Novel coumarin derivatives as potential antidyslipidemic agents

Article abstract of DOI:10.1016/j.bmcl.2010.05.023

A series of novel benzocoumarin derivatives were synthesized and evaluated for their in vivo antidyslipidemic and in vitro antioxidant activities. Among 11 compounds tested, 2 compounds showed potent antidyslipidemic activity and 3 compounds showed potent antioxidant activity.

Full text of DOI:10.1016/j.bmcl.2010.05.023

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4248–4251
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel coumarin derivatives as potential antidyslipidemic agents
a
a
b
b
b
Koneni V. Sashidhara a, , Abdhesh Kumar , Manoj Kumar , Ravi Sonkar , Gitika Bhatia , A. K. Khanna
*
a Medicinal and Process Chemistry Division, Central Drug Research Institute, CSIR, Lucknow 226 001, India
b Biochemistry Division, Central Drug Research Institute, CSIR, Lucknow 226 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel benzocoumarin derivatives were synthesized and evaluated for their in vivo antidyslip-
idemic and in vitro antioxidant activities. Among 11 compounds tested, 2 compounds showed potent
antidyslipidemic activity and 3 compounds showed potent antioxidant activity.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 11 March 2010
Revised 29 April 2010
Accepted 11 May 2010
Available online 21 May 2010
Keywords:
Synthesis
Benzocoumarins
Cholesterol lowering
Antidyslipidemic
Antioxidant
resulting in the formation of OÅ2 and OH radicals which further
accelerates the risk of cardiac diseases in dyslipidemic subjects.5
Therefore, it is envisaged that, beside cholesterol lowering prop-
erty, a hypolipidemic agent that incorporates antioxidant activity
will be able to protect endothelial and myocardial function could
serve as a better anti-atherosclerotic agent.
Å
Atherosclerotic cardiovascular disease is the leading cause of
death in both developed and developing countries. Epidemiological
studies have indicated that dyslipidemia and coagulation distur-
bances are among the most significant risk factors of the develop-
ment of atherosclerotic condition.1
Current therapies mostly focus on lowering LDL-cholesterol and
statins (HMG-CoA reductase inhibitors) used for this purpose are
pretty effective. However, most patients still experience adverse
coronary events despite statin therapy. In addition, recent reports
of undesirable side effects (myopathy) of some ‘super statins’ indi-
cate that the scope of improving the potency of this class of drugs
Coumarins are an elite class of oxygen heterocycles, which oc-
cupy a special role in nature. Coumarins and their derivatives have
attracted intense interest in recent years because of their diverse
pharmacological properties.6–11 In addition many coumarin deriv-
atives have the special ability to scavenge reactive oxygen species
(ROS) and to influence processes involving free radical injury.12
Furthermore, coumarins (umbelliferone) and its derivatives are
shown to have lipid lowering potential.13,14 The coumarins are ex-
tremely variable in structure, due to the various types of substitu-
tions in their basic structure, which in turn can influence their
biological activity.
Recently, we have reported the synthesis and biological evalua-
tion of benzocoumarin derivatives as potential antioxidant and
lipid lowering activity.15 Our initial studies towards the identifica-
tion of a new lead for antidyslipidemia indicated compound 3
(1-hydroxy-4-methyl-naphthalene-2-carbaldehyde) as biologically
important scaffold (compound 3 significantly lowered the TC, PL
and TG by 24%, 25%, 23%, respectively) thus a series of novel deriv-
atives of benzocoumarins from compound 3 were synthesized and
all the derivatives were evaluated for their potential lipid lowering
activity in vivo and antioxidant activity in vitro.
may be modest.2 Fibrate class (PPAR
a agonists) of drugs, which are
mostly used to treat hyper triglyceridemia and low HDL-choles-
terol, requires high doses to show significant efficacy.3 In addition,
a combination of fibrate and statins has met with serious safety
concerns as exemplified by the withdrawal of Cerivastatin in
2001. Therefore, there is a constant need for a different class of po-
tent compounds to treat dyslipidemia without severe side effects.
Figure 1 shows the chemical structures of some clinically used
fibrates.
Hyperlipidemia may also induce other abnormalities like oxida-
tion of free fatty acids, leading to the formation of ketone bodies as
well as masking liver and muscles resistance to insulin which ini-
tiates the progress of diabetes in patients.4 Furthermore, in hyper-
glycemic patients, several non-enzymatic glycosylations occurs
accompanied by glucose oxidation catalyzed by Cu2+ and Fe2+
The Duff reaction on naphthalen-1-ol resulted in expected
compound 2 and unexpected compound 3 that was character-
ized ambiguously by us.16 As previously revealed compound 3
* Corresponding author. Tel.: +91 9919317940; fax: +91 522 2623405.
E-mail
addresses:
sashidhar123@gmail.com,
kv_sashidhara@cdri.res.in,
sashidhar123@rediffmail.com (K.V. Sashidhara).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.023

K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4248–4251
4249
Figure 1. Structures of fibrates and benzocoumarin (compound 6) as lipid lowering agents.
(1-hydroxy-4-methyl-naphthalene-2-carbaldehyde) exhibited
promising antidyslipidemic activity, it prompted us to take the
compound 3 as a molecular template and an active pharmacophore
for further diversification. The Knoevenagel condensation on 3 that
was catalyzed by piperidine resulted in the formation of its benzo-
coumarin derivatives (4–8).17 Furthermore, compound 8 on alka-
line hydrolysis furnished compound 9, which on its esterification
resulted in compounds 10–13 (Scheme 1). All the compounds were
characterized using 1H NMR, 13C NMR, Mass and IR spectroscopy.
The purity of these compounds was ascertained by TLC and spec-
tral analysis (see Supplementary data).
The antidyslipidemic and post heparin lipolytic activity of benzo-
coumarin derivatives 4–13 were evaluated in an in vivo Triton mod-
el.18–20 Administration of Triton WR-1339 in rats induced marked
hyperlipidemia as evidenced by increase in the plasma level of total
cholesterol TC (4.05-fold), phospholipids PL (3.31-fold) and triglyc-
eride TG (2.67-fold) as compared to control. Triton induced rats
caused inhibition of post heparin lipolytic activity plasma PHLA
(À28%) as compared to control.21 Treatment of hyperlipidemic rats
with benzocoumarin derivatives at the dose of 100 mg/kg po re-
versed the plasma levels of lipid with varying extents.22
49% and microsomal lipid peroxidation inhibition by 41% and 38%,
respectively. The standard drug Alloperinol at 20 g/mL showed
80% inhibition in superoxide anions. Manitol and -Tocopherol at
the dose of 100 g/ml showed 52% and 47% inhibition of hydroxyl
ions and microsomal lipid peroxidation, respectively. The scaveng-
ing potential of other derivatives were modest. The wide variation
in the free radical scavenging potential may be due to the variation
in the proton-electron transfer by the derivatives due to difference
in their structures and stability.
The frequently prescribed hypolipidemic agent, Gemfibrozil
having a 5-phenoxypentanoic acid moiety instead of the fibric acid
moiety is also considered a fibrate because of having almost similar
pharmacological properties as the other classical fibrates.24 The
l
a
l
fibrates primary mode of action is to selectively activate the a-iso-
type of the receptors peroxisome proliferator-activated receptors
(PPARs).25 Though efforts are ongoing to delineate the precise
mechanism of action of the synthesized compounds, we believe
it to be acting on the same target as Gemfibrozil.
In terms of SAR, the minimum structural requirement for binding
of the derivatives to the target includes an aromatic or heteroaro-
matic ring (ring B, C), which is believed to participate in
P–P stack-
The synthesized derivatives inhibited cholesterol biosynthesis
and potentiated the activity of lipolytic enzymes to early clearance
of lipids from circulation in triton—induced hyperlipidemia. Com-
pound 3 significantly lowered the TC, PL and TG by 24%, 25%,
23% and however, the compound 6 (the benzocoumarin derivative
of 3 having a chlorine at position 3) was the most potent in the ser-
ies as it showed 27%, 26% and 26% lowering in TC, PL and TG,
respectively, while compounds 4, 5, 7–13 showed mild activity
(Fig. 2). These data are comparable with standard drug Gemfibrozil
at the dose of 100 mg/kg which decreased level of TC, PL and TG in
plasma by 34%, 38% and 35%, respectively. In PHLA enzyme activ-
ity, again compound 3 and 6 showed significant reversal of PHLA
in plasma of hyperlipidemic rats by 26% and 25%, respectively,
comparable to Gemfibrozil, which caused 28% reversal of activity
of this enzyme as compared to control group.
ing with aromatic amino acids residue of the receptor. Substituents
in ring B have varied effect on binding. It is generally true, however
that an electron releasing group (–CH3) substituted at the 7 position
as in compound 3 markedly increases the antidyslipidemic activity,
as in the compound 2 having electron withdrawing group (–CHO)
was found to be inactive. In the ring A neither the acid or ester group
at position 3 is required for in vivo dyslipidemic activity. Though the
electron withdrawing group at position 3 is desired, the lack of activ-
ity for compound 5 having –CN group might be due to its conversion
back to –C@N. Substitution of Cl for CN increases the activity which
we believe is due to the better selectivity.
Compound 6 exhibited interesting most promising activity in
both lipid lowering and antioxidant experiments. Initial studies
indicate compound 6 to be devoid of cytotoxicity in normal cells.
Further studies on 6 including dose optimization are in progress
and will be published in the future.
In conclusion, a series of novel substituted benzocoumarin
derivatives (4–13) have been synthesized from compound 3
(1-hydroxy-4-methyl-naphthalene-2-carbaldehyde) by Knoevena-
gel condensation. Among the synthesized compounds, compound
6 was found to be the most active hypolipidemic agent in addition
to having potent antioxidant and thus represent a new class of
antidyslipidemic agents.
In another experiment, antioxidant activities of compounds
3–13 were evaluated by generating free radicals in vitro in the ab-
sence and presence of these compounds.23 The scavenging potential
of benzocoumarins 3–13 at 200 l
g/mL against formation of OÅ2 and
ÅOH in non-enzymic systems were studied. Further, their effect on
lipid peroxidation in microsomes were also studied (Fig. 3). Com-
pounds 3 and 6 showed significant decrease in superoxide anions
inhibition by 27% and 32%, hydroxyl radicals inhibition by 41% and

4250
K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4248–4251
Scheme 1. Synthesis of novel benzocoumarin derivatives 4–13.
Figure 2. The lipid lowering activity of novel benzocoumarin derivatives (100 mg/kg) in Triton treated hyperlipidemic rats. Triton treated group is compared with control and
drug treated group is compared with triton group (units—mg/dL). Values are mean SD of six animals.

K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4248–4251
4251
Figure 3. The effect of novel benzocoumarin derivatives (200
peroxidation in microsomes (nmol MDA formed/mg protein) is shown (standard drugs for superoxide anions-Alloperinol (20
l
g/mL) on superoxide ion (nmol formazone formed/min), hydroxyl ion (nmol MDA formed/h) and lipid
g/mL), hydroxyl ions-Manitol and for
l
microsomal lipid peroxidation-a-tocopherol (100 lg/mL) were used). Values are mean SD of six animals.
and concentrated through high vacuum. The crude product thus obtained was
purified by column chromatography (60–120 mesh) to furnish (147 mg, 92%
Acknowledgments
yield) of pure
yellow solid.
18. Buccolo, G.; David, H. Clin. Chem. 1973, 19, 476.
19. Deeg, R.; Ziegehorn, J. Clin. Chem. 1983, 29, 1798.
20. Ghosh, S.; Misra, A. K.; Bhatia, G.; Khan, M. M.; Khanna, A. K. Bioorg. Med. Chem.
Lett. 2009, 19, 386.
4 (3-acetyl-6-methyl-2H-benzo[h]chromen-2-one) as light
The authors are grateful to the Director, CDRI, Lucknow, India
for constant encouragement in drug development program, S.P.
Singh for technical support, SAIF for NMR, IR, and Mass spectral
data. A.K., M.K. & R.S. are thankful to CSIR New Delhi, India for
financial support. This is CDRI publication number 7937.
21. (a) Wing, D. R.; Robinson, D. F. Biochem. J. 1968, 109, 841; (b) Mosinger, F. J.
Lipid Res. 1965, 6, 157.
22. Lipid lowering activity: Adult male Charles Foster rats (200–225 g) bred in the
animal house of the institute were used for the lipid lowering activity. Rats
were divided in control, triton induced, triton plus compounds and Gemfibrozil
(100 mg/kg) treated groups containing six rats in each. Hyperlipidemia was
developed by administration of Triton WR-1339 (Sigma chemical Co., St. Louis,
USA) at a dose of 400 mg/kg body wt. intraperitoneally to animals of all groups
except the control. Compounds 3–13 were macerated with gum acacia (0.2%
w/v), suspended in water and fed simultaneously with triton at a dose of
100 mg/kg po to the animals of treated groups. Animals of the control and
triton group without treatment with test compounds were given same amount
of gum acacia suspension (vehicle). After 18 h of treatment (50 mg/kg b. wt.)
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 4 °C for 10 min and the plasma was separated. Plasma was diluted
with normal saline (ratio 1:3) and used for analysis of total cholesterol (TC),
phospholipids (PL), triglycerides (Tg) and post heparin lipolytic activity (PHLA)
using spectrophotometer, Beckmann auto-analyzer and standard kits
purchased from Beckmann Coulter International, USA.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.05.023.
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KH2PO4 buffer (pH 7.4) to
a
final volume of 2.5 mL. The above reaction
g/mL) were
mixtures in the absence or presence of test compounds (200
l
incubated at 37 °C for 90 min. The test compounds were also studied for their
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To an equivalent mixture of 1-hydroxy-4-methyl-naphthalene-2-carbaldehyde
3
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dissolved in ethanol (20 mL) and to this piperidine (0.1 mL) was added and
the reaction mixture was refluxed for 30 min. After completion of the reaction,
ethanol was removed through high vacuo. The residue was neutralized with
acetic acid, diluted with water and extracted with CHCl3 (75 mL Â 3). The
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