Coumarin chalcone fibrates: A new structural class of lipid lowering agents

Article abstract of DOI:10.1016/j.ejmech.2013.04.026

In our continuing search for safe and efficacious antidyslipidemic agents, structurally interesting coumarin-chalcone fibrates were synthesized and evaluated in triton WR-1339 induced hyperlipidemic rats. The most active compound 41 decreased the total cholesterol (TC), phospholipids (PL) and triglycerides (TG), of hyperlipidemic rats by 26, 24, and 25% respectively. In addition, the compound 41 significantly reversed the levels of VLDL, LDL HDL and also increased the LPL activity. Altogether, our data suggests that these novel hybrids would be a potential new class of therapeutic agents against dyslipidemia.

Full text of DOI:10.1016/j.ejmech.2013.04.026

Accepted Manuscript
Coumarin chalcone fibrates: a new structural class of lipid lowering agents
Koneni V. Sashidhara, Gopala Reddy Palnati, Ravi Sonkar, Srinivasa Rao Avula,
Chetan Awasthi, Gitika Bhatia
PII:
S0223-5234(13)00258-4
10.1016/j.ejmech.2013.04.026
EJMECH 6136
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 February 2013
Revised Date: 8 April 2013
Accepted Date: 10 April 2013
Please cite this article as: K.V. Sashidhara, G.R. Palnati, R. Sonkar, S.R. Avula, C. Awasthi, G. Bhatia,
Coumarin chalcone fibrates: a new structural class of lipid lowering agents, European Journal of
Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.04.026.
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ACCEPTED MANUSCRIPT
Graphical abstract
Coumarin chalcone fibrates: a new structural class of lipid
lowering agents*
Koneni V. Sashidhara *, Gopala Reddy Palnati, Ravi Sonkar, Srinivasa Rao, Chetan Awasthi, Gitika Bhatia
The hybrid 41 significantly decreased the plasma lipids and considerably reversed the
levels of VLDL, LDL and HDL activity, representing a new structural class of lipid
lowering agent.

ACCEPTED MANUSCRIPT
Coumarin chalcone fibrates: a new structural class of lipid
lowering agents*
a,
a
b
a
Koneni V. Sashidhara *, Gopala Reddy Palnati , Ravi Sonkar , Srinivasa Rao Avula ,
Chetan Awasthi b, Gitika Bhatia b
Medicinal and Process Chemistry Divisiona, CSIR-Central Drug Research Institute,
Lucknow, 226 001, India
Biochemistry Divisionb, CSIR-Central Drug Research Institute, Lucknow, 226 001, India
Abstract
In our continuing search for safe and efficacious antidyslipidemic agents, structurally
interesting coumarin-chalcone fibrates were synthesized and evaluated in triton WR-1339
induced hyperlipidemic rats. The most active compound 41 decreased the total cholesterol
(TC), phospholipids (PL) and triglycerides (TG), of hyperlipidemic rats by 26, 24, and 25%
respectively. In addition, the compound 41 significantly reversed the levels of VLDL, LDL
HDL and also increased the LPL activity. Altogether, our data suggests that these novel
hybrids would be a potential new class of therapeutic agents against dyslipidemia.
Keywords: Coumarin chalcone fibrates, lipid lowering activity, lipoprotein metabolism, and
LPL activity, triton model.
* Part XXIII in the series, “Advances in drug design and discovery”.
*Corresponding authors. Tel.: +91 9919317940; Fax: +91-522-2628493.
Email: sashidhar123@gmail.com, kv_sashidhara@cdri.res.in.
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1. Introduction
Metabolic syndrome (MS) remains one of the leading causes of mortality and has become a
major global health challenge of the 21st century. It is associated with many pathological
conditions related to insulin resistance, obesity, impaired glucose tolerance, type 2 diabetes, a
high level of triglycerides, a low level of HDL cholesterol, and elevated blood pressure.
Hence, such metabolic modifications lead to a marked increase in cardiovascular risk [1].
Gemfibrozil and fenofibrate are the couple of prominent FDA-approved agents that
reduce the level of triglycerides in the blood plasma thereby decreasing the risk of
hyperlipidemia. Peroxisome proliferator-activated receptor α (PPARα) is the molecular target
of fibrates, including clofibrate (1 figure 1) and bezafibrate (2 figure 1) [2-5]. Statins (HMG-
CoA reductase inhibitors) can hardly normalize the high density lipoprotein abnormality and
significant residual cardiovascular risk remains in most patients despite statin therapy. In
addition, reports of undesirable side effects (myopathy) of some ‘super statins’ and in light of
the recent warning by the US FDA, that statins may increase the risk of diabetes mellitus [6-
7], calls for an urgent need for novel therapeutic strategies that could halt or reverse the
underlying disease process in lipid disorders, which could have a profound impact on their
long-term clinical management.
In the search for novel drug leads, the hybrid approach is a promising one since it can
effectively target multi factorial diseases like metabolic syndrome. “Due to the high potential
of natural products to exhibit pronounced biological activities, natural products have been
one of the major sources of components in hybrid molecules” [8]. Coumarins and chalcones
are a family of natural and synthetic compounds that have recently drawn much attention due
to its broad pharmacological activities [9-13]. Esculetin (3 figure 1) a coumarin derivative has
been shown to inhibit oxidative LDL [14]. Chalcones like licochalcone A (4 figure 1) and
xanthohumol [15a] (4A figure 1) exhibit interesting biological activities including anti-
inflammatory, [15b], antioxidant, [16] and antihyperglycemic [17]. In addition coumarin and
chalcone derivatives have the unique ability to scavenge reactive oxygen species (ROS) and
to protect the influence of free radical damage [13, 18]. Hybrid molecules that contain
multiple structural units of different nature generally possess improved biological activities
[19-24]. Inspired by the hybridization concept and our previous results [25-30] we have
designed and synthesized a novel series of compounds that have coumarin, chalcone and
fibrate entities in one frame (5 figure 1) and have evaluated them for their lipid profile
activity. Also, we recently discovered that coumarin-fibrate hybrids were found to ameliorate
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the symptoms associated with hyperlipidemia and that they have potential benefits in the
prevention of obesity and liver dysfunction (unpublished results).
Figure 1 shows chemical structure of important fibrates, coumarin, chalcones and our
synthesized fused prototype containing fragments endowed with interesting and
complimentary activities.
2. Chemistry
The detailed synthetic route for the preparation of the target compounds (27-38 & 39-42) is
summarized in scheme 1. Following reported procedures, treating 4-hydroxy acetophenone
with substituted bromoesters in the presence of K2CO3 afforded the corresponding ester
derivatives (6-10). Commercially available 2-alkyl phenols underwent the Duff formylation
in the presence of hexamethylenetetramine (HMTA) and TFA at 120 ºC to furnish the
compounds (11-14). These aromatic dialdehydes on subsequent reaction with para-
substituted acetophenones (6-10) in the presence of a catalytic amount of conc.HCl in
dioxane gave the regioselective chalcones (15-26) [23]. These were then engaged in a
Knoevenagel condensation with diethylmalonate in the presence of a catalytic amount of
piperidine resulted in the formation of coumarin-chalcone fibrates (27–38). Some of these
diester derivatives on subsequent alkaline hydrolysis furnished their corresponding acid
derivatives (39–42) (Scheme 1) in good yields. The structures of all the compounds were
established by using 1H-NMR, 13C-NMR, IR spectroscopy and mass spectrometry.
3. Pharmacology
3.1. Animals used.
Rats (Charles Foster strain, male, adult, body weight 200–225g) were kept in a room with
controlled temperature (25–260C), humidity (60–80%) and12/12 h light/dark cycle (light on
from 8.00 A.M. to 8.00 P.M.) under hygienic conditions. Animals, which were acclimatized
for one week before starting the experiment, had free access to the normal diet and water.
3.2. Lipid lowering and Post heparin lipolytic activity
Rats were divided into twelve groups control, triton induced, triton plus 19, 20, 27-42 and
gemfibrozil (100 mg/kg) treated groups, containing six rats in each group. In this experiment
of 18h, hyperlipidemia was developed by administration of triton WR-1339 (Sigma chemical
company, St. Louis, MO, USA) at a dose of 400 mg/kg body weight intraperitoneally to
animals of all the groups except the control. These derivatives were macerated with gum
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acacia (0.2% w/v), suspended in water and fed simultaneously with triton with a dose of 100
mg/kg p.o. to the animals of treated group and the diet being withdrawn. Animals of control
and triton group without treatment with hybrid compounds were given same amount of gum
acacia suspension (vehicle). After 18h of treatment the animals were anaesthetized with
thiopentone solution (50 mg/kg b.w.) prepared in normal saline and then 1.0 ml blood was
withdrawn from retro-orbital sinus using glass capillary in EDTA coated eppendorf tube (3.0
mg/ml blood). The blood was centrifuged (at 2500g) at 40C for 10 min and plasma was
separated. Plasma was diluted with normal saline (ratio of 1:3) and used for analysis of total
cholesterol (TC), triglycerides (TG) and phospholipids (PL) by standard enzymatic methods
[31] and post heparin lipolytic activity (PHLA) were assayed [32] using spectrophotometer,
Beckmann auto-analyzer and standard kits purchased from Beckmann Coulter International,
USA.
3.3. Lipoprotein measurement in blood plasma of triton induced hyperlipidemic rats.
Plasma was fractionated into very low density lipoprotein (VLDL), low density lipoprotein
(LDL) and high density lipoprotein (HDL) by poly anionic precipitation methods. Plasma and
lipoproteins were analyzed for their total cholesterol (TC), phospholipid (PL), and
triglyceride (TG) by standard procedures reported earlier [33].
3.4. Lipoprotein lipase activity in liver of triton induced hyperlipidemic rats.
Liver was homogenized (10%, w/v) in cold 100 mM phosphate buffer pH 7.2 and used for
the assay of total lipolytic activity of lipoprotein lipase (LPL) [34].
3.5. Statistical evaluation
Data were analyzed using student’s t-test. The hyperlipidemic groups were compared with
control drug treated groups. P<0.05 was considered to be significant.
4. Results and Discussion
4.1. The lipid lowering activity of coumarin-chalcone fibrates
The present study has been undertaken to evaluate the lipid lowering activity of coumarin-
chalcone fibrates (19, 20 & 27-42) in an acute triton induced hyperlipidemic model. Triton
administration caused significant increase in plasma TC, PL and TG levels by (2.12, 2.28 and
2.49 folds respectively) followed by decrease in PHLA (post heparin lipolytic activity) by (-
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33%). Treatment of hyperlipidemic rats with coumarin-chalcone fibrates (19, 20 & 27-42) at
the dose of 100 mg/kg p.o. reversed the plasma levels of lipid with varying extents. The
synthesized hybrids inhibited the biosynthesis of cholesterol and enhanced the activity of
lipolytic enzymes to facilitate early clearance of lipids from circulation in triton induced
hyperlipidemia. Compound 41 was found to be the most persuasive in the series as it showed
26%, 24% and 25%, lowering in TC, PL and TG respectively, while compounds 35 and 28
showed good activity and compound 36 showed moderate activity. These data were
comparable with standard hypolipidemic drug gemfibrozil which at the dose of 100 mg/kg
decreased levels of TC, PL and TG in plasma by 31%, 33% and 33%, respectively.
Compounds 41, 28 and 35 showed significant reversal of PHLA in plasma of hyperlipidemic
rats by 17%, 15% and 15% respectively, comparable to gemfibrozil, which caused 19%
reversal of activity of this enzyme as compared to control group and the results are
summarised in Table 1. In terms of structure activity relationship of the hybrids synthesized
following observations can be made, first the coumarin moiety is essential as compounds
without coumarin nucleus were inactive (19 and 20). Secondly, though in general most
hybrids exhibited activity (28, 35 & 36) the acid derivative (41) was found to be more active
than ester analogues, and seems to confer potency and solubility.
4.2. Effect of compounds 41 and 35 on lipid lowering at different doses in triton induced
hyperlipidemic rat.
After the confirmation of most active compounds in primary screening the efficacy of active
compounds 41 and 35 at different doses in 50 to 200 mg/kg body weight was studied.
Compound 41 lowered the TC by 16% to 27%, PL by 15% to 24% and TG by 14% to 26%
followed by increase in PHLA level by 12% to 17%. Similarly compound 35 lowered the TC
by 18% to 23%, PL by 16% to 24% and TG by 16% to 25% followed by increase in PHLA
level by 13% to 15% respectively. The results are summarised in table 2.
4.3. Effect of compound 41 on plasma lipoprotein lipids in triton induced hyperlipidemic rats.
Next, the most active compound 41 was tested for its effect on plasma lipoprotein lipids. As
shown in figure 2, the analysis of hyperlipidemic plasma of triton administered rats showed
marked increase in the level of lipoprotein lipids and these effects were pronounced for
VLDL(figure 2A) and LDL (figure 2B) followed by a decrease in HDL (figure 2C) as
compared to control rats. Interestingly, the treatment with compound 41, significantly
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reversed the levels of VLDL, LDL and HDL, the complete lipid profile results are shown in
figure 2.
4.4. Effect of compound 41 on liver-LPL activity in triton induced hyperlipidemic rats.
Furthermore the compound 41 was studied for liver-LPL activity in triton induced
hyperlipidemic rats. Administration of triton in rats markedly decreases the LPL activity in
liver as shown in figure 3. However, the treatment with compound 41 significantly increased
the LPL activity, which underscores the utility of the hybrid (figure 3). The results were
comparable with the standard drug gemfibrozil, which at the same dose (100mg/kg) caused
marked increase in the LPL activity.
5. Conclusion
In conclusion, the designed hybrid 41 decreased the total cholesterol (TC), phospholipids
(PL) and triglycerides (TG), of hyperlipidemic rats by 26, 24, and 25%, respectively, at 100
mg/kg body weight in triton WR-1339 induced model. In addition, treatment with compound
41 significantly reversed the levels of VLDL, LDL and HDL also increased the LPL activity.
These results suggest that these coumarin-chalcone fibrates constitute a new prototype of
lipid lowering agents that act by modulating lipoprotein lipase, further experimental and
mechanistic studies are however required to advance this series in preclinical development.
6. Experimental
6.1. General Information:
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 TLC;
1
silica gel plates with fluorescence F254 were used. Melting points were uncorrected. The H
NMR, 2D-NMR (COSY, HMBC, HSQC) and 13C NMR spectra were determined on 200,
300, MHz and 50, 75, MHz, respectively, using CDCl3 and DMSO- d6 as solvents and TMS
as internal standard. All chemical shifts were given in ppm. IR spectra were recorded on in
the range of 500~4000 cm-1, and multiplicity (s = singlet, brs = broad singlet, d = doublet, brd
= broad doublet, dd = double doublet, t = triplet, q = quartet, m = multiplet).
6.2. General synthetic procedure for preparation of ester compounds (6-10):
An oven dried 50-mL round bottom flask was charged with 4-hydroxy acetophenone (1.0
equiv.), substituted bromoester compound (1.1 equiv.) and acetonitrile (25 mL). K2CO3 (10.0
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equiv.) was then added, and the reaction mixture was heated to 90 0C for 4 h. The acetonitrile
was evaporated in vacuum, and the residue was purified by silica gel (100-200) column
chromatography (20% EtoAc/Hexane) to afford ester compounds 6-10 in good yields.
6.2.1. Ethyl 2-(4-acetylphenoxy) propionate (7):
From 4-hydroxy acetophenone and ethyl 2-bromopropanoate; white crystalline solid; yield:
0
1
92%; mp: 147 – 149 C; IR(KBr,cm–1): 2925, 2352, 1677, 1566, 1207, 1073, 760; H NMR
(CDCl3, 300 MHz) δ :7.93 (d, J = 8.7Hz, 2H ) 6.91(d, J = 8.8, 2H) 4.83 (q , J = 6.6Hz, 1H)
4.23 (q , J = 7.1 Hz, 2H) 2.55 (s, 3H) 1.66 (d, J = 6.7, 3H) 1.26 (t , J = 7.1Hz, 3H); ESI – MS:
(m/z): 237 (M+H)+.
6.2.2. Ethyl 2-(4-acetylphenoxy) butanoate (10):
From 4-hydroxy acetophenone and ethyl 4-bromobutanoate; white crystalline solid; yield:
0
1
90%; mp: 157 – 159 C; IR(KBr,cm–1): 2935, 2352, 1675, 1566, 1207, 1073, 770; H NMR
(CDCl3, 300 MHz) δ :7.93 (d, J = 8.7Hz, 2H ) 6.92(d, J = 8.7, 2H) 4.16 (q , J = 7.1Hz, 2H)
4.09 (t , J = 6.1 Hz, 2H) 2.56 – 2.50 (m, 5H) 2.19 – 2.10 (m, 2H) 1.27 (t, J = 7.1, 3H); ESI –
MS: (m/z): 251 (M+H)+.
6.3. General synthetic procedure for preparation of 4-hydroxy-5-alkyl isophthalaldehydes
(11-14):
2-Alkyl phenol (1.0 equiv.) and hexamethylenetetramine (1.2 equiv.) were dissolved in TFA
(25 mL) and the solution was heated at 120 ºC for 3 h. After cooling to room temperature 10
% aq.H2SO4 (25 mL) was added and again the temperature maintained (at 90-100 ºC) for two
more hours. After completion the solution was basified with Na2CO3 to pH 8 and extracted 3-
fold with 25 mL of CHCl3. The combined organic layers were dried on Na2SO4, filtered, and
concentrated to dryness under reduced pressure. The crude product was purified on silica gel
column (100-200 mesh) using hexane-ethyl acetate (12:88, v/v) as eluent to afford
compounds 11-14 in good yields.
6.3.1. 4-hydroxy-5-methylisophthalaldehyde (11):
From 2-methyl phenol and HMT; white solid; Yield: 60%; mp: 125 – 127 0C; IR (neat):
3262, 2865, 1703, 1626, 1013 cm-1; 1H NMR (CDCl3, 300 MHz) δ: 11.82 (s, 1H), 9.97 (s,
1H), 9.90 (s, 1H), 7.97 (d, J = 1.8, 1H), 7.93 (brs, 1H), 2.33 (s, 3H); ESI-MS: m/z: 164
(M+H)+.
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6.3.2. 5-tert-butyl-4-hydroxyisophthalaldehyde (13):
From 2-tertbutyl phenol and HMT; Oily; Yield: 65%; IR (neat): 3252, 2865, 1703, 1626,
1013 cm-1; 1H NMR (CDCl3, 300 MHz) δ: 12.39 (s, 1H), 9.99 (s, 1H), 9.93 (s, 1H), 8.07 (brs,
1H), 7.99 (brs, 1H), 1.46 (s, 9H); ESI-MS: m/z: 207 (M+H)+.
6.4. General synthetic procedure for preparation of para substituted chalcones (15-26):
To a solution of 4-hydroxy-5-alkyl isophthalaldehyde 11-14 (1.0 equiv.) and 4-substutited
acetophenone 6-10 (1.0 equiv.) in dioxane (15 mL) was treated with conc.HCl (2 mL). The
solution was refluxed for 8-10 h. after completion of required time the reaction mixture was
cooled and most of the excess dioxane was evaporated under reduced pressure, and the
residue was suspended in water (50 mL) and extracted 3-fold with CHCl3 (50 mL). The
combined organic layers were dried on Na2SO4, filtered, and concentrated to dryness under
reduced pressure. The residue was purified on a silica gel (230-400 mesh) column
chromatography using hexane-ethyl acetate (90:10, v/v) as eluent to afford regioselective
para chalcones 15-26 in moderate yields.
6.4.1. (E)-ethyl2-(4-(3-(3-formyl-4-hydroxy-5-methylphenyl) acryloyl) phenoxy) propanoate
(19):
From 7 and 11; white solid; yield: 72%; mp: 145 – 147 0C; IR(KBr,cm–1): 3445, 2925, 2352,
1718, 1677, 1566, 1504, 1207, 1073, 760; 1H NMR (CDCl3, 300 MHz) δ :11.50 (s,OH) 9.93
(s, CHO) 8.02 (d, J = 8.8Hz, 2H ) 7.77 - 7.72 (m, 2H) 7.64 (d, J = 1.9, 1H) 7.44 (d, J =
15.5, 1H) 6.75 (d, J = 8.8, 2H) 4.48 (q , J = 6.7Hz, 1H) 4.23 (q , J = 7.1 Hz, 2H) 2.32 (s, 3H)
1.66 (d, J = 6.7, 3H) 1.25 (t , J = 7.1Hz, 3H); ESI – MS: (m/z): 383 (M+H)+.
6.4.2. (E)-ethyl2-(4-(3-(3-sec-butyl-5-formyl-4-hydroxyphenyl)acryloyl)phenoxy)propanoate
(20):
From 7 and 14; white solid; yield: 74%; mp: 142 – 144 0C; IR(KBr,cm–1): 3419, 2965, 2358,
1
1744, 1654, 1600, 1505, 1340, 1218, 1027, 769; H NMR (CDCl3, 300 MHz) δ :11.59
(s,OH) 9.94 (s, CHO) 8.02 (d, J = 8.8Hz, 2H ) 7.76 (d, J = 15.6Hz, 1H)7.69 (s, 1H) 7.67 (s,
1H) 7.43 (d, J = 15.6, 1H) 6.96 (d, J = 8.8, 2H) 4.48 (q , J = 6.7Hz, 1H) 4.22 (q , J = 7.1 Hz,
2H) 3.20 – 3.13 (m, 1H) 1.75 -1.63 (m, 6H) 1.28 -1.23 (m, 6H) .88 (t , J = 7.3Hz, 3H); ESI –
MS: (m/z): 425 (M+H)+.
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6.5. General synthetic procedure for preparation coumarin-chalcone fibrates (27-38):
An oven dried 50-mL round bottom flask was charged with chalcone 15-26 (1.0 mmol),
diethylmalonate (1.2 mmol) and absolute ethanol (15 mL) .This was treated with piperidine
(0.30 mL) and refluxed for 30 min. after completion of reaction (monitor by TLC) the
reaction mixture was cooled and evaporated under reduced pressure, the residue was acidified
with acetic acid and extracted 3-fold with CHCl3 (20 mL). The combined organic layers were
dried on Na2SO4, filtered, and concentrated to dryness under reduced pressure. The crude
product was washed with ethanol gave pure coumarin chalcone fibrates (27-38) in good
yields.
6.5.1. (E)-Ethyl 6-(3-(4-(2-ethoxy-2-oxoethoxy) phenyl)-3-oxoprop-1-enyl)-8-methyl-2-oxo-
2H-chromene-3-carboxylate (27):
0
From 15 and diethylmalonate; white solid; yield: 84%; mp: 168 – 170 C; IR(KBr,cm–1):
2965, 2358, 1744, 1654, 1600, 1505, 1340, 1216, 1027, 767; 1H NMR (CDCl3, 300 MHz) δ :
8.51 (s, 1H) 8.04 (d, J = 8.7Hz, 2H )7.79 – 7.74 (m, 2H) 7.65 (s, 1H) 7.52 (d, J = 15.6Hz,
1H) 7.00 (d, J = 8.7, 2H) 4.71 (s, 2H) 4.42 (q , J = 7.0Hz, 2H) 4.29 (q , J = 7.1 Hz, 2H) 2.50
(s, 3H) 1.41 (t , J = 7.0Hz, 3H) 1.30 (t , J = 7.1Hz, 3H); 13C NMR (CDCl3 75 MHz) δ:188.0,
168.2, 162.8, 161.7, 156.3, 154.6, 148.4, 141.7, 134.3, 131.7, 131.5, 130.9,127.5, 127.3,
122.5, 118.7, 118.0, 114.6, 65.3, 62.1, 61.7, 15.6, 14.3, 14.2 ; ESI – MS: (m/z): 465 (M+H)+.
6.5.2. (E)-Ethyl6-(3-(4-(2-ethoxy-2-oxoethoxy)phenyl)-3-oxoprop-1-enyl)-8-isopropyl-2-oxo-
2H-chromene-3-carboxylate (28):
From 16 and diethylmalonate; white solid; yield: 79%; mp: 163 – 1650C; IR(KBr,cm–1):
2972, 2368, 1757, 1659, 1611, 1515, 1376, 1288, 1079, 775; 1H NMR (CDCl3, 300 MHz) δ :
8.52 (s, 1H) 8.04 (d, J = 8.8Hz, 2H )7.81 – 7.76 (m, 2H) 7.67 (d, J = 1.6Hz 1H) 7.51 (d, J =
15.6Hz, 1H) 7.00 (d, J = 8.8, 2H) 4.71(s, 2H) 4.42 (q , J = 7.1Hz, 2H) 4.29 (q , J = 7.1 Hz,
2H) 3.67 – 3.58 (m, 1H) 1.41 (t , J = 7.1Hz, 3H) 1.36 (m , 9H); 13C NMR (CDCl3 75 MHz)
δ:188.1, 168.2, 162.8, 161.6, 156.2, 153.5, 148.6, 142.0, 137.5, 131.6, 130.8, 130.5,127.1,
122.5, 118.6, 118.0, 114.5, 65.2, 62.0, 61.5, 26.7, 22.4, 14.2, 14.1 ; ESI – MS: (m/z): 493
(M+H)+.
6.5.3. (E)-Ethyl8-tert-butyl-6-(3-(4-(2-ethoxy-2-oxoethoxy)phenyl)-3-oxoprop-1-enyl)-2-oxo-
2H-chromene-3-carboxylate (29):
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From 17 and diethylmalonate; light brown solid; yield: 85%; mp: 165 – 1670C; IR(KBr,cm–
1): 2985, 2359, 1767, 1665, 1621, 1518, 1366, 1298, 1079, 760; 1H NMR (CDCl3, 300 MHz)
δ : 8.52 (s, 1H) 8.28 (d, J = 8.8Hz, 2H ) 7.85 (d, J = 1.8, 1H) 7.78 (d, J = 15.6Hz, 1H) 7.70
(d, J = 1.8Hz, 1H) 7.50 (d, J = 15.6Hz, 1H)7.00 (d, J = 8.8, 2H) 4.71 (s, 2H) 4.42 (q , J =
7.1Hz, 2H) 4.28 (q , J = 7.1 Hz, 2H) 1.53 (s, 9H) 1.41 (t , J = 7.1Hz, 3H) 1.30 (t , J = 7.1Hz,
3H); 13C NMR (CDCl3 75 MHz) δ:188.1, 168.2, 162.9, 161.6, 155.7, 154.9, 148.9, 142.2,
138.9, 131.6, 131.3, 131.2, 130.8, 127.5, 122.4, 118.7, 118.2, 114.5, 65.2, 62.0, 61.6, 35.1,
29.6, 14.2, 14.1 ; ESI – MS: (m/z): 507 (M+H)+.
6.5.4. (E)-Ethyl8-sec-butyl-6-(3-(4-(2-ethoxy-2-oxoethoxy)phenyl)-3-oxoprop-1-enyl)-2-oxo-
2H-chromene-3-carboxylate (30):
From 18 and diethylmalonate; white solid; yield: 90%; mp: 164 – 1660C; IR(KBr,cm–1):
1
2970, 2368, 1757, 1659, 1601, 1459, 1376, 1288, 1213, 1027, 767; H NMR (CDCl3, 300
MHz) δ : 8.52 (s, 1H) 8.04 (d, J = 8.8Hz, 2H ) 7.81 – 7.75 (m, 2H) 7.68 (s, 1H) 7.51 (d, J =
15.6Hz 1H) 7.00 (d, J = 8.8Hz, 2H) 4.71 (s, 2H) 4.42 (q , J = 7.0Hz, 2H) 4.29 (q , J = 7.1Hz,
2H) 3.45 – 3.38 (m, 1H) 1.77 – 1.68 (m, 2H) 1.41 (t , J = 7.1 Hz, 3H) 1.33 – 1.28 (m, 6H)
.88 (t , J = 7.3Hz, 3H); 13C NMR (CDCl3 75 MHz) δ:188.1, 168.1, 162.8, 161.6, 156.2, 153.8,
148.6, 142.0, 136.6, 131.6, 131.3, 130.8,127.0, 122.5, 118.6, 118.1, 114.5, 65.2, 62.0, 61.6,
33.4, 29.6, 20.4, 14.2, 14.1, 11.9; ESI – MS: (m/z): 507 (M+H)+.
6.5.5. (E)-Ethyl 6-(3-(4-(1-ethoxy-1-oxopropan-2-yloxy) phenyl)-3-oxoprop-1-enyl)-8-methyl-
2-oxo-2H-chromene-3-carboxylate (31):
0
From 19 and diethylmalonate; white solid; yield: 80%; mp: 160 – 162 C; IR(KBr,cm–1):
2970, 2368, 1757, 1659, 1601, 1510, 1376, 1288, 1079, 762; 1H NMR (CDCl3, 300 MHz) δ :
8.51 (s, 1H) 8.02 (d, J = 8.8Hz, 2H )7.78 – 7.73 (m, 2H) 7.65 (s, 1H) 7.52 (d, J = 15.6Hz,
1H) 6.95 (d, J = 8.8, 2H) 4.84 (q , J = 6.7Hz, 1H) 4.41 (q , J = 7.1 Hz, 2H) 4.29 (q , J = 7.0
Hz, 2H) 2.49 (s, 3H) 1.66 (d, J = 6.7, 3H) 1.40 (t , J = 7.1Hz, 3H) 1.25 (t , J = 7.1Hz, 3H); 13C
NMR (CDCl3 75 MHz) δ:187.9, 171.4, 162.8, 161.5, 156.2, 154.5, 148.3, 141.6, 134.2, 131.4,
131.3, 130.8,127.4, 127.2, 122.5, 118.6, 117.9, 114.8, 72.5, 62.0, 61.5, 18.4, 15.5, 14.2, 14.1
; ESI – MS: (m/z): 479 (M+H)+.
6.5.6.
(E)-Ethyl8-sec-butyl-6-(3-(4-(1-ethoxy-1-oxopropan-2-yloxy)phenyl)-3-oxoprop-1-
enyl)-2-oxo-2H-chromene-3-carboxylate (32):
10

ACCEPTED MANUSCRIPT
From 20 and diethylmalonate; white solid; yield: 85%; mp: 153 – 1550C; IR(KBr,cm–1):
1
2973, 2358, 1757, 1659, 1611, 1459, 1366, 1288, 1223, 1027, 757; H NMR (CDCl3, 300
MHz) δ : 8.52 (s, 1H) 8.03 (d, J = 8.8Hz, 2H ) 7.81 – 7.74 (m, 2H) 7.68 (d, J = 1.7Hz,
1H)7.51 (d, J = 15.6Hz 1H) 6.97 (d, J = 8.8Hz, 2H) 4.85 (q , J = 6.7Hz, 1H) 4.42 (q , J =
7.0Hz, 2H) 4.23 (q , J = 7.1Hz, 2H) 3.45 – 3.38 (m, 1H) 1.75 – 1.65 (m, 5H) 1.41 (t , J = 7.1
Hz, 3H) 1.33 – 1.24 (m, 6H) 0.88 (t , J = 7.2Hz, 3H); 13C NMR (CDCl3 75 MHz) δ:188.1,
171.4, 162.8, 161.5, 156.2, 153.8, 148.5, 141.9, 136.6, 131.6, 131.4, 131.3, 130.8,126.9,
122.6, 118.7, 118.1, 114.8, 72.5, 62.0, 61.5, 33.4, 29.6, 20.4, 18.4, 14.2, 14.1, 11.9; ESI –
MS: (m/z): 521 (M+H)+.
6.5.7. (E)-Ethyl 6-(3-(4-(1-ethoxy-1-oxobutan-2-yloxy)phenyl)-3-oxoprop-1-enyl)-8-methyl-2-
oxo-2H-chromene-3-carboxylate (33):
From 21 and diethylmalonate; pale yellow solid; yield: 70%; mp: 154 – 1560C; IR(KBr,cm–
1): 2963, 2356, 1754, 1654, 1640, 1505, 1347, 1216, 1027, 767; 1H NMR (CDCl3, 300 MHz)
δ : 8.54 (s, 1H) 8.02 (d, J = 8.7Hz, 2H )7.81 – 7.76 (m, 2H) 7.68 (s, 1H) 7.55 (d, J = 15.5Hz,
1H) 6.99 (d, J = 8.8, 2H) 4.68 (t , J = 6.1Hz, 1H) 4.44 (q , J = 7.1 Hz, 2H) 4.25 (q , J = 7.1
Hz, 2H) 2.53 (s, 3H) 2.10 – 2.01 (m, 2H) 1.43 (t, J = 7.0, 3H) 1.27 (t , J = 7.0Hz, 3H) 1.12 (t ,
J = 7.3Hz, 3H); ESI – MS: (m/z): 493 (M+H)+.
6.5.8. (E)-Ethyl 6-(3-(4-(1-ethoxy-2-methyl-1-oxopropan-2-yloxy)phenyl)-3-oxoprop-1-enyl)-
8-methyl-2-oxo-2H-chromene-3-carboxylate (34):
From 22 and diethylmalonate; white solid; yield: 75%; mp: 171 – 1730C; IR(KBr,cm–1):
2971, 2368, 1757, 1659, 1611, 1510, 1376, 1288, 1079, 765; 1H NMR (CDCl3, 300 MHz) δ :
8.51 (s, 1H) 7.98 (d, J = 8.8Hz, 2H )7.79 – 7.74 (m, 2H) 7.65 (s, 1H) 7.52 (d, J = 15.6Hz,
1H) 6.89 (d, J = 8.8, 2H) 4.42 (q , J = 7.1Hz, 2H) 4.23 (q , J = 7.1 Hz, 2H) 2.51 (s, 3H) 1.67
(s, 6H) 1.41 (t , J = 7.1Hz, 3H) 1.23 (t , J = 7.1Hz, 3H); 13C NMR (CDCl3 75 MHz) δ:188.1,
173.7, 162.8, 160.0, 156.3, 154.6, 148.4, 141.6, 134.3, 131.5, 131.3, 130.5,127.5, 127.3,
122.6, 118.7, 118.0, 117.6, 79.5, 62.1, 61.7, 25.4, 15.6, 14.3, 14.1 ; ESI – MS: (m/z): 479
(M+H)+.
6.5.9.
(E)-Ethyl8-sec-butyl-6-(3-(4-(1-ethoxy-2-methyl-1-oxopropan-2-yloxy)phenyl)-3-
oxoprop-1-enyl)-2-oxo-2H-chromene-3-carboxylate (35):
From 23 and diethylmalonate; white solid; yield: 90%; mp: 154 – 1560C; IR(KBr,cm–1):
2985, 2359, 1767, 1665, 1621, 1518, 1366, 1298, 1079, 760; 1H NMR (CDCl3, 300 MHz) δ :
11

ACCEPTED MANUSCRIPT
8.53 (s, 1H) 7.99 (d, J = 8.7Hz, 2H ) 7.81 – 7.75 (m, 2H) 7.68 (d, J = 1.5Hz, 1H) 7.51 (d, J =
15.6Hz, 1H) 6.89 (d, J = 8.7Hz, 2H) 4.42 (q , J = 7.1Hz, 2H) 4.23 (q , J = 7.1Hz, 2H) 3.48 –
3.38 (m, 1H) 1.77 – 1.67 (m, 8H) 1.41(t , J = 7.1 Hz, 3H) 1.31(d , J = 6.9 Hz, 3H) 1.22(t , J =
7.1 Hz, 3H) 1.41(t , J = 7.1 Hz, 3H) .88 (t , J = 7.2Hz, 3H); 13C NMR (CDCl3 75 MHz)
δ:188.2, 173.7, 162.9, 160.0, 156.3, 153.9, 148.6, 142.0, 136.7, 131.8, 131.4, 130.5,
127.0,122.7, 118.8, 118.2, 117.7, 79.5, 62.1, 61.7, 33.5, 29.7, 25.5, 20.5, 14.3, 14.1, 12.0; ESI
– MS: (m/z): 535 (M+H)+.
6.5.10. (E)-Ethyl 6-(3-(4-(4-ethoxy-4-oxobutoxy) phenyl)-3-oxoprop-1-enyl)-8-methyl-2-oxo-
2H-chromene-3-carboxylate (36):
0
From 24 and diethylmalonate; white solid; yield: 74%; mp: 178 – 180 C; IR(KBr,cm–1):
2965, 2358, 1744, 1654, 1600, 1505, 1340, 1216, 1027, 767; 1H NMR (CDCl3, 300 MHz) δ :
8.51 (s, 1H) 8.03 (d, J = 8.6Hz, 2H )7.78 – 7.73 (m, 2H) 7.65 (s, 1H) 7.53 (d, J = 15.5Hz,
1H) 6.97 (d, J = 8.5, 2H) 4.41 (q , J = 7.1Hz, 2H) 4.18 – 4.08(m, 4H) 2.55 – 2.50 (m, 5H)
2.16 (q , J = 6.3 Hz, 2H) 1.41 (t , J = 7.1Hz, 3H) 1.26 (t , J = 7.0Hz, 3H); 13C NMR (CDCl3
75 MHz) δ:187.8, 173.0, 162.9, 162.7, 156.2, 154.4, 148.3, 141.4, 134.2, 131.5, 130.8,
130.7,127.4, 127.2, 122.5, 118.5, 117.9, 114.3, 67.0, 62.0, 60.5, 30.6, 24.4, 15.5, 14.2 ; ESI –
MS: (m/z): 493 (M+H)+.
6.5.11.
(E)-Ethyl8-tert-butyl-6-(3-(4-(4-ethoxy-4-oxobutoxy)phenyl)-3-oxoprop-1-enyl)-2-
oxo-2H-chromene-3-carboxylate (37):
From 25 and diethylmalonate; white solid; yield: 69%; mp: 172 – 1740C; IR(KBr,cm–1):
2972, 2368, 1757, 1659, 1611, 1515, 1376, 1288, 1079, 775; 1H NMR (CDCl3, 300 MHz) δ :
8.52 (s, 1H) 8.03 (d, J = 8.6Hz, 2H ) 7.85 (s, 1H) 7.78 (d, J = 15.6Hz 1H)7.71 (s, 1H) 7.52
(d, J = 15.5Hz, 1H) 6.98 (d, J = 8.6, 2H) 4.42 (q , J = 7.1Hz, 2H)4.19 -4.09 (m, 4H) 2.53 (t , J
= 7.2 Hz, 2H) 2.19 – 2.11 (m, 2H) 1.54 (s, 9H) 1.41 (t , J = 7.0Hz, 3H) 1.26 (t , J = 7.1Hz,
3H); 13C NMR (CDCl3 75 MHz) δ:188.1, 173.0, 162.9, 155.7, 154.8, 148.9, 141.9, 138.9,
131.3, 131.2, 130.8, 130.7,127.4, 122.6, 118.7, 118.2, 114.4, 67.0, 61.9, 60.5, 35.1, 30.6,
29.6, 24.4 14.2; ESI – MS: (m/z): 535 (M+H)+.
6.5.12.
(E)-Ethyl8-sec-butyl-6-(3-(4-(4-ethoxy-4-oxobutoxy)phenyl)-3-oxoprop-1-enyl)-2-
oxo-2H-chromene-3-carboxylate (38):
From 26 and diethylmalonate; pale brown solid; yield: 80%; mp: 168 – 1700C; IR(KBr,cm–1):
2995, 2369, 1767, 1655, 1621, 1508, 1356, 1298, 1074, 766; 1H NMR (CDCl3, 300 MHz) δ :
12

ACCEPTED MANUSCRIPT
8.52 (s, 1H) 8.03 (d, J = 8.7Hz, 2H)7.81 – 7.75 (m, 2H) 7.68 (d, J = 1.9Hz, 1H) 7.53 (d, J =
15.6Hz, 1H) 6.98 (d, J = 8.8Hz, 2H) 4.42 (q , J = 7.2Hz, 2H) 4.19 – 4.09 (m, 4H) 3.46 – 3.39
(m, 1H) 2.53 (t , J = 7.2Hz, 2H) 2.17 – 2.13 (m, 2H) 1.73 (t , J = 7.4 Hz, 2H) 1.41(t , J = 7.1
Hz, 3H) 1.33 – 1.24 (m, 6H) .89 (t , J = 7.3Hz, 3H); 13C NMR (CDCl3 75 MHz) δ:188.1,
173.0, 162.9, 162.8, 156.2, 153.7, 148.6, 141.8, 136.5, 131.7, 131.3, 130.8,
130.7,126.9,122.6, 118.6, 118.1, 114.4, 67.0, 62.0, 60.5, 33.4, 30.6, 29.6, 24.4, 20.4, 14.2,
12.0; ESI – MS: (m/z): 535 (M+H)+.
6.6. General synthetic procedure for preparation of acids (39-42):
A 50 ml round bottom flask was charged with coumarin- chalcones diester (28, 30, 31 and
35) and methanol (15 ml). To this 10% aq KOH was added and stirred at room temperature
for 5-6 h. After completion of reaction (conformed by TLC), excess methanol was removed
under reduce pressure and acidified with dil. HCl the resulted solid was filtered and dried
under vacuum to get compounds 39-42 in good yields.
6.6.1.
(E)-6-(3-(4-(carboxymethoxy)phenyl)-3-oxoprop-1-enyl)-8-isopropyl-2-oxo-2H-
chromene-3-carboxylic acid (39):
From 28; white solid; yield: 82%; mp: 265 – 2670C; IR(KBr,cm–1):3749, 3661, 3038, 2365,
1
1742, 1664, 1584, 1275, 1225, 1019, 839; H NMR (DMSO-d6, 300 MHz) δ : 8.67 (s, 1H)
8.25 - 8.15(m, 4H) 7.99 (d, J = 15.6Hz, 1H ) 7.73 (d, J = 15.5Hz, 1H) 7.09 (d, J = 8.7Hz, 2H)
4.84 (s , 2H) 4.34 – 4.27(m, 1H)1.33(d, J = 6.7 Hz, 6H); ESI – MS: (m/z): 437 (M+H)+.
6.6.2.
(E)-8-sec-butyl-6-(3-(4-(carboxymethoxy)phenyl)-3-oxoprop-1-enyl)-2-oxo-2H-
chromene-3-carboxylic acid (40):
From 30; pale yellow solid; yield: 85%; mp: 270 – 2720C; IR(KBr,cm–1):3749, 3651, 3028,
1
2365, 1752, 1664, 1584, 1375, 1222, 1029, 829; H NMR (DMSO-d6, 300 MHz) δ : 13.17
(bs, 1H) 8.69 (d, J = 7.9Hz, 1H) 8.24 - 8.15(m, 4H) 7.98 (d, J = 15.0Hz, 1H) 7.73 (d, J =
15.6Hz, 1H) 7.39 (d, J = 8.6Hz, 2H) 4.83 (s, 2H) 4.34 – 4.27 (m, 1H) 1.73 -1.72 (m, 2H)
1.31(d, J = 6.9 Hz, 3H) .83(t, J = 6.9 Hz, 3H); ESI – MS: (m/z): 423 (M+H)+.
6.6.3.
(E)-6-(3-(4-(1-carboxyethoxy)phenyl)-3-oxoprop-1-enyl)-8-methyl-2-oxo-2H-
chromene-3-carboxylic acid (41):
13

ACCEPTED MANUSCRIPT
From 31; pale yellow solid; yield: 80%; mp: 260 – 2620C; IR(KBr,cm–1):3749, 3651, 3028,
2365, 1752, 1664, 1584, 1375, 1222, 1029, 829; 1H NMR (DMSO-d6, 300 MHz) δ : 8.63 (s,
1H) 8.17 - 8.11(m, 4H) 7.92 (d, J = 15.4Hz, 1H ) 7.63 (d, J = 15.5Hz, 1H) 7.01 (d, J = 8.6Hz,
2H) 5.03 (q , J = 6.3Hz, 1H) 2.39 (s, 3H) 1.55(d, J = 6.6 Hz, 3H); 13C NMR (DMSO-d6, 75
MHz) δ: 187.4, 173.0, 164.2, 161.9, 156.7, 154.3, 148.7, 141.7, 134.8, 131.4, 131.3, 131.1,
129.2, 126.3, 122.9, 118.9, 118.4, 115.1, 72.1, 18.5, 15.3; ESI – MS: (m/z): 423 (M+H)+.
6.6.4. (E)-8-sec-butyl-6-(3-(4-(2-carboxypropan-2-yloxy)phenyl)-3-oxoprop-1-enyl)-2-oxo-
2H-chromene-3-carboxylic acid (42):
From 35; yellow solid; yield: 75%; mp: 274 – 2760C; IR(KBr,cm–1):3749, 3661, 3038, 2365,
1
1742, 1664, 1584, 1275, 1225, 1019, 839; H NMR (DMSO-d6, 300 MHz) δ : 8.63 (s, 1H)
8.20 - 8.12(m, 4H) 7.97 (d, J = 15.6Hz, 1H ) 7.85 (d, J = 15.5Hz, 1H) 7.94 (d, J = 8.5Hz, 2H)
3.32 – 3.20(m, 1H) 1.74 – 1.71 (m, 2H) 1.60 (s, 6H) 1.31(d, J = 6.8 Hz, 3H) 0.86 - 0.81(m,
3H); ESI – MS: (m/z): 478 (M+H)+.
Acknowledgements
We wish to thank Dr Tushar Kanti Chakraborty (Director, CSIR-CDRI) for his constant
support and encouragement in drug development program, S.P. Singh for technical support,
SAIF for NMR, IR, and Mass spectral data. G.R.P., S.R.A. and R.S. are thankful to CSIR,
New Delhi, India for financial support. This is CSIR-CDRI communication number
2013/KVS.
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Captions to illustrations.
Table 1 - Percentage (%) change of plasma lipids with the treatment of coumarin - chalcone
fibrate hybrid molecules (19-42) in triton-induced hyperlipidemic rats at the dose of 100
mg/kg body weight.
Table 2 - Percentage (%) change of plasma lipids with the treatment of compounds 41 and 35
in triton-induced hyperlipidemic rats at the different doses.
Figure 1 - Chemical structures of some representative lipid lowering fibrates, coumarin,
chalcones and our synthesized prototype.
Figure 2 - Effect of compound 41 on lipoprotein metabolism in triton induced
hyperlipidemic rats. Blood was drawn after 18 hours of hyperlipidemia was developed by
administration of triton WR-1339 with and without compound 41 and gemfibrozil (100
mg/kg). Compounds 41 and gemfibrozil improve lipoprotein lipids profile level in triton
induced hyperlipidemic rats. Values are expressed as the mean ± SD (n = 6). *P < 0.05; **P
a
< 0.01; ***P < 0.001; NS (non significant) compared to triton treated only and P < 0.05; bP
< 0.01; cP < 0.001 comparison triton and triton plus Compound 41 treated groups.
Figure 3 - Effect of compound 41 on LPL activity in triton induced hyperlipidemic rats.
Animals were sacrificed after 18 hours of hyperlipidemia was developed by administration of
triton WR-1339 with and without compound 41 and gemfibrozil (100 mg/kg). Compound 41
and gemfibrozil improve LPL activity in triton induced hyperlipidemic rats. Values are
expressed as the mean ± SD (n = 6). *P < 0.05; **P < 0.01; NS (non significant) compared to
a
triton treated only and P < 0.05 comparison triton and triton plus compound 41 treated
groups.
Scheme 1- Synthesis of novel coumarin-chalcone fibrates. Reagents and conditions: (i)
K2CO3, CH3CN, 100 0C, 4h (ii) (a) HMTA / TFA, 120 ºC, 3h; (b) 10% H2SO4, 90-100 ºC, 2h
(iii) Conc.HCl, dioxane, 110 0C, 8-10 h.(iv) Diethylmalonate, cat. Piperidine, Ethanol, 90 0C,
1h.(v) 20% aq.KOH, Methanol, r.t, 5-6h.
17

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Table 1
Percentage (%) change of plasma lipids with the treatment of coumarin - chalcone fibrate hybrid molecules (19-
42) in triton-induced hyperlipidemic rats at the dose of 100 mg/kg body weight
Compound
no.
Lipid profile
Structure
PHLAb
Total cholesterol
(TC)a
Phospholipids Triglyceride
(PL)a
(TG)a
O
O
O
O
O
19
20
OHC
HO
-12*
-13*
-6NS
-13*
-11*
-8NS
-11*
+6 NS
O
H3C
O
O
OHC
-11*
-8NS
+5 NS
+2 NS
+15*
HO
O
O
O
O
O
27
28
O
O
O
H3
C
O
O
O
O
O
O
O
-21**
-20**
-22**
O
H3
C
CH3
O
O
O
O
O
O
29
30
-13*
-11*
-11*
-12*
-11*
-11*
+5 NS
O
O
H3C
CH3
CH3
O
O
O
O
O
O
O
+8 NS
O
H3C
CH3
O
O
O
O
O
O
O
31
32
-10*
-10*
-12*
-9NS
-11*
-9NS
+6 NS
O
H3C
O
O
O
O
O
O
O
+5 NS
O
H3C
CH3
18

ACCEPTED MANUSCRIPT
O
O
O
O
O
O
O
33
-14*
-15*
-14*
+10*
O
H3C
O
O
O
O
O
O
34
35
-10*
-10*
-8NS
+5 NS
+15*
O
O
H3
C
O
O
O
O
O
O
-22**
-23***
-23***
O
C
O
H3
H3C
O
O
O
36
O
O
-19*
-17*
-16*
+12*
O
O
O
H3C
O
O
O
O
O
O
37
38
-12*
-13*
-11*
-12*
-11*
-12*
+5 NS
O
O
H3C
H3C CH3
O
O
O
+7 NS
O
O
O
O
O
H3C
CH3
O
OH
O
O
39
40
HO
O
-9 NS
-7 NS
-8NS
+3 NS
O
O
H3C
CH3
O
OH
O
O
O
HO
O
-8 NS
-9 NS
-9 NS
+3 NS
O
H3
C
CH3
O
OH
O
O
O
HO
41
42
-26***
-8 NS
-24***
-8 NS
-25***
-9 NS
+17 *
O
O
H3C
O
OH
O
O
HO
O
+4 NS
O
C
O
H3
CH3
19

ACCEPTED MANUSCRIPT
O
O
OH
-32***
-30***
-33***
+19*
Gemfibrozil
a: mg/dl; b: n mol of free fatty acids formed/h/ml of plasma; Values are mean ± SD of six rats, ***p <0.001; **p
<0.01; *p <0.05; NS = Non significant.
20

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Table 2
Percentage (%) change of plasma lipids with the treatment of compounds 41 and 35 in triton-induced
hyperlipidemic rats at the different doses.
Compound no.
Compound 41
50(mg/kg)
Lipids profilea
PL
PHLA b
TC
TG
-16 *
-15 *
-14 *
+12 *
100(mg/kg)
200(mg/kg)
Compound 35
50(mg/kg)
-26 ***
-27 ***
-24 ***
-24 ***
-25 ***
-26 ***
+17 ***
+17 ***
-18 *
-16 *
-16 *
+13 *
100(mg/kg)
200(mg/kg)
Gemfibrozil
100(mg/kg)
-22 **
-23 ***
-23 ***
-24 ***
-23 ***
-25 ***
+15 **
+15 **
-32***
-30***
-33***
+19 ***
a: mg/dl; b: n mol of free fatty acids formed/h/ml of plasma; Values are mean ± SD of six rats, ***p <0.001; **p
<0.01; *p <0.05.
21

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Figure 1: Chemical structures of some representative lipid lowering fibrates, coumarin,
chalcones and our synthesized prototype.
22

ACCEPTED MANUSCRIPT
Figure 2:
Effect of compound 41 on lipoprotein metabolism in triton induced hyperlipidemic rats.
Blood was drawn after 18 hours of hyperlipidemia was developed by administration of triton
WR-1339 with and without compound 41 and Gemfibrozil (100 mg/kg). Compound 41 and
Gemfibrozil improve lipoprotein lipids profile level in triton induced hyperlipidemic rats.
Values are expressed as the mean ± SD (n = 6). *P < 0.05; **P < 0.01; ***P < 0.001; NS
a
b
c
(non significant) compared to triton treated only and P < 0.05; P < 0.01; P < 0.001
comparison triton and triton plus compound 41 treated groups.
23

ACCEPTED MANUSCRIPT
Figure 3:
Effect of compound 41 on LPL activity in triton induced hyperlipidemic rats. Animals
were sacrificed after 18 hours of hyperlipidemia was developed by administration of triton
WR-1339 with and without compound 41 and Gemfibrozil (100 mg/kg). Compound 41 and
Gemfibrozil improve the LPL activity in triton induced hyperlipidemic rats. Values are
expressed as the mean ± SD (n = 6). *P < 0.05; **P < 0.01; NS (non significant) compared to
a
triton treated only and P < 0.05 comparison triton and triton plus compound 41 treated
groups.
24

ACCEPTED MANUSCRIPT
Scheme 1: Synthesis of novel coumarin-chalcone fibrates. Reagents and conditions: (i)
K2CO3, CH3CN, 100 0C, 4h (ii) (a) HMTA / TFA, 120 ºC, 3h; (b) 10% H2SO4, 90-100 ºC, 2h
0
0
(iii) Conc.HCl, dioxane, 110 C, 8-10 h.(iv) Diethylmalonate, cat.Piperidine, Ethanol, 90 C,
1h.(v) 20% aq.KOH, Methanol, r.t, 5-6h.
25

ACCEPTED MANUSCRIPT
Research Highlights
•
•
Discovery of lipid lowering activity of coumarin chalcone fibrates hybrids.
Hybrid 41 produced striking reduction in serum
levels of total lipids.
•
Hybrids represent new class of lipid lowering
agents.

ACCEPTED MANUSCRIPT
Supporting information
Coumarin chalcone fibrates: a new structural class of lipid
lowering agents*
a,
a
b
a
Koneni V. Sashidhara *, Gopala Reddy Palnati , Ravi Sonkar , Srinivasa Rao Avula ,
Chetan Awasthi b, Gitika Bhatia b
a
Medicinal and Process Chemistry Division , CSIR-Central Drug Research Institute,
Lucknow, 226 001, India
Biochemistry Divisionb, CSIR-Central Drug Research Institute, Lucknow, 226 001, India
Experimental Part –General Information ………………………… S2
Compounds Spectra………………………………………………… S3-S22
Compound 7 (1H)………………………………………………… S3
Compound 10 (1H)………………………………………………… S3
Compound 11 (1H)………………………………………………… S4
Compound 13 (1H)………………………………………………… S4
Compound 19 (1H)………………………………………………… S5
Compound 20 (1H)…………………………………………
Compound 27 (1H & 13C)..........................................................
S5
S6
Compound 27 (DEPT 90 and 135)...................................................... S7
Compound 27 (COSY)…………………………………………… S8
Compound 27 (HSQC)……………………………………………… S8
Compound 27 (HMBC)……………………………………………… S9
Compound 28 (1H & 13C)…………………………………………… S9 & S10
Compound 29 (1H &13C)………………………………………………S10 & S11
Compound 30 (1H & 13C)…………………………………………… S11 & S12
Compound 31 (1H & 13C)…………………………………………… S12 & S13
Compound 32(1H & 13C)…………………………………………… S13 &S14
S1

ACCEPTED MANUSCRIPT
Compound 33 (1H)……………………………………………………S14
Compound 34 (1H & 13C)…………………………………………… S15
Compound 35 (1H & 13C)…………………………………………… S16
Compound 36 (1H & 13C)…………………………………………… S17
Compound 37 (1H & 13C)…………………………………………… S18
Compound 38 (1H & 13C)…………………………………………… S19
Compound 39 (1H)…………………………………………………… S20
Compound 40 (1H)…………………………………………………… S20
Compound 41 (1H & 13C)……………………………………………… S21
Compound 38 (1H)………………………………………………………S22
General Information:
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 TLC; silica gel
1
plates with fluorescence F254 were used. Melting points were uncorrected. The H NMR, 2D-NMR
(COSY, HMBC, HSQC) and 13C NMR spectra were determined on 200, 300, MHz and 50, 75, MHz,
respectively, using CDCl3 and DMSO- d6 as solvents and TMS as internal standard. All chemical
shifts were given in ppm. IR spectra were recorded on in the range of 500~4000 cm-1, and multiplicity
(s = singlet, brs = broad singlet, d = doublet, brd = broad doublet, dd = double doublet, t = triplet, q =
quartet, m = multiplet).
S2