Synthesis and anti-inflammatory activity of novel biscoumarin-chalcone hybrids

Article abstract of DOI:10.1016/j.bmcl.2011.06.002

A series of synthesized novel biscoumarin-chalcone hybrids were evaluated for their anti-inflammatory and antioxidant activity. The tested compounds significantly inhibit the carrageenin induced paw oedema in albino rats and also exhibit important scaveng

Full text of DOI:10.1016/j.bmcl.2011.06.002

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4480–4484
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anti-inflammatory activity of novel biscoumarin–chalcone
hybrids q
Koneni V. Sashidhara a, , Manoj Kumar a, Ram K. Modukuri a, Ravi Sonkar b, Gitika Bhatia b, A. K. Khanna b,
⇑
Shivika Rai c, Rakesh Shukla c
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
c Pharmacology 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:
A series of synthesized novel biscoumarin–chalcone hybrids were evaluated for their anti-inflammatory
and antioxidant activity. The tested compounds significantly inhibit the carrageenin induced paw
oedema in albino rats and also exhibit important scavenging activities. These compounds thus constitute
an interesting template for the design of new therapeutic tools against inflammation.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 15 March 2011
Revised 18 May 2011
Accepted 1 June 2011
Available online 12 June 2011
Keywords:
Synthesis
Biscoumarin–chalcone
Anti-inflammatory
TNF-a
Antioxidant
Inflammation is a self-protective response of body, which in-
activities may lead to the development of drugs with an improved
therapeutic index. This has already been established for a number
of commercially available nonsteroidal anti-inflammatory drugs
(NSAIDs), which simultaneously possess radical scavenging
properties.6
duces physiological adaptations to reduce tissue damage and to
eliminate the pathogenic infections. It is a usual symptom covering
different pathologies and causes a large number of diseases,
amongst this, some commonest are rheumatoid arthritis (RA),
inflammatory bowel disease, psoriasis and multiple sclerosis.1,2
Cytokines have been recognised as necessary components in acute
Coumarin and chalcone are important class of compounds hav-
ing versatile biological activities.7,8 Both of these pharmacophores
are well known for their antioxidant and anti-inflammatory poten-
tial.9 The coumarin nucleus incorporates the styryl carbonyl moi-
ety known to possess appreciable anti-inflammatory activity
while various chalcones (like naturally occurring butein, sappan-
chalcone and licochalcones) are known to exhibit potent scaveng-
ing activity (Fig. 1).10–13 The combination of the distinct
pharmacophores of two different biologically active compounds
in the same structure (medicinal chemistry hybridization), has
been previously reported in literature and is highly likely to lead
to hybrid compounds with significant activity.14 On the basis of
these observations, we wanted to design and synthesize molecules
which include both coumarin and chalcone pharmacophore in one
frame. In continuation of our efforts to explore the chemical diver-
sity space around coumarin scaffold, we have synthesized diverse
class of coumarin compounds with good pharmacological profile.15
In this Letter, we report here our attempt on antioxidant and anti-
inflammatory evaluation of biscoumarin derivatives. The general
synthetic strategy was employed to prepare biscoumarin–chalcone
hybrid is based on methodology perfected in our lab.16 The
synthetic route used to synthesize title compounds is outlined in
and chronic inflammatory processes. TNF-
chronic inflammation and it can stimulate secretion of cytokines
such as IL-1, IL-6, and IL-10.3 Additionally, TNF-
inhibitors can
be used to cure many diseases like rheumatoid arthritis, Alzhei-
mer’s disease, tumor, and obesity.4 Most of the marketed TNF-
a is a major mediator of
a
a
inhibitors are monoclonal antibodies (Adalimumab, Infliximab,
Cimzia and Golimumab) that exhibit severe side effects. Therefore,
there is a constant need for small molecules that selectively inhibit
TNF-a.
Oxidative stress has been associated with the inflammation
process. Reactive oxygen species like superoxide radical anion,
hydrogen peroxide, and hydroxyl radical, are produced during
the inflammation process by macrophages.5 Thus, the discovery
of molecules which combine anti-inflammatory and antioxidant
q
Part XI in the series, ‘Advances in drug design and discovery’. CDRI publication
no. 8075.
Corresponding author. Tel.: +91 9919317940; fax: +91 522 2623405.
E-mail addresses: sashidhar123@gmail.com, kv_sashidhara@cdri.res.in (K.V.
Sashidhara).
⇑
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.002

K.V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4480–4484
4481
Figure 1. Few naturally occurring anti-inflammatory chalcone.
Scheme 1. Thus, the Pechmann reaction on 1,5-dihydroxynaphthe-
lene (1), furnished 7-hydroxy-4-methylbenzo(h)chromene-2-one
(2) which on the Duff formylation in the presence of hexamethyl-
enetetraamine (HMTA) and TFA at 120 °C gave dicarbaldehyde (3),
which on coupling with different appropriate acetophenones in
refluxing dioxane, in the presence of a catalytic amount of concd
HCl afforded regioselective para-condensed chalcones (4–7) in
good yields. These chalcone derivatives on subsequent Knoevena-
gel-type condensation with different active methylene compounds
furnished biscoumarin–chalcone hybrids in high yields (8–17). It
was confirmed that the synthesized chalcones were in trans-con-
figuration, as evidenced by their coupling constant. Structures of
the compounds were substantiated by 1H NMR, 13C NMR, 2D
NMR, Mass spectrometry and IR spectroscopy. The purity of these
compounds was ascertained by TLC and spectral analysis (Supple-
mentary data).17
planter aponeurosis. The volume of the paw was measured ple-
thysmographically immediately after and 3 h after the injection
of the irritant. The difference in volume gave the amount of oede-
ma developed. Percent inhibition of the oedema between the con-
trol group and compound-treated groups was calculated and
compared with the group receiving a standard drug. Anti-inflam-
matory activity of compounds (8–17) screened is presented in Ta-
ble 1. Out of all compounds evaluated three compounds showed
significant activity. The Compounds 8, 13 and 17 at 100 mg/kg po
exhibited 29%, 26%, and 33% activity, respectively, while the refer-
ence drug, ibuprofen exhibited 59.5% protection at an equivalent
dose. While the compounds 11, 12 and 14 showed moderate activ-
ities. The in vivo examined compounds did not show any toxic ef-
fects in different doses studied and in almost all the cases, the
animals survived (80–90%) and looked normal both macroscopi-
cally and by autopsy, demonstrating low general toxicity of the
synthesized compounds.
Anti-inflammatory activity. In the present study, we carried out
experiments to investigate the anti-inflammatory activity18 of bis-
coumarin–chalcone hybrids (8–17) in carrageenin-induced paw
oedema in albino rats. Oedema in one of the hind paws was
induced by injection of carrageenin solution (0.1 ml of 1%) into
Both coumarin and chalcones are well known inhibitors of TNF-
a 19,20
.
In the next step, the active compounds 8, 13 and 17 were
evaluated for their potential to inhibit TNF- inhibition using
whole blood assay.21 All the said compounds showed TNF-
inhibi-
a
a
Compound
R1
R2
% Yield Compound
R1
R2
% Yield
80
8
85
81
79
86
78
13
14
15
16
17
H3C
H3C
H3C
O
O
O
9
C2H5
CH3
C2H5
CH3
C2H5
CH3
C2H5
CH3
83
80
75
78
10
11
12
S
S
Scheme 1. Synthesis of novel bisccoumarin–chalcone hybrids (8–17). Reagents and conditions: (a) ethyl acetoacetate, p-toluene sulphonic acid, 75 °C, 8 h; (b) (i)
hexamethylenetetramine, trifluoroacetic acid, 120 °C, 4 h; (ii) aq H2SO4, 100 °C, 1 h; (c) R1-COCH3, concd HCl, dioxane, reflux, 5 h; (d) CH2(COOR2)2 , piperidine, EtOH, reflux,
30 min ; (e) ethyl acetoacetate , piperidine, EtOH, reflux, 30 min.

4482
K.V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4480–4484
Table 1
In vivo anti-inflammatory activity of biscoumarin–chalcone hybrids (8–17) in carrageenin-induced paw oedema in albino rats and TNF-
a
inhibition
Compound
Structure
% Anti-inflammatory activitya
% Inhibition TNF-a
O
O
O
O
Dose (
200
400
l
g/mL)
% Inhibition
33
47
8
29
O
O
O
O
O
O
O
O
9
NA
ND
O
O
O
O
10
NA
ND
O
O
O
O
O
O
S
O
O
O
11
12
13
14
8.77
3.5
26
ND
O
S
O
O
O
O
O
ND
O
O
O
O
O
O
O
Dose (
200
400
l
g/mL)
% Inhibition
25
43
O
O
O
O
O
O
O
O
13
ND
O
O
O

K.V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4480–4484
4483
Table 1 (continued)
Compound
Structure
% Anti-inflammatory activitya
% Inhibition TNF-a
O
O
O
O
15
NA
ND
O
O
O
O
O
O
O
O
16
NA
ND
O
O
O
O
O
O
Dose (
l
g/mL)
% Inhibition
17
33
200
400
04
21
O
O
O
O
Ibuprofen
59.5
Pent(5 mM) + LPS (5
lg)
57
NA—Not active.
ND—Not done.
a
Dose 100 mg/kg po.
Compounds
Figure 2. The effect of novel bisccoumarin–chalcone (200
peroxidation in microsomes (nmol MDA formed/mg protein) is shown (standard drugs for superoxide anions—Alloperinol (20
microsomal lipid peroxidation- -tocopherol (100 g/mL) were used). Each value is mean SD of six values.
l
g/mL) on superoxide ion (nmol formazone formed/min), hydroxyl ion (nmol MDA formed/h) and lipid
lg/mL), hydroxyl ions—Manitol and for
a
l
tion in dose dependent manner. Compounds 8, 13 and 17 at a dose
of 400 g/mL showed 47%, 43% and 21% inhibition (Table 1).
Antioxidant activity. Antioxidant activities of compounds 8–17
were evaluated by generating free radicals in vitro in the absence
and presence of these compounds.22 The scavenging potential of
respectively. The scavenging potentials of other derivatives were
modest. The propensity of radical formation and stabilization, abil-
ity of metal complexation and lipophilicity are important factors
for the antioxidant activity. The wide variation in the free radical
scavenging potential for the tested compounds may be due to
the variation in the proton–electron transfer by the derivatives
due to difference in their structures and stability.
In conclusion, a series of novel substituted biscoumarin–chal-
cone hybrids (8–17) have been synthesized in four steps starting
from 1,5-dihydroxynaphthalene. Among the synthesized com-
pounds, compound 17 was found to be the most active anti-inflam-
matory agent in addition to having potent antioxidant activity,
suggesting that the anti-inflammatory property of the compound
might be partly due to its radical scavenging activity. Thus, the
compound 17 provides a useful starting point for the rational
l
biscoumarins–chalcones 8–17 at 200 lg/mL against formation of
OÁ2 and ÅOH in non-enzymic systems were studied. Further, their ef-
fects on lipid peroxidation in microsomes were also studied
(Fig. 2). Compounds 11, 14 and 17 showed significant decrease in
superoxide anions inhibition by 24%, 30% and 29%, hydroxyl radi-
cals inhibition by 26%, 21% and 27% and microsomal lipid peroxida-
tion inhibition by 27%, 24% and 23%, respectively (each value is
mean SD of six values P <0.001). The standard drug Alloperinol
at 20
and
inhibition of hydroxyl ions and microsomal lipid peroxidation,
l
g/mL showed 42% inhibition in superoxide anions. Manitol
a
-tocopherol at the dose of 100 g/mL showed 40% and 41%
l

4484
K.V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4480–4484
Med. Chem. Lett. 2010, 20, 7205; (e) Sashidhara, K. V.; Kumar, A.; Kumar, M.;
Srivastava, A.; Puri, A. Bioorg. Med. Chem. Lett. 2010, 20, 6504; (f) Sashidhara, K.
V.; Kumar, A.; Chatterjee, M.; Rao, K. B.; Singh, S.; Verma, A. K.; Palit, G. Bioorg.
Med. Chem. Lett. 2011, 21, 1937.
design of anti-inflammatory agents with improved pharmacoki-
netics and pharmacodynamics properties.
16. (a) Sashidhara, K. V.; Rosaiah, J. N.; Narender, T. Tetrahedron Lett. 2007, 48,
1699; (b) Sashidhara, K. V.; Rosaiah, J. N.; Kumar, M.; Gara, R. K.; Nayak, L. V.;
Srivastava, K.; Bid, H. K.; Konwar, R. Bioorg. Med. Chem. Lett. 2010, 20, 7127.
Acknowledgments
Instrumentation facilities from SAIF, CDRI and financial support
from CSIR, New Delhi, India to A.K., R.K.M. and S.R. are gratefully
acknowledged. This is CSIR-CDRI contribution number 8075.
17. (E)-Methyl
dihydrochromeno[8,7-h]chromene-2-carboxylate (17). Yellow solid, yield: 78%;
mp 137–138 °C; IR (KBr): 3015, 1726, 1548, 1217, 755 cmÀ1 1H NMR (TFA d,
7-methyl-3,9-dioxo-11-(3-oxo-3-phenylprop-1-enyl)-3,9-
;
300 MHz) d: 9.06 (s, 1H), 8.98 (d, J = 7.7 Hz, 1H), 8.59 (d, J = 4.3 Hz, 1H) 8.06 (br
s, 4H), 7.62–7.49 (m, 4H), 6.71 (s, 1H), 4.05 (s, 3H), 2.65 (s, 3H): 13C NMR (TFA d,
75 MHz) d: 196.8, 164.6, 163.8, 157.5, 152.6, 151.6, 149.9, 148.4, 135.6, 134.5,
131.5, 128.9, 128.5, 126.8, 125.6, 124.6, 123.5, 123.4, 120.8, 119.8, 116.6, 115.2,
114.6, 53.0, 17.6: DEPT 135 (TFA d, 75 MHz) d: 151.6, 148.4, 134.5, 128.9,
Supplementary data
128.5, 126.8, 125.6, 123.4, 119.7, 114.6, 53.0, 17.6 : ESI-MS: (m/z): 467 (M+H)+
:
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.06.002.
HRMS m/z calcd for C28H18O7 (M+H)+ 467.1132, found 467.1103.
18. Winter, C. A.; Risley, E. A.; Nuss, G. W. Proc. Soc. Exp. Biol. Med. 1962, 111, 544.
19. Upadhyay, K.; Bavishi, A.; Thakrar, S.; Radadiya, A.; Vala, H.; Parekh, S.;
Bhavsar, D.; Savant, M.; Parmar, M.; Adlakha, P.; Shah, A. Bioorg. Med. Chem.
Lett. 2011, doi:10.1016/j.bmcl.2011.02.016.
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nitroblue tetrazolium (320
lM) in absence or presence of compounds (8–17)
(200 g/mL) in 100 mM phosphate buffer (pH 8.2). Fractions were sonicated
l
well in phosphate buffer before use. The reaction mixtures were incubated at
37 °C and after 30 min the reaction was stopped by adding 0.5 mL glacial acetic
acid. The amount of formazone formed was calculated spectrophotometrically.
In another set of experiment effect of compounds on the generation of
hydroxyl radical was also studied by non-enzymatic reactants. Briefly, OH-
were generated in a non-enzymatic system comprising deoxy ribose (2.8 mM),
FeSO4Á7H2O (2 mM), sodium ascorbate (2.0 mM) and H2O2 (2.8 mM) in 50 mM
KH2PO4 buffer (pH 7.4) to
mixtures in the absence or presence of test compounds (200
a
final volume of 2.5 mL. The above reaction
g/mL) were
l
incubated at 37 °C for 90 min. The test compounds were also studied for their
inhibitory action against microsomal lipid peroxidation in vitro by non-
enzymatic inducer. Reference tubes and reagents blanks were also run
simultaneously. Malondialdehyde (MDA) contents in both experimental and
reference tubes were estimated spectrophotometrically by thiobarbituric acid
as mentioned (Okhawa, H.; Ohishi, N.; Yagi, K. Anal. Biochem. 1978, 95, 351.).
Alloprinol, Mannitol and
a-tocopherol were used as standard drugs for
superoxide, hydroxylations and microsomal lipid peroxidation. All
experimental data were analyzed using Student’s t-test. Oxidized LDL was
compared with the test compounds treated oxidized LDL. The generation of
oxygen free radicals was compared in the presence and absence of test
compounds.