Novel thiocoumarins as inhibitors of TNF-α induced ICAM-1 expression on human umbilical vein endothelial cells (HUVECs) and microsomal lipid peroxidation

Article abstract of DOI:10.1016/j.bmc.2004.12.013

Different coumarin/thiocoumarin derivatives, that is, 7-hydroxy-4- methylcoumarin, 7,8-dihydroxy-4-methylcoumarin, 7-acetoxy-4-methylcoumarin, 7,8-diacetoxy-4-methylcoumarin, 7-hydroxy-4-methylthiocoumarin, 7,8-dihydroxy-4-methylthiocoumarin, 7-acetoxy-4-methylthiocoumarin and 7,8-diacetoxy-4-methylthiocoumarin were synthesized and evaluated for their effects on TNF-α induced expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells and on NADPH-catalyzed rat liver microsomal lipid peroxidation with a view to identify modulators for expression of cell adhesion molecules and to establish structure-activity relationship. We found that dihydroxy and diacetoxy derivatives of thiocoumarin were more potent in comparison to the corresponding coumarin derivatives in inhibiting TNF-α-induced expression of ICAM-1. However, coumarin derivatives were found to be more potent in comparison to the corresponding thiocoumarins in inhibiting microsomal lipid peroxidation. We have also tested the intermediate compounds 7,8-dibenzyloxy-4-methylcoumarin and 7,8-dibenzyloxy-4- methylthiocoumarin for their inhibitory activity on TNF-α-induced ICMA-1 expression. We found that dibenzyloxy-4-methylthiocoumarin is better than dibenzyloxy-4-methylcoumarin. The mechanisms underlying the observed activities of coumarins and thiocoumarins have been discussed with reference to their structures. Such structure-function relationship studies may help in developing molecules with better anti-inflammatory and anti-oxidant activities.

Full text of DOI:10.1016/j.bmc.2004.12.013

Bioorganic & Medicinal Chemistry 13 (2005) 1605–1613
Novel thiocoumarins as inhibitors of TNF-a induced
ICAM-1 expression on human umbilical vein endothelial
cells (HUVECs) and microsomal lipid peroxidationq,qq
Sarvesh Kumar,a Brajendra K. Singh,b Neerja Kalra,b Vineet Kumar,b Ajit Kumar,c
Ashok K. Prasad,b Hanumantharao G. Raj,c Virinder S. Parmarb and Balaram Ghosha,*
aMolecular Immunogenetics Laboratory, Institute of Genomics and Integrative Biology, Mall Road, Delhi-110 007, India
bBioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi-110 007, India
cDepartment of Biochemistry, V.P. Chest Institute, University of Delhi, Delhi-110 007, India
Received 28 October 2004; revised 7 December 2004; accepted 8 December 2004
Abstract—Different coumarin/thiocoumarin derivatives, that is, 7-hydroxy-4-methylcoumarin, 7,8-dihydroxy-4-methylcoumarin, 7-
acetoxy-4-methylcoumarin, 7,8-diacetoxy-4-methylcoumarin, 7-hydroxy-4-methylthiocoumarin, 7,8-dihydroxy-4-methylthiocouma-
rin, 7-acetoxy-4-methylthiocoumarin and 7,8-diacetoxy-4-methylthiocoumarin were synthesized and evaluated for their effects on
TNF-a induced expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells and on NADPH-catalyzed rat liver
microsomal lipid peroxidation with a view to identify modulators for expression of cell adhesion molecules and to establish struc-
ture–activity relationship. We found that dihydroxy and diacetoxy derivatives of thiocoumarin were more potent in comparison to
the corresponding coumarin derivatives in inhibiting TNF-a-induced expression of ICAM-1. However, coumarin derivatives were
found to be more potent in comparison to the corresponding thiocoumarins in inhibiting microsomal lipid peroxidation. We have
also tested the intermediate compounds 7,8-dibenzyloxy-4-methylcoumarin and 7,8-dibenzyloxy-4-methylthiocoumarin for their
inhibitory activity on TNF-a-induced ICMA-1 expression. We found that dibenzyloxy-4-methylthiocoumarin is better than dibenz-
yloxy-4-methylcoumarin. The mechanisms underlying the observed activities of coumarins and thiocoumarins have been discussed
with reference to their structures. Such structure–function relationship studies may help in developing molecules with better anti-
inflammatory and anti-oxidant activities.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
kocytes requires an increased expression of cell adhesion
molecules on the surface of endothelial cells that interact
with their corresponding receptors on the surface of leu-
kocytes.1 Various inflammatory mediators, for example,
cytokines like TNF-a, IL-1b and bacterial lipopolysac-
charides increase the expression of cell adhesion mole-
cules, namely intercellular adhesion molecule-1
(ICAM-1), vascular cell adhesion molecule-1 (VCAM-
1) and E-selectin on the endothelial cells.2–5 The in-
creased expression of cell adhesion molecules alters the
adhesive property of the vasculature leading to indis-
criminate infiltration of the leukocytes across the blood
vessels and hence causes inflammation. A regulated
expression of cell adhesion molecules is therefore essen-
tial for maintaining the body fluidity. Inhibition of cell
adhesion molecules has shown to be a useful therapeutic
approach to regulate inflammatory response. Monoclo-
nal antibodies (mAbs) specific to cell adhesion molecules
and small molecules from natural sources and synthetic
The adhesion of leukocytes to the endothelium is among
the earliest and essential processes during any inflamma-
tory response. The transendothelial migration of the leu-
Abbreviations: ICAM-1, Intercellular adhesion molecule-1; TNF-a,
Tumor necrosis factor-a; NADPH, Nicotinamide adenine dinucleotide
phosphate; NF-jB, Nuclear factor-jB.
Keywords: Coumarins; Thiocoumarins; TNF-a; ICAM-1; Anti-oxi-
dant; Lipid peroxidation.
q Council of Scientific and Industrial Research, New Delhi, India,
supported this work.
qq Reprint requests may be sent to Dr. B. Ghosh, Molecular
Immunogenetics Laboratory, Institute of Genomics and Integrative
Biology, University of Delhi Campus (North), Mall Road, Delhi
1100 07, India.
*
Corresponding author. Tel.: +91 11 2766 2580; fax: +91 11 2766
7471; e-mail: bghosh@igib.res.in
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.12.013

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S. Kumar et al. / Bioorg. Med. Chem. 13 (2005) 1605–1613
routes have been used successfully for downregulating
the induced expression of cell adhesion molecules both
in vitro and in vivo.6–8 However, treatment with mAbs
is found to be limited because of problems with endo-
toxin contamination, secondary antibody formation,
serum sickness and anaphylaxis.9
agent.25,26 The thio-analogue 6 of coumarin 1 was ob-
tained in good yield, but because of the poor solubility
of coumarin 2 in toluene, its thio-analogue was obtained
in very poor yield. To improve the solubility of 7,8-dihy-
droxy-4-methylcoumarin (2), the two hydroxyl groups
were protected with benzyl group by refluxing 2 with
K2CO3 and BnBr in acetone.27 The dibenzyl derivative
5 was converted to its thio-analogue 7, which was debenz-
ylated by using AlCl3 and N,N-dimethylaniline28 to
afford the dihydroxythiocoumarin 8. The corresponding
acetates, 7-acetoxy-4-methylthiocoumarin (9) and 7,8-
diacetoxy-4-methylthiocoumarin (10) were synthesized
by the acetylation of 6 and 8, respectively, by the acetic
anhydride–pyridine–DMAP method.
Coumarins belong to class of polyphenolic compounds
that abundantly occur in the plant kingdom. Many com-
pounds of this class are reported to exhibit different bio-
logical activities,10 for example, 4-methylcoumarins
have been found to possess choleretic,11 analgesic,12
anti-spermatogenic13 and diuretic properties.14 7,8-
Dihydroxy-4-methylcoumarin and 7,8-diacetoxy-4-
methylcoumarin were reported to exhibit anti-oxidant
properties on three counts: (i) efficient scavenging of
the oxygen radicals,15 (ii) prevention of the formation
of ADP-perferryl leading to the cessation of the forma-
tion of the oxygen radicals16 and (iii) inhibition of cyto-
chrome P-450-linked mixed function oxidases.17 The
simple coumarins/thiones exhibit hypnotic,18 hypother-
mal19 and insecticidal activities.20
2.1. Coumarin and thiocoumarin derivatives inhibit the
TNF-a-induced expression of ICAM-1 on endothelial
cells
The effect of 10 different coumarins and thiocoumarins
1–10 (Fig. 1) has been examined on the modulation of
cytokine-induced expression of ICAM-1 in human
endothelial cells (Fig. 2). The endothelial cells plated
to confluence in 96 well plates were incubated with vary-
ing concentrations of these compounds (Table 1). The
effects of these compounds on the viability (determined
by trypan blue exclusion test) and the morphology of
the endothelial cells (observed under microscope) were
also examined. The maximal tolerable concentrations
were found to be different for different compounds.
For further analysis, the concentrations at the maximal
tolerable range were used. The effects of the compounds
1–10 on TNF-a-induced ICAM-1 expression were seen
using cell-ELISA as detailed in the Experimental
Section.
Although several activities of coumarins/thiocoumarins
have been reported, but not much is known regarding
their effects on cytokine-induced cell adhesion molecule
expression, which is involved in many inflammatory
conditions. Many compounds that inhibit lipid peroxi-
dation and influence the generation of ROS, in turn lead
to decrease in the expression of the cell adhesion mole-
cules and subsequently decrease inflammation, hence
are found to be useful therapeutic agents in various
inflammatory diseases.21 Treatment of endothelial cells
with anti-oxidants is also shown to downregulate the
expression of ICAM-1 on endothelial cells.22 In the pres-
ent study, for understanding the mechanisms underlying
the anti-inflammatory activities of compounds of this
class and for establishing structure–activity relationship,
we have synthesized several oxygenated coumarins and
corresponding thiocoumarins and have studied their
inhibitory activities on TNF-a-induced expression of
ICAM-1 on HUVECs, and on NADPH-catalyzed liver
microsomal lipid peroxidation.
Our results using cell-ELISA demonstrate that ICAM-1
was expressed at low levels on unstimulated endothelial
cells and there was over fivefold increase in its expres-
sion upon stimulation with TNF-a (data not shown).
Pretreatment of endothelial cells with 1–10 had no effect
on the constitutively expressed levels of ICAM-1 (data
not shown), while they had varying effects on TNF-a in-
duced ICAM-1 expression (Table 1). As the maximum
tolerable concentrations used in these experiments are
to some degree different, a direct comparison cannot
be made. However, it has been found that the two thio-
coumarins, 7,8-dihydroxy-4-methylthiocoumarin (8)
and 7,8-diacetoxy-4-methylthiocoumarin (10) inhibited
the TNF-a induced ICAM-1 expression by 92% and
82%, respectively, at a concentration of 100 lg/mL.
The inhibition of ICAM-1 expression by 7,8-dihy-
droxy-4-methylcoumarin (2) and 7,8-diacetoxy-4-meth-
ylcoumarin (4) at concentrations of 100 and 17.5 lg/
mL (maximum tolerable concentrations) was 75% and
60%, respectively, which is less than the corresponding
thiocoumarins 8 and 10 (Table 1). The monohydroxy
and corresponding monoacetoxy coumarins 1 and 3
and thiocoumarins 6 and 9 did not show appreciable
inhibition of TNF-a-induced ICAM-1 expression at
the maximum tolerable concentration, viz. 100 lg/mL
(Table 1). We have also tested the intermediate
compounds 7,8-dibenzyloxy-4-methylcoumarin (5) and
2. Results
7-Hydroxy-4-methylcoumarin (1) and 7,8-dihydroxy-4-
methylcoumarin (2) were synthesized in quantitative
yields by Pechmann23 condensation of resorcinol and
pyrogallol, respectively, with ethyl acetoacetate in the
presence of concentrated sulfuric acid. The correspond-
ing acetates, that is, 7-acetoxy-4-methylcoumarin (3)
and 7,8-diacetoxy-4-methylcoumarin (4) were synthe-
sized by acetylation of compounds 1 and 2 with acetic
anhydride in pyridine in the presence of catalytic
amount of dimethylaminopyridine (DMAP) in quantita-
tive yields. The structures of coumarins 1–4 were estab-
lished on the basis of their spectral (1H, 13C NMR and
mass) data and comparison of their melting points and
spectral data with those reported in the literature.17,24
Thionation of the coumarins 1 and 2 was attempted
by refluxing them in toluene with LawessonÕs re-

S. Kumar et al. / Bioorg. Med. Chem. 13 (2005) 1605–1613
OH
1607
1
8
HO
O
O
AcO
O
O
HO
O
O
3
6
5
CH3
CH3
CH3
1
3
2
OAc
OBn
AcO
O
O
HO
O
S
BnO
O
O
CH3
CH3
6
4
5
CH3
OH
OBn
AcO
O
S
HO
O
S
BnO
O
S
CH3
CH3
CH3
9
7
8
OAc
AcO
O
S
CH3
10
Figure 1. Structures of coumarin and thiocoumarin derivatives.
Figure 2. Comparison of inhibition of NADPH catalyzed initiation of lipid peroxidation and TNF-a induced ICAM-1 expression on endothelial
cells by coumarins and thiocoumarins.
7,8-dibenzyloxy-4-methylthiocoumarin (7) for their
inhibitory activity on TNF-a induced ICMA-1 expres-
sion. Compound 7 was found to inhibit the ICAM-1
expression by 45%, whereas 5 inhibited the ICAM-1
expression by 30%, thus showing that the intermediate
compound dibenzyloxy-4-methylthiocoumarin is better
than dibenzyloxy-4-methylcoumarin (Table 1).
2.2. Coumarin and thiocoumarin derivatives inhibit lipid
peroxidation
Reactive oxygen species are primary signaling molecules
in regulating the expression of ICAM-1 on endothelial
cells and hence play an important role in various inflam-
matory diseases. We have observed that the coumarin

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S. Kumar et al. / Bioorg. Med. Chem. 13 (2005) 1605–1613
Table 1. Effect of coumarins 1–5 and thiocoumarins 6–10 on the TNF-a-induced expression of ICAM-1 on endothelial cells
Compounds
Concentrationa
% Inhibition
lg/mL
lM
7-Hydroxy-4-methylcoumarin (1)
7,8-Dihydroxy-4-methylcoumarin (2)
7-Acetoxy-4-methylcoumarin (3)
100
100
100
17.5
30
568.2
520.8
458.7
63.3
7
75
5
7,8-Diacetoxy-4-methylcoumarin (4)
7,8-Dibenzyloxy-4-methylcoumarin (5)
7-Hydroxy-4-methylthiocoumarin (6)
7,8-Dibenzyloxy-4-methylthiocoumarin (7)
7,8-Dihydroxy-4-methylthiocoumarin (8)
7-Acetoxy-4-methylthiocoumarin (9)
7,8-Diacetoxy-4-methylthiocoumarin (10)
60
30
10
45
92
7
75.9
100
30
520.8
77.09
478.4
427.3
341.2
100
100
100
82
The data presented are representative of three independent experiments. Values shown are mean SD of quadruplicate wells.
a The concentration levels of different compounds are based on their maximum tolerable concentration by the cells.
and thiocoumarin derivatives 1–10 inhibit ICAM-1
expression, while some of these compounds viz. 1–4, 6
and 8–10 also inhibit lipid peroxidation.
methylthiocoumarin (10) to terminate the lipid peroxi-
dation. The compounds were added to the reaction mix-
ture of lipid peroxidation at different time intervals after
initiation of the reaction (Table 3). 7,8-Dihydroxy- and
7,8-diacetoxy-4-methylthiocoumarins 8 and 10 were
found to terminate the lipid peroxidation up to an extent
of 47% and 45%, respectively, when added after 30 min
of initiation of lipid peroxidation. This simply demon-
strated the excellent anti-oxidant activity of dihydroxy-
and diactetoxy-4-methylthiocoumarins.
We have already reported the inhibitory effects of cou-
marin derivatives 2–4 on lipid peroxidation.15 It has
been found that 7,8-dihydroxy-4-methylcoumarin (2)
and 7,8-diacetoxy-4-methylcoumarin (4) profoundly in-
hibit the rat liver microsomal lipid peroxidation (Table
2). The monohydroxycoumarin 1 and its acetyl deriva-
tive 3 did not have any significant microsomal lipid per-
oxidation inhibitory activity. Herein we have examined
the effect of thiocoumarins 6, 8, 9 and 10 on initiation
of lipid peroxidation in rat liver microsomes. The results
presented in Table 2 illustrate the influence of these com-
pounds on the enzymatic initiation of lipid peroxidation.
Again as in the case of coumarins, the monohydroxy 4-
methylthiocoumarin 6 and monoacetoxy 4-methylthio-
coumarin 9 were found to exhibit insignificant inhibition
of initiation of lipid peroxidation in rat liver micro-
somes. However, the other two thiocoumarins, that is,
7,8-dihydroxy-4-methylthiocoumarin (8) and 7,8-diacet-
oxy-4-methylthiocoumarin (10) exhibited much effective
inhibition of lipid peroxidation in rat liver microsomes,
both these compounds inhibited rat liver microsomal
lipid peroxidation initiated enzymatically by the addi-
tion of NADPH to the reaction mixture to an extent
of 60% (Table 2).
The radical scavenging abilities of 4-methylthiocouma-
rin derivatives 6, 8, 9 and 10 based on the determination
of drop in the absorption of stable radical of DPPH
(1,1-diphenyl-1-picrylhydrazyl) have been determined
and the results are documented in Table 4; dihydroxy-
4-methylthiocoumarin (8) scavenged DPPH to the
extent of 99.97% at a concentration of 100 lM. The
Table 3. Termination of lipid peroxidation initiation by 4-
methylthiocoumarins
Compound
Time (min)
15
9.75
5
10
6.94
30
6
3.50
11.10
46.79
10.75
44.66
8
13.21
3.23
13.51
26.73
5.57
25.67
37.23
9.25
39.61
9
10
The data presented are representative of three independent
experiments.
Efforts were made to evaluate the activity of 7,8-dihy-
droxy-4-methylthiocoumarin (8) and 7,8-diacetoxy-4-
Table 2. Effect of 4-methylcoumarin and thiocoumarin derivatives (at 100 lM concentration) on NADPH-catalyzed rat liver microsomal lipid
peroxidation initiation
Compound
% Inhibition of NADPH-catalyzed lipid peroxidation
Ratio ICAM-1/lipid peroxidation
7-Hydroxy-4-methylcoumarin (1)
7,8-Dihydroxy-4-methylcoumarin (2)
7-Acetoxy-4-methylcoumarin (3)
8
89a
0a
87a
11
60
14
59
0.875
0.843
0
7,8-Diacetoxy-4-methylcoumarin (4)
7-Hydroxy-4-methylthiocoumarin (6)
7,8-Dihydroxy-4-methylthiocoumarin (8)
7-Acetoxy-4-methylthiocoumarin (9)
7,8-Diacetoxy-4-methylthiocoumarin(10)
0.689
0.909
1.53
0.5
1.39
The data presented are representative of three independent experiments.
a This data has been reported by us previously.15

S. Kumar et al. / Bioorg. Med. Chem. 13 (2005) 1605–1613
1609
Table 4. Radical scavenging potential of 4-methylthiocoumarins at
100 lM concentration
compared to monohydroxy compounds in both, the
coumarin and thiocoumarin series. This indicates that
increase in the number of hydroxy groups on the couma-
rin or thiocoumarin nucleus enhances the anti-oxidant
as well as ICAM-1 inhibitory activity. The activity of
dihydroxy and diacetoxy coumarin and thiocoumarin
derivatives may be because of two reasons: (a) facile oxi-
disable nature of such compounds resulting in the for-
mation of quinones having stable quinonoid structures
(Fig. 3), and (b) the ability to form stable phenoxy rad-
icals. This proposition is further supported by the obser-
vation that dihydroxythiocoumarins have better activity
as compared to diacetoxythiocoumarins, this may be
due to the higher rate constant for the formation of
phenoxyl redical by hydroxy derivatives as compare to
acetoxy derivatives. We propose that these compounds
inhibit NADPH-catalyzed liver microsomal lipid perox-
idation and ICAM-1 expression on human endothelial
cells by getting oxidized to quinones. A similar trend
of the two activities (Fig. 2) supports the proposition
that hydroxycoumarins and hydroxythiocoumarins,
particularly those that can lead to the formation of sta-
ble quinonoid structures are more active in the present
system of investigation. We have also tested the interme-
diate compounds 7,8-dibenzyloxy-4-methylocoumarin
(5) and 7,8-dibenzyloxy-4-methylthiocoumarin (7) and
found that comparatively they show much less activity.
The possible reason for their less activity may be that
they are not capable of forming the stable quinonoid
forms as the benzyl moieties, being joined through ether
linkages cannot be converted into free phenolic ana-
logues in the animal systems (Fig. 3). In Figure 3, acetyl
derivatives first transformed to hydroxy derivatives be-
fore forming the stable quinonoid form.29 Investigations
by several groups of researchers on polyphenolic com-
pounds demonstrated the fact that the presence of phe-
nolic groups contributes greatly to the anti-oxidant
potential of the compounds30–32 as observed by us in
present study (Table 2). It is noteworthy that dihydroxy
compounds are more effective as compared to diacetoxy
compounds.
Compound
Scavenging of
DPPH (in %)a
7-Hydroxy-4-methylthiocoumarin (6)
7,8-Dihydroxy-4-methylthiocoumarin (8)
7-Acetoxy-4-methylthiocoumarin (9)
7,8-Diacetoxy-4-methylthiocoumarin (10)
23.28
99.97
14.38
47.51
The data presented are representative of three independent experi-
ments.
a The decolourization of DPPH by various test compounds was carried
out as described in Experimental Section.
radical scavenging activity of 7,8-diacetoxy-4-methyl-
thiocoumarin (10) was found to be almost half of the
activity of thiocoumarin 8, 7-hydroxy- and 7-acetoxy-
4-methylthiocoumarins (6) and (9) did not exhibit
appreciable activity at a concentration of 100 lM.
It has been observed that dihydroxy and diacetoxy thio-
coumarins 8 and 10 were more potent in comparison
to the corresponding coumarin analogues 2 and 4 in
inhibiting TNF-a induced expression of ICAM-1. How-
ever, coumarins 2 and 4 were found to be more potent in
comparison to the thiocoumarins 8 and 10 in inhibiting
microsomal lipid peroxidation. In separate set of experi-
ments, we have observed that thiocoumarin acetates
were more potent in inhibiting protein kinase C (PKC)
catalyzed by the purified rat liver microsomal transacet-
ylase (TAase) (unpublished data). Also, thiocoumarin
acetates exhibited high specificities with TAsae and
hence highly effective in causing TAase mediated biolog-
ical action (unpublished data). As PKC is associated in
the activation of TNF-a induced ICAM-1 expression,
thiocoumarins could be more potent in inhibiting
ICAM-1 expression than their corresponding coumarin
derivatives. The monohydroxy and monoacetoxy cou-
marins 1 and 3, and thiocoumarins 6 and 9 did not ex-
hibit appreciable activity either for the inhibition of
TNF-a-induced expression of ICAM-1 or for the inhibi-
tion of NADPH-catalyzed rat liver microsomal lipid
peroxidation. Although the inhibition patterns of
NADPH-catalyzed lipid peroxidation and TNF-a-in-
duced ICAM-1 expression follow very similar trend,
however, we have noted that dihydroxy- and diacet-
oxythiocoumarins 8 and 10 are more potent in inhibiting
TNF-a-induced ICAM-1 expression (Fig. 2).
Our earlier work has demonstrated that the initiating
reactive oxygen radical interacts with the acetoxy group
of 7,8-diacetoxy-4-methylcoumarin (4) leading to the
formation of phenoxy radical with the possible loss of
acetyl carbocation.33 This mechanism seems to be true
in the case of thiocoumarin derivatives as well and there-
by renders them good anti-oxidants. Recent studies
suggest that increased circulating lipid peroxides in
pre-eclemptic women are responsible for increased
ICAM-1 expression on endothelial cells,34 so the com-
pounds which can inhibit the lipid peroxidation, could
also inhibit the ICAM-1 expression on endothelial
cells,35 our results support the earlier observations. As
the formation of reactive oxygen intermediates and acti-
vation of cell adhesion molecules is involved in various
other pathways involved in inflammation, the results re-
ported here may explain the mechanism underlying the
observed activities of coumarins and thiocoumarins.
Such structure–function relationship studies can help
in developing better molecules with anti-oxidant and
anti-inflammatory activities.
3. Discussion
In the present investigation, coumarins 1–5 and thio-
coumarins 6–10 have been evaluated for their ability
to modulate TNF-a induced ICAM-1 expression and
for the inhibition and termination of initiation and
propagation steps of NADPH-catalyzed microsomal
lipid peroxidation, respectively, in order to examine
their anti-oxidant property. As shown in Tables 1 and
2, hydroxy substituent on the coumarin and thiocouma-
rin nucleus is required for both, anti-oxidant activity
and ICAM-1 inhibitory activity. For example, dihy-
droxy compounds showed high inhibitory activity as

1610
S. Kumar et al. / Bioorg. Med. Chem. 13 (2005) 1605–1613
OH
O
O
O
O
O
HO
HO
O
O
O
[O]
[O]
Quinonoid form I
Quinonoid form not possible
OH
OCOCH3
O
O
O
HO
HO
H3COCO
AcO
O
Enzymatic
[O]
I
Deacetylation
O
O
O
O
Enzymatic
[O]
Quinonoid form
not possible
deacetylation
OH
O
O
S
O
HO
HO
O
S
[O]
[O]
Quinonoid form II
O
S
Quinonoid form not possible
OH
OCOCH3
O
O
O
S
HO
HO
H3COCO
H3COCO
S
[O]
II
Enzymatic
deacetylation
S
O
S
[O]
Enzymatic
Quinonoid form
not possible
deacetylation
Figure 3. Proposed mechanism of conversion of coumarins and thiocoumarins to their respective quinonoid forms.
4. Experimental section
and anti-mouse IgG-HRP were purchased from Sigma
Chemical Co., USA. Foetal calf serum was purchased
from Biological Industries, Israel. NADPH, ADP and
trichloroacetic acid (TCA) were obtained from Sisco Re-
search Laboratory (Mumbai, India).
4.1. Chemicals
The organic solvents (acetone, toluene, CH2Cl2, pyr-
idine) were dried and distilled prior to their use. Analyt-
ical TLCs were performed on precoated Merck silica gel
60 F254 plates; the spots were visualized under UV light.
Melting points were determined in a sulfuric acid bath
and are uncorrected. The IR spectra were recorded on
a Perkin–Elmer model 2000 FT-IR spectrophotometer.
4.2. 7,8-Dibenzyloxy-4-methylcoumarin (5)
To a mixture of 7,8-dihydroxy-4-methylcoumarin (2)
(8 g, 42.0 mmol) and benzyl bromide (15.7 g, 10.9 mL)
in dry acetone (100 mL) was added anhydrous K2CO3
(17.3 g) and the reaction mixture was refluxed for 10 h.
The progress of the reaction was monitored by TLC.
On completion, the solvent was removed under vacuum
and the residue was poured into ice-cold water (75 mL).
The aqueous reaction mixture was extracted with ethyl
acetate (3 · 50 mL) and the combined organic layer
was dried over anhydrous Na2SO4 and concentrated un-
der reduced pressure. The residue, thus obtained was
washed with petroleum ether and purified by column
chromatography on silica gel using gradient solvent
1
The H NMR and 13C NMR spectra were recorded on
a Bruker Avance instrument at 300 and 75.5 MHz,
respectively, using TMS as internal standard. The chem-
ical shift values are on d scale and the coupling constant
values (J) are in Hz. The HRMS were recorded on a
TMS-AX 505 W instrument. Anti-ICAM-1 antibody
and TNF-a were purchased from Pharmingen, USA.
M199, L-glutamine, penicillin, streptomycin, amphoteri-
cin, endothelial cell growth factor, trypsin, Pucks saline,
HEPES, DMSO, o-phenylenediamine dihydrochloride

S. Kumar et al. / Bioorg. Med. Chem. 13 (2005) 1605–1613
1611
system of ethyl acetate and petroleum ether to afford
7,8-dibenzyloxy-4-methylcoumarin (5) as a white solid
(13.0 g) in 84% yield, mp 152 ꢁC; Rf: 0.46 (petroleum
ether/ethyl acetate, 4:1); IR (KBr): 2925, 2361, 1712
fied by column chromatography using gradient system
of ethyl acetate–petroleum ether to afford 7,8-dihy-
droxy-4-methylthiocoumarin (8) as a yellow solid
(0.30 g) in 56% yield, mp 230–235 ꢁC; Rf: 0.48 (petro-
leum ether/ethyl acetate, 3:2); IR (KBr): 3312 (OH),
1
(C@O), 1607, 1293, 1088, 981 and 697 cmÀ1; H NMR
1
(CDCl3, 300 MHz): d 2.35 (3H, s, CH3), 5.17 and 5.19
(4H, 2s, 2H each, 2 · OCH2C6H5), 6.13 (1H, s, C-3H),
6.90 (1H, d, J = 8.9 Hz, C-6H), 7.21–7.51 (11H, m, C-
5H and 2 · OCH2C6H5); 13C NMR (CDCl3,
75.5 MHz): 20.01 (CH3), 72.63 and 76.94 (2 · O–CH2–
C6H5), 111.38 (C-6), 113.95 (C-3), 116.43 (C-10),
120.79 (C-5), 128.69, 129.40, 129.52, 129.55, 129.97
( 2 · OCH2C6H5), 137.56 and 138.42 (C-7 and C-8),
153.7 (C-4), 156.06 (C-9) and 161.78 (C-2). HRMS calcd
for C24H20O4 [M+Na]+ 395.1254, found 395.1243.
2925, 2855, 1561, 1434, 1366, 1254 and 1095 cmÀ1; H
NMR (DMSO-d6 + CDCl3, 300 MHz): d 2.34 (3H, s,
CH3), 6.95 (1H, d, J = 8.6Hz, C-6H), 7.02 (1H, s, C-
3H), 7.09 (1H, d, J = 8.6 Hz, C-5H), 8.83 and 8.94
(2H, 2s, 1H each, 2 · OH); 13C NMR (DMSO-
d6 + CDCl3, 75.5 MHz): d 20.68 (CH3), 116.42 (C-6),
117.72 (C-10), 118.07 (C-5), 118.31 (C-3), 134.23 (C-4),
148.55 and 148.93 (C-7 and C-8), 151.96 (C-9) and
199.31 (C-2); HRMS calcd for C10H9O3S [M+H]+
209.0272, found 209.0260.
4.3. General procedure for the thionation of coumarins 1
and 5
4.6. General procedure for the acetylation of thiocouma-
rins 6 and 8
A mixture of coumarin 1 or 5 (5 g, 28.4 or 13.4 mmol)
and LawessonÕs reagent25,26 (5.7 or 2.71 g, 14.2–
6.7 mmol) was refluxed in toluene (40 mL) for 24 h.
The progress of the reaction was monitored by TLC.
On completion, the reaction mixture was allowed to cool
to room temperature and the solvent evaporated to dry-
ness in vacuum, the residue was purified by column
chromatography on silica gel using gradient solvent
system of ethyl acetate–petroleum ether to afford 7-
hydroxy-4-methylthiocoumarin (6) and 7,8-dibenzyl-
oxy-4-methylthiocoumarin (7) as yellow solids in 44%
and 29% yields, respectively. 7-Hydroxy-4-methylthio-
coumarin (6) was identified on the basis of the spectro-
scopic data, which was found to be identical with the
spectroscopic data reported in the literature.36
To a solution of the thiocoumarin 6 or 8 (0.2 g, 1.04 or
0.96 mmol) in acetic anhydride (1.1 equiv) and pyridine
(2 equiv) was added a catalytic amount of DMAP and
the reaction mixture was stirred at 25–28 ꢁC for 6–8 h.
The progress of the reaction was monitored by TLC.
After completion, the reaction mixture was poured into
ice-cold water, the solid that precipitated was filtered
and washed with petroleum ether, dried and recrystal-
lized from CHCl3 to afford the corresponding acetates
9 and 10 in 54 or 83% yields, respectively. The structure
of monoacetoxycoumarin 9 was established on the basis
of physical and spectral data analysis and by comparing
it with the data reported in the literature.36
4.7. 7,8-Diacetoxy-4-methylthiocoumarin (10)
4.4. 7,8-Dibenzyloxy-4-methylthiocoumarin (7)
It was obtained as a yellow solid (0.15 g) in 83% yield,
mp 190–195 ꢁC; Rf: 0.52 (petroleum ether/ethyl acetate,
2:1); IR (KBr): 2925, 2854, 2361, 1774 (C@O), 1562,
It was obtained as a yellow solid (1.5 g) in 29% yield, mp
130–135 ꢁC; Rf: (petroleum ether/ethyl acetate, 4:1); IR
1
1431, 1376, 1292, 1163, 1096, 1015 and 873 cmÀ1; H
(KBr): 2924, 1596, 1378, 1103, 965 cmÀ1
;
1H NMR
NMR (CDCl3, 300 MHz): d 2.34 and 2.36 (6H, 2s, 3H
each, 2 · OCOCH3), d 2.43 (3H, s, CH3), 7.12 (1H, s,
C-3H), 7.20 (1H, d, J = 8.5Hz, C-5H) and 7.50 (1H, d,
J = 8.5 Hz, C-6H); 13C NMR (CDCl3, 75.5 MHz): d
18.36 (CH3), 20.65 and 21.00 (2 · OCOCH3), 120.04
(C-6), 120.82 (C-10), 121.79 (C-5), 129.47 (C-3), 143.16
(C-7 and C-8), 145.77 (C-4 and C-9), 167.75 and
167.93 (2 · C@O) and 196.15 (C@S); HRMS calcd for
C14H13O5S [M+H]+ 293.0484, found 293.0514.
(CDCl3, 300 MHz): d 2.27 (3H, s, CH3), 5.19 and 5.25
(4H, 2s, 2H each, 2 · OCH2C6H5) and 6.92–7.54 (13H,
m, C-3H, C-5H, C-6H and 2 · OCH2C6H5); 13C NMR
(CDCl3, 75.5 MHz): 18.39 (CH3), 71.77 and 76.20
(2 · O–CH2–C6H5), 111.74 (C-6), 116.99 (C-10), 119.77
(C-5), 127.40, 127.73, 128.55, 128.64, 129.07, 129.27
(C-3, 2 · O–CH2–C6H5), 136.45, 137.26 (C-7, C-8),
144.61 (C-4), 155.29 (C-9), 197.16 (C-2); HRMS calcd
for C24H21O3S [M+H]+ 389.1211, found 389.1221.
4.8. Cells and cell culture
4.5. Synthesis of 7,8-Dihydroxy-4-methylthiocoumarin (8)
The primary endothelial cells were isolated from the
umbilical cord by mild trypsinisation.8 Cells were main-
tained in gelatin coated tissue culture flasks in M 199
medium supplemented with 20% heat inactivated foetal
calf serum, 2 mM L-glutamine, 100 units/mL penicillin,
100 lg/mL streptomycin, 0.25 lg/mL amphotericin,
endothelial cell growth factor (50 lg/mL) and heparin
(5 U/mL). The cells were sub-cultured by dislodging
with 0.125% trypsin–0.01 M EDTA solution in Pucks
saline and HEPES buffer. For the present analysis, cells
were used between passages three to four and the viabil-
ity of cells was determined by trypan blue exclusion test.
To a solution of the thiocoumarin 7 (1.0 g, 2.58 mmol)
and N,N-dimethylaniline (1.25 g, 10.32 mmol) in
CH2Cl2 (20 mL), powdered AlCl3 (1.03 g, 7.74 mmol)
was added in portions. The reaction mixture was stirred
at room temperature for 45 min and on completion, the
reaction was quenched by the addition of 1 N HCl
(4.5 mL). The aqueous reaction mixture was extracted
with ethyl acetate (3 · 30 mL), combined organic layer
was washed with 5% NaHCO3 solution, dried over
anhydrous Na2SO4 and the solvent concentrated under
reduced pressure. The residue, thus obtained was puri-

1612
S. Kumar et al. / Bioorg. Med. Chem. 13 (2005) 1605–1613
E-selectin expression was employed to determine the
purity of endothelial cells.
4.12. Assay of DPPH radical scavenging
A solution of test compounds in methanol (4 mL) at
concentration of 100 lM was added to 1 mL of DPPH
solution in methanol (0.15 mM). The contents were vig-
orously mixed and allowed to stand at 20 ꢁC for 30 min
and the absorbance was taken at 517 nm.
4.9. Modified ELISA for measurement of ICAM-1
The expression of ICAM-1 on surface of endothelial
cells was quantified using cell-ELISA.8 Endothelial cells
plated to confluence in gelatin coated 96 well plates were
incubated with or without compounds at desired con-
centrations for 1 h, followed by treatment with TNF-a
(10 ng/mL) for 16 h. The cells were fixed with 1.0% glu-
taraldehyde and nonspecific binding of antibody was
blocked by using non-fat dry milk (3.0% in PBS). The
cells were incubated overnight at 40 ꢁC with ICAM-1
mAb or control IgG Ab (0.25 lg/mL, diluted in block-
ing buffer), followed by washing with PBS and incuba-
tion with peroxidase-conjugated goat anti-mouse
secondary Ab (1:1000 diluted in PBS). The cells were
again washed with PBS and exposed to the peroxidase
substrate (ortho-phenylenediamine dihydrochloride
40 mg/100 mL in citrate phosphate buffer, pH 4.5), 2 N
sulfuric acid was added to stop the reaction and absor-
bance at 490 nm was measured using an automated
microplate reader (Spectramax 190, Molecular Devices,
USA).
Acknowledgements
The financial assistance from the Council of Scientific
and Industrial Research (CSIR), New Delhi, Govt. of
India, is gratefully acknowledged. S.K., N.K. and
V.K. thank the Council of Scientific and Industrial
Research (CSIR), New Delhi, for the award of
Fellowships.
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