Discovery and synthesis of novel substituted benzocoumarins as orally active lipid modulating agents

Article abstract of DOI:10.1016/j.bmcl.2011.09.053

The synthesis of a series of benzocoumarin keto-enamine schiff bases is reported. The novel compounds were evaluated for their antihyperlipidemic activity in the hyperlipidemic hamster model. The compound 11 at a dose of 10 mg/kg body weight significantly

Full text of DOI:10.1016/j.bmcl.2011.09.053

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6709–6713
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and synthesis of novel substituted benzocoumarins as orally
active lipid modulating agents q
Koneni V. Sashidhara a, , Manoj Kumar a, Ram K. Modukuri a, Anuj Srivastava b, Anju Puri b
⇑
a Medicinal and Process Chemistry Division, Central Drug Research Institute (CSIR-CDRI), Lucknow 226 001, India
b Biochemistry Division, Central Drug Research Institute (CSIR-CDRI), 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:
Received 24 June 2011
Revised 2 September 2011
Accepted 15 September 2011
Available online 20 September 2011
The synthesis of a series of benzocoumarin keto-enamine schiff bases is reported. The novel compounds
were evaluated for their antihyperlipidemic activity in the hyperlipidemic hamster model. The com-
pound 11 at a dose of 10 mg/kg body weight significantly lowered the plasma triglyceride levels (TG)
by 70%, total cholesterol (TC) by 47%, accompanied by an increase in HDL-C/TC ratio by 80% in hyperlipi-
demic hamsters to a greater degree than the reference drugs atorvastatin and lovastatin.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Synthesis
Benzocoumarin
Statins
Antidyslipidemic
Dyslipidemia and coagulation disturbances are among the most
significant risk factors of the development of atherosclerotic condi-
tion.1 Elevated plasma concentration of cholesterol, especially low-
density lipoprotein (LDL) and triglyceride is recognized as a leading
cause in the development of atherosclerosis and coronary heart
disease.2 Furthermore, hypertriglyceridemia is the most frequent
form of hyperlipidemia observed in type 2 diabetes, because it
co-exists with hyperinsulinemia in the general population.
previously we have synthesized 3 and 4 substituted coumarins
and some of which have shown good lipid lowering profile in
comparison to standard drug.5 That study revealed that, such at-
tempts on benzocoumarins based pharmacophores which is a
biologically important scaffold might result in identification of
new lead for antidyslipidemia.
Furthermore, in the synthesis of a series of keto-enamine schiff
bases of benzocoumarin and its evaluation as potential lipid lower-
ing agents, indicated that the keto-enamine side chain is crucial for
activity.6 However, it remained unclear as to what substituents
would be desirable to enhance the activity. To answer this ques-
tion, we synthesized derivatives with different side chain modifica-
tions, that took into account the homologation, chain branching,
ring chain transformed analogs, etc. and studying the impact of
such modification on the hypolipidemic activity. This study under-
scores the importance of the side chain modification on coumarin
scaffold that seem to modulate the activity dramatically. In this
communication, we describe the details of this study. A graphical
representation of the evolution of our work on coumarins resulting
in the current series of molecules is depicted in Figure 1.
The synthetic strategy started with Pechmann reaction between
naphthalene 1,5-diol 1, and ethyl acetoacetate to give 7-hydroxy-4-
methyl-benzo(h)chromene-2-one 2. The coumarin 2 was subjected
to the Duff formylation to furnish 7-hydroxyl-4-methyl-2-oxo-2H-
benzo(h)chromene-8,10 dicarbaldehyde 3. This dicarbaldehyde
was then condensed with various primary amines to give final com-
pounds (4–13).7 The detailed synthetic methodology of which has
been previously reported by us and the reaction conditions are out-
line in Scheme 1. The structures of all the novel compounds were
Statins and fibrate class of drugs are the most widely used can-
didates for treatment of dyslipidemia. Statins (HMG-CoA reductase
inhibitors) used for lowering LDL-cholesterol are pretty effective.
However, most patients still experience adverse coronary events
despite statin therapy. Furthermore, current reports of unwanted
side effects (myopathy) of some ‘super statins’ point out that the
scope of improving the potency of this class of drugs may be mod-
est.3 The fibrate class (PPAR
a agonists) of drugs, which are mostly
used to treat hypertriglyceridemia and low HDL-cholesterol, re-
quires high doses to show significant efficacy.4 Additionally, a com-
bination of fibrate and statins has met with serious safety concerns
as exemplified by the withdrawal of Cerivastatin in 2001. There-
fore, there is a constant need for a different class of potent com-
pounds to treat dyslipidemia without severe side effects.
In continuation of our efforts to explore the chemical diver-
sity space around coumarin scaffold as antidyslipidemic agents,
q
Part XIII in the series, ‘Advances in drug design and discovery’. CDRI MS 8128.
⇑
Corresponding author. Tel.: +91 9919317940; fax: +91 522 2623405.
E-mail
addresses:
kv_sashidhara@cdri.res.in,
sashidhar123@gmail.com
(K.V. Sashidhara).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.053

6710
K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6709–6713
Figure 1. Graphical depiction of the evolution of our work on coumarins leading to the current series of substituted coumarins.
Scheme 1. Synthesis of benzocoumarins (4–13).
confirmed by NMR and Mass Spectrometry (see Supplementary
data).
(Table 1). No significant differences were observed in the food in-
take and weight gain between the groups (data not shown). As
such, the compounds (4–13) supplement did not apparently ad-
versely affect the hamsters. All the compounds were administered
orally at the dose of 10 mg/kg body weight for seven consecutive
days. Normal hamsters fed with HFD and given drug vehicle
(gum acacia) only served as control animals.
The role of amino alkane is important in metabolic disorders like
diabetes and dyslipidemia.11 In our current series diversification
with alkane amine side chain shown interesting biological profile
with desired parameters. The analysis of the lipid profile data (TC,
TG, HDL, LDL, and H/C) of different groups clearly suggests that
most of the synthesized compounds exerted excellent hypolipi-
demic activity. The most promising compound in the series was
In the present study, we have carried out experiments to inves-
tigate the antidyslipidemic activity of benzocoumarins (4–13) in
the high fat diet (HFD) fed dyslipidemic hamster model,8 which
has been reported as an ideal in vivo model for evaluating antidy-
slipidemic drugs. The development of dyslipidemic hamster model
is based on diet induced dyslipidemia in normal animals with an
intact metabolism. The high fat diet (HFD) in combination with
fructose is found to give rapidly induced dyslipidemia with
hyper-cholesteremia and hyper triglyceridemia.9,10 In the high fat
diet (HFD) fed dyslipidemic hamsters, the administration of ben-
zocoumarins caused a distinct decrease in the serum triglyceride,
total cholesterol, LDL and increase in HDL and HDL-C/TC ratio

K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6709–6713
6711
Table 1
Percentage (%) decrease/increase of plasma lipids with the treatment of compound (4–13) in dyslipidemic hamsters at the dose of 10 mg/kg body weight
Compounds
Structures
TG
TC
HDL
LDL
H/C
O
O
H
O
4
À47***
À17
+19
À14
+9
H
N
Cl
O
O
O
H
O
5
6
À40*
À13
À26
À1
À10
À21
+15
+39
H
N
Br
O
O
O
H
O
À50***
+3
H
N
HO
HO
O
O
O
H
O
7
8
9
À49***
À48***
À62***
À36*
À48***
À14
+1
+1
+1
À36
+61
+93
+17
H
N
O
O
O
H
O
À59***
H
N
O
O
O
O
H
O
O
À10
H
N
O
O
H
10
À42***
À3
+15
+12
+19
H
N
O
OH
O
O
H
MeO
O
11
12
13
À70***
À52***
À16
À47***
À47***
À30*
+1
+3
+1
À54*
+80
+93
+43
H
N
O
H
O
O
OMe
O
O
À59***
H
N
O
H
O
O
À40***
H
N
O
Atorvastatin
Lovastatina
À67***
À29
À25
À9
+2
+3
À23
À11
+83
+12
Values represented are % change with respect to HFD-fed hamster group (group of eight animals).
H/C = HDL/TC.
a
Lovastatin at the dose of 25 mg/kg body weight.
p <0.05.
p <0.001.
*
***
the compound 11 which at a oral dose of 10 mg/kg body weight sig-
nificantly lowered the plasma triglyceride levels (TG) by 70%, total
cholesterol (TC) by 47%, low density lipoprotein-cholesterol (LDL-C)
by 54% and increased HDL-C/TC ratio by 80%, in comparison to ator-
vastatin at the same dose lowered TG by 67%, TC by 25%, LDL-C by
23% and increased HDL-C/TC ratio by 83%, while lovastatin at the

6712
K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6709–6713
Table 2
Antioxidant potential of compounds (4–13) using the TEAC
assays
Compounds
TEAC value (mM)
4
5
6
7
8
9
10
11
0.38
0.10
1.46
1.90
0.99
0.30
0.36
1.36
1.26
0.99
3.13
12
13
Ascorbic acid
Figure 3. In vitro dose response study of compound 11 and lovastatin for HMG-CoA
inhibitory activity.
higher dose of 25 mg/kg body weight in the same model, decreased
the level of TG by 29%, total cholesterol by 9% and increase in HDL-
C/TC ratio by 12%. Additionally compounds 7, 8, and 12 lowered TG
by 49%, 48%, and 52%, TC by 36%, 48%, and 47%, LDL by 36%, 59%, and
59% increased the HDL-C/TC ratio by 61%, 93%, and 93% respec-
tively, which is considered a beneficiary effect in the treatment of
dyslipidemia condition. The reduction of the level of LDL-C by the
compounds 8, 11, and 12 by 59%, 54%, and 59% respectively is an
interesting finding because the LDL fraction is generally thought
to carry cholesterol to the tissues and is responsible for the arther-
ogenesis process.12 The higher TG levels and lower the HDL-C in-
crease the risk of coronary heart disease. In latter case, the high
density lipoproteins (HDL) mediate the reverse transport of choles-
terol from peripheral tissues to the liver, which will disallow the
slow accumulation of lipids in artery walls. The compound 11
exhibited both the above beneficiary effects as it has decreased
the TG by 70% and increased the HDL-C/TC ratio by 80% at a dose
of 10 mg/kg body weight. Furthermore, these synthesized com-
pounds were also evaluated for their antioxidant potential using
TEAC assay.13 Antioxidant activities of benzocoumarins in compar-
ison to ascorbic acid are given in Table 2. Oxidative stress is one of
the causative factors that link hypercholesterolemia with athero-
genesis. However, the antioxidant potential of the synthesized
compound was found to be modest.
substituted derivatives as compound 11 with 4-methoxy benzyl
amine and compound 12 with 3-methoxy benzyl amine group
showed better activity than reference drug atorvastatin and
lovastatin.
With these promising activities in hand, dose dependent (differ-
ent doses 2.5, 5, 10, and 25 mg/kg body weight) study on the com-
pound 11 was performed on a different set of experimental
animals which exhibited concentration dependent effects (Fig. 2).
To investigate the mechanistic pathway of the synthesized ben-
zocoumarins, we have carried out HMG-CoA inhibitory activity8
of compound 11 in comparison to standard drug lovastatin. The
inhibitory activity was evaluated at five different concentrations
ranging from 5 to 100
inhibition of HMG-CoA reductase at 5
l
M (Fig. 3). Compound 11 showed 26.10%
M concentration which
l
was similar to lovastatin, thus suggesting the plasma lipid lower-
ing action of the compound 11 seemed partly due to the inhibition
of HMG-CoA reductase. Somewhat parallel results were obtained
at further higher concentrations of 10, 25, 50, and 100
lM. Maxi-
mum inhibition of 63.80% was shown by 11 at 100 M concentra-
l
tion. However, more extensive investigation of compound 11 is
still required to further elucidate the mechanism and usefulness
of this synthetic derivative in different animal models and humans.
In conclusion, novel substituted benzocoumarins derivatives
were prepared and their hypolipidemic activity was screened in
hyperlipidemic hamsters. Among the synthesized compounds,
the compound 11 was found to be most active hypolipidemic agent
affording significant hypocholesterolemic and hypotriglyceridemic
Structure–activity relationship reveals that incorporation of
halogen atom (like compounds
4 and 5) to benzocoumarin
showed mild protection. Whereas, increased lipophilicity as in
compound 8 with adamantyl group proved admirable activity.
But most interesting results were obtained in benzyl amine
Figure 2. In vivo dose response study of compound 11 on TG, TC, LDL, and HDL-C/TC of hamster model.

K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6709–6713
6713
(br s, 1H), 2.56 (br s, 3H), 2.30 (s, 3H), 2.03 (br s, 6H), 1.86–1.73 (m, 6H), 13C
NMR (CDCl3, 75 MHz) d 191.5, 179.1, 159.8, 159.2, 153, 137.3, 132.9, 124.6,
121.9, 120.9, 120.1, 119.9, 115.7, 108.7, 56.1, 42.6, 35.6, 29.1, 19.5, ESI-MS: (m/
z): 416 (M+H)+.
activities and seems to be a good candidate for developing a new
lead with good hypolipidemic and antiatherosclerotic benefits.
Further studies on compound 11 are under progress to advance it
as a novel antidyslipidemic lead compound.
8. Animals: Golden Syrian hamsters (Mesocricetus auratus), male, 12 week old, wt
110 10 g were used. Animals were kept in a room-controlled temperature at
25–26 °C, relative humidity 60–80% and 12:12 h light/dark cycle light (on from
8.00 AM to 8.00 PM) under hygienic conditions. The animal had free access to
the diet and water ad libitum. Experimental protocols were approved by our
institutional ethical committee, which follows guidelines of Committee for the
Purpose of Control and Supervision of Experiments on Animals (CPCSEA),
which complies with international norms of INSA.
Acknowledgments
Instrumentation facilities from SAIF, CDRI are gratefully
acknowledged. Manoj and Ram are thankful to CSIR, New Delhi, In-
dia for financial support. This is CSIR-CDRI communication number
8128.
Antidyslipidemic activity in high fat diet fed hamster model: Feeding with high fat
diet- (HFD) developed hyperlipidemia in hamster. Hamsters were divided into
–
HFD fed, HFD fed, and benzocoumarins treated group containing eight
animals in each group. High fat diet was given daily from day 1 to 10 to animals
of all the groups. Hamsters were treated with benzocoumarins orally at a dose
of 10 mg/kg body weight once a day for seven consecutive days, from day 4 to
10. HFD fed animals treated with vehicle (0.1% gum acacia) served as controls.
Body weight of animals was recorded daily.
Collection of blood samples and biochemical analysis from plasma: After the last
day of treatment blood was collected in EDTA coated tubes from the retro
orbital plexus of the hamsters. The samples were centrifuged at 4000 rpm for
5 min and plasma was separated. The plasma samples were used for the assay
of total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL)-
cholesterol (HDL-C) and low-density lipoprotein (LDL)-cholesterol by standard
enzymatic methods using Synchron CX-5 Clinical System Beckmann Coulter
auto analyzer. All assay kits were purchased from Beckman Coulter
International, USA.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.053.
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1315 cmÀ1 1H NMR (CDCl3 300 MHz) d 13.76 (br s, 1H, ANH), 11.13 (s, 1H,
,
ACHO), 8.42 (d, J = 8.6 Hz, 1H), 8.14–8.08 (m, 2H), 7.62 (d, J = 8.6 Hz, 1H), 6.44