Novel natural product-based cinnamates and their thio and thiono analogs as potent inhibitors of cell adhesion molecules on human endothelial cells

Article abstract of DOI:10.1016/j.ejmech.2011.09.008

In the present study, we report the design and synthesis of novel analogs of cinnamates, thiocinnamates and thionocinnamates and evaluated the potencies of these analogs to inhibit TNF-α induced ICAM-1 expression on human endothelial cells. By using whole cell-ELISA, our screening data demonstrated that ethyl 3′,4′,5′-trimethoxythionocinnamate (ETMTC) is the most potent inhibitor of TNF-α induced ICAM-1, VCAM-1 and E-selectin. As functional consequences, ETMTC abrogated TNF-α induced adhesion of neutrophils to the endothelial monolayer. Structure-activity relationship studies revealed the critical role of the chain-length of the alkyl group in the alcohol moiety, number of methoxy groups in the aromatic ring of the cinnamoyl moiety and the presence of the α, β- C-C double bond in the thiocinnamates and thionocinnamates.

Full text of DOI:10.1016/j.ejmech.2011.09.008

European Journal of Medicinal Chemistry 46 (2011) 5498e5511
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel natural product-based cinnamates and their thio and thiono analogs
as potent inhibitors of cell adhesion molecules on human endothelial cells
,
,
Sarvesh Kumara b, Brajendra K. Singhc d, Pragya Aryac, Shashwat Malhotrac, Rajesh Thimmulappab,
Ashok K. Prasadc, Erik Van der Eyckend, Carl E. Olsene, Anthony L. DePassf, Shyam Biswalb,
**
Virinder S. Parmarc , Balaram Ghosh
,
a,
*
a Molecular Immunogenetics Laboratory, CSIR-Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007, India
b Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA
c Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India
d Laboratory for Organic Synthesis, Department of Chemistry, University of Leuven, Celeslinjenlaan 200F, B-3001 Heverlee, Belgium
e Department of Basic Sciences and Environment, University of Copenhagen, DK-1871 Frederiksberg C, Denmark
f Department of Biology, Long Island University, DeKalb Avenue, Brooklyn, NY 11201, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 May 2011
Received in revised form
7 September 2011
Accepted 8 September 2011
Available online 16 September 2011
In the present study, we report the design and synthesis of novel analogs of cinnamates, thiocinnamates
and thionocinnamates and evaluated the potencies of these analogs to inhibit TNF-
expression on human endothelial cells. By using whole cell-ELISA, our screening data demonstrated
that ethyl 30,40,50-trimethoxythionocinnamate (ETMTC) is the most potent inhibitor of TNF-
induced
ICAM-1, VCAM-1 and E-selectin. As functional consequences, ETMTC abrogated TNF- induced adhesion
a induced ICAM-1
a
a
of neutrophils to the endothelial monolayer. Structureeactivity relationship studies revealed the critical
role of the chain-length of the alkyl group in the alcohol moiety, number of methoxy groups in the
Keywords:
Cinnamates
Thiocinnamates thionocinnamates
Cell adhesion molecules
Endothelial cells
aromatic ring of the cinnamoyl moiety and the presence of the
cinnamates and thionocinnamates.
a, b- CeC double bond in the thio-
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
The endothelium plays a crucial role in inflammation by
providing key signals for leukocyte migration and extravasation. In
response to tissue injury caused by environmental toxicants
(pathogens and non-pathogens), endothelial cells are activated by
extravascular stimuli inducing the surface expression of cell
adhesion molecules (CAMs) that include E-selectin, ICAM-1 and
VCAM-1 [8]. Interaction of CAMs on the surface of endothelial cell
and leukocytes facilitates rolling and extravasations of leuckocytes
to the sites of inflammation. Therefore, pharmacological inhibition
of CAMs on endothelial cells is a promising strategy for therapeutic
intervention of inflammatory disorders [9].
Previously, we have reported the isolation and characterization of
a novel aromatic ester from the chloroform extract of Piper longum
that exhibited potent ICAM-1 inhibitory activity on human umbilical
vein endothelial cells [10]. In another study on coumarins/thio-
nocoumarins, we have shown that the sulfur analogs (thio-
nocoumarins), when compared to the oxygenated analogs
Leukocyte mediated inflammatory response is the key event in
multiple inflammatory diseases including asthma, acute respiratory
distress syndrome, rheumatoid arthritis, and atherosclerosis [1e3].
Leukocytes in the inflamed tissue secrete proinflammatory media-
tors, reactive oxygen species and proteases that cause tissue damage,
inflammation and disease pathogenesis [4e6]. Pharmaceutical
companies are designing drugs to limit leukocyte mediated inflam-
matory response. However, current anti-inflammatory drugs show
limited efficacy and exhibit severe side effects. Therefore, more
specific and potent anti-inflammatory drugs are needed urgently [7].
Abbreviations: ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular cell
adhesion molecule-1; TNF-a, tumor necrosis factor a; NF-kB, nuclear factor kB;
HUVECs, human umbilical cord vein endothelial cells; ETMC, ethyl 30,40,50-trime-
thoxycinnamate; ETMTC, ethyl 30,40,50-trimethoxythionocinnamate.
* Corresponding author. Tel.: þ91 11 2766 2580; fax: þ91 11 2766 7471.
** Corresponding author. Fax: þ91 11 2766 7206.
(coumarins), are more potent inhibitors of TNF-
a induced expression
of ICAM-1 on endothelial cells [11]. In view of these findings, in the
present study, we have designed, synthesized and evaluated the
ICAM-1 inhibitory activity of libraries of cinnamates, thiocinnamates
E-mail addresses: virparmar@gmail.com (V.S. Parmar), bghosh@igib.res.in
(B. Ghosh).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.008

S. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 5498e5511
5499
Table 2
and thionocinnamates. We for the first time report novel thio and
ICAM-1 expression inhibitory activities of the thiocinnamates 29e44.
thiono analogs of the newcinnamic acid esters that exhibit potent cell
adhesion molecules inhibitory activity on human endothelial cells.
Further, detailed structureeactivity relationship of these novel cin-
namates reveal that the chain-length of the alkyl group in the alcohol
Entry Compound Conc.
%
IC50
(m
g/mL)a Inhibition (
m
g/mL)
1
2
3
4
5
6
7
8
Ethyl 30,40,50-trimethoxythiocinnamate (29) 30
Propyl 30,40,50-trimethoxythiocinnamate (30) 70
Octyl 30,40,50-trimethoxythiocinnamate (31) 70
Phenyl 30,40,50-trimethoxythiocinnamate (32) 50
Benzyl 30,40,50-trimethoxythiocinnamate (33) 40
65
50
46
85
75
70
55
30
50
55
45
65
40
50
25
70
e
moiety, substituents in the aromatic ring and the
bond of the thionocinnamates have significant effect on the inhibition
of TNF- induced expression of ICAM-1 on endothelial cells.
a, b- CeC double
30
20
35
45
e
a
Ethyl 20,50-dimethoxythiocinnamate (34)
Propyl 20,50-dimethoxythiocinnamate (35)
Octyl 20,50-dimethoxythiocinnamate (36)
Benzyl 20,50-dimethoxythiocinnamate (37)
Ethyl thiocinnamate (38)
50
50
50
60
50
60
70
60
60
2. Biological results
9
60
47
e
2.1. Cinnamates are non-toxic to the endothelial cells
10
11
12
13
14
Octyl thiocinnamate (39)
Phenyl thiocinnamate (40)
55
e
All cinnamate analogs were examined for their cytotoxic effect
on human endothelial cells as described in the experimental
procedure section, and were found to be non-toxic to endothelial
cells (data not shown) as more than 95% cells were viable at
maximum tolerable concentrations. This implied that these deriv-
atives are safe to use at indicated concentrations. The maximal
tolerable concentration was found to be different for different
compounds. For all further analyses, the maximum tolerable
concentration was used as shown in Tables 1e3.
Benzyl thiocinnamate (41)
Ethyl 30,40,50-trimethoxydihydro
-thiocinnamate (42)
60
15
16
Octyl 30,40,50-trimethoxydihydro
-thiocinnamate (43)
80
60
40
45
e
e
Benzyl 30,40,50-trimethoxydihydro
-thiocinnamate (44)
‘e’ These compounds did not reach up to 50% inhibition even at maximum tolerable
concentration.
a
Highest concentration tested that showed more than 95% cells viability.
2.2. Cinnamates, thiocinnamates and thionocinnamates inhibit
TNF-a induced expression of ICAM-1 on endothelial cells
various sub-lethal concentrations there was a significant inhibition in
TNF-
a induced expression of ICAM-1. The IC50 value of each
Cinnamates, thiocinnamates and thionocinnamates were evalu-
ated for their ability to inhibit TNF- induced expression of ICAM-1
using high-throughput whole cell-ELISA assay. Endothelial cells
were pre-incubated for 2 h with or without cinnamates, thio-
cinnamates and thionocinnamates at various concentrations followed
compound was calculated (Tables 1e3) separately from its respective
activityeconcentration graph. Also, the levels of maximum % inhibi-
tion of ICAM-1 expression are reported at their maximum tolerable
concentration where cells viability was not affected by the tested
compounds (Tables 1e3). Of the 36 compounds tested, ethyl 30,40,50-
trimethoxythionocinnamate (ETMTC, 54) was found to be the most
a
by induction with TNF-
induction in ICAM-1 expression by TNF-
a
(10 ng/mL) for 16 h. There was 3-fold higher
compared to unstimulated
a
potent inhibitor of the TNF-a induced ICAM-1 expression. Further-
more, the inhibition of ICAM-1 by ETMTC was concentration depen-
dent (Fig. 1).
cells (data not shown). However, when endothelial cells were pre-
treated with cinnamates, thiocinnamates and thionocinnamates at
Table 1
2.3. ETMTC inhibits TNF-
E-selectin
a induced expression of VCAM-1 and
ICAM-1 expression inhibitory activities of the cinnamates 10e17 and 20e23.
Entry Compound
Conc.
%
IC50
(
m
g/mL)a Inhibition Concentration
Next, we investigated the effect of ETMTC on the TNF-a induced
(mg/mL)
expression of two other important cell adhesion molecules,
VCAM-1 and E-selectin by using whole cell-ELISA assay. Human
endothelial cells were pre-treatment for 2 h followed by induction
1
2
DMSO (Vehicle)
0.25% No
No
45
Isopropyl 30,40,50
65
80
40
35
55
30
30
-trimethoxycinnamate (10)
Phenyl 30,40,50
-trimethoxycinnamate (11)
Propyl 20,50
with TNF-
a for 4 h to measure E-selectin and 16 h to measure
3
4
5
6
7
45
e
120
120
60
e
-dimethoxycinnamate (12)
Table 3
Propyl 20,50
110
e
ICAM-1 expression inhibitory activities of the thionocinnamates 54e61.
-dimethoxycinnamate (13)
Phenyl 20,50
Entry Compound Conc.
%
IC50
g/mL)
(
m
g/mL)a Inhibition
(m
-dimethoxycinnamate (14)
Isopropyl 20-hydroxy-30
-methoxycinnamate (15)
Octyl cinnamate (16)
Benzyl cinnamate (17)
Propyl 30,40,50
120
e
1
2
3
4
Ethyl 30,40,50
20
30
25
40
95
80
80
70
10
17
15
25
-trimethoxythionocinnamate (54)b
Propyl 30,40,50
8
9
10
70
70
120
35
50
62
e
70
110
-trimethoxythionocinnamate (55)
Ethyl 20,50
-trimethoxydihydrocinnamate (20)
-dimethoxythionocinnamate (56)
Propyl 20,50
11
12
13
Isopropyl 30,40,50
-trimethoxydihydrocinnamate (21)
Propyl 30,40
65
100
110
40
55
30
e
-dimethoxythionocinnamate (57)
Ethyl thionocinnamate (58)
Propyl thionocinnamate (59)
Isopropyl thionocinnamate (60)
Butyl thionocinnamate (61)
90
e
5
6
7
8
50
80
35
85
90
75
80
70
22
50
20
55
-dimethoxydihydrocinnamate (22)
Isopropyl 30,40
-dimethoxydihydrocinnamate (23)
a
‘e’ These compounds did not reach up to 50% inhibition even at maximum tolerable
Highest concentration tested that showed more than 95% cells viability.
Thionocinnamate ETMTC 54 was prepared by using the natural ETMC 47 from
b
concentration.
a
Highest concentration tested that showed more than 95% cells viability.
Piper longum.

5500
S. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 5498e5511
Fig. 1. Concentration dependent inhibition of TNF-a induced ICAM-1, VCAM-1 and
E-selectin expression on endothelial cells by ETMTC. Endothelial cells were incubated
with or without indicated concentration of ETMTC for 2 h followed by co-incubation
with or without TNF-a (10 ng/mL). E-selectin was measured after 4 h while ICAM-1
and VCAM-1 were measured after 16 h by cell based-ELISA as described in experi-
mental procedure. Data are mean ꢀ SD of 3 independent experiments. Significant
compared to vehicle control at P < 0.05.
VCAM-1. Pretreatment of endothelial cells with ETMTC significantly
attenuated TNF-
a induced expression of VCAM-1 on endothelial
cells. The inhibition of VCAM-1 by ETMTC was concentration
dependent (Fig. 1). Likewise, ETMTC pre-treatment markedly
inhibited the TNF-a induced expression of E-selectin on endothelial
cells (Fig. 1). Similar inhibition pattern of adhesion molecules on
endothelial cells was observed by ETMTC in response to LPS (data
not shown).
The inhibitory activity of ETMTC on ICAM-1, VCAM-1 and E-
selectin expression was further confirmed by flow cytometry. In
response to TNF-a stimulation, a substantial increase (5e6 folds) in
the expression of all these three adhesion molecules was observed
(Fig. 2AeC). However, pre-treatment of endothelial cells with
ETMTC markedly inhibited the TNF-a induced expression of ICAM-
1, VCAM-1 and E-selectin (by more than 95%, Fig. 2AeC).
Fig. 2. Flow cytometric analysis of inhibition of TNF-
E-selectin expression on endothelial cells by ETMTC: Endothelial cells were pretreated
with or without ETMTC (20 g/mL) for 2 h followed by TNF- (10 ng/mL) stimulation
a induced ICAM-1, VCAM-1 and
2.4. ETMTC inhibits neutrophils adhesion to endothelial monolayer
m
a
for 4 h for E-selectin measurement and 16 h for ICAM-1 and VCAM-1 measurement by
flow cytometry as described in experimental procedures. The data presented as
mean þ S.D. of three independent experiments. Cell Quest software was used for
statistical analysis (P < 0.05).
Next we assessed the functional implications of inhibition of
adhesion molecules by ETMTC using neutrophil-endothelial
adhesion assay. Endothelial cells were incubated with or
without ETMTC at various concentrations for
2 h prior to
induction with TNF- (10 ng/mL) for 6 h. As measured by color-
a
imetric assay, unstimulated endothelial cells showed low adher-
ence of neutrophils, however there was a three-fold increase in
neutrophil adhesion in response to TNF-
shown). The pre-treatment of endothelial cells by ETMTC
significantly inhibited the TNF- induced neutrophil adhesion in
concentration dependent manner. The maximum inhibition of
TNF- induced neutrophils adhesion was calculated by more than
a stimulation (data not
a
a
95% (Fig. 3).
3. Discussion
We have identified ethyl 30,40,50-trimethoxythionocinnamate
(54, ETMTC) as most potent inhibitor of TNF-a induced cell adhe-
sion molecules (ICAM-1, VCAM-1 and E-selectin) from a series of
thirty six cinnamate esters studied in the present report. Ex-vivo
inhibition of neutrophils adhesion to endothelial monolayer by
ETMTC substantiated its anti-inflammatory function. Clinical
studies using monoclonal antibodies directed against either VCAM-
Fig. 3. ETMTC inhibits adhesion of neutrophils to endothelial monolayer. Endothelial
cells were grown to confluency and incubated with or without ETMTC at various
concentrations. After 2 h, cells were stimulated with TNF-a (10 ng/mL) for 6 h.
Adherent neutrophils were assayed colorimetrically as described in experimental
procedure. Values shown are mean ꢀ SD of 3 independent experiments.

S. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 5498e5511
5501
1 and/or ICAM-1 have indicated critical role of these two adhesion
molecules in inhibiting neutrophil adhesion and subsequent
protection from inflammatory response in multiple inflammatory
diseases including organ transplantation. On the contrary, our lead
small molecule, ETMTC coordinatively inhibits ICAM-1, VCAM-1
and E-selectin expression that may result in robust protection from
inflammation.
To examine the role of different modifications, i.e (i) effect of
alkyl chain in the alcohol part of esters, (ii) number of methoxy
groups in the benzene ring, (iii) saturation of the a, b- alkene bond
expression inhibitory activity of ethyl 30,40,50-trimethox-
ycinnamate (47) having two carbon atom alkyl group in the
alcohol moiety is much higher than of propyl 30,40,50-trime-
thoxycinnamate (48) having three carbon atom alkyl group (90%
and 62% ICAM-1 expression inhibition values, respectively,
Table 4, Fig. 7)
Further, the esters having branched alkyl chains or those
carrying aryl moieties had low-to-moderate ICAM-1 expression
inhibitory activity values as revealed by the % inhibition values of
ICAM-1 expression for these esters in Table 1.
in the cinnamoyl moiety and (iv) replacement of hetero atom
(sulfur in place of oxygen in the ester moiety; both in the carbonyl
part in the cinnamoyl moiety to give the thiono esters and the
oxygen of the alkoxy group in the alcoholic moiety to give the thio
3.1.2. Effect of the number of methoxy groups in the benzene ring
The ICAM-1 inhibitory activity decreases with the decrease in
the number of methoxy groups present in the benzene ring of
cinnamates 10e17 and 20e23. Thus, the ICAM-1 expression inhi-
bition value of isopropyl 30,40,50-trimethoxycinnamate (10, 80%)
having three methoxy groups is higher than that of the propyl 20,50-
dimethoxycinnamate (13, 55% inhibition) having two methoxy
groups, further the compound propyl 20-hydroxy-30-methox-
ycinnamate (15) having only one methoxy group shows only 30%
ICAM-1 expression inhibition (Table 1). Also the esters lacking any
methoxy group in the benzene ring have very low activity as ICAM-
1 expression inhibitors (Table 1).
esters) in inhibiting the TNF-a induced expression of ICAM-1, we
have synthesized different oxygenated cinnamates 10e17 and
20e23 (Fig. 4), and the corresponding thiocinnamates 29e44
(Fig. 5) and the thionocinnamates 54e61 (Fig. 6). The experimental
activity data shown in Tables 1e3 revealed that the following
functional components are critical for the ICAM-1 expression
inhibitory activities among all the three tested classes of
compounds.
3.1. Oxygenated cinnamates 10e17 and 20e23
3.1.3. Effect of the saturation of the alkene bond
It has been observed that the dihydro derivatives of cinnamates
were less effective in inhibiting the TNF-a induced expression of
3.1.1. Effect of the length of the alkyl group in the alcohol moiety
The ICAM-1 inhibitory activity data given in Table 1 revealed
that activity of the cinnamates 10e17 and 20e23 significantly
decreases with the increase in the length of the alkyl chain in the
alcohol part. Earlier we have reported that ethyl cinnamate (51),
propyl cinnamate (52) and the butyl cinnamate (53), having
increasingly longer alkyl chains in the alcohol moiety inhibit 85%,
ICAM-1. Thus, as can be seen in Table 1, the ICAM-1 expression
inhibition value of isopropyl 30,40,50-trimethoxycinnamate (10, 80%)
is higher than that of its dihydro analog, i.e. the isopropyl 30,40,50-
trimethoxydihydrocinnamate (21, 40%). Similarly, the ICAM-1
expression inhibitory activity of ethyl 30,40,50-trimethoxycinnamate
(47, 90%) is much higher than that of its dihydro analog, ethyl 30,40,50-
trimethoxydihydrocinnamate (62, 50%, Table 4, Fig. 7). Again the
ICAM-1 expression was inhibited to the extent of 55% by propyl 20,50-
dimethoxycinnamate (13) and its dihydro analog, isopropyl 30,40-
dimethoxydihydrocinnamate (23) has the inhibitory activity value of
30% only (Table 1)
70% and 55%, respectively (Table 4, Fig. 7) the TNF-
a induced
expression of ICAM-1 in human endothelial cells [10]. In the
present study, we have found that further increase in the chain
length, i.e. the octyl cinnamate (16) having eight carbon atoms in
the alkyl chain showed insignificant ICAM-1 expression inhibi-
tory activity (35% inhibition only, Table 1). Similarly, the ICAM-1
Fig. 4. Structures of alkyl and aryl cinnamates.

5502
S. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 5498e5511
Fig. 5. Structures of alkyl and aryl thiocinnamates.
3.2. Thiocinnamates 29e44
in the alcohol moiety in the thiocinnamates 29e44. A significant
fall in the ICAM-1 inhibitory activities was observed in increasing
the chain length from ethyl to propyl to octyl in the alcohol part of
the thiocinnamate esters 29e31, similar trend was observed in the
compounds 34e36, 38e39 and 42e43 also (Table 2).
3.2.1. Effect of the length of the alkyl groups in the alcohol moiety
It is very clearly evident from Table 2 that ICAM-1 inhibitory
activity decreases with an increase in the length of the alkyl chain
Fig. 6. Structures of alkyl and aryl thionocinnamates.

S. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 5498e5511
5503
Table 4
25
m
g/mL), and 59 (IC50 50
mg/mL) and 61 (IC50 55
mg/mL),
ICAM-1 expression inhibitory activities of the compounds already reported by us
[10] for comparison to establish the SAR study in the current manuscript.
respectively (Table 3).
Compound
Concentration % ICAM-1
IC50 (mg/mL)
3.3.2. Effect of the number of methoxy groups in the benzene ring
As revealed from Table 3, similar to the cases of the cinnamates
10e17 and 20e23 and thiocinnamates 29e44, the ICAM-1 inhibi-
tory activity values of the thionocinnamates 54e61 decrease with
the decrease in the number of methoxy groups present in the
benzene rings of the thionocinnamates as well. Thus, the IC50
values of the ethyl thionocinnamates: ethyl 30,40,50-trimethox-
(
m
g/mL)a
expression
inhibition
Ethyl 30,40,50-tri
-methoxycinnamate (ETMC, 47)
Propyl 30,40,50
-trimethoxycinnamate (48)
Ethyl cinnamate (51)
Propyl cinnamate (52)
Butyl cinnamate (53)
Ethyl 30,40,50-trimethoxy
-dihydrocinnamate (62)
50
90
25
120
62
100
130
140
140
110
85
70
55
50
95
100
125
110
ythionocinnamate (54, ETMTC, 10
groups is lower than that of the ethyl 20,50-dimethox-
ythionocinnamate (56, 15 g/mL) having two methoxy groups, and
that of ethyl thionocinnamate (58, 22 g/mL) lacking the presence
mg/mL) having three methoxy
m
m
a
Highest concentration tested that showed more than 95% cells viability.
of any methoxy group (Table 3). The same trend prevailed in the
case of propyl thionocinnamates, thus the IC50 value of propyl
30,40,50-trimethoxythionocinnamate having three methoxy groups
3.2.2. Effect of the number of methoxy groups in the benzene ring
As revealed from Table 2, the ICAM-1 inhibitory activity
decreases with the decrease in the number of methoxy groups
present in the benzene ring of the thiocinnamates 29e44. The IC50
(55, 17
ythionocinnamate having two methoxy groups (57, 25
which in turn is lower than that of propyl thionocinnamate (59,
50 g/mL) lacking the presence of any methoxy group (Table 3).
We have earlier reported that the replacement of the lactone
m
g/mL) is lower than that of propyl 20,50-dimethox-
mg/mL),
m
value of ethyl 30,40,50-trimethoxythiocinnamate (29, 25
m
g/mL) is
lower than that of the ethyl 20,50-dimethoxythiocinnamate (34,
35 g/mL). The lack of the presence of any methoxy groups in the
benzene ring further decreases the ICAM-1 expression lowering
activity as the IC50 value (50 g/mL) of ethyl thiocinnamate (38)
lacking any methoxy group is much higher as compared to those of
compounds 29 (25 g/mL) and 34 (35 g/mL) having three and two
methoxy groups, respectively (Table 2).
ring carbonyl oxygen atom by sulfur in the case of coumarins makes
the corresponding thionocoumarins more potent than the parent
coumarins in inhibiting the ICAM-1 expression [11]. Taking clue
from this finding, we have in the present study, synthesized new
analogs 10e17 and 20e23 and different novel thio and thiono
analogs 29e44 and 54e61, respectively of the naturally occurring
cinnamate 47 [10]. On comparing the ICAM-1 inhibitory activity
values of the new oxygen containing cinnamate analogs 10e17 and
20e23 of the natural cinnamate 47 (Table 1) with those of the
thiocinnamates (Table 2) and thionocinnamates (Table 3), we have
observed that sulfur containing compounds are more potent than
their oxygen containing counterparts. When the oxygen atom of
the alkoxy moiety was replaced by sulfur atom, the resulting thi-
ocinnamates 29e44 (Table 2) showed better ICAM-1 expression
inhibition than the corresponding oxygenated cinnamates 10e17
and 20e23 (Table 1). However, the ICAM-1 expression lowering
activities of the thionocinnamates 54e61 having the ketonic
oxygen of the cinnamoyl moiety substituted by the sulfur atom,
unlike the substitution of the alkoxy oxygen of the alcoholic chain
by sulfur to give the thiocinnamates 29e44 (Table 2) were much
higher (Table 3). A brief comparison of the ICAM-1 inhibitory
activity of cinnamates, thiocinnamates and thionocinnamates is
presented in Fig. 8, it can clearly be seen that there is inhibition in
ICAM-1 expression, shown by the three different classes of cinna-
mates follows the order: thionocinnamates > thiocinnamates > the
oxygenated cinnamates. Further it is noteworthy to see that all the
m
m
m
m
3.2.3. Effect of the saturation of the alkene bond
The dihydro analogs 42, 43 and 44 of the thiocinnamates 29, 31
and 33, respectively when tested for ICAM-1 inhibitory activity
were found to be much less effective in inhibiting the TNF-
a
induced expression of ICAM-1 (Table 2).
3.3. Thionocinnamates 54e61
3.3.1. Effect of the length of the alkyl group in the alcohol moiety
Again the same trend as in the cases of cinnamates 10e17 and
20e23 and the thiocinnamates 29e44 was observed in the case of
the thionocinnamates 54e61 as well, viz. the ICAM-1 inhibitory
activity of the compounds 54e61 decreases with the increase in the
length of the alkyl group of the alcohol moiety. Thus the IC50 value
of ethyl 30,40,50-trimethoxythionocinnamate (54, ETMTC, 10
ethyl 20,50-dimethoxythionocinnamate (56, 15
g/mL) and ethyl
thionocinnamate (58, 22 g/mL) are lower than those of their
corresponding propyl and butyl analogs 55 (IC50 17 g/mL), 57 (IC50
mg/mL),
m
m
m
Fig. 7. Structures of the compounds already reported by us earlier [10] for comparison to establish SAR study in the current manuscript.

5504
S. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 5498e5511
advantage of this stability by using disulfide bonds as structural
features of proteins. The presence of thiols in their structures could
be responsible for their better biological activities including anti-
oxidant properties [14,15]. Furthermore, in comparison with the
known anti-inflammatory clinical drugs, such as aspirin (IC50
6000
(IC50 500
inhibiting the TNF-
m
M), mesalamine (IC50 16,000
M), ETMTC (IC50, 40 M) showed greater potency in
induced expression of adhesion molecules
mM) and phenyl methimazole
m
m
a
[16e18]. Likewise, when compared with the clinically used anti-
inflammatory drugs, such as diclofenac, N-acetylcysteine and pyr-
rolidone dithiocarbamate that are effective at concentrations of
750 mM, 100 mM and 1000 mM, respectively, [19,20] ETMTC (54)
shows robust inhibition at very low concentration. Summing up
these results indicate that our novel compound (ETMTC, 54) is
potentially effective and therefore is, worthy of further pharma-
ceutical studies.
Previously, we have reported that inhibition of cell adhesion
molecules by different natural compounds was mediated by NF-kB
[21e23]. Although, in the current study, the mechanism of cell
adhesion molecule expression inhibition by ETMTC and its analogs
is not investigated, it is tempting to speculate that they may be
Fig. 8. Comparison of ICAM-1 inhibitory activity of selected cinnamates, thio-
cinnamates and thionocinnamates. IC50 values of some cinnamates, thiocinnamates
and thionocinnamates are compared to demonstrate the effect of replacement of
oxygen atom with that of sulfur. Compound numbers are shown on x-axis and IC50
values are represented on y-axis. y The most active compound (54) was prepared from
the natural product 47 isolated from Piper longum. z did not show any IC50 values
interfering with the NF-kB activation pathway. Furthermore,
eight thionocinnamates 54e61 showed very high activities as they
all exhibited % inhibition of ICAM-1 expression values in the range
nuclear factor E2p45-related factor (Nrf2) is a central transcription
factor that regulates the anti-oxidant defense system and acts as
a modifier of several inflammatory diseases that involve oxidative
stress and inflammation. Nrf2 activating agents are also known for
70e95% with IC50 values ranging between 10 and 55
mg/mL
(Table 3), as compared to those of the thiocinnamates 29e44
(having % inhibition values ranging between 30 and 85% with IC50
NF-kB inhibitors [24e26]. These findings prompt us to examine the
values ranging between 20 and 70
m
g/mL and only ten compounds
effect of cinnamate analogs on Nrf2 activation. Our unpublished
data from ARE-reporter based assay showed a similar structur-
eeactivity pattern for Nrf2 activation by these esters. In any event,
the exact mechanism of action responsible for inhibition of cell
adhesion molecules expression remains to be elucidated and would
be the subject of a future publication.
out of sixteen showed the IC50 values, Table 2) and those of the
oxygenated cinnamates 10e17 and 20e23 having % inhibition
values ranging between 30 and 80% and the IC50 values ranging
between 70 and 110 mg/mL and only five compounds out of twelve
showed the IC50 values (Table 1), In other words, all the thio-
nocinnamates 54e61 were moderate to highly active as each of the
eight thionocinnamates showed significant values of % inhibition of
ICAM-1 expression and IC50 values (Table 3) unlike the thio-
cinnamates 29e44 (Table 2) and the oxygenated cinnamates 10e17
and 20e23 (Table 1). Taken together, our results suggest that the
ICAM-1 expression inhibitory activity of the thionocinnamate
esters is better when:
4. Conclusion
Our
results
of
identifying
ethyl
30,40,50-trimethox-
ythionocinnamate (ETMTC, 54) and its analogs have implications in
discovering therapeutically valuable lead molecules against various
inflammatory diseases. Importantly, these findings will open new
avenues for research on ethyl 30,40,50-trimethoxythionocinnamate
as a lead pharmaceutical molecule.
(i) thionocinnamate ester has alcohol moiety of shorter chain
length (ethyl is the best),
(ii) more number of methoxy groups are present in the aromatic
ring,
(iii) the
a
,b
-unsaturation is present in the cinnamoyl moiety, and
5. Experimental procedures
(iv) sulfur atom is present in the place of oxygen atom in the
ketonic moiety, i.e. the cinnamoyl part of the ester (viz. the
thionocinnamates).
5.1. Reagents and chemicals
The organic solvents (dioxane, petroleum ether and ethyl
acetate) used were dried and distilled prior to their use. All other
chemical like alcohols, thiols and cinnamic/dihydrocinnamic acids
used were purchased from SigmaeAldrich, USA and were used
without any further purification. The analytical TLCs were per-
formed on precoated Merck silica gel 60 F254 plates; the spots were
visualized under UV light. The 1H NMR and 13C NMR spectra were
recorded on a Bruker Avance at 300 MHz and 75.5 MHz, respec-
tively using TMS as internal standard. The chemical shift values are
The efficacy of our thiono derivative ETMTC (54) was found to be
200 times more than that of the natural parent compound from
which it was prepared, present in P. longum, i.e. ETMC (47) [10]. In
short, thionocinnamates and thiocinnamates have better inhibitory
activities than cinnamates because of sulfur in their structures. It
has been well documented in literature that sulfur-containing
compounds are biologically more active then oxygen containing
counterparts [12,13]. As members of the same periodic group,
sulfur and oxygen share similarities in chemical reactivity.
However, sulfur’s position lower in the periodic table endows its
compounds with distinct properties that are advantageous to bio-
logical systems. For example, thiols are superior nucleophiles
compared with alcohols and also serve as versatile activating
groups in thioester biochemistry. Disulfide bonds (RSeSR) are more
stable than peroxide bonds (ROeOR), and biology has taken
on
were recorded on a JEOL JMS-AX505W instrument. Anti-ICAM-1, E-
selectin and TNF- were purchased from Pharmingen, USA. M-199
media, -glutamine, endothelial cell growth factor, trypsin, Pucks
d scale and the coupling constant values (J) are in Hz. The HRMS
a
L
saline, HEPES, o-phenylenediamine and anti-mouse IgG-HRP were
purchased from Sigma Chemical Co., USA. Fetal calf serum was
purchased from Biological Industries, Israel.

S. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 5498e5511
5505
5.2. Chemistry
(75.5 MHz, CDCl3):
d
21.71 [CH(CH3)2], 56.21 (2ꢁ OCH3), 70.43
[OCH(CH3)2], 111.51 (C-60), 114.49 (C-40), 115.20 (C-30), 116.0 (C-10),
116.39 (C-2), 138.94 (C-1), 151.3 (C-20) 152.81 (C-50) and 166.51 (CO);
HRMS calcd for C14H18O4 m/z 250.1205, observed m/z 250.1207.
5.2.1. General procedure for the synthesis of the cinnamates 10e17
and 20e23
A mixture of the commercially available cinnamic acid (4)/
substituted cinnamic acids 1e3/dihydrocinnamic acids (18/19,
5 mmol) and the corresponding commercially available alcohols
5e9, i.e. propyl (7)/isopropyl (5)/octyl (8)/phenyl (6)/benzyl alcohol
(9) (25 mL) and concentrated sulfuric acid (0.2 mL) was refluxed for
4e5 h (Schemes 1 and 2). On completion of the reactions as
observed from TLC examination, the reaction mixture was neutral-
ized by the addition of 10% aqueous sodium hydrogen carbonate
solution (w/v) and then extracted with chloroform (2 ꢁ 25 mL), the
combined organic layer was separated and dried over anhydrous
Na2SO4. The crude product obtained after evaporation of organic
solvent was loaded on a small silica gel column and eluted with
chloroform to obtain the pure esters in 71e99% yields. The struc-
tures of all the cinnamates 10e17 and 20e23 were unambiguously
established on the basis of their spectral (IR, 1H NMR, 13C NMR
spectra and HRMS) analysis. The structures of the known esters 11,
16 and 17 were further confirmed by comparison of their melting
points with those reported in the literature [27,28] (Table 5).
5.2.4. Propyl 20,50-dimethoxycinnamate (13)
It was obtained as light yellow oil in 96% yield. IR (Thin film):
2982, 1712, 1638, 1496, 1312, 1271, 1176, 1039, 980 cmꢂ1 1H NMR
;
(300 MHz, CDCl3):
d
0.91 (3H, t, J ¼ 7.3 Hz, CH2CH2CH3), 1.61 (2H, m,
CH2CH2CH3), 3.83 and 3.85 (6H, s, 2ꢁ OCH3), 4.03 (2H, t, J ¼ 6.7 Hz,
OCH2CH2CH3), 6.35 (1H, d, J ¼ 15.9, Ce2H), 6.77 (3H, m, Ce30H, C-40
and C-60) and 7.59 (1H, d, J ¼ 15.9 Hz, Ce1H). 13C NMR (75.5 MHz,
CDCl3): 10.91 (CH2CH2CH3), 21.84 (CH2CH2CH3), 55.63 and 55.73 (C-
20 and C-50) 65.84 (OCH2CH2CH3), 111.28 (C-60), 114.62 (C-40), 115.19
(C-30), 116.0 (C-10), 116.22 (C-2), 138.61 (C-1), 151.51 (C-20), 152.58
(C-50) and 170.81 (CO); HRMS calcd for C14H18O4 m/z 250.1205,
observed m/z 250.1202.
5.2.5. Phenyl 20,50-dimethoxycinnamate (14)
It was obtained as white solid (mp 92e95 ꢃC) in 99% yield. IR
(Thin film): 2837, 1720, 1630, 1494, 1261, 1142, 1046, 988 cmꢂ1 1H
NMR (300 MHz, CDCl3):
d
3.81 and 3.71 (6H, s, 2ꢁ OCH3), 6.69 (1H, d,
J ¼ 16.08, Ce2H), 7.42e6.82 (8H, m, AreH), 8.14 (1H, d, J ¼ 16.08 Hz,
5.2.2. Isopropyl 30,40,50-trimethoxycinnamate (10)
Ce1H); 13C NMR (75.5 MHz, CDCl3):
d
55.71 and 55.98 (2ꢁ OCH3),
It was obtained as pale yellow oil in 95% yield. IR (thin film):
112.45 (C-60), 113.48 (C-40), 117.58 (C-30), 117.94 (C-2), 121.65 (C-200
and C-600), 123.59 (C-10), 125.6 (C-400), 129.32 (C-300 and C-500), 141.71
(C-1), 150.91 (C-100), 153.49 (C-20 and C-50), 165.60 (CO). HRMS calcd
for C17H16O4 m/z 284.1049, observed m/z 284.1051.
2981, 2930, 1713, 1638, 1450, 1311, 1176, 1039, 980, 768 cmꢂ1 1H
;
NMR (300 MHz, CDCl3):
d
1.30 [6H, d, J ¼ 6.2 Hz, CH(CH3)2], 3.88
(9H, s, 3ꢁ OCH3), 5.14 [1H, m, CH(CH3)2], 6.35 (1H, d, J ¼ 15.9,
Ce2H), 6.75 (2H, s, C-20H and C-60H), 7.58 and (1H, d, J ¼ 15.9 Hz,
Ce1H). 13C NMR (75.5 MHz, CDCl3):
d
21.64 [CH(CH3)2], 55.84 (2ꢁ
5.2.6. Isopropyl 20-hydroxy-30-methoxycinnamate (15)
OCH3), 60.62 (OCH3), 67.46 [OCH(CH3)2], 104.99 (C-20 and C-60),
117.78 (C-2), 129.73 (C-10) 140.54 (C-40), 143.94 (C-21), 153.14 (C-30
and C-50) and 166.11 (CO); HRMS calcd for C15H20O5 m/z 280.1311,
observed m/z 280.1314.
It was obtained as yellow viscous oil in 8 yield. IR (thin
film):2838, 1728, 1590, 1459, 1240, 1109, 1010 cmꢂ1 1H NMR
;
(300 MHz, CDCl3):
d
1.31 [6H, d, J ¼ 6.2 Hz, CH(CH3)2], 3.90 (3H, s,
OCH3), 5.13 [1H, m, OCH(CH3)2], 6.27 (1H, d, J ¼ 15.9 Hz, Ce2H), 6.80
(1H, d, J ¼ 8.2 Hz, H-40), 7.02 (1H, dd, J ¼ 8.3 and 1.8 Hz, H-50), 7.13
(1H, d, J ¼ 1.8 Hz, H-60) and 7.57 (1H, d, J ¼ 15.9 Hz, Ce1H). 13C NMR
5.2.3. Isopropyl 20,50-dimethoxycinnamate (12)
It was obtained as light yellow oil in 97% yield. IR (Thin film): 2966,
(75.5 MHz, CDCl3): d 22.35 CH(CH3)2, 56.30 (OCH3), 68.07
1709,1633,1497,1318,1258,1174,1046, 988 cmꢂ1; 1H NMR (300 MHz,
OCH(CH3)2, 111.96, 113.47 and 117.12 (C-40, C-50 and C-60), 122.10 (C-
2), 128.47 (C-10), 144.63 (C-1), 146.26 and 148.93 (C-20 and C-30) and
167.31 (CO); HRMS calcd for C13H16O4 m/z 236.1049, observed m/z
236.1049.
CDCl3)
d
1.26 [6H, d, J ¼ 6.2 Hz, CH(CH3)2], 3.89 (6H, s, 2ꢁ OCH3), 5.11
[1H, m, CH(CH3)2], 6.37 (1H, d, J ¼ 15.9, Ce2H), 6.78(2H, m, Ce30H and
Ce40H), 7.0 (1H, s, Ce60H) and 7.58 (1H, d, J ¼ 15.9 Hz, Ce1H).13C NMR
Product
R1
H
H
OCH3
OCH3
OCH3
OH
H
R2
R3
OCH3
OCH3
H
H
H
H
H
H
R4
R5
H
H
H
H
H
H
H
H
R6
CH(CH3)2
C6H5
CH(CH3)2
C3H7
C6H5
CH(CH3)2
C8H17
CH2C6H5
% Yield
95
71
97
96
99
86
86
95
OCH3
OCH3
H
H
H
OCH3
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
H
10
11
12
13
14
15
16
17
H
H
H
Scheme 1.

5506
S. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 5498e5511
Product
R1 R2
R3
R4
R5 R6
% Yield
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
C3H7
CH(CH3)2
C3H7
95
97
96
98
20
21
22
23
OCH3
OCH3
OCH3
H
CH(CH3)2
Scheme 2.
5.2.7. Propyl 30,40,50-trimethoxydihydrocinnamate (20)
It was obtained as colorless oil in 95% yield. IR (thin film): 1714,
1638, 1451, 1311, 1215, 1067, 980, 756 cmꢂ1 1H NMR (300 MHz,
CDCl3):
CH2CH2CH3), 2.63 (2H, t, J ¼ 7.9 Hz, Ce2H), 2.90 (2H, t, J ¼ 7.5 Hz,
Ce1H), 3.81 and 3.83 (9H, s, 3ꢁ OCH3), 4.04 (2H, t, J ¼ 6.7 Hz,
OCH2CH2CH3) and 6.74 (2H, s, C-20 and C-60) 13C NMR (75.5 MHz,
m, C-20, C-50 and C-60H). 13C NMR (75.5 MHz, CDCl3): 10.91
(CH2CH2CH3), 21.84 (CH2CH2CH3), 30.48 (C-2), 36.01 (C-1), 55.63
(C-30OCH3), 55.73 (C-40OCH3) 65.84 (OCH2CH2CH3), 111.28 (C-20),
111.62 (C-50), 111.99 (C-60), 133.11 (C-10), 147.38 (C-40), 148.78 (C-30),
172.81 (CO). HRMS calcd for C14H20O4 m/z 252.1362, observed m/z
252.1361.
;
d
0.91 (3H, t, J ¼ 7.3 Hz, CH2CH2CH3), 1.64 (2H, m,
CDCl3):
d
10.65 (CH2CH2CH3), 22.30 (CH2CH2CH3), 31.70 (C-2), 36.34
5.2.10. Isopropyl 30,40-dimethoxydihydrocinnamate (23)
(C-1), 56.35 (C-30OCH3 and C-50OCH3), 61.05 (C-40OCH3), 66.38
(OCH2CH2CH3), 105.63 (C-20 and C-60), 136.69 (C-10), 153.51 (C-30, C-
40, C-50) and 173.20 (CO); HRMS calcd for C15H22O5 m/z 282.1467,
observed m/z 282.1465.
It was obtained as yellow viscous oil in 98% yield. IR (thin film):
1713, 1638, 1451, 1313, 1173, 1062, 981 cmꢂ1 1H NMR (300 MHz,
;
CDCl3):
d
1.21 [6H, d, J ¼ 6.2 Hz, CH(CH3)2], 2.56 (2H, t, J ¼ 8.0 Hz,
Ce2H), 2.89 (2H, t, J ¼ 7.5 Hz, Ce1H), 3.85 and 3.86 (6H, s, 2ꢁ
OCH3), 5.02 [1H, m, OCH(CH3)2] and 6.78 (3H, m, C-20, C-50 and C-
5.2.8. Isopropyl 30,40,50-trimethoxydihydrocinnamate (21)
60H). 13C NMR (75.5 MHz, CDCl3):
d 21.60 [CH(CH3)2], 30.16 (C-2),
It was obtained as colorless oil in 97% yield. IR (thin film): 1713,
36.56 (C-1), 55.55 (C-30OCH3), 55.66 (C-40OCH3) 67.47 [OCH(CH3)2],
111.15 (C-20), 111.53 (C-50), 111.99 (C-60), 133.07 (C-10), 147.27 (C-40),
148.67 (C-30) and 172.31 (CO). HRMS calcd for C14H20O4 m/z
252.1362, observed m/z 252.1360.
1638, 1451, 1311, 1256, 1172, 980, 768 cmꢂ1 1H NMR (300 MHz,
.
CDCl3):
d
0.86 [6H, d, J ¼ 6.2 Hz, CH(CH3)2], 2.23 (2H, t, J ¼ 7.3 Hz,
Ce2H), 2.53 (2H, t, J ¼ 7.6 Hz, Ce1H), 3.45 and 3.48 (9H, s, 3ꢁ
OCH3), 4.65 [1H, m, OCH(CH3)2] and 6.08 (2H, s, C-20H, and C-60H).
13C NMR (75.5 MHz, CDCl3): 22.07 [CH(CH3)2], 31.66 (C-2), 36.57 (C-
1), 56.27 (2ꢁ OCH3), 60.95 (OCH3), 67.91 [OCH(CH3)2], 105.65 (C-20
and C-60), 136.70 (C-10), 153.45 (C-30, C-40, C-50) and 172.53 (CO).
HRMS calcd for C15H22O5 m/z 282.1467, observed m/z 282.1465.
5.3. Synthesis of the thiocinnamates 29e44
A mixture of the commercially available carboxylic acids (1, 2, 4
and 18, 3 mmol), the appropriate commercially available thiol
(24e28, 3.1 mmol) and polyphosphate ester (PPE, 2 mL) was stirred
at room temperature for 0.5e11 h (Schemes 3 and 4). After
completion of the reaction, the mixture was treated with saturated
aqueous sodium hydrogen carbonate solution (20 mL) and extrac-
ted with chloroform (3 ꢁ 20 mL). The combined chloroform extract
was dried over sodium sulfate and evaporated in a rotary vacuum
evaporator. The crude compounds were passed through a silica-gel
column, using pure petroleum ether as eluent, the desired
compounds were obtained as colorless viscous oils in 92e98%
yields. The structures of all the thio esters were unambiguously
established on the basis of their spectral (IR, 1H NMR, 13C NMR
spectra and HRMS) analysis. The structure of the known esters
38e41 were further confirmed by comparison of their melting
points with those reported in the literature [29e33] (Table 5).
5.2.9. Propyl 30,40-dimethoxydihydrocinnamate (22)
It was obtained as colorless oil in 96% yield. IR (thin film): 1715,
1639, 1455, 1345, 1180, 1062, 979 cmꢂ1; 1H NMR (300 MHz, CDCl3):
d
0.91 (3H, t, J ¼ 7.3 Hz, CH2CH2CH3), 1.61 (2H, m, CH2CH2CH3), 2.60
(2H, t, J ¼ 7.9 Hz, Ce2H), 2.89 (2H, t, J ¼ 7.5 Hz, Ce1H), 3.83 and 3.85
(6H, s, 2ꢁ OCH3), 4.03 (2H, t, J ¼ 6.7 Hz, OCH2CH2CH3) and 6.77 (3H,
Table 5
Melting points of the known compounds.
Compound
% Yield Mp (ꢃC)
Lit. (ref.)
Obs.
Phenyl 30,40,50-trimethoxycinnamate (11)
Octyl cinnamate (16)
Benzyl cinnamate (17)
71
86
95
95e97 ꢃC (23) 94e96 ꢃC
Oil (24) Oil
33e35 ꢃC (24) Oil
5.3.1. Ethyl 30,40,50-trimethoxythiocinnamate (29)
Ethyl thiocinnamate (38)
Octyl thiocinnamate (39)
Phenyl thiocinnamate (40)
Benzyl thiocinnamate (41)
100
94
98
Oil (25)
Oil (26)
Oil
Oil
It was obtained as colorless oil in 98% yield. IR (thin film): 1635
(C]O), 1464, 1421, 1287, 1126, 1005 cmꢂ1; 1H NMR data (300 MHz,
78e80 ꢃC (27) 80e81 ꢃC
67e69 ꢃC (28) 67e68 ꢃC
CDCl3);
d
1.31 (3H, t, J ¼ 7.2 Hz, CH2CH3), 3.04 (2H, t, J ¼ 7.2 Hz,
92
SCH2), 3.86 (9H, s, 3ꢁ OCH3), 6.61 (1H, d, J ¼ 15.7 Hz, Ce2H), 6.81

S. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 5498e5511
5507
Product
29
30
31
32
33
34
35
36
37
38
39
40
R1
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
H
H
H
H
R2
R3
R4
R5 R6
% Yield
98
98
93
97
98
98
97
97
98
100
94
98
92
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
C2H5
C3H7
C8H17
C6H5
CH2C6H5
C2H5
C3H7
C8H17
CH2C6H5
C2H5
C8H17
C6H5
H
H
H
H
H
H
H
H
H
CH2C6H5
41
Scheme 3.
(2H, brs, C-20H and C-60H) and 7.53 (1H, d, J ¼ 15.7 Hz, Ce1H). 13C
5.3.3. Octyl 30,40,50-trimethoxythiocinnamate (31)
It was obtained as colorless viscous oil in 93% yield. IR (Thin
film):1664, 1580, 1457, 1341, 1266, 1127, 1034 cmꢂ1 1H NMR
NMR data (75.5 MHz, CDCl3):
d 14.94 (SCH2CH3), 22.70 (SCH2CH3),
61.11 and 61.26 (3ꢁ OCH3), 105.80 (C-20 and C-60), 126.58, 131.76,
138.83 (C-2, C-10 or/C-40), 142.42 (C-1), 153.76 (C-30 and C-50),
189.94 (C]O); HRMS: Calcd for C14H18O4S [M]þ 282.1082, found
282.1080.
;
(300 MHz, CDCl3):
d
0.88 (3H, t, J ¼ 7.0 Hz, CH2CH3), 1.27e1.55 (2H,
m, CH2CH3), 2.87e2.89 (10H, m, C-200H-C-600H), 3.00 (2H, t, J ¼ 7.1 Hz,
SCH2), 3.88 (9H, s, 3ꢁ OCH3), 6.64 (1H, d, J ¼ 15.8 Hz, Ce2H), 6.76
(2H, s, C-20H and C-60H), 7.49 (1H, d, J ¼ 15.8 Hz, Ce1H); 13C NMR
5.3.2. Propyl 30,40,50-trimethoxythiocinnamate (30)
(75.5 MHz, CDCl3): d 10.9 (CH3), 13.92 (CH2CH3), 22.87, 23.80, 28.88,
It was obtained as viscous oil in 98% yield. IR (Thin film): 1635
30.42 and 33.4 (C-600, C-500, C-400, C-300 and C-200), 38.82 (C-100), 66.86
(3ꢁ OCH2), 118.29 (C-1), 127.92 (C-20 and C-60), 128.73 (C-30 and C-
50), 130.04 (C-40), 134.44 (C-2), 144.35 (C-1), 191.98 (CO), HRMS calcd
for C20H30O4S m/z 366.1865, observed m/z 366.1863.
(C]O), 1464, 1421, 1287, 1126, 1005 cmꢂ1
;
1H NMR data
(300 MHz, CDCl3);
d
1.01 (3H, t, J ¼ 7.3 Hz, CH2CH2CH3), 1.70 (2H,
m, CH2CH2CH3), 3.01 (2H, t, J ¼ 7.8 Hz, SCH2), 3.85 (9H, s, 3ꢁ
OCH3), 6.62 (1H, d, J ¼ 15.7 Hz, Ce2H), 6.76 (2H, brs, C-20H and C-
60H) and 7.52 (1H, d, J ¼ 15.7 Hz, Ce1H). 13C NMR data (75.5 MHz,
5.3.4. Phenyl 30,40,50-trimethoxythiocinnamate (32)
CDCl3):
d
13.24 (CH2CH2CH3), 22.66 and 30.84 (CH2CH3 and
It was obtained as green solid mp 74e76 ꢃC in 97% yield. IR (Thin
SCH2), 56.07 (3ꢁ OCH3), 105.48 (C-20 and C-60), 124.35, 129.51,
138.83 (C-2, C-10 or/C-40), 140.11 (C-1), 153.35 (C-30 and C-50),
189.54 (C]O); HRMS: Calcd for C15H20O4S [M þ Na]þ 319.0975,
found 319.0972.
film): 1669, 1581, 1464, 1342, 1266, 1129, 1023 cmꢂ1 1H NMR
;
(300 MHz, CDCl3):
d
3.86e3.89 (9H, s, 3ꢁ OCH3), 6.56 (1H, d,
J ¼ 15.85 Hz, Ce2H), 6.80 (2H, s, Ce20H and C-60H), 7.17e7.41 (5H,
m, C-200H-C-600H), 7.75 (1H, d, J ¼ 15.85, Ce1H); 13C NMR (75.5 MHz,
Product
R1 R2
R3
R4
R5 R6
% Yield
98
98
H
H
H
OCH3
OCH3
OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
H
H
H
C2H5
C8H17
CH2C6H5 98
42
43
44
Scheme 4.

5508
S. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 5498e5511
CDCl3):
d
58.3 and 63.08 (3ꢁ OCH3), 107.76 (C-20 and C-60), 118.65
189.3 (CO); HRMS calcd for C18H18O3S m/z 314.0977, observed
314.0974.
(C-2), 123.77 (C-500 and C-600), 127.89 (C-10), 131.79 (C-200, C-300 and
C-400), 143.55 (C-40), 148.66 (C-1), 153.24 (C-30 and C-50), 155.66 (C-
100), 189.45 (CO); HRMS calcd for C18H18O4S m/z 330.0926, observed
m/z 330.0929.
5.3.10. Ethyl 30,40,50-trimethoxydihydrothiocinnamate (42)
It was obtained as colorless oil in 98% yield. IR (thin film): 1688,
1590, 1455, 1346, 1239, 1129, 982, 755 cmꢂ1 1H NMR (300 MHz,
;
5.3.5. Benzyl 30,40,50-trimethoxythiocinnamate (33)
CDCl3):
d
0.99 (3H, t, J ¼ 7.41 Hz, SCH2CH3), 2.59e2.67 (4H, m, 2ꢁ
It was obtained as light green oil in 98% yield. IR (Thin film):
CH2), 3.02 (2H, q, J ¼ 7.38 Hz, SCH2CH3), 3.58 (9H, s, 3ꢁ OCH3), 6.15
(2H, s, aromatic protons); 13C NMR (75.5 MHz, CDCl3): 16.88 (CH3),
33.9 (C-2), 47.7 (C-1), 58.2 (3ꢁ OCH3), 62.9 (SCH2), 107.5 (C-20 and
C-60), 127.6 (C-10) 138.06 (C-30 and 50), 155.36 C-40) and 200.7 (CO),
HRMS calcd for C14H20O4S m/z 284.1082, observed 284.1080.
1662, 1580, 1454, 1341, 1268, 1126, 1035 cmꢂ1; 1H NMR (300 MHz,
CDCl3):
d
3.87e3.88 (9H, m, 3ꢁ OCH3), 4.26 (2H, s, SCH2), 6.60 (1H,
d, J ¼ 15.68 Hz, Ce2H), 6.75 (2H, s, C-20H and C-60H), 7.27e7.33 (5H,
m, C-200H-C-600H), 7.59 (1H, d, J ¼ 15.68, Ce1H); 13C NMR (75.5 MHz,
CDCl3):
d
35.36 (SCH2), 58.33 and 63.08 (3ꢁ OCH3), 107.85 (C-20 and
C-60), 126.03 (C-400), 129.43 (C-500 and C-600), 130.78 (C-2, C-40, C-200
and C-300), 131.65 (C-10), 139.78 (C-100), 143.14 (C-30 and C-50), 155.64
(C-1), 190.94 (CO); HRMS calcd for C19H20O4S m/z 344.1139,
observed 344.1139.
5.3.11. Octyl 30,40,50-trimethoxydihydrothiocinnamate (43)
It was obtained as colorless oil in 98% IR (thin film): 1688, 1591,
1463, 1345, 1239, 1130, 981, 757 cmꢂ1; 1H NMR (300 MHz, CDCl3):
d
0.88 (3H, t, J ¼ 7.35 Hz, CH3), 1.27e1.55 (12H, m, C-200H, C-300H, C-
400H, C-500H, C-600 and C-700H), 2.87e2.89 (6H, m, C-100H, Ce1H,
5.3.6. Ethyl 20,50-dimethoxythiocinnamate (34)
Ce2H), 3.8 (9H, s, 3ꢁ OCH3), and 6.4 (2H, s, aromatic protons); 13
C
It was obtained as light green viscous oil in 98% yield. IR (Thin
NMR (75.5 MHz, CDCl3): 13.9 (CH3), 28.6, 28.8, 28.9, 28.98, 29.4 and
29.5 (C-200, C-300, C-400, C-500, C-600, C-700), 31.7 (C-2), 45.4 (C-1), 55.9
(3ꢁ OCH3), 60.6 (SCH2), 105.2 (C-20 and C-60), 127.6 (C-10) 135.7 (C-
40), 153.0 (C-30 and C-50) and 198.4 (CO); HRMS calcd for C20H32O4S
m/z 368.1941, observed m/z 368.1940.
film): 1663, 1605, 1496, 1464, 1288, 1222, 1140, 1027 cmꢂ1; 1H NMR
data (300 MHz, CDCl3); 1H NMR data (300 MHz, CDCl3);
d 1.0 (3H, t,
J ¼ 6.8 Hz, CH2CH3), 3.0 (2H, q, J ¼ 7.04 Hz, CH2CH3), 3.7 (6H, s, 2ꢁ
OCH3), 6.7 (1H, s, AreH) 6.8 (1H, d, J ¼ 12.4 Hz, Ce2H), 6.87 (1H, s,
AreH), 7.0 (1H, s, AreH) and 7.8 (1H, d, J ¼ 15.9 Hz, Ce1H); 13C NMR
data (75.5 MHz, CDCl3):
d
14.8 (CH2CH3), 23.5 (CH2CH3), 56.32(2ꢁ
5.3.12. Benzyl 30,40,50-trimethoxydihydrothiocinnamate (44)
It was obtained as light green oil in 98% yield. IR (thin film):
OCH3), 112.7 (C-2), 113 (C-30), 117.6 (C-60), 123.9 (C-10), 126.2 (C-40),
135.87 (C-1), 153.49 (C-50), 153.8(C-20) and 190.4 (C]O); HRMS
calcd for C13H16O3S m/z 252.0820, observed 252.0818.
1687, 1590, 1458, 1346, 1239, 1129, 928, 755 cmꢂ1 1H NMR
.
(300 MHz, CDCl3): d 2.87e2.89 (4H, m, Ce1H and Ce2H), 3.80 (9H,
m, 3ꢁ OCH3), 4.1 (2H, s, SCH2), 6.37 (2H, s, C-20 and C-60), 7.23e7.25
(5H, m, aromatic protons); 13CNMR (75.5 MHz, CDCl3): 31.6 (C-2),
45.1 (C-1), 55.9 (3ꢁ OCH3), 60.6 (SCH2), 105.35 (C-20 and C-60), 127.1
(C-400), 128.51 and 128.65 (C-200, C-600, C-300 and C-500), 135.6 (C-40),
143.31 (C-100), 153.1 (C-30 and C-50) and 197.5 (CO); HRMS calcd for
C19H22O4S m/z 346.1240, observed m/z 346.1244.
5.3.7. Propyl 20,50-dimethoxythiocinnamate (35)
It was obtained as light green viscous oil in 97% yield. IR (Thin
film): 1664, 1607, 1497, 1463, 1289, 1223, 1140, 1030 cmꢂ1; 1H NMR
data (300 MHz, CDCl3);
d
1.0 (3H, t, J ¼ 7.2 Hz, CH2CH2CH3),1.66 (2H,
m, CH2CH2CH3), 2.97 (2H, t, J ¼ 7.0 Hz, CH2CH2CH3), 3.7 (3H, s,
OCH3), 3.8 (3H, s, OCH3), 6.77 (1H, d, J ¼ 16.0 Hz, Ce2H), 6.82e7.02
(3H, m, AreH) and 7.89 (1H, d, J ¼ 15.9 Hz, Ce1H); 13C NMR data
5.4. Synthesis of the thionocinnamates 54e61
(75.5 MHz, CDCl3):
d 13.6 (CH2CH3CH3), 13.6(CH2CH2CH3), 23.3
(CH2CH2CH3), 56.0 and 56.3 (2ꢁ OCH3), 112.7 (C-2), 113.6 (C-30),
117.6 (C-60), 123.9 (C-10) 126.2 (C-40), 135.8 (C-1), 153.5 (C-50), 153.8
(C-20) and 190.44 (C]O); HRMS calcd for C14H18O3S m/z 266.0977,
observed 266.0972.
The thionocinnamates 541e61 were prepared by the sulfonation
of their corresponding oxygenated cinnamates 13 and 47e53,
[10,34] which in turn were prepared from the corresponding
commercially available cinnamic acids and alcohols using P2S5 with
little modification (Scheme 5) to the literature reported procedure
[35]. All the eight cinnamates (13 and 47e53) were freshly
recrystallized prior to their use, and the solvent dioxane was freshly
dried and distilled. A mixture of the oxygenated cinnamate 13/
47e53 (100 mg) and P2S5 (1.5 equivalent) was refluxed in anhy-
drous dioxane (10 mL), the progress of the reaction was monitored
by TLC. After completion of the reaction, the crude reaction mixture
was poured into ice-cold water and extracted with ethyl acetate
(3 ꢁ 30 mL). The combined organic layer was dried over sodium
sulfate and evaporated in vacuo, and the residual oil was chroma-
tographed on a silica gel column using petroleum ether. All the
novel thionocinnamate 54e61 were fully and unambiguously
identified on the basis of their spectral (IR, 1H NMR, 13C NMR
spectra and HRMS) analysis. It may be mentioned that though the
compounds 58 [35,36] and 54 [37] are mentioned in the literature,
but neither of these literature reports have the spectral data of the
compounds for comparison, one of them does not have even the
correct structure. For compound 54, no IR, UV, 13C NMR, MS or
HRMS, or elemental analysis, or Rf (TLC) values are given, only the
5.3.8. Octyl 20,50-dimethoxythiocinnamate (36)
It was obtained as colorless oil in 93% yield. IR (Thin film): 1665,
1607, 1496, 1464, 1288, 1222, 1140, 1031 cmꢂ1 1H NMR (300 MHz,
;
CDCl3):
d
0.88 (3H, t, J ¼ 7.37 Hz, CH3), 1.28e1.39 (10H, m, 5ꢁ CH2),
1.63 (2H, m, SCH2CH2), 2.99 (2H, t, J ¼ 7.3 Hz, SCH2CH2), 3.83 (6H, s,
2ꢁ OCH3), 6.71 (1H, d, J ¼ 15.7 Hz, Ce2H), 6.80e7.0 (3H, m, AreH),
7.86 (1H, d, J ¼ 15.8 Hz, Ce1H) 13C NMR (75.5 MHz, CDCl3): 13.9
(CH3), 22.5 (C-700), 28.8, 29.0 and 29.5 (C-200, C-300, C-400, C-500, C-600),
31.6 (C-100), 55.9 (2ꢁ OCH3), 112.4 (C-2), 113.26 and 117.3 (C-30 and
C-40), 123.63 (C-10), 125.86 (C-60), 135.4 (C-1), 153.4 (C-20 and C-50)
and 191.04 (C]O); HRMS calcd for C19H28O3S m/z 336.1759,
observed m/z 336.1762.
5.3.9. Benzyl 20,50-dimethoxythiocinnamate (37)
It was obtained as light green oil in 98% yield. IR (thin film):
1665, 1607, 1496, 1464, 1288, 1222, 1140, 1031 cmꢂ1 1H NMR
;
(300 MHz, CDCl3):
d
3.76 (6H, s, 2ꢁ OCH3), 4.22 (2H, s, CH2),
6.73e7.31 (9H, m, aromatic protons and Ce2H) and 7.92 (1H, d,
J ¼ 15.8, Ce1H); 13C NMR (75.5 MHz, CDCl3): 33.12 (CH2), 55.69 and
55.98 (2ꢁ OCH3), 112.44, 113.0 and 1117.5 (C-30, C-40 and C-60),
123.48 (C-10), 123.4, 125.2, 127.1, 128. 5 and 128.8 (C-200, C-300, C-400,
C-500, C-600 and C-2), 136.2 (C-1), 137.78 (C-100), 153.5 (C-20 and C-50),
1
The thionocinnamate 54 (ETMTC) was prepared by using the natural product 47
(ETMC) isolated from Piper longum.

S. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 5498e5511
5509
R2
R5
R2
R2
R3
R4
R1
R3
R4
R1
R3
R4
R1
P2S5
H2SO4, 70 o
R6OH
5, 7, 45 & 46
C
OR6
OH
Dioxane, reflux
OR6
S
R5
O
R5
13 & 47-53
O
45 R6 = C2H5
46 R6 = C4H9
1, 2 & 4
63-89 %
54-61
49 R1 = R4 = OCH3, R2 = R3 = R5 = H
50 R1 = R2 = R3 = R4 = R5 = H
Starting
Product
Thiono-
cinnamate
%
Yield
Oxygenated
Cinnamate
4710,†
4810
R1
R2
R3
R4
R5 R6
H
H
OCH3
OCH3
H
H
H
H
OCH3
OCH3
H
H
H
H
H
H
OCH3
OCH3
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
C2H5
C3H7
C2H5
C3H7
C2H5
C3H7
CH(CH3)2
C4H9
63
69
78
79
86
88
88
89
54
55
56
57
58
59
60
61
4930
13*
5110
5210
5010
5310
†The natural product isolated from Piper longum was used
*We have prepared 13 as a novel compound (Scheme 1)
Scheme 5.
1H NMR spectrum is reported, that too is not correct, e.g. the two
NMR (300 MHz, CDCl3):
d
1.47 (3H, t, J ¼ 6.6 Hz, OCH2CH3), 3.78 and
aromatic protons are reported as a singlet at
d
1.13 ppm. Further, no
3.85 (6H, s, 2ꢁ OCH3), 4.63 (2H, q, J ¼ 6.5 Hz, OCH2CH3), 6.91 (1H, d,
detailed method of preparation or characterization or any cross
reference is given. So we strongly suspect that this is not the correct
compound reported by a pharmacy group, and the publication is in
an obscure non-stream chemistry journal which is not well circu-
lated [37]. There is no other report in the literature on the
compound 54 and we have synthesized completely and unambig-
uously characterized novel compound, viz the most active thio-
nocinnamate 54. Though there are two literature references [35,36]
for the compound 58, but no spectral data is given for comparison
in any of them.
J ¼ 14.9 Hz, Ce2H), 7.03e7.08 (3H, Ce30H, Ce40H and Ce60H) and
8.02 (1H, d, J ¼ 15.9 Hz, Ce1H); 13CNMR (75.5 MHz, CDCl3):
d 14.21
(OCH2CH3), 56.22 and 56.53 (2ꢁ OCH3), 68.24 (OCH2CH3), 112.91
(C-60), 113.13 (C-40), 117.92 (C-30), 124.61 (C-2), 130.12 (C-10), 135.51
(C-1), 153.52 (C-20), 154.01 (C-50) and 211.2 (C]S). HRMS: Calcd for
C13H17O3S [M þ H]þ 253.0820, found 253.0818.
5.4.4. Propyl 20,50-dimethoxythionocinnamate (57)
It was obtained as an orange red viscous oil in 79% yield. IR (Thin
film): 1664 (C]S),1605,1496,1462,1277,1222,1175, 1048 cmꢂ1; 1H
NMR (300 MHz, CDCl3):
d
1.05 (3H, t, J ¼ 7.4 Hz, OCH2CH2CH3), 1.90
5.4.1. Ethyl 30,40,50-trimethoxythionocinnamate (54)
(2H, m, OCH2CH2CH3), 3.80 and 3.85 (6H, s, 2ꢁ OCH3), 4.53 (2H, t,
J ¼ 6.6 Hz, OCH2CH2CH3), 6.90 (1H, d, J ¼ 14.7 Hz, Ce2H), 7.04e7.09
(3H, m, Ce30H, Ce50H and Ce60H) and 8.02 (1H, d, J ¼ 15.9, Ce1H);
It was obtained as an orange solid having melting point 90e91 ꢃC
in 63% yield. IR (KBr): 1633 (C]S), 1580, 1464, 1421, 1287, 1126,
1005 cmꢂ1 1H NMR (300 MHz, CDCl3):
d
1.93 (3H, t, J ¼ 7.4 Hz,
13CNMR (75.5 MHz, CDCl3):
d 18.21 (OCH2CH2CH3), 29.4
OCH2CH3), 3.81, 3.84 and 3.90 (9H, s, 3ꢁ OCH3), 4.53 (2H, q, J ¼ 6.5 Hz,
OCH2CH3), 6.73 (2H, s, Ce20H and Ce60H), 6.94 (1H, d, J ¼ 15.6 Hz,
Ce2H), and 7.60 (1H, d, J ¼ 15.6, Ce1H). 13CNMR (75.5 MHz, CDCl3):
(OCH2CH2CH3), 63.82 (2ꢁ OCH3), 81.22 (OCH2CH2CH3), 120.13 (C-
2),120.31 (C-60), 125.26 (C-40),131.82 (C-10), 137.31 (C-30),142.90 (C-
1), 160.81 (C-20), 161.24 (C-50) and 218.62 (C]S); HRMS: Calcd for
C14H19O3S [M þ H]þ 267.1055, found 267.1055.
d
11.02 (OCH2CH3), 56.58 (2ꢁ OCH3), 61.36 (OCH3), 74.10 (OCH2CH3),
106.05(C-20 and C-60),128.72 (C-2),130.53 (C-10),131.20 (C-40),140.70
(C-1), 153.86 (C-30 and C-50), and 210.73 (C]S). HRMS Calcd for
C14H19O4S [M þ H]þ 283.3819, found 283.3820.
5.4.5. Ethyl thionocinnamate (58)
It was obtained as an orange red oil in 86% yield. IR (Thin film):
1613 (C]S), 1447, 1373, 1328, 1289, 1257, 1185, 1093 cmꢂ1; 1H NMR
5.4.2. Propyl 30,40,50-trimethoxythionocinnamate (55)
(300 MHz, CDCl3):
d
1.41 (3H, t, J ¼ 6.14 Hz, OCH2CH3), 5.76 (2H, q,
It was obtained as an orange red viscous oil in 69% yield. IR (Thin
J ¼ 6.24 Hz, OCH2CH3), 7.0 (1H, d, J ¼ 15.74 Hz, Ce2H), 7.35e7.52
film): 1635 (C]S), 1580, 1464, 1421, 1287, 1126, 1005 cmꢂ1 1H NMR
(5H, m, Ce20H, Ce30H, Ce40H, Ce50H and Ce60H) and 7.63 (1H, d,
(300 MHz, CDCl3):
d
1.07 (3H, t, J ¼ 7.3 Hz, OCH2CH2CH3), 1.91 (2H,
J ¼ 15.75, Ce1H); 13CNMR (75.5 MHz, CDCl3):
d 21.71 (OCH2CH3),
m, OCH2CH2CH3), 3.82, 3.84 and 3.89 (9H, s, 3ꢁ OCH3), 4.53 (2H, t,
J ¼ 6.5 Hz, OCH2CH2CH3), 6.70 (2H, s, C-20H and C-60H), 6.92 (1H, d,
J ¼ 15.6 Hz, Ce2H), and 7.61 (1H, d, J ¼ 15.6, Ce1H). 13CNMR
75.21 (OCH2CH3), 128.7 (C-2), 129.3 (C-20 and C-60), 130 (C-40),
130.57 (C-30 and C-50), 135.24 (C-10), 140.53 (C-1) and 209.9 (C]S).
HRMS: Calcd for C11H13OS [M þ H]þ 193.0609, found 193.0610.
(75.5 MHz, CDCl3):
d 11.04 (OCH2CH2CH3), 22.14 (OCH2CH2CH3),
56.60 (2ꢁ OCH3), 61.33 (OCH3), 74.0 (OCH2CH2CH3), 106.0 (C-20 and
C-60), 128.73 (C-2), 130.57 (C-10), 131.16 (C-40), 140.72 (C-1), 153.88
(C-30 and C-50), and 210.70 (C]S). HRMS Calcd for C15H21O4S
[M þ H]þ 297.3819, found 297.3820.
5.4.6. Propyl thionocinnamate (59)
It was obtained as orange red oil in 88% yield. IR (thin film): 1613
(C]S),1450,1202,1170, 979 cmꢂ1. 1H NMR (300 MHz, CDCl3):
d 1.05
(3H, t, J ¼ 7.3 Hz, OCH2CH2CH3), 1.9 (2H, m, OCH2CH2CH3), 4.5 (2H, t,
J ¼ 6.5 Hz, OCH2CH2CH3), 7.04 (1H, d, J ¼ 15.8 Hz, Ce2H), 7.30e7.54
(5H, m, Ce20H, Ce30H, Ce40H, Ce50H and Ce60H) and 7.68 (1H, d,
5.4.3. Ethyl 20,50-dimethoxythionocinnamate (56)
It was obtained as an orange red viscous oil in 78% yield. IR (Thin
J
¼
15.8 Hz, Ce1H). 13CNMR (75.5 MHz, CDCl3):
d
10.23
film): 1663 (C]S), 1595, 1496, 1464, 1340, 1278, 1120, 1040 cmꢂ1 1H
(OCH2CH2CH3), 21.4 (OCH2CH2CH3), 73.2 (OCH2CH2CH3), 128.0 (C-

5510
S. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 5498e5511
2), 128.5 (C-20 and C-60), 128.7 (C-40), 129.9 (C-30 and C-50), 134.4 (C-
10), 139.95 (C-1) and 210.2 (C]S). HRMS: Calcd for C12H15OS
[M þ H]þ, 207.0844 found. 207.0845.
antibodies for ICAM-1, VCAM-1 and or E-selectin (Santa Cruz, USA).
Cells were washed with PBS and incubated with peroxidase-
conjugated goat anti-mouse secondary Abs. After a further wash
with PBS, cells were incubated with peroxidase substrate (o-phe-
nylenediamine dihydrochloride 40 mg/100 mL in citrate phosphate
buffer, pH 4.5). The color development reaction was stopped by the
addition of 2 N sulfuric acid and absorbance at 490 nm was
measured using an automated microplate reader (680 Bio-Rad,
USA).
5.4.7. Isopropyl thionocinnamate (60)
It was obtained as orange red viscous oil in 88% yield. IR (thin
film): 1613 (C]S), 1447, 1257, 1093, 971, 752 cmꢂ1 1H NMR
(300 MHz, CDCl3):
d
1.41 [6H, d, J ¼ 6.14 Hz, CH(CH3)2], 5.76 (1H, m,
CH(CH3)2), 7.0 (1H, d, J ¼ 15.8 Hz, Ce2H), 7.35e7.52 (5H, m, Ce20H,
Ce30H, Ce40H, Ce50H and Ce60H) and 7.63 (1H, d, J ¼ 15.8 Hz,
Ce1H). 13CNMR (75.5 MHz, CDCl3):
d
21.71 (CH(CH3)2), 75.21
5.8. Determination of IC50
(CH(CH3)2), 128.7 (C-2), 129.3 (C-40), 130.06 (C-30 and C-50), 130.57
(C-20 and C-60), 135.24 (C-10), 140.53 (C-1) and 209.9 (C]S). HRMS:
Calcd for C12H15OS [M þ H]þ 207.0844, found 207.0843.
The percentage inhibitions of each compound at its various log
concentrations were measured and plotted graphically from three
independent experiments. From the graph plotted, the concentra-
tion at which a compound showed 50% inhibition was taken as its
IC50 value.
5.4.8. Butyl thionocinnamate (61)
It was obtained as orange red oil in 89% yield. IR (thin film): 1656
(C]S), 1618, 1452, 1215, 1040, 756 cmꢂ1 1H NMR (300 MHz, CDCl3):
d
1.02 (3H, t,
J
¼
7.2 Hz, OCH2CH2CH2CH3), 1.52 (2H, m,
5.9. Flow cytometry analysis
OCH2CH2CH2CH3), 1.83 (2H, m, OCH2CH2CH2CH3), 4.61 (2H, t,
J ¼ 6.2 Hz, OCH2CH2CH2CH3), 7.02 (1H, d, J ¼ 15.8 Hz, Ce2H), 7.3e7.5
(5H, m, Ce20H, Ce30H, Ce40H, Ce50H and Ce60H) and 7.67 (1H, d,
The cell surface expression of ICAM-1, VCAM-1 and E-selectin on
endothelial cells was further confirmed by flow cytometry [10].
Briefly, endothelial cells were incubated with or without ethyl
30,40,50-trimethoxythionocinnamate (54) for 2 h followed by TNF-
J¼ 15.8 Hz, Ce1H).13CNMR (75.5 MHz, CDCl3):
d 13.12 (OCH2CH2CH2C-
H3), 18.74 (OCH2CH2CH2CH3), 27.75 (OCH2CH2CH2CH3), 71.34
(OCH2CH2CH2CH3), 127.72 (C-2), 128.35 (C-40), 128.41 (C-30 and C-50),
129.62 (C-20 and C-60) 134.97 (C-10), 139.62 (C-1) and 209.9 (C]S).
HRMS: Calcd for C13H17OS [M þ H]þ 221.3305, found 221.303.
a
(10 ng/mL). E-selectin was measured at 4 h whereas ICAM-1 and
VCAM-1 expression was measured 16 h after TNF- treatment. The
a
cells were washed with PBS and dislodged, following which they
were incubated with anti-ICAM-1, anti-VCAM-1, anti-E-selectin or
5.5. Cell culture
control IgG antibody (1.0 m
g/106 cells, 30 min, 4 ꢃC). After incuba-
tion, the cells were washed with PBS and then stained with FITC-
conjugated goat anti-mouse IgG for 30 min at 4 ꢃC. Finally, the
cells were fixed with 1.0% paraformaldehyde and analyzed for the
expression of cell adhesion molecules using a flow cytometer (FACS
Vantage, Becton & Dickinson, USA). For each sample, 20,000 events
were acquired. Analysis was carried out by using Cell Quest Soft-
ware (Becton Dickinson, USA). The auto-fluorescence intensity was
subtracted from treated conditions and mean fluorescence inten-
sity was estimated from three independent experiments and bar
diagrams were plotted.
Primary endothelial cells were isolated from human umbilical
cord by mild trypsinization [10]. The cells were grown in M-199
medium supplemented with 15% heat inactivated fetal calf serum,
2 mM
mycin, 0.25
(50 g/mL) and heparin (5 U/mL) in 5% CO2 in humidified water
L-glutamine, 100 units/mL penicillin, 100
mg/mL strepto-
m
g/mL amphotericin B, endothelial cell growth factor
m
jacketed incubator. The purity of endothelial cells was determined
by FACS analysis of E-selectin expression.
5.6. Cell viability assay
5.10. Neutrophils isolation
The cytotoxicity of ethyl 30,40,50-trimethoxythionocinnamate
(54) and other analogs was analyzed by using trypan blue exclusion
test as described [21], and was further confirmed by colorimetric
MTT assay as described [10]. Briefly, endothelial cells were plated
onto 96-microwell plates and allowed to attach overnight. Next
day, the cells were treated either with DMSO (0.25%) or tested
Neutrophils were isolated from peripheral blood of healthy
individuals [19]. Briefly, blood was collected in heparin solution
(20 U/mL) and erythrocytes were removed by sedimentation
against 6% dextrin solution. Plasma rich white blood cells were
layered over ficoll-hypaque solution followed by centrifugation
(300 g for 20 min, 20 ꢃC). The top saline layer and the ficoll-hypaque
layer were aspirated leaving the neutrophils/RBC pellet. The
residual red blood cells were removed by hypotonic lysis. Isolated
cells were washed with PBS and resuspended in PBS containing
5 mM glucose, 1 mM CaCl2 and 1 mM MgCl2 at a final concentration
of 6 ꢁ 105 cells/mL. This procedure usually resulted in approxi-
mately 95% neutrophils and the cell viability was more than 95% as
detected by trypan blue exclusion test.
compounds for 24 h followed incubation with 100
mL in PBS) for additional 4 h. Finally cells were washed with PBS,
and incubated with 100 l DMSO to dissolve water insoluble MTT-
ml of MTT (5 mg/
m
formazan crystals. Absorbance was recorded at 570 nm in an ELISA
reader (BIO RAD, Model 680, and USA). All experiments were per-
formed at least 3 times in triplicate.
5.7. Cell ELISA for measurement of ICAM-1, VCAM-1 and E-selectin
Expression of ICAM-1, VCAM-1 and E-selectin was quantified by
whole cell-ELISA [10]. Briefly, endothelial cells were pre-incubated
with or without the tested compounds at desired concentrations
5.11. Neutrophils adhesion assay
Neutrophils adhesion assay was performed under static condi-
tions as described previously [19]. Endothelial cells were plated to
confluence in 96-well culture plates and were incubated with or
without ethyl 30,40,50-trimethoxythionocinnamate (54) for 2 h fol-
for 2 h followed by induction with TNF-a (10 ng/mL) or LPS (1 mg/
mL) for 16 h for ICAM-1 and VCAM-1 expression and 4 h for E-
selectin expression. After incubation, the cells were fixed imme-
diately within the plate with 1.0% glutaraldehyde. Nonspecific
protein binding was blocked subsequently using non-fat dry milk
(3.0% in PBS). Cells were incubated overnight at 4 ꢃC with primary
lowed by induction with TNF-a (10 ng/mL) for 6 h. After washing,
the endothelial monolayer was incubated with neutrophils
(6 ꢁ 104/well) for 1 h at 37 ꢃC. The non-adherent neutrophils were

S. Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 5498e5511
5511
[9] M.R. Weiser, S.A.L. Gibbs, H.B. Hechtman, in: L.C. Paul, T.B. Issekutz (Eds.),
Adhesion Molecules in Health and Disease, Marcel Dekker Inc., NewYork,
1997, pp. 55e86.
[10] S. Kumar, P. Arya, C. Mukherjee, B.K. Singh, N. Singh, V.S. Parmar, A.K. Prasad,
B. Ghosh, Biochemistry 44 (2005) 15944e15952.
[11] S. Kumar, B.K. Singh, N. Kalra, V. Kumar, A. Kumar, A.K. Prasad, H.G. Raj,
V.S. Parmar, B. Ghosh, Bioorg. Med. Chem. 13 (2005) 1605e1613.
[12] R. Singh, G.M. Whitesides, S. Patai, Z. Rappoport, The Chemistry of Sulphur-
containing Functional Group. John Wiley and Sons, Chichester, UK, 1993,
pp. 82e89.
removed by washing the wells with PBS and neutrophils bound to
endothelial cells were assayed by adding a substrate solution con-
sisting of o-phenylenediamine dihydrochloride (40 mg/100 mL in
citrate phosphate buffer, pH 4.5), 0.1% cetitrimethyl ammonium
bromide and 3-amino-1,2,4 triazole (1 mM). The absorbance was
determined at 490 nm using an automated microplate reader
(Spectramax 190, Molecular Devices, USA).
[13] S. Chevion, M. Roberts, M. Chevion, Free Radic. Biol. Med. 28 (2000) 860e870.
[14] E.V. Dorozhko, I. Korotkova, Pharma. Chem. J. 44 (2011) 581e584.
[15] I. Rahman, S.K. Biswas, P.A. Kirkham, Biochem. Pharmacol. 72 (2006)
1439e1452.
[16] C. Weber, W. Erl, A. Pietsch, P.C. Weber, Circulation 91 (1995) 1914e1917.
[17] L.J. Egan, D.C. Mays, C.J. Huntoon, M.P. Bell, M.G. Pike, W.J. Sandborn,
J.J. Lipsky, D.J. McKean, J. Biol. Chem. 274 (1999) 26448e26453.
[18] N.M. Dagia, N. Harii, A.E. Meli, X. Sun, C.J. Lewis, L.D. Kohn, D.J. Goetz,
J. Immunol. 173 (2004) 2041e2049.
6. Statistical analysis
Results are given as means ꢀ SD. Independent two-tailed
Student’s t test was performed. Differences were considered
statistically significant for p < 0.05. The statistical analysis was
performed using Microcal Origin software (ver 3.0; Microcal Soft-
ware Inc, Northampton, MA and Cell Quest Software, Bec-
toneDickinson, USA).
[19] A. Sakai, Life Sci. 58 (1996) 2377e2387.
[20] C. Weber, W. Erl, A. Pietsch, M. Strobel, H.W.L. Ziegler-Heitbrock, P.C. Weber,
Arterioscl. Thromb. Vascl. Biol. 14 (1994) 1665e1673.
[21] S. Kumar, V. Singhal, R. Roshan, A. Sharma, G.W. Rembhotkar, B. Ghosh, Eur. J.
Pharmacol. 575 (2007) 177e186.
[22] S. Kumar, A. Sharma, B. Madan, V. Singhal, B. Ghosh, Biochem. Pharmacol. 73
(2007) 1602e1612.
Acknowledgments
[23] S. Kumar, B.K. Singh, A.K. Pandey, A. Kumar, S.K. Sharma, H.G. Raj, A.K. Prasad,
E. Van der Eycken, V.S. Parmar, B. Ghosh, Bioorg. Med. Chem. 15 (2007)
2952e2962.
[24] S.K. Tusi, N. Ansari, M. Amini, A.D. Amirabad, A. Shafiee, F. Khodagholi,
Apoptosis 15 (2010) 738e751.
[25] E. Heiss, C. Herhaus, K. Klimo, H. Bartsch, C. Gerhauser, J. Biol. Chem. 276
(2001) 32008e32015.
[26] A. Gopalakrishnan, A.N. Tony Kong, Food Chem. Toxicol. 46 (2008) 1257e1270.
[27] M. Sova, A. Perdih, M. Kotnik, K. Kristan, L.T. Rizner, T. Solmajer, S. Gobeca,
Bioorg. Med. Chem. 14 (2006) 7404e7418.
Authors acknowledge the help of St. Stephen’s Hospital, Delhi
for providing the umbilical cord. Authors acknowledge the help
provided by Ms. Gauri Awasthi and Reema Roshan. This work was
partly funded by the Council of Scientific and Industrial Research
(CSIR, India) grant NWP0033 (to B.G.), NIH grants- P50HL084945,
R33 33AI080541 P50ES015903, P01 ES018176 and ES03819 (SB) and
by the University of Delhi to Professor VS Parmar, Professor Ashok K
Prasad and Dr BK Singh.
[28] A.K. Sinha, A. Sharma, A. Swaroop, V. Kumar, Tetrahedron 63 (2006)
1000e1007.
[29] W. Pollmann, G. Schramm, Biochim. Biophys. Acta 80 (1964) 1e7.
[30] G.J. Braude, Org. Chem. 22 (1957) 1675e1678.
References
[31] M. Arisawa, T. Kubota, M. Yamaguchi, Tet. Lett. 49 (2008) 1975e1978.
[32] R.D. Mazery, M. Pullez, F. López, S.R. Harutyunyan, A.J. Minnaard, B.J. Feringa,
J. Am. Chem. Soc. 127 (2005) 9966e9967.
[33] T. Takido, M. Toriyama, K. Itabashi, Synthesis (1988) 404e406.
[34] H.B. Bang, S.Y. Han, D.H. Choi, J.W. Hwang, J.G. Jun, ARKIVOC ii (2009)
112e125.
[35] T.J. Curphey, J. Org. Chem. 67 (2002) 6461e6473.
[36] W.H. Bunnelle, B.R. McKinnis, B.A. Narayanan, J. Org. Chem. 55 (1989)
768e770.
[1] T.A. Springer, Cell 76 (1994) 301e314.
[2] A. Davidson, B. Diamond, New Engl. J. Med. 345 (2001) 340e350.
[3] P. Vanderslice, R.J. Biediger, D.G. Woodside, K.L. Berens, G.W. Holand,
R.A. Dixon, Pulm. Pharmacol. Ther. 17 (2004) 1e10.
[4] H. Ulbrich, E.E. Einar Eriksson, L. Lindbom, Trends Pharmacol. Sci. 24 (2003)
640e647.
[5] A. Gorski, Immunol. Today 15 (1994) 251e255.
[6] W.A. Muller, Lab. Invest. 82 (2002) 521e533.
[7] J.M. Harlan, R.K. Winn, Crit. Care Med. 30 (2002) S214eS219.
[8] A. Mantovani, F. Bussolino, M. Introna, Immunol. Today 18 (1997) 231e240.
[37] F. Guerrera, M.C. Sarva, M.A. Siracusa, G. Ronsisvalle, G. Conforto, S. Dibennardo,
G. Failla, M. Matera, Farmaco Edizione Scientifica 43 (1988) 551e558.