel coumarinꢀindole hybrids as
20 potential antidyslipidemic agents.
Results and discussion
Chemistry
The synthesis of targeted hybrids was achieved as shown in
Scheme 1. The synthesis of dicarbaldehyde intermediate (5ꢀ8)
25 was achieved by the Duff reaction on commercially available
orthoꢀsubstituted
phenols
in
the
presence
of
hexamethylenetetraamine (HMTA) and trifluoroacetic acid (TFA)
Fig.2 In vitro HMGꢀCoA activity assay: (A) Percentage inhibition of
at 120°C.18 Subsequent condensation of these dicarbaldehyde 55 HMGꢀCoA reductase activity in the presence of coumarinꢀindole hybrids
intermediates (5ꢀ8) with the appropriately substituted
30 phenylacetic acids in the presence of cyanuric chloride and Nꢀ
methyl morpholine (NMM) in DMF for 1 hr gave the 3ꢀ
arylcoumarin intermediates (9ꢀ17) in good yields.19 Further, these
3ꢀarylcoumarin intermediates (9ꢀ17) were subjected to
electrophilic substitution with substituted indoles using iodine in
35 acetonitrile for 30 min at room temperature, cleanly furnished the
desired coumarin–indole hybrids (18ꢀ29).20The structures of all
the compounds were confirmed by 1H NMR, 13C NMR, and Mass
spectrometry (Supporting information).
and atorvastatin at a dose of 10 µM. (B) Percentage inhibition of HMGꢀ
CoA reductase activity at different doses (1.0ꢀ100 µM) of active
compounds (19 and 26) and atorvastatin. **P < 0.01; ***P < 0.001 vs
control.
60
The complete doseꢀresponse study at different concentrations
ranging between 1 µM to 100 µM is presented in Fig. 2B.From
the inhibition results, it is observed that up to 10 µM
concentration, activity remains similar to that of standard drug
65 atorvastatin but at higher concentrations wide variation in
inhibition was observed. Both active compounds 26 and 19
exhibited significant 63% and 58% inhibition at 20 µM
concentrations, 74% and 59% of inhibition at 50 µM
concentrations respectively, whereas atorvastatin displayed 69%
Biological evaluation
40 Inhibition of HMG-CoA reductase enzyme
Initially, to determine the effects of the synthesized coumarinꢀ 70 and 76% inhibition at 20 µM and 50 µM concentrations
indole hybrids in cholesterol biosynthesis, we evaluated them for
their HMGꢀCoA reductase inhibition using standard protocol.21
The percentages of inhibitory activities of hybrids at a dose of
45 10ꢁM are depicted in Fig. 2A, and results compared with the
respectively. Interestingly, at 100 µM concentration the active
compounds 26 and 19 inhibited 81% and 62% of enzyme
respectively, while atorvastatin showed 83% of inhibition. From
the primary findings, it is clear that compounds 26 and 19
standard drug atorvastatin. Interestingly, compounds 26 and 19 75 inhibited HMGꢀCoA reductase enzyme in a doseꢀ dependent
showed similar potency to that of standard drug atorvastatin as
they are exhibiting 56% and 55% of inhibition compared to that
of standard drug atorvastatin, which showed 60% of inhibition. In
manner. However, since the concentrations shown to inhibit the
in vitro HMGꢀCoA are on the higher side, other underlying
mechanism of these hybrids cannot be ruled out.
80
2
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Scheme 1 Synthesis of coumarinꢀindole derivatives. Reagents and conditions: (i) HMTA, TFA, 120°C, 4h; (ii) aq H2SO4, 100°C, 2 h; (iii) Phenylacetic
acid derivatives, cyanuric chloride, NꢀMethylmorpholine, DMF, reflux, 1 h; (iv) Substituted indoles, I2, CH3CN, rt, 30 min.
5
Effect of coumarin-indole hybrids in triton induced
hyperlipidemic rats
lipidemic indices, comparable to that of both standard drugs.
Although compound 19 was somewhat less active than compound
26, both compounds were considered as lead molecules among
the series. These remarkable results guided and motivated us to
30 carry out further doseꢀresponse studies on these two lead
compounds.
Due to the reliability and simplicity of Triton WRꢀ1339 model, it
has been widely used for screening antihyperlipidemic drugs.22
Thus, to investigate the potential of the coumarinꢀindole
10 derivatives on lipid parameters, we employed Triton WRꢀ1339
which induced acute hyperlipidemia in test animals. The data in
Table 1 showed that intraperitoneal (I.P) injection of triton WRꢀ
1339 (400 mg/kg b.w) led to a remarkable raise in the level of
total cholesterol (TC), phospholipids (PL) and triglycerides (TG)
15 approximately by 3.5, 2.9 and 3.1 folds respectively. After
administration of coumarinꢀindole hybrids at dose a of 100 mg/kg
in triton induced hyperlipidemic rats, compounds 19 and 26
registered potent activity profile compared with the standard
drugs atorvastatin and fenofibrate. Compounds 19 and 26
20 diminished the levels of TC by 30% and 32%, PL by 31% and
33%, TG by 29% and 33%, respectively. Whereas under the same
experimental conditions, atorvastatin (at a dose of 10 mg/kg/d)
and fenofibrate (100 mg/kg/d) diminished the levels of TC by
36% and 35%, PL by 35% and 37%, TG by 35% and 35%,
25 respectively. Compound 26 had a noteworthy effect on all
Table 2 represents hypolipidemic activities of active lead
compounds in triton induce hyperlipidemic rats treated with doses
of 50 mg/kg, 100 mg/kg and 150 mg/kg body weight. The results
35 revealed the dose dependent activity of both the lead compounds
in all the studied lipid parameters. However of the two leads, 26
was preferred choice for further detailed studies.
Thus, compound 26 was next evaluated in
a chronic
hyperlipidemia model in HFDꢀfed rats, as the feeding behavior of
40 these animals is similar to humans. A series of experiments were
carried out including the study of their effects on body weight
gain, plasma and hepatic lipid profile, receptor mediate
catabolism of LDL, and fecal bile acid excretions in HFDꢀfed
hyperlipidemic rats. Findings of these experiments are discussed
45 next.
Table 1.Hypolipidemic activity of synthetic compounds in triton induce hyperlipidemic rats at 100 mg/kg dose
Serum lipid profile
Compound no.
TC (mg/dL)
PL (mg/dL)
TG (mg/dL)
Control
Triton (T) + Vehicle
T+18
84.37±6.92
300.62±25.17c
80.47±6.12
86.28±8.00
270.17±17.30c
(+3.56 F)
240.61±18.64c (+2.99 F)
210.75±13.70 (ꢀ12)*
166.10±12.60 (ꢀ31)***
225.18±20.17 (ꢀ6) NS
200.83±18.88 (ꢀ16)*
222.22±19.30 (ꢀ8) NS
217.55±16.08 (ꢀ10)*
216.66±17.17 (ꢀ10)*
211.11±17.00 (ꢀ12)*
160.43±13.00 (ꢀ33)***
(+3.13 F)
260.38±22.77 (ꢀ13)*
210.63±16.77 (ꢀ30)***
253.37±21.11 (ꢀ16)*
270.92±21.93 (ꢀ10)*
266.66±20.00 (ꢀ11)*
277.50±24.84 (ꢀ8)NS
260.52±23.07 (ꢀ13)*
266.21±24.00 (ꢀ11)*
205.10±1.63 (ꢀ32)***
225.55±23.00 (ꢀ17)*
190.92±17.17 (ꢀ29)***
250.30±21.14 (ꢀ7) NS
232.20±18.37 (ꢀ14)*
240.30±18.99 (ꢀ11)*
248.62±20.69 (ꢀ8) NS
240.14±23.10 (ꢀ11)*
250.44±20.70 (ꢀ7) NS
180.22±18.93 (ꢀ33)***
T+19
T+20
T+21
T+22
T+23
T+24
T+25
T+26
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T+27
270.66±20.51 (ꢀ10)*
280.93±25.17 (ꢀ7) NS
262.20±23.44 (ꢀ13)*
190.17±16.77 (ꢀ36)***
206.12±18.10 (ꢀ14)*
233.31±18.99 (ꢀ14)*
244.39±20.10 (ꢀ9) NS
234.40±21.18 (ꢀ13)*
175.18±13.00 (ꢀ35)***
T+28
212.63±16.08 (ꢀ12)*
210.60±15.00 (ꢀ12)*
155.10±12.14 (ꢀ35)***
T+29
T+Atorvastatin (At)
(10 mg/Kg)
T+Fenofibrate
(100 mg/kg)
195.40± 15.83 (ꢀ35)***
151.58± 11.31(ꢀ37)***
175.14±14.26 (ꢀ35)***
Each parameter represents pooled data from 6 rats/group and values are expressed as mean±S.D.cP< 0.001, Triton treated + Vehicle rats 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)
5
Table 2.Hypolipidemic activity of active compounds 26 and 19 in triton induced hyperlipidemic rats.
Compound no.
and dose
Lipid parametersa
HDL-C (mg/dL)
TC (mg/dL)
PL (mg/dL)
TG (mg/dL)
80.31±7.04
280.71±25.51c (3.50 Folds)
98.88±4.41
73.35±2.82c
(ꢀ26%)
83.73±8.04
253.12±19.62c(3.02
81.24±7.32
250.49±21.18c
(3.08 Folds)
Control
Triton
Folds)
26 50 mg/kg
224.56±19.42** (ꢀ20)
190.88±21.06*** (ꢀ32)
188.07±12.54*** (ꢀ33)
84.35±3.83* (+15)
90.22±3.44*** (+23)
91.68±4.37*** (+25)
197.43±18.88** (22)
200.39±18.82** (ꢀ20)
167.82±12.41***
(ꢀ33)
169.59±11.26***
(ꢀ
100 mg/kg
33)
164.52±15.92***
35)
(ꢀ
168.12±15.92***
(ꢀ33)
150 mg/kg
19 50 mg/kg
221.76±21.04**
196.49±17.28***
191.13±20.08***
(ꢀ21)
(ꢀ30)
(ꢀ32)
82.15±3.32* (+12)
89.48±3.49** (+22)
89.76±4.04** (+22)
205.02±19.62*
(ꢀ19)
(ꢀ
200.21±18.66**
(ꢀ20)
174.65±13.81***
100 mg/kg
150 mg/kg
31) 174.37±17.35***
(ꢀ31)
177.84±10.52***
175.34±18.28***
(ꢀ29)
(ꢀ30)
Atorvastatin (10
179.65±15.36***
182.46±12.72***
(ꢀ36)
(ꢀ35)
89.59±4.61** (+22)
164.14±14.82***
35)
(ꢀ
(ꢀ
162.81±14.84***
162.61±15.05***
(ꢀ35)
(ꢀ35)
mg/kg)
Fenofibrate(100
93.15±3.80*** (+27)
159.46±15.56***
37)
mg/kg)
Each parameter represents pooled data from 6 rats/group and values are expressed as mean±S.D.cP< 0.001, Triton treated +Vehicle rats 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
Effect of compound 26andatorvastatinon body weight in 15 weight gain compared to the value for the HFD rats without
10 hyperlipidemic rats
change in food intake during treatment period (Table 3).
Charles Foster (CF) rats fed with HFD attained significant
increase in their body weight to induce hyperlipidemia in 4
weeks. Oral supplementation of compound 26 (100 mg/kg b.w/d)
for next 4 weeks along with HFD significantly reduced the body
Effect of compound 26 and atorvastatin on serum
biochemistry and lipoprotein metabolism in HFD-fed rat
Compound 26 showed significantly lower serum concentrations
20 of TC (ꢀ24%), TG (ꢀ26%), AST (ꢀ28%) and ALT (ꢀ26%) in
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comparison with the values for HFDꢀfed rats (Table 4).
Compound 26 reversed the detrimental effects of HFD feeding on
the hepatic accumulation of TC (ꢀ27%) and TG (ꢀ29%)
the treatment of compound 26 by +31% and +39% respectively in
HFDꢀfed hyperlipidemic rats, the similar effect was also observed
in atorvastatin treated rats. HFD administered rat serum, showed
respectively. Additionally, continuous feeding of HFD in rats 10 significantly increase in the contents of cholesterol and TG in
caused a remarkable reduction in the fecal bile acids excretion of
cholic and deoxycholic acids and these levels were improved by
5
VLDL and LDL respectively.
Table 3.Effect of Compound 26 and atorvastatin on body weight and food intake in hyperlipidemic rats.
ND+Vehicle
HFD+Vehicle
HFD+Compound26
HFD+Atorvastatin
(100 mg/kg)
(10 mg/kg)
15
186.38±13.86 263.52±21.56*** over
ND+Vehicle
228.19±19.28**
over HFD+Vehicle
9.63±0.78NS over
HFD+Vehicle
255.73±22.61NSover
HFD+Vehicle
9.71±0.59NS over
Body weight
(g)
12.68±1.03
9.81±0.61** over
ND+Vehicle
Food intake
(g/d)
HFD+Vehicle
20
Each parameter represents pooled data from 6 rats per group and values are expressed as mean ± S.D, ***P< 0.001; **P< 0.01 and NS non significant.
Table 4.Effect of compound 26 and atorvastatin on serum and hepatic biochemical parameters and fecal bile acids excretion in
hyperlipidemicrats.
25
HFD+ Comp 26
ND+Vehicle
HFD+Vehicle
HFD+ At
(100 mg/kg)
(10 mg/kg)
Serum
TC (mg/dL)
83.26±6.31
88.10±4.92
75.38±5.83
41.08±2.91
188.33±15.62*** (+2.2 fold
over ND)
143.13±11.62***
24% over HFD)
143.16±10.39***
26% over HFD)
93.15±6.26***
28% over HFD)
57.21±3.82***
26%) over HFD
(ꢀ
(ꢀ
(ꢀ
(ꢀ
141.24±9.81***
(ꢀ25% over HFD)
TG (mg/dL)
AST (IU/L)
ALT ((IU/L)
193.46±14.73*** (+2.1 fold
over ND)
143.10±12.71*** (ꢀ26%
over HFD)
129.38±10.61*** (+1.7 fold
over ND)
91.85±6.39***
over HFD)
57.99±4.13***
over HFD
(ꢀ29%
77.32±4.91*** (+1.8 fold
over ND)
(ꢀ25%)
Liver
TC (mg/g)
9.06±0.86
17.73±1.43*** (+1.9 fold
over ND)
12.94±1.08***
27% over HFD)
18.39±1.41***
29%) over HFD
(ꢀ
(ꢀ
12.76±0.96***
over HFD)
18.13±1.48***
over HFD
(ꢀ28%
TG (mg/g)
14.75±1.04
25.91±1.86***
ND)
(ꢀ1.7 over
(ꢀ30%)
Fecal bile acids
Cholic acid (µ/g)
71.63±4.26
43.82±3.68
33.28±2.51*** (ꢀ54% over
43.46±2.93*** (+31%
over HFD)
44.26±3.86*** (+33% over
ND)
29.39±2.08*** (ꢀ33% over
ND)
HFD)
41.14±2.61*** (+40% over
HFD)
Deoxycholic acid
(µ/g)
40.85±3.61*** (+39%
over HFD)
Each parameter represents pooled data from 6 rats per group and values are expressed as mean ± S.D, ***P< 0.001.
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Treatment of compound 26 and atorvastatin considerably
reversed the levels of TC and TG in VLDL and LDL, in HFDꢀfed
hyperlipidemic rats (Fig 3A and B). Interestingly, the decreased
levels of HDLꢀTC and HDLꢀTG in these animals were partially
improved (Fig 3C). HFD feeding caused significant decrease in
serum LCAT activity and lipolytic activity of LPL in liver (Fig3
D and E). Treatment of compound 26 and atorvastatin moderately
reactivated this lipolytic action in serum and liver of HFDꢀfed
animals. Additionally, increase in hepatic and circulatory lipids in
10 HFDꢀfed rats suppressed receptor mediate catabolism of LDL in
the liver membrane. Compound 26 and atorvastatin significantly
reversed the receptor mediated catabolism of LDL in HFDꢀfed
rats (Fig 3 F).
35 Effects of compound 26 and atorvastatin on hepatic HMG-
CoA reductase activity in HFD-induced hyperlipidemic rats
The HMGꢀCoA reductase activity was measured as HMG CoAꢀ
Mevalonate ratio in order to determine whether the decrease in
cholesterol levels was due to the suppression of HMGꢀCoA
40 reductase activity. As expected, feeding of HFD significantly
increased the activity of HMGꢀCoA reductase (Figure 4) in HFDꢀ
fed hyperlipidemic rats. However the treatment with compound
26 and atorvastatin significantly reduced the enzymatic activity in
HFDꢀfed hyperlipidemic rats (Figure 4).
5
45
50
55
Fig 4.Effect of Compound 26 (100 mg/kg/d) and At (10 mg/kg/d) on
hepatic HMGꢀCoA reductase activity of HFDꢀfed hyperlipidemic rats.
Values are expressed as the mean ± SD of six animals per group. *P <
60 0.05; ***P < 0.001 vsND+Vehicle and ##P < 0.01 vsHFD+Vehicle groups.
In-silico binding mode prediction of compound 26
Next, the docking studies were carried out to corroborate the
above findings and investigate the possible bound conformation
of compound 26 in the active site of HMGꢀCoA. Conformation of
65 atorvastatin generated after reꢀdocking was found to be almost
identical to coꢀcrystallized conformation (Figure 5A), indicating
that the docking protocol was good enough to generate the correct
binding pose of atorvastatin and can be used further to predict the
binding mode of compound 26 under study. Proposed binding
70 mode of compound 26 is shown in Figure 5B. Surfelx docking
score of compound 26 was lower (7.21) than the atorvastatin
(15.92), which shows that compound 26 binds to the target with
lesser affinity than the standard drug atorvastatin.
Fig 3.Compound 26 (100 mg/kg/d) and At (atorvastatin) (10 mg/kg/d)
15 improve serum lipoprotein metabolism in HFDꢀfed rat. (A) Serum
VLDLꢀTC and VLDLꢀTG, (B) Serum LDLꢀTC and LDLꢀ TG, (C) Serum
HDLꢀ TC and HDLꢀ TG, (D) LPL activity, (E) LCAT activity, and (F)
I125ꢀLDL catabolism in the liver plasma membrane of HFDꢀinduced
hyperlipidemic rats, Values are expressed as mean±SD of six animals per
20 group. **P< 0.01; ***P< 0.001 vsND+Vehicle and ##P < 0.01; ###P<
0.001 vsHFD+Vehicle groups
.
25
30
Fig 5. Overlay of docked (magenta) and coꢀcrystallized (cyan) conformations of atrovastatin (A) and proposed binding mode of compound 26 (orange)
75
(B) in the active site of HMGꢀCoA reductase. For clear representation chain A (grey) and chain B (sky blue) are shown in different colours.
6
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Detailed interaction study revealed that the bound conformation
enzymes in digestive fluid and plasma but was subject to
50 metabolism in the liver microsomes. Apparent mean residence
time (MRT) of compound 26 was found to be 4.46 hr in plasma,
which indicated prolong efficacy following single dose
administration. Thus, the pharmacokinetic behavior of
compound26 is favorable for its oral administration and supports
is mostly stabilized by hydrophobic interaction contributed by
residues Leu562_A, Leu853_A, Ala856_A and Leu857_A. One
of the indole rings of compound 26 was involved in Hꢀbonding
with Asp690_A. Compound 26 showed many similar
hydrophobic interactions as seen in the case of atorvastatin.
Coumarin moiety occupies the same position as the pyrrole ring
5
of atorvastatin, also one of the hydrophobic indole rings project 55 its efficacy for antiꢀhyperlipidemic activity.
10 towards Leu857_A similar to the 4ꢀflurophenyl ring of
atorvastatin and other indole ring occupies the deep pocket where
HMG like moiety of atorvastatin is present. We also observed
possible cationꢀπ interactions between the Arg590_B residue and
two indole rings of the compound 26, which may stabilize its
15 binding within the HMGꢀCoA active site.
Stability in simulated gastrointestinal fluids
SGF and SIF stability studies were next carried out in order to
evaluate the compound stability in gastroꢀintestinal (GI) fluids
before membrane permeation. Compound 26 was found to be
20 stable in SGF and SIF fluids (Figure 6) and this data supports its
oral administration.
Fig 7.Metabolic Stability of compound 26 of in rat liver microsomes
(RLM). Metabolic elimination profiles (% turnover or amount remaining
vs. incubation time) for: (A) without NADPH and (B) with NADPH. Data
are shown as mean ± S.D (n=3)
.
Fig 6.Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF)
stability of compound 26. Data are shown as mean ± S.D (n=3)
25 In Vitro Metabolic Stability
Drug loss in the body is mostly due to its metabolism in the liver.
Liver metabolism plays significant role in clearance of the drug
from the body. The first pass metabolism in the liver is one of the
factors for low oral bioavailability. Compound 26 was found
30 stable in the control reaction in absence of cofactor confirmed the
compound stability as well as cofactor dependent degradation
(Figure 7).The calculated microsomal intrinsic clearance (CLint,
vitro) of compound 26 was 0.0058 mL/min/mg microsomal
protein. The predicted in vitro hepatic intrinsic clearance CLint
35 was 12.81 ± 0.53 mL/min* kg of body weight. Compound 26 was
stable in simulated gastric and intestinal fluid and it was mainly
metabolized by the enzymes involved in major metabolizing
cellular organelles such as hepatocytes or enterocyte.
60 Fig 8. Mean plasma concentration−time profile of compound 26, after a
single oral administration of (100 mg/kg). Data are shown as mean ± S.D
(n=3)
.
Discussion
The compound 26 exhibited potent antihyperlipidemic activity in
65 both models of triton WRꢀ1339 and HFD induced hyperlipidemic
rats. Atorvastatin (well known inhibitor of the HMGꢀCoA
reductase) was used as positive control in this study [23]. The
weight gain in the rats treated with atorvastatin was similar to that
of HFDꢀinduced hyperlipidemic rats, while in contrast compound
70 26 significantly reduced the gain in body weight when compared
to the hyperlipidemic rats. Both compound 26 and atorvastatin
significantly decreased biochemical parameters of TC, PL, TG,
VLDL, LDL, AST and ALT and significantly increased HDL
concentration in the HFDꢀinduced hyperlipidemic rats(Figure 3,
75 Table 4). As expected due to the complications that follow the
obesity induced hyperlipidemia, the serum lipids and lipoproteins
In vivo pharmacokinetic studies in male Sprague-Dawley rats
40 Plasma concentration−time profile obtained after administration
of compound 26 as a single oral 100 mg/kg dose was given in
Figure 8. The main pharmacokinetic parameters are summarized
in Table 5. The mean peak concentration (Cmax) 78.97 ± 6.47
ng/mL was achieved at 4.0 hr after oral administration. The
45 apparent clearance (Cl/F) was found to be 241.37 ± 18.10. The
liver and peripheral tissue are usually active site for
antihyperlipidmic drugs, thus compound 26 may have good
distribution at target site. The compound 26 was resistant to
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level of rats was found to be elevated in this study. However,
Conclusion
compound 26 reduced the development of diet induced obesity as
indicated by the decreased body weight without change in food
intake pattern. The rats fed with HFD showed increased levels of
serum TC, TG, LDLꢀTC, VLDLꢀTC and decreased level of HDLꢀ
TC. Administration of compound 26 to HFDꢀinduced
hyperlipidemic rats significantly decreased the level of serum
lipids TC, TG, LDLꢀTC, VLDLꢀTC and increased the HDLꢀC.
In conclusion, a series of coumarinꢀindole derivatives have been
designed, synthesized and evaluated using the HMGꢀCoA
50 reductase inhibitor assay. The hypolipidemic activity of the most
potent compound 26 was confirmed in two different animal
models. Compound 26 remarkably reduced the body weight, liver
mass, plasma and hepatic lipid level followed by the increase in
excretion of fecal bile acids without change in food intake of
55 experimental rats. It also balanced the lipoprotein metabolism by
5
increasing the LPL, LCAT, receptor mediated catabolism of I125
ꢀ
10 Table 5. Pharmacokinetic estimation of compound 26 in rat after
single oral 100 mg/kg dose administration, each value represents
Mean ± SD
LDL. We are now in the process of elucidating the precise
molecular mechanism of action of this compound and also
synthesize analogs of 26 for better understanding of the structure
60 activity relationships, so as to advance this new prototype as a
new class of hypolipidemic agents. The exquisite potency,
favorable pharmacokinetic and structural novelty of 26 suggests it
to be a new class of lipid lowering agent.
Parameter
Cmax
Units
ng/mL
h
Estimates
78.97 ± 6.47
4.0 ± 0.0
Tmax
65 Experimental
AUC0ꢀ∞
Cl/F
h*ng/mL
L/h/kg
L/kg
h
416.57 ± 30.56
241.37 ± 18.10
683.91 ± 93.83
4.46 ± 0.14
Analysis and instruments
All reagents were commercial and were used without further
purification. Chromatography was carried on silica gel (60ꢀ120
70 and 100ꢀ200 mesh). All reactions were monitored by thinꢀlayer
chromatography (TLC), silica gel plates with fluorescence F254
were used. Melting points were taken in open capillaries on
Complab melting point apparatus and are presented uncorrected.
Infrared spectra were recorded on a PerkinꢀElmer FTꢀIR RXI
75 spectrophotometer. 1H NMR and 13C NMR spectra were recorded
using BrukerSupercon Magnet DRXꢀ300 spectrometer (operating
at 300, 400MHz for 1H and 100, 75MHz for 13C) using DMSOꢀd6
or CDCl3 as solvent and tetramethylsilane (TMS) as internal
standard. Chemical shifts are reported in parts per million.
80 Electrospray ionization mass spectra (ESIꢀMS) were recorded on
Thermo Lcq Advantage MaxꢀIT. High resolution mass spectra
(HRMS) were recorded on 6520 Agilent Q T of LC MS/MS
(Accurate mass). Additionally, purity of compounds was
measured by a RPꢀ HPLC with the following conditions, and
85 purity of the compounds was >95%. The Waters HPLC system,
Milford USA consisted of a binary pump (model Waters 515
HPLC pump), auto sampler (model 717 plus Auto sampler) and
PDA detector (Waters 2996). Data collection and analysis were
performed using Empower pro 2 software. The resolution and
90 better peak shape were achieved on a SymmetryꢀShield C18
(5µm, 4.6 X 150 mm) column, protected with a C18 guard
column. The system was analyzed in isocratic mode with a
mobile phase consisting of Methanol: 0.5% aqueous formic acid
Vd/F
MRT
Moreover the LPL activity in liver of rats fed with HFD were
15 found to be decreased. Compound 26 caused significant increase
in LPL activity, which separates free fatty acids from TGs present
in VLDL and chylomicrones. Serum LCAT activity was also
increased by compound 26, which is the main modulator of HDLꢀ
TC and plays an important role in the reverse cholesterol
20 transport system. The bovine serum albumin (BSA) interaction
studies indicate that the lead compound has strong affinity for
albumin protein and forms a ground state complex (supporting
information). These drug like effects clearly underscore the
antihyperlipidemic potential of compound 26.
25 The antihyperlipidemic activity of compound 26 may be due to
reduced anabolism of cholesterol in the liver. Specifically it may
be due to the decrease in the activity of hepatic HMGꢀCoA
reductase. The present results showed that treatment of
Compound 26 in hyperlipidemic rat caused significant reduction
30 in hepatic HMGꢀCoA reductase activity, which strongly suggests
that the activity is mediated by inhibition of HMGꢀCoA reductase
in the hyperlipidemic rat liver and the results corroborated well
with in vitro data and molecular docking analysis. The schematic
representation of mechanism of action of compound 26 is shown
35 in Figure 9.
(52: 48, v/v) at a flow rate of 1.0 mL/min.
95 General procedure for the synthesis of compounds (5–8)
Synthesis of dicarbaldehyde substrates (5ꢀ8) were achieved by
our previously reported protocol.18
4-hydroxy-5-methylisophthalaldehyde (5)
White solid, Yield: 60%; mp 125ꢀ127ºC; IR (KBr) : 3262, 1703,
100 1626, 745 cmꢀ1; H NMR (CDCl3, 400MHz) δ :11.83 (s, 1H),
1
40
9.98 (s, 1H), 9.90 (s, 1H), 7.97(d, J = 1.96 Hz, 1H), 7.93 (brs,
1H), 2.34 (s, 3H) ; 13C NMR (CDCl3, 100MHz): 196.66, 189.97,
165.20, 137.46, 134.93, 128.91, 119.96, 15.40 ; ESIꢀMS (m/z):
165 [M+H]+.
105 4-hydroxy-5-isopropylisophthalaldehyde (6)
White solid, Yield: 65%; mp 163ꢀ164ºC; IR (KBr) : 3023, 1693,
45 Fig 9.Coumarinꢀindole derivative may regulate lipoprotein metabolism
1655, 758 cmꢀ1; 1H NMR (CDCl3, 400MHz) δ:11.94 (s, 1H), 9.97
via inhibition on HMGꢀCoA reductase activity in liver.
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(s, 1H), 9.91 (s, 1H), 7.99 (brs, 1H), 7.96 (s, 1H), 3.41ꢀ3.34 (m,
3-(4-methoxyphenyl)-8-methyl-2-oxo-2H-chromene-6-
carbaldehyde (12)
1H), 1.27 (d, J = 6.92 Hz, 6H); 13C NMR (CDCl3, 75MHz):
196.62, 189.90, 164.34, 138.90, 134.86, 133.28, 129.06, 119.94,
26.54, 22.11; ESIꢀMS (m/z): 193 [M+H]+.
Light yellow solid, yield: 65%; mp 148ꢀ150ºC; IR (KBr): 3033,
1725, 1705, 1639, 1021 cmꢀ1; 1H NMR (CDCl3, 300MHz) δ: 9.98
65 (s, 1H), 7.86 (d, J = 4.6 Hz, 2H), 7.78 (s, 1H), 7.68 (d, J = 8.8 Hz,
2H), 6.96 (d, J = 8.8 Hz, 2H), 3.84 (s, 3H), 2.53 (s, 3H); 13C
NMR (CDCl3, 75MHz) δ: 190.55, 160.53, 159.94, 155.38,
137.94, 132.43, 132.30, 129.90, 128.63, 128.07, 127.26, 126.48,
119.84, 114.10, 55.44, 15.58; ESIꢀMS: m/z: 295 [M+H]+.
70 3-(3,4-dimethoxyphenyl)-8-methyl-2-oxo-2H-chromene-6-
carbaldehyde (13)
5
5-tert-butyl-4-hydroxyisophthalaldehyde (7)
1
Oily, Yield: 60%; IR (KBr) : 3381, 1692, 1653, 778 cmꢀ1; H
NMR (CDCl3, 300MHz) δ:12.39 (s, 1H), 9.97 (s, 1H), 9.91 (s,
1H), 8.05 (d, J =1.4 Hz, 1H), 7.97(d, J = 1.5 Hz, 1H), 1.44 (s,
9H); 13C NMR (CDCl3, 75MHz): 196.62, 189.90, 164.34, 138.90,
10 134.86, 133.28, 129.06, 119.94, 26.54, 22.11 ; ESIꢀMS (m/z): 207
[M+H]+.
5-(sec-butyl)-4-hydroxyisophthalaldehyde (8)
Light yellow solid yield: 60%; mp 178ꢀ180ºC; IR (KBr): 3053,
1730, 1695, 1634, 1026 cmꢀ1; 1H NMR (CDCl3, 300MHz) δ:
10.01 (s, 1H), 7.89 (d, J = 7.2 Hz, 2H), 7.83 (s, 1H), 7.31–7.28
75 (m, 2H), 6.95 (d, J = 8.9 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H), 2.56
(s, 3H); 13C NMR (CDCl3, 75MHz) δ: 190.49, 159.88, 155.35,
150.16, 148.89, 138.17, 132.45, 132.39, 128.66, 128.03, 127.26,
126.80, 121.40, 119.78, 111.84, 111.18, 56.10, 56.04, 15.54; ESIꢀ
MS: m/z: 325 [M+H]+.
Oily, Yield 65%, IR (neat): 3267, 3062, 1709, 1622, 1018 cmꢀ1;
1H NMR (CDCl3, 200MHz) δ: 11.99 (s, 1H), 10.51 (s, 1H), 9.96
15 (s, 1H), 8.08 (brs, 1H), 7.99 (s, 1H), 3.27ꢀ3.10 (m, 1H), 1.74–1.57
(m, 2H), 1.25 (d, J = 7.0 Hz, 3H), 0.87 (t, J = 7.3 Hz, 3H); 13
NMR: (CDCl3, 75MHz) δ: 196.47, 189.43, 163.98, 137.19,
134.50, 133.49, 128.70, 119.64, 32.77, 28.89, 19.39, 11.58; ESIꢀ
MS: m/z: 207 [M+H]+.
C
20 General procedure for the synthesis of 3-phenylcoumarins
80 8-methyl-2-oxo-3-(3,4,5-trimethoxyphenyl)-2H-chromene-6-
carbaldehyde (14)
(9-17).19
To
a solution of cyanuric chloride (4.85 mmol) in Nꢀ
White solid, yield: 60%; mp 196ꢀ198ºC; IR (KBr): 3074, 1728,
methylmorholine (7.28 mmol) was added phenylacetic acid (4.85
mmol), and the solution was stirred at room temperature for 10
25 min. To the above solution was added DMF solution of
dialdehyde (4.85 mmol), and the reaction mixture was stirred 100
°C for 1.5 h. The mixture was extracted with EtOAc (50 mL × 3).
The combined EtOAc extracts were dried (Na2SO4) and
concentrated to yield the the crude products, which were purified
30 by column chromatography on silica gel yielded the desired
compounds.
1
1695, 1632, 1010 cmꢀ1; H NMR (CDCl3, 300MHz) δ: 10.02 (s,
1H), 7.93ꢀ7.91 (m, 2H), 7.86 (s, 1H), 6.95 (s, 2H), 3.93 (s, 6H),
85 3.90 (s, 3H), 2.57 (s, 3H); 13C NMR (CDCl3, 75MHz) δ: 190.48,
159.82, 155.53, 153.36, 139.38, 139.38, 132.77, 132.58, 129.62,
129.04, 128.19, 127.44, 119.67, 106.20, 61.04, 56.44, 15.63; ESIꢀ
MS: m/z: 355 [M+H]+.
3-(3,4-dimethoxyphenyl)-8-isopropyl-2-oxo-2H-chromene-6-
90 carbaldehyde (15)
8-(tert-butyl)-3-(4-methoxyphenyl)-2-oxo-2H-chromene-6-
carbaldehyde (9)
Pale yellow solid, yield: 79%; mp 144ꢀ145ºC; IR (KBr): 3078,
1
1647, 1229, 746 cmꢀ1; H NMR (CDCl3, 400MHz) δ:10.01 (s,
White solid, yield: 63%; mp 154ꢀ155ºC; IR (KBr): 3014, 1705,
1H), 7.95 (s, 1H), 7.89 (s, 1H), 7.84 (s, 1H), 7.30ꢀ7.25 (m, 2H),
6.93 (d, J = 8.0 Hz, 1H), 3.93 (s, 3H), 3.91 (s, 3H), 3.71ꢀ3.64 (m,
95 1H), 1.35 (d, J = 6.8 Hz, 6H);13C NMR (CDCl3, 100MHz):
190.71, 159.90, 154.44, 150.16, 148.90, 138.47, 137.72, 132.73,
128.65, 128.39, 128.11, 126.86, 121.39, 119.96, 111.84, 111.20,
56.12, 56.07, 26.76, 22.59; ESIꢀMS (m/z): 353 [M+H]+.
8-isopropyl-2-oxo-3-(3,4,5-trimethoxyphenyl)-2H-chromene-
100 6-carbaldehyde (16)
1
35 1285, 776 cmꢀ1; H NMR (CDCl3, 400MHz) δ: 10.03 (s, 1H),
8.02 (d, J = 1.8 Hz, 1H), 7.91 (d, J = 1.8 Hz, 1H), 7.82 (s, 1H),
7.71 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H), 3.86 (s, 3H),
1.58 (s, 9H); 13C NMR: (CDCl3, 75MHz) δ: 190.87, 160.58,
159.41, 155.90, 139.14, 138.47, 132.27, 129.93, 128.88, 128.85,
40 128.17, 126.49, 120.68, 114.18, 55.50, 35.35, 29.82; ESIꢀMS:
m/z: 337 [M+H]+.
8-tert-butyl-3-(3,4-dimethoxyphenyl)-2-oxo-2H-chromene-6-
carbaldehyde (10)
Pale yellow solid, yield: 75%; mp 131ꢀ132ºC; IR (KBr): 3028,
1
1677, 1229, 768 cmꢀ1; H NMR (CDCl3, 400MHz) δ:10.02 (s,
White solid, yield: 75%; mp 163ꢀ164ºC; IR (KBr): 3020, 1697,
45 1272, 757 cmꢀ1; 1H NMR (CDCl3, 400MHz) δ:10.01 (s, 1H), 8.01
(d, J = 1.7 Hz, 1H), 7.92 (d, J = 1.6 Hz, 1H), 7.85 (s, 1H), 7.35ꢀ
7.29 (m, 2H), 6.94 (d, J = 8.4 Hz, 1H), 3.93 (s, 3H), 3.92 (s, 3H),
1.56 (s, 9H); 13C NMR (CDCl3, 100MHz): 190.82, 159.40,
155.84, 150.16, 148.90, 139.13, 138.70, 132.28, 128.96, 128.84,
50 128.13, 126.78, 121.31, 120.60, 111.84, 111.22, 56.12, 56.08,
35.33, 29.79; ESIꢀMS (m/z): 367 [M+H]+.
1H), 7.97 (s, 1H), 7.92 (s, 1H), 7.87 (s, 1H), 6.94 (s, 2H), 3.91 (s,
6H), 3.88 (s, 3H), 3.71ꢀ3.64 (m, 1H), 1.36 (d, J = 6.9 Hz, 6H);13C
105 NMR (CDCl3, 100MHz): 190.65, 159.76, 154.54, 153.33, 139.36,
139.29, 137.81, 132.80, 129.62, 128.90, 128.70, 128.24, 119.79,
106.13, 61.03, 56.41, 26.81, 22.59; ESIꢀMS (m/z): 383 [M+H]+.
8-sec-butyl-3-(3,4-dimethoxyphenyl)-2-oxo-2H-chromene-6-
carbaldehyde (17)
110 Light yellow solid, yield: 59%;mp 140ꢀ142ºC; IR (KBr): 3065,
1728, 1697, 1607, 1032 cmꢀ1; 1H NMR (CDCl3, 300MHz) δ:
10.03 (s, 1H), 7.92 (d, J = 1.6 Hz, 2H), 7.86 (s, 1H), 7.32–7.29
(m, 2H), 6.97 (d, J = 8.5 Hz, 1H), 3.96 (s, 3H), 3.94 (s, 3H),
3.53–3.44 (m, 1H), 1.81–1.71 (m, 2H), 1.34 (d, J = 6.9 Hz, 3H),
115 0.90 (t, J = 7.3 Hz, 3H) ; 13C NMR (CDCl3, 75MHz) δ: 190.75,
159.96, 154.79, 150.24, 148.98, 138.55, 136.81, 132.77, 129.19,
128.77, 128.02, 126.94, 121.44, 120.06, 111.94, 111.28, 56.18,
8-tert-butyl-2-oxo-3-(3,4,5-trimethoxyphenyl)-2H-chromene-
6-carbaldehyde (11)
Pale yellow solid, yield: 69%; mp 181ꢀ182ºC; IR (KBr): 3042,
1
55 1647, 1242, 754 cmꢀ1; H NMR (CDCl3, 400MHz) δ: 10.02 (s,
1H), 8.03 (d, J = 1.8 Hz, 1H), 7.94 (d, J =1.8 Hz, 1H), 7.87 (s,
1H), 6.97 (s, 2H), 3.91 (s, 6H), 3.89 (s, 3H), 1.56 (s, 9H); 13C
NMR (CDCl3, 100MHz): 190.75, 159.26, 155.92, 153.33, 139.58,
139.28, 139.22, 132.35, 129.51, 129.25, 128.96, 128.34, 120.42,
60 106.08, 61.04, 56.41, 35.35, 29.78; ESIꢀMS (m/z): 397 [M+H]+.
56.13, 33.54, 29.77, 20.58, 12.14; ESIꢀMS: m/z: 367 [M]+
.
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General procedure for the synthesis of final compounds (18–
29)
111.49, 111.33, 55.59, 55.53, 26.38, 22.40; HRMS (m/z): calcd
for C37H32N2O4[M+H]+ 569.2440, found: 569.2437.
6-(bis(2-methyl-1H-indol-3-yl)methyl)-8-isopropyl-3-(3,4,5-
70 trimethoxyphenyl)-2H-chromen-2-one (23)
To a magnetically stirred solution of 3ꢀphenylcoumarins (9ꢀ17)
(1.06 mmol) in acetonitrile (20 mL) were added different
substituted indoles (2.12 mmol) and iodine (0.21 mmol) and
stirred for 30min at rt. The formed solids were collected by
filtration and obtained solids (18ꢀ29) were dried invacuo.
6-(di(1H-indol-3-yl)methyl)-3-(4-methoxyphenyl)-8-methyl-
2H-chromen-2-one (18)
5
Yellow solid, yield: 66%;mp 140ꢀ142ºC; IR (KBr) : 3028, 2936,
1663, 1566, 1218, 701 cmꢀ1; 1H NMR (DMSOꢀd6, 300MHz)
δ:10.77 (s, 2H), 8.14 (s, 1H), 7.48 (s, 1H), 7.30 (s, 1H), 7.22 (d,
J=7.9 Hz, 2H), 7.04 (s, 2H), 6.92ꢀ6.83 (m, 4H), 6.71ꢀ6.67 (m,
10 Yellow solid, yield: 68%; mp 168ꢀ169ºC; IR (KBr) : 3008, 2915, 75 2H), 6.03 (s, 1H), 3.78 (s, 6H), 3.68 (s, 3H), 3.39 (brs, 1H), 2.07
1
1648, 1570, 1559, 736 cmꢀ1; H NMR (DMSOꢀd6, 300MHz) δ:
10.85 (s, 2H), 8.08 (s, 1H), 7.68ꢀ7.65 (d, J = 8.7 Hz, 2H), 7.51 (d,
J = 9.6 Hz, 2H) , 7.37ꢀ7.30 (m, 4H), 7.07ꢀ7.02 (m, 2H), 6.99 (s,
2H), 6.90ꢀ6.85 (m, 4H), 5.89 (s, 1H), 3.78 (s, 3H), 2.34 (s,
15 3H);13C NMR (DMSOꢀd6, 75MHz): 160.02, 159.43, 149.45,
140.91, 139.59, 136.61, 132.73, 129.72, 126.92, 126.50, 125.76,
125.09, 124.17, 123.75, 120.95, 118.98, 118.27, 117.70, 113.57,
(s, 6H), 1.16 (d, J= 6.8 Hz, 6H);13C NMR (DMSOꢀd6, 75MHz):
159.74, 152.45, 148.46, 141.03, 140.39, 137.88, 135.09, 134.36,
132.27, 130.04, 129.69, 128.14, 125.89, 125.32, 119.64, 118.85,
118.32, 117.99, 111.74, 110.39, 106.23, 60.02, 55.97, 38.14,
80 26.35, 22.32, 12.03; HRMS (m/z): calcd for C40H38N2O5[M+H]+
627.2859, found: 627.2854.
111.49, 55.16, 15.02
;
HRMS (m/z): calcd for
6-(bis(2-methyl-1H-indol-3-yl)methyl)-8-tert-butyl-3-(4-
methoxyphenyl)-2H-chromen-2-one (24)
C34H26N2O3[M+H]+ 511.2022, found: 511.2027
20 6-(bis(5-nitro-1H-indol-3-yl)methyl)-3-(4-methoxyphenyl)-8-
methyl-2H-chromen-2-one (19)
White solid, yield: 69%;mp 253ꢀ254ºC;IR (KBr): 3021, 1705,
85 1645, 1595, 1285, 768 cmꢀ1; 1H NMR (DMSOꢀd6,
400MHz)δ:10.80 (s, 2H), 8.06 (s, 1H), 7.70(d, J= 8.8 Hz, 2H),
7.54 (d, J=1.8 Hz, 1H), 7.36 (s, 1H), 7.24 (d, J= 8.0 Hz, 2H), 6.96
(d, J=7.0 Hz, 2H), 6.94ꢀ6.87 (m, 4H), 6.72ꢀ6.68 (m, 3H), 3.77 (s,
3H), 2.09 (s, 6H), 1.35 (s, 9H); 13C NMR (DMSOꢀd6, 75MHz):
90 159.57, 159.42, 149.69, 140.21, 139.74, 135.65, 135.08, 132.18,
129.71, 128.10, 126.70, 125.96, 125.13, 119.61, 118.32, 117.94,
113.55, 111.84, 110.36, 55.12, 38.21, 34.32, 29.49, 12.04; HRMS
(m/z): calcd for C39H36N2O3[M+H]+ 581.2804, found: 581.2810.
6-(bis(5-nitro-1H-indol-3-yl)methyl)-8-tert-butyl-3-(4-
95 methoxyphenyl)-2H-chromen-2-one (25)
Pale yellow solid, yield: 75%; mp 180ꢀ181ºC;IR (KBr) : 3047,
2928, 1680, 1555, 1512, 758 cmꢀ1; 1H NMR (DMSOꢀd6,
400MHz)δ: 11.71 (s, 2H), 8.38 (d, J = 2.2 Hz, 2H), 8.13 (s, 1H),
25 7.99 (dd, J1,3 = 8.9 Hz, J1,2 = 2.2 Hz, 2H), 7.68 (d, J = 8.9 Hz,
2H), 7.57 (s, 1H), 7.55 (m, 3H), 7.15 (d, J = 1.8 Hz, 2H), 6.99 (d,
J = 8.9 Hz, 2H), 6.30 (s, 1H), 3.79 (s, 3H), 2.38 (s, 3H);13C NMR
(DMSOꢀd6, 100MHz): 159.95, 159.49, 149.78, 140.31, 139.85,
139.67, 139.45, 132.51, 129.74, 127.89, 126.86, 126.01, 125.72,
30 125.06, 124.70, 120.28, 119.28, 116.69, 116.12, 113.57, 112.14,
55.14, 37.73, 15.03; HRMS (m/z): calcd for C34H24N4O7[M+H]+
601.1723, found: 601.1719.
6-(di(1H-indol-3-yl)methyl)-3-(3,4-dimethoxyphenyl)-8-
Yellow solid, yield: 69%;mp 209ꢀ210ºC;IR (KBr): 3021, 2927,
1645, 1550, 1285, 768 cmꢀ1; 1H NMR (DMSOꢀd6,
300MHz)δ:11.69 (s, 2H), 8.37 (s, 2H), 8.13 (s, 1H ), 8.00ꢀ7.96
(m, 2H), 7.75 (s, 1H), 7.69 (d, J = 8.7 Hz, 2H), 7.59 (s, 1H ),
100 7.57ꢀ7.54 (m, 2H), 7.22 (s, 2H), 6.99 (d, J= 8.7 Hz, 2H), 6.34 (s,
1H), 3.79 (s, 3H), 1.44 (s, 9H); 13C NMR (DMSOꢀd6, 75MHz):
159.49, 159.46, 149.91, 140.25, 140.13, 139.85, 139.48, 136.14,
129.71, 128.98, 127.65, 126.73, 126.07, 125.74, 125.37, 120.32,
120.15, 116.66, 116.26, 113.61, 112.14, 55.14, 38.12, 34.52,
105 29.57; HRMS (m/z): calcd for C37H30N4O7[M+H]+ 643.2193,
found: 643.2198.
methyl-2H-chromen-2-one (20)
35 Orange solid, yield: 73%;mp 155ꢀ156ºC;IR (KBr) : 3012, 2938,
1645, 1588, 1527, 785 cmꢀ1; 1H NMR (DMSOꢀd6, 300 MHz)
δ:10.85 (s, 2H), 8.15 (s, 1H), 7.52 (s, 2H), 7.37ꢀ7.31 (m, 6H),
7.07ꢀ6.98 (m, 2H), 6.91 (s, 1H), 6.88 (s, 2H), 6.86 (s, 2H), 5.90
(s, 1H), 3.78 (s, 6H), 2.35 (s, 3H);13C NMR (DMSOꢀd6, 75MHz):
40 159.96, 149.42, 149.18, 148.31, 140.94, 139.77, 136.63, 132.82,
127.08, 126.51, 125.79, 125.06, 124.18, 123.78, 121.23, 120.98,
118.98, 118.30, 117.71, 112.09, 111.51, 111.31, 55.58, 55.53,
15.04; HRMS (m/z): calcd for C35H28N2O4[M+H]+ 541.2127,
found: 541.2171.
45 6-(bis(2-methyl-1H-indol-3-yl)methyl)-8-methyl-3-(3,4,5-
trimethoxyphenyl)-2H-chromen-2-one (21)
8-tert-butyl-6-(di(1H-indol-3-yl)methyl)-3-(3,4-
dimethoxyphenyl)-2H-chromen-2-one (26)
Red solid, yield: 65%;mp 170ꢀ171ºC; IR (KBr): 3036, 2948,
1
1548, 1600, 1209, 720 cmꢀ1; H NMR (DMSOꢀd6, 300MHz)δ:
White solid, yield: 65%;mp 272ꢀ273ºC;IR (KBr); 3015, 2935,
10.79 (s, 2H), 8.17 (s, 1H), 7.34 (d, J = 10.5 Hz, 2H), 7.23 (d,
50 J=7.9 Hz, 2H), 7.05 (s, 2H), 6.93ꢀ6.84 (m, 4H), 6.72ꢀ6.67 (m,
2H), 6.00 (s, 1H), 3.79 (s, 6H), 3.68 (s, 3H), 2.32 (s, 3H), 2.07 (s,
6H); 13C NMR (DMSOꢀd6, 75MHz): 159.85, 152.47, 149.54,
140.85, 140.42, 137.85, 135.10, 133.56, 132.39, 130.08, 128.20,
125.98, 125.39, 124.19, 119.66, 118.70, 118.31, 118.12, 111.59,
55 110.44, 106.18, 60.06, 55.98, 38.03, 15.07, 12.05; HRMS (m/z):
calcd for C38H34N2O5[M+H]+ 599.2546, found: 599.2542
6-(di(1H-indol-3-yl)methyl)-3-(3,4-dimethoxyphenyl)-8-
isopropyl-2H-chromen-2-one (22)
1
110 1662, 1570, 1274, 756 cmꢀ1; H NMR (DMSOꢀd6, 400MHz) δ:
10.84 (s, 2H), 8.17 (s, 1H ), 7.67 (s, 1H), 7.56 (s, 1H), 7.38ꢀ7.32
(m, 6H), 7.07ꢀ6.98 (m, 3H), 6.90ꢀ6.87 (m, 4H), 5.95 (s, 1H ),
3.79 (s, 6H), 1.43 (s, 9H); 13C NMR (DMSOꢀd6, 75MHz): 159.62,
149.70, 149.26, 148.40, 140.76, 140.47, 136.72, 135.88, 129.26,
115 127.03, 126.62, 125.95, 125.29, 123.76, 121.28, 121.09, 119.85,
119.15, 118.38, 117.95, 112.14, 111.62, 111.43, 55.67, 55.60,
34.50, 29.69; HRMS (m/z): calcd for C38H34N2O4 [M+H]+
583.2597, found: 583.2593.
6-(bis(2-methyl-1H-indol-3-yl)methyl)-8-tert-butyl-3-(3,4-
120 dimethoxyphenyl)-2H-chromen-2-one (27)
Pale yellow solid, yield: 75%; mp 180ꢀ181ºC; IR (KBr) : 3047,
60 2928, 1680, 1555, 1512, 758 cmꢀ1; 1H NMR (DMSOꢀd6,
300MHz)δ:10.83 (s, 2H), 8.15 (s, 1H), 7.63 (s, 1H), 7.51 (s, 1H),
7.37ꢀ7.29 (m, 6H), 7.07ꢀ6.98 (m, 3H), 6.90ꢀ6.85 (m, 4H), 5.93 (s,
Pale yellow solid, yield: 69%;mp 248ꢀ249ºC;IR (KBr): 3021,
2927, 1645, 1585, 1285, 768 cmꢀ1; 1H NMR (DMSOꢀd6,
400MHz) δ : 10.80 (s, 2H), 8.13(s, 1H), 7.54 (d, J= 1.8 Hz, 1H),
7.38ꢀ7.34 (m, 3H), 7.23 (d, J = 8.0 Hz, 2H), 6.98 (d, J= 8.5 Hz,
125 1H), 6.92ꢀ6.85 (m, 4H), 6.72ꢀ6.68 (m, 2H), 6.04 (s, 1H), 3.77 (s,
6H), 2.09 (s, 6H), 1.36 (s, 9H);13C NMR (DMSOꢀd6, 75MHz):
1H), 3.78 (s, 6H), 3.46ꢀ3.42 (m, 1H), 1.24 (d, J= 6.8 Hz, 6H); 13
C
NMR (DMSOꢀd6, 75MHz): 159.86, 149.17, 148.31, 141.08,
65 140.01, 136.63, 134.32, 128.79, 127.08, 126.52, 125.68, 125.16,
123.67, 121.22, 120.95, 119.15, 119.02, 118.24, 117.83, 112.12,
10|
Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 11 of 13
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DOI: 10.1039/C6MD00283H
65 7. P.D. Thompson, P. Clarkson, R.H. Karas, JamaꢀJ. Am. Med. Assoc.,
2003, 289,1681ꢀ1690.
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Dallongeville, J. Auwerx, K. Schoonjans, E. Leitersdorf, J.G.
159.50, 149.66, 149.16, 148.31, 140.37, 139.76, 135.66, 135.09,
132.21, 129.81, 128.11, 126.89, 125.92, 125.21, 121.19, 119.62,
118.33, 117.95, 112.13, 111.82, 111.32, 110.37, 55.57, 55.51,
38.21, 34.32, 29.49, 12.04; HRMS (m/z): calcd for
5
C40H38N2O4[M+H]+ 611.2910, found: 611.2917.
70
75
Fruchart, Circulation, 1998, 98, 2088ꢀ2093.
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dimethoxyphenyl)-2H-chromen-2-one (28)
Lancet, 2005, 365, 1389ꢀ1397
.
Pale yellow solid, yield: 75%;mp 160ꢀ161ºC; IR (KBr); 3043,
2912, 1670, 1532, 1246, 751 cmꢀ1; 1H NMR (DMSOꢀd6,
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Soubhye, I. Aldib, B. Elfving, M. Gelbcke, P.G. Furtmuller, M.
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10 300MHz) δ : 11.69 (s, 2H), 8.36 (s, 2H ), 8.17 (s, 1H), 8.00ꢀ7.97
(m, 2H), 7.75 (s, 1H), 7.57 (s, 2H), 7.54 (s, 1H), 7.36ꢀ7.33(m,
2H), 7.21 (s, 2H), 7.01ꢀ6.99 (m, 1H), 6.34 (s, 1H), 3.78 (s, 6H),
1.44 (s, 9H) ;13C NMR (DMSOꢀd6, 75MHz): 159.38, 149.86,
149.22, 148.31, 140.25, 139.85, 139.50, 136.13, 129.03, 127.66,
15 126.89, 126.04, 125.73, 125.40, 121.19, 120.32, 120.12, 116.66,
116.25, 112.15, 112.08, 111.34, 55.58, 55.51, 38.10, 34.52, 29.57
; HRMS (m/z): calcd for C38H32N4O8[M+H]+ 673.2298, found:
673.2284.
80
6-(bis(5-nitro-1H-indol-3-yl)methyl)-8-sec-butyl-3-(3,4-
20 dimethoxyphenyl)-2H-chromen-2-one (29)
85
Pale yellow solid, yield: 76%; mp 130ꢀ131ºC; IR (KBr): 3012,
2921, 1710, 1627, 1210, 719 cmꢀ1; 1H NMR (DMSOꢀd6,
300MHz) δ :11.70 (s, 2H), 8.34 (s, 2H), 7.99 (d, J = 8.6 Hz, 2H ),
7.67ꢀ7.54 (m, 4H), 7.35ꢀ7.31 (m, 2H), 7.19 (s, 2H), 6.99 (d, J =
25 8.4 Hz, 1H), 6.33 (s, 1H), 5.75 (s, 1H), 3.78 (s, 6H), 1.66ꢀ1.61
(m, 2H), 1.22 (d, J = 6.8 Hz, 3H), 0.75 (t, J = 7.1 Hz, 3H,) ;13C
NMR (DMSOꢀd6, 75MHz): 159.81, 149.20, 148.91, 148.30,
140.24, 139.87, 133.41, 129.29, 127.71, 127.64, 127.04, 125.96,
125.74, 125.38, 121.25, 120.37, 120.22, 119.57, 116.70, 116.24,
30 112.20, 112.10, 111.31, 55.58, 55.54, 38.04, 33.13, 28.99, 20.47,
11.74; HRMS (m/z): calcd for C38H32N4O8 [M+H]+ 673.2298,
found: 673.2293.
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Hameed, V. Shinde, C. Bathula, V. Humnabadkar, N. Kumar, J.
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90
95
100
105
110
115
120
Ethical statement
35 In vivo experiments were conducted as per the guidelines
provided by the animal ethics committee (IAEC) of the Central
Drug Research Institute, Lucknow, India.
14. H.Y. Lee, S.L. Pan, M.C. Su, Y.M. Liu, C.C. Kuo, Y.T. Chang, J.S.
Wu, C.Y. Nien, S. Mehndiratta, C.Y. Chang, S.Y. Wu, M.J. Lai, J.Y.
Chang, J.P. Liou, J. Med. Chem., 2013, 56,8008ꢀ8018.
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40 Instrumentation facilities from SAIF, CDRI are gratefully
acknowledged. K.B.R., R.K.M., and R.S. are thankful to CSIR
and P.K. is thankful to UGC, New Delhi, India, for financial
support. S.P.S. is acknowledged for technical support. The
authors are grateful to the Director, CDRI, Lucknow, India, for
45 constant encouragement in the drug development program. This
is CSIRꢀCDRI Communication No. 9270
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DOI: 10.1039/C6MD00283H
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DOI: 10.1039/C6MD00283H
Hybrids of coumarin-indole: Design, synthesis and biological
evaluation in triton WR-1339 and high-fat diet induced
hyperlipidemic rat models
Koneni V. Sashidhara,*a K. Bhaskara Rao,§a Ravi Sonkar ,§b Ram K. Modukuri,a Yashpal S.
Chhonker, c Pragati Kushwaha,a Hardik Chandasana, c A. K. Khanna, b Rabi S. Bhatta, c Gitika
b
b
Bhatia,b Manish Kumar Suthar, Jitendra Kumar Saxena, Vikash Kumar,d Mohammad
Imran Siddiqi. d
Herein, we have described the in vitro and in vivo investigations of novel coumarin-indole
hybrids and showed their promising lipid lowering activity.