Synthesis and biological evaluation of novel 2′,4′,5′- trimethoxyflavonol derivatives as anti-inflammatory and antimicrobial agents

Article abstract of DOI:10.1007/s00044-013-0651-z

A series of novel 3-hydroxy-2-(2,4,5-trimethoxyphenyl)-4H-chromen-4-one (flavonol) derivatives (2a-u) of biological interest have been prepared via CLAISEN-SCHMIDT condensation followed by ALGAR-FLYNN-OYAMADA reaction and to search for the potent nonsteroidal anti-inflammatory agents from this novel series. All the synthesized compounds have been screened for their in vitro proinflammatory cytokines tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) inhibitory activity along with antimicrobial activity. As many as three compounds viz. 2h, 2l, and 2q from this novel series were found to be potent TNF-α and IL-6 inhibitor (up to 72-81 % TNF-α and 86-92 % IL-6 inhibitory activity) but at 10 μM concentration as compared with the standard dexamethasone (71 % TNF-α and 84 % IL-6 inhibitory activities at 1 μM concentration). While the compounds 2d, 2m, 2n, and 2s were found to be potent antimicrobial agent showing even 2-2.5-fold more potency than that of standard ciprofloxacin and miconazole at the same MIC value of 10 μg/mL.

Full text of DOI:10.1007/s00044-013-0651-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0651-z
ORIGINAL RESEARCH
Synthesis and biological evaluation of novel 20,40,50-
trimethoxyflavonol derivatives as anti-inflammatory
and antimicrobial agents
•
Girish D. Hatnapure Ashish P. Keche Atish H. Rodge
•
•
•
•
•
Rajesh H. Tale Satish S. Birajdar Mahendra J. Pawar
Vandana M. Kamble
Received: 28 February 2013 / Accepted: 25 May 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of novel 3-hydroxy-2-(2,4,5-trimethoxy-
phenyl)-4H-chromen-4-one (flavonol) derivatives (2a–u) of
biological interest have been prepared via CLAISEN–
SCHMIDT condensation followed by ALGAR–FLYNN–
OYAMADA reaction and to search for the potent nonste-
roidal anti-inflammatory agents from this novel series. All
the synthesized compounds have been screened for their
in vitro proinflammatory cytokines tumor necrosis factor
(TNF-a) and interleukin-6 (IL-6) inhibitory activity along
with antimicrobial activity. As many as three compounds
viz. 2h, 2l, and 2q from this novel series were found to be
potent TNF-a and IL-6 inhibitor (up to 72–81 % TNF-a and
86–92 % IL-6 inhibitory activity) but at 10 lM concentra-
tion as compared with the standard dexamethasone (71 %
TNF-a and 84 % IL-6 inhibitory activities at 1 lM con-
centration). While the compounds 2d, 2m, 2n, and 2s
were found to be potent antimicrobial agent showing even
2–2.5-fold more potency than that of standard ciprofloxacin
and miconazole at the same MIC value of 10 lg/mL.
Keywords Flavonoid ꢀ NSAIDs ꢀ
ALGAR–FLYNN–OYAMADA anti-inflammatory
and antimicrobial activities
Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) have been
recognized as a vital therapeutic agents for the alleviation
of pain and inflammation associated with a numerous
pathologic conditions viz. arthritis, bursitis, and tendinitis.
However, chronic administration of NSAIDs has been
associated with clinically significant complications such as
gastrointestinal (GI) symptoms including mucosal damage,
bleeding, nausea, heartburn, dyspepsia, abdominal pain,
and renal toxicity, etc. Nowadays available polytherapy for
inflammatory conditions associated with microbial infec-
tions increases the risk for developing NSAID-related
complications especially in elderly, patients with prior
history of peptic ulcer disease, patients with impaired liver
or kidney functions, and patients taking anticoagulants,
corticosteroids, etc. Hence, there is a pressing need for the
drugs having both anti-inflammatory and antimicrobial
activities, both from the pharmacoeconomic as well as the
patient compliance point of view. (Allison et al., 1992;
Flower, 2003; Bekhit and Abdel-Azeim, 2004).
G. D. Hatnapure ꢀ A. P. Keche ꢀ S. S. Birajdar
Department of Chemistry, Maharashtra Udayagiri
Mahavidyalaya, Udgir, India
V. M. Kamble (&)
Department of Chemistry, The Institute of Science,
Mumbai, India
e-mail: m.vandana248@gmail.com
The flavonoids are important bioactive molecules hav-
ing a group of natural products present in a wide variety of
plants reported to have potent anti-inflammatory activity in
both (in vitro and in vivo) although not fully understood
(Harborne and Williams, 2000; Middleton et al., 2000).
The several action mechanisms are proposed to explain
in vivo anti-inflammatory action of flavonoids depending
on their chemical structures. The unique action mecha-
nisms and significant in vivo activity make flavonoids a
A. H. Rodge ꢀ R. H. Tale
Dendrimer and Bio-organic Chemistry Laboratory, School
of Chemical Science, S. R. T. M. University, Nanded, India
M. J. Pawar
Department of Nanotechnology, Art, Commerce and Science
College, Amravati, India
123

Med Chem Res
reasonable candidate for new anti-inflammatory drugs.
Flavonoids exhibit a broad range of biological activities,
including antiviral, anti-inflammatory, antioxidant, antial-
lergic, hepatoprotective, antithrombotic and antitumoral
actions (Manthey et al., 2001; Rice-Evans and Miller,
1996; Hollman et al., 1996).
molecular targets for the treatment of the above-mentioned
diseases. The available biopharmaceuticals [TNF-soluble
receptor (Enbrel TM) and TNF antibody (Remicade TM)] are
expensive, difficult to administer orally and have major side
effects on prolonged clinical use. Therefore, there is an
urgent medical need to discover small-molecule agents to
deal with higher levels production of TNF-a.
On going through the literature, we found some flavo-
noids such as luteolin, galangin, etc., inflammatory inhib-
itors, when their structural activity relationships were
compared (Fig. 1), the important moieties are the C-2,
3-double bond, A-ring 5, 7-hydroxyl groups, and B-ring 40-
or 30,40-hydroxyl groups. The C-3 hydroxyl group as in
flavonol is favorable for lipoxygenase (LOX) inhibition
and oral anti-inflammatory activity (Bauman et al., 1980;
Landolfi et al., 1984).
However, the naturally found flavonoids like baicalein,
quercetin, and myricetin have the –OH group in their
structure, but in recent studies it is noteworthy that the
O-alkylation of these phenolic –OH has considerable
attention. Because of converting the trihydroxyl groups to
the alkoxy groups on the ‘‘A’’ ring of baicalein resulting in
increased activity on P-gp 170 inhibition (Lee et al., 2004).
´
Fernandez et al. analyzed inhibition of aldose reductase by
33 baicalein derivatives with modifications on the ‘‘A’’ and
‘‘B’’ rings, leading to the conclusion that compounds with
either three hydroxyl or methoxyl functionalities on the
‘‘A’’ ring are effective inhibitors of aldose reductase
(Fernandez et al., 2005). Furthermore, Morita et al. showed
that the increased lipophilicity of phenolic hydroxyls via
alkylation may enhance the therapeutic potential to
X-linked adrenoleukodystrophy (X-ALD) (Morita et al.,
2005).
In flavones and flavonol of the same type, flavonol
showed greater inhibition than flavones. The tri methoxy
flavonol derivatives have not hitherto tested as anti-
inflammatory agents, in particular against the TNF-a and
IL-6 and also their antimicrobial activities. In order to
improve the anti-inflammatory and antimicrobial activity
of the compound based on quercetin and myricetin core
(Cho et al., 2003). We envisage the new scaffold by
incorporating O-alkylation on ring ‘‘B’’ and different sub-
stituents on ring ‘‘A’’ hitherto remained untested (Fig. 2).
We set out to test our notion by design, synthesis, and
biological evaluation of novel flavonol derivatives.
Nowadays, our research group also engaged in anti-
inflammatory activity study program of novel urea and
thiourea derivatives (Keche et al., 2012a, b; Tale et al.,
2011). Herein, we presenting results on the synthesis and
discovery of potent TNF-a and IL-6 inhibitor-based
20,40,50-trimethoxyflavonol scaffold. The libraries of 21
different compounds (2a–u) are obtained according to
Scheme 1 with good yield.
Based on the aforementioned information, the combi-
nation of the biologically effective –O alkylation with a
flavonol skeleton in continuation with our previous study
(Hatnapure et al., 2012), herein, we reporting a novel series
of 3-hydroxy-2-(2,4,5-trimethoxyphenyl)-4H-chromen-4-
one derivatives and have been screened for their in vitro
proinflammatory cytokines TNF-a and IL-6 inhibitory
activity.
The cytokines are intercellular messengers responsible
for host defense mechanisms as inflammatory, immune and
hematogenic responses. One of the key proinflammatory
cytokine tumor necrosis factor-a (TNF-a) is mainly pro-
duced by the activated macrophages and monocytes, which
further induces the production of the several inflammatory
cytokines such as interleukin-1b (IL-1b), interleukin-6 (IL-
6), and granulocyte–macrophage colony-stimulating factor
(GM-CSF). It is also a multitude of biological activities
linked to pathology of autoimmune diseases such as rheu-
matoid arthritis (RA) (Macnaul et al., 1990; Brennan et al.,
1992), Crohn’s disease (van Dullemen et al., 1995), sys-
temic lupus erythematosus (Maury and Teppo, 1989), mul-
tiple sclerosis (Sharief and Hentges, 1991; Beck et al., 1988;
Gallo et al., 1989), septic shock (Lechner et al., 1992), and
AIDS (Makonkawkeyoon et al., 1993). On the other hand,
cytokine IL-6 (from the series of cytokine-signaling path-
way) contributes to the initiation and extension of the
inflammatory process and considered as a central mediator
in a range of inflammatory diseases but has not received the
desired attention in drug discovery (Dominic and Raj, 2009).
TNF-a and IL-6 are thus pharmaceutically important
Our synthetic strategy for 3-hydroxy-2-(2,4,5-trime-
thoxyphenyl)-4H-chromen-4-one derivatives have been
planned by employing the general synthetic path of reac-
tion. The 21 different 3-hydroxyflavones have been pre-
pared by CLAISEN–SCHMIDT condensation followed by
ALGAR–FLYNN–OYAMADA reaction (Scheme 1) (Li
et al., 2008). The key intermediates 1-(2-hydroxyphenyl)-
OH
3'
OH
4'
1
O
B
B
8
HO
O
O
HO
2
7
A
A
6
OH
3
5
OH
4
OH
O
galangin
luteolin
Fig. 1 Structure of luteolin and galangin
123

Med Chem Res
Fig. 2 New design scaffold
Active core
Active core
OH
OH
OH
OH
OH
HO
O
O
HO
O
O
OH
Quercetin
OH
Myricetin
OH
OH
O
O
O
4'
2'
B
8
1
O
2
7
5'
R
A
C
6
OH
3
4
5
O
O
O
O
O
O
O
O
a
H
R
R
OH
O
OH
O
1a-u
b
O
O
O
O
R
O
OH
2a-u
R = 2a : 5-Cl
2k : 6-F, 8-F
2b : 5-Cl, 7-CH3 2l : 5-F, 7-F
2c : 8-Cl
2m: 6-NO2, 8-F
2n : 6-CN, 8-F
2o : 7-CH3 ,
2d : 6-Cl, 8-Cl
2e 6-CN, 8-Cl
2f : 5-Br
2p : 6-CH3, 8 NO2
2g : 6-Br, 8-NO2 2q : 6-CF3
2h : 5-F
2i : 7-F
2j : 8-F
2r : 6-NO2
2s : 6-SO2Me
2t : 8-CN
2u : 5-CN
Reagents and conditions:- (a) EtOH,60% KOH, 0-rt 6-8h: (b) 15% aq NaOH/ 16% H2O2 (v/ v 1:1) 5-6h.
Scheme 1 Synthesis of 3-hydroxy-2-(2,4,5-trimethoxyphenyl)-4H-chromen-4-one derivatives
3-(2,4,5-trimethoxyphenyl) prop-2-en-1-one (1) derivatives
have been synthesized by the CLAISEN–SCHMIDT con-
densation between substituted 2-hydroxyacetophenones
and 2,4,5-trimethoxybenzaldehyde in moderate to good
yields (60–70 %). The treatment of 1 with hydrogen per-
oxide and base in ethanol under ALGAR–FLYNN–OYA-
MADA conditions afforded the corresponding flavonol
(2a–u) in (50–70 % yields). The desired products were
purified by column chromatography (silica gel with 20 %
ethyl acetate/hexane). The purity of the compounds was
and MS of the newly synthesized compounds have been in
full agreement with their proposed structures.
Having secured the structurally diverse novel 3-hydroxy-
2-(2,4,5-trimethoxyphenyl)-4H-chromen-4-one derivatives,
next in order to search for their proinflammatory cytokines,
TNF-a, and IL-6 production by lipopolysaccharide (LPS) in
THP-1 cells was measured according to the method
described by Hwang et al. (1993). The results of the TNF-a
and IL-6 inhibitory activity are shown in Table 1. As many
as 3 compounds from this library have been found to be
potent TNF-a and IL-6 inhibitor with reference standard
1
checked by TLC and HPLC. Spectral data H, C13 NMR,
123

Med Chem Res
drug dexamethasone. While the compounds 2d, 2m, 2n, and
2s found to be potent antimicrobial agent showing even
2–2.5-fold more potency than that of standard ciprofloxacin
and miconazole at the same MIC value of 10 lg/mL by
using agar-well diffusion method (Sridhar et al., 2004).
Thus the compounds 2h, 2l, and 2q from the series
found to be promising TNF-a and IL-6 inhibitors (up to
72–81 % TNF-a and 82–92 % IL-6 inhibitory activity but
at 10 lM concentration), when compared with standard
dexamethasone (71 % TNF-a and 84 % IL-6 inhibitory
activities at 1 lM concentration). Notably, the compound
2h exhibited much higher TNF-a or IL-6 (81 and 92 %)
inhibitory activity. The compounds 2c, 2e, 2i, 2j, and 2k
exhibited moderate activity even as remaining compounds
found to have low, very low, or no activity at the same
level of concentration against the proinflammatory cyto-
kines TNF-a and IL-6. As a result, the most potent
compound 2h could prove to be a promising candidate for
drug discovery.
In order to search for the potent antimicrobial agent from
these newly synthesized compounds (2a–u) have been
evaluated for in vitro antibacterial and antifungal activity
against various Gram-positive, Gram-negative bacteria, and
fungal strains (Tables 2, 3). As can be seen from obtained
results, many compounds from the newly synthesized series
found to be potent antibacterial and antifungal agents. Thus
the compounds 2d, 2m, 2n, and 2s exhibited comparable to
or even higher antimicrobial activity than the standard
ciprofloxacin and miconazole, respectively, almost against
all the tested bacteria or fungi. The remaining compounds of
this series have been found to be moderate, low, or no
activity. Moreover, the compounds 2d and 2s have more
potent antibacterial agents than the standard drug against
some bacteria viz. Staphylococcus aureus, Bacillus subtilis,
and Salmonella typhimurium. Also the compound 2d found
to be better antifungal agents as compared with standard
miconazole.
Table 1 Anti-inflammatory activity of 3-hydroxy-2-(2,4,5-trime-
thoxyphenyl)-4H-chromen-4-one derivatives
The SAR of 3-hydroxy-2-(2,4,5-trimethoxyphenyl)-4H-
chromen-4-one derivatives are shown in Tables 1, 2, and 3,
some interesting trend has been observed as the effect of
substituent present on ring ‘‘A’’ along with the O-methyl-
ation of ‘‘B’’ ring on taken activities that is the nature of the
substituent affecting the biological activity of the synthe-
sized analogues. From the TNF-a and IL-6 inhibitory
activity data (Table 1), it is observed that a majority of the
analogues of this series found to be active as IL-6 inhibitor
while very few exhibited TNF-a inhibitory activity. So it is
clear from the results that the position of the substituent on
‘‘A’’ ring of flavonol moiety has profound effect on the
activity.
O
O
8
O
7
R
6
O
OH
5
O
Compound 2(a–u)
R
% inhibition at 10 lM
TNF-a
IL-6
2a
2b
2c
2d
2e
2f
5-Cl
45
52
60
30
65
17
–
39
46
65
28
78
22
–
5-Cl, 7-CH3
8-Cl
6-Cl, 8-Cl
6-CN, 8-Cl
5-Br
The 5-, 6-, and 7-positions are favorable sites for the
higher potency. Evidently, the compounds 2h and 2l with F
at 5 and 7 positions or both as well as CF3 at 6-positions
exhibiting highest TNF-a and IL-6 inhibitory activity,
while presence of Cl, Br, CH3, CN, and NO2 at same
position (compounds 2a, 2g, 2p, 2r, 2o, and 2u) exhibits no
TNF-a or IL-6 inhibitory activity. However, surprisingly
the compounds 2c and 2e have shown moderate activity
with chloro and cyano groups at 8- and 6- or 8-positions of
‘‘A’’ ring, whereas, the fluoro group at 7- and 6-, 8-posi-
tions is moderately potent with (64–67 % TNF-a and
76–77 % IL-6 inhibitory activity). The compound 2h
shows better TNF-a (81 % and IL-6 92 %) activity at
10 lM concentration. None of the compounds exhibits no
or very low anti-inflammatory activity. It clears from the
SAR fluoro group at 5-, 6-, and 7-positions tolerate the
procytokine activity.
2g
2h
2i
6-Br, 8-NO2
5-F
81
67
60
64
72
28
27
15
–
92
74
75
76
86
41
49
21
–
7-F
2j
8-F
2k
2l
6-F, 8-F
5-F, 7-F
6-NO2, 8-F
6-CN, 8-F
7-CH3
2m
2n
2o
2p
2q
2r
2s
6-CH3, 8-NO2
6-CF3
75
–
82
–
6-NO2
6-SO2Me
8-CN
16
08
–
30
11
–
2t
The antimicrobial activity data are presented in Tables 2
and 3. As shown in our results, some analogues of this series
have been found to be more potent than the standard drug
2u
Ref-1
5-CN
Dexamethasone (1 lM) 71
84
123

Med Chem Res
chromen-4-one derivatives of antibacterial activity strongly
correlates with their SAR of antifungal activity. Again the
positions 6 and 8 of ring ‘‘A’’ are favorable sites for high
activity. The compounds 2d and 2s were found to be 2–2.5-
fold more potent than the standard drug miconazole, while
2m and 2n exhibited comparable antifungal activity similar
to the antibacterial activity trend, polar lipophilic groups
such as Cl, SO2Me, NO2, F at 6- and 8-position or both at
same level of concentration 10 lg/mL. Apart from the
compounds 2a, 2b, 2g, 2t, and 2u have no major effect on
the antifungal activity.
Table 2 Antibacterial activity of 3-hydroxy-2-(2,4,5-trimethoxy-
phenyl)-4H-chromen-4-one derivatives
Compounds Gram-positive
Gram-negative
Staphylococcus Bacillus Escherichia Salmonella
subtilis coli
aureus
typhimurium
2a
2b
2c
2d
2e
2f
35
30
85
10
90
80
40
90
65
–
30
45
–
25
35
80
15
75
–
40
50
90
10
80
75
35
80
50
90
80
–
10
80
65
35
–
It reveals from our SAR studied, the presence of lipophilic
F and CF3 groups with trimethoxy (O-alkylation) tolerates
the procytokine activity because of fluorine imparts the
special characteristics that enhance therapeutic efficacy and
improved pharmacological properties in bioactive mole-
cules. While the fluoro is only slightly more lipophilic than
hydrogen and trifluoromethyl is much more lipophilic than
methyl or chloro, this factor is often the most significant in
improving the pharmacological activity.
2g
2h
2i
45
75
45
60
–
55
80
85
90
15
15
85
–
2j
2k
2l
75
80
20
25
90
–
85
15
10
–
2m
2n
2o
2p
2q
2r
2s
20
25
80
–
In conclusion, we have developed the novel approach for
the synthesis of structurally diverse 3-hydroxy-2-(2,4,5-tri-
methoxyphenyl)-4H-chromen-4-onederivatives(2a–u) using
simple reaction sequence under mild conditions and their
TNF-a and IL-6 inhibitory and antimicrobial activity have
been evaluated. This study on the novel flavonol derivative
bearing lipophilic fluoro group at 5-, 6-, and 7-positions and
presence of trimethoxy group on ‘‘B’’ ring has lead to the
discovery of novel and potent TNF-a and IL-6 inhibitors viz.
2h, 2l, and 2q and could prove to be the valuable platform for
the search of nonsteroidal anti inflammatory agents based on
such a scaffold in vivo. While the presence of chloro, cyano or
sulfonyl groups on 6- and 8-positions or both on ring ‘‘A’’
found to be an effective potent antimicrobial agents viz.
compounds 2d, 2m, 2n, and 2s.
85
90
90
20
25
60
15
85
85
15
40
35
70
90
10
30
45
20
90
90
10
30
40
20
2t
2u
Ciprofloxacin 25
MIC values are the average of three reading (lg/mL)
– No activity was observed up to 200 lg/mL
ciprofloxacin, while some of them have comparable
potency. Interestingly none of the compounds with high
anti-inflammatory activity found to be potent antibacterial
or antifungal agents. The compounds 2d, 2m, 2n, and 2s
bearing Cl, NO2, CN, and SO2Me groups, respectively, at
6-position with Cl, F, at 8-position or at both have higher
potency than the compounds bearing such a group at 5- or
7-positions or at both. It is clear from our results that 6- or
8-position is the favorable site for high-antibacterial activ-
ity. The high potency of 2d, 2m, 2n, and 2s may be
attributed to the presence of lipophilic or H-bond acceptor
type group’s placement such as F, Cl, CN, SO2Me, and NO2
at 6- or 8-positions, but unfortunately this functional group
presence on 5, 7 or both shows moderate or no activity with
respect to standard drug against the tested strains. No
activity has been observed in case of compounds 2o–2r up
to concentration of 200 lg/mL against some bacteria and
fungi.
Experimental
Chemistry
General techniques
All commercial chemicals and solvents are of reagents
grade and have been used without further purification. The
thin layer chromatography was performed on Merck pre-
coated silica gel 60 F254 plates, with visualization under
UV light. 1H NMR spectra have been recorded with Bruker
300 MHz AVANCE instrument and J values are in Hertz
and chemical shifts (d) are reported in ppm relative to
internal tetramethylsilane. The mass spectra have been
measured with Thermo Finnigan-TSQ Quarter Ultra (triple
Quad). The purity of all compounds was determined by
It is clear from activity data (Table 2 vs. Table 3) that
the SAR of 3-hydroxy-2-(2,4,5-dimethoxyphenyl)-4H-
123

Med Chem Res
General procedure for the synthesis of 3-hydroxy-2-(2,4,5-
trimethoxyphenyl)-4H-chromen-4-one derivatives (2a–u)
Table 3 Antifungal activity of 3-hydroxy-2-(2,4,5-trimethoxy-
phenyl)-4H-chromen-4-one derivatives
Compounds Candida
albicans
Aspergillus Fusarium
niger
Aspergillus
flavus
solani
To a well-stirred solution of 2-hydroxychalcones 1 (1 eqi.)
in MeOH (20 mL) and aq. KOH (10 mL, 20 %), cooled at
0–5 °C, 30 % H2O2 (10 mL) was added drop wise for
30 min. The reaction mixture was further stirred for 4–5 h.
The resulting light-yellow reaction mixture was poured on
crushed ice and neutralized with dil. HCl. The light-yellow
solid thus obtained was filtered, washed with water and
dried. The crude product was purified by chromatography
on SiO2 (100–200 silica gel) (EtOAc/petroleum ether
v/v = 2:8) to give the desired compounds as solids. The
physical, analytical, and spectral data of new 3-hydroxy-2-
(2,4,5-trimethoxyphenyl)-4-H-chromen-4-one analogues
are given below.
2a
2b
2c
2d
2e
2f
25
30
–
40
25
80
10
85
–
35
20
90
15
–
45
35
80
10
90
–
10
75
–
90
25
85
85
80
75
–
2g
2h
2i
45
–
30
90
65
–
40
70
90
95
80
–
70
90
–
2j
2k
2l
80
80
15
20
80
90
70
–
65
20
25
–
2m
2n
2o
2p
2q
2r
2s
20
15
90
–
10
25
85
–
5-Chloro-3-hydroxy-2-(2,4,5-trimethoxyphenyl)-4H-chro-
men-4-one (2a) White solid: yield 66 %; mp: 191–193 °C.
1H NMR (CDCl3, 300 MHz): d 9.05 (bs, 1H), 7.77 (d,
J = 8.5 Hz, 1H), 7.51 (d, J = 9 Hz, 1H), 7.35 (m, 1H),
7.10 (s, 1H), 6.61 (s, 1H), 3.90 (s, 3H), 3.83 (s, 3H), 3.81 (s,
3H); 13C NMR (CDCl3, 300 MHz, d ppm): 172.49(C=O),
165.71(C-2), 164.14 (C-9), 155.19(C-20), 152.29(C-40),
151.19(C-50), 144.25(C-5), 143.31(–OH/C-3), 139.12(C-7),
127. 2 3(C-10), 118.16(C-6), 113.35(C-8), 111.14 (C-60),
104.54(C-10), 97.72 (C-30), 57.16, 56.28, 56.23 (OMe-C);
MS (APCI); m/z 363.1 [M?H]?; HPLC: 95.96 %.
–
60
90
15
40
25
75
80
10
25
15
15
85
–
10
30
30
20
20
30
25
20
2t
2u
Miconazole 20
MIC values are the average of three reading (lg/mL)
– No activity was observed up to 200 lg/mL
5-Chloro-3-hydroxy-7-methyl-2-(2,4,5-trimethoxyphenyl)-
4H-chromen-4-one (2b) Off white solid: yield 67 %; mp:
187–189 °C .1H NMR (CDCl3, 300 MHz): d 9.25 (bs, 1H),
8.00 (s, 1H), 7.63 (s, 1H), 7.05 (s, 1H), 6.79 (s, 1H), 3.84 (s,
3H), 3.77 (s, 3H), 3.69 (s, 3H), 2.96 (s, 3H); 13C NMR
(CDCl3, 300 MHz, d ppm): 173.21(C=O), 157.10(C-2), 152.91
(C-9), 150.31(C-20), 148.63(C-40), 147.37(C-50),143.10(C-7),
141.21(C-5), 140.31(–OH/C-3), 125.41(C-10), 120.51(C-6),
118.12(C-8), 112.33(C-60), 110.10(C-10), 98.10(C-30), 57.33,
56.46, 56.23 (OMe-C), 22.23 (Me/C); MS (APCI); m/z 377.1
[M?H]?; HPLC: 96.89 %.
HPLC (Waters 2695 Alliance) using column Kromasil
C18, solvent acetonitrile and buffer (0.01 M ammonium
acetate ? 0.5 % triethylamine, pH 5.0 with acetic acid).
Melting points were determined with (PEW-340 MP)
melting point apparatus and are uncorrected.
General procedure for the synthesis of 1-(2-
hydroxyphenyl)-3-(2,4,5-trimethoxyphenyl) prop-2-en-1-
one derivatives (1a–u)
Different substitutes (2-hydroxyacetophenone) (1 eqi.) and
2,4,5-trimethoxy benzaldehyde (1 eqi.) in EtOH (15 mL)
were added and a 60 % of KOH (5 mL) solution was added
drop wise at 0 °C. The reaction mixture was stirred at room
temperature for 6–8 h. The reaction mixture was cooled
and poured into ice water, neutralized using 1 N HCl. The
light-yellow solid thus obtained was filtered and purified
using silica gel column chromatography (with 1:9 ethyl
acetate in hexane) or recrystallized from EtOH, yielded the
desired 2,4,5-trimethoxy chalcone analogues (1a–u) with
an average yield of 60–70 %.
8-Chloro-3-hydroxy-2-(2,4,5-trimethoxyphenyl)-4H-chro-
men-4-one (2c) White solid: yield 64 %; mp: 196–198 °C.
1H NMR (CDCl3, 300 MHz): d 9.05 (bs, 1H), 7.77 (d,
J = 8.5 Hz, 1H), 7.51 (d, J = 9 Hz, 1H), 7.35 (m, 1H),
7.10 (s, 1H), 6.61 (s, 1H), 3.90 (s, 3H), 3.83 (s, 3H), 3.81 (s,
3H); 13C NMR (CDCl3, 300 MHz, d ppm): 172.59(C=O),
165.71(C-2), 164.14 (C-9), 155.19(C-20), 152.29(C-40),
151.19(C-50), 143.25(C-8), 142.31(–OH/C-3), 138.12(C-7),
127.33(C-10), 117.16(C-6), 115.35(C-5), 112.14 (C-60),
105.54(C-10), 98.72 (C-30), 56.30, 56.28, 56.23 (OMe-C);
MS (APCI); m/z 363.1 [M?H]?; HPLC: 93.96 %.
123

Med Chem Res
163.71(C-2), 164.14 (C-9), 155.19(C-20), 152.29(C-40),
151.19(C-50), 143.25(C-5), 141.31(–OH/C-3), 139.12(C-7),
127. 2 3(C-10), 118.16(C-6), 113.35(C-8), 111.14 (C-60),
102.54(C-10), 95.72(C-30), 57.28, 56.27, 56. 23 (OMe-C); MS
(APCI); m/z 347.1 [M?2H]?; HPLC: 97.39 %.
6,8-Dichloro-3-hydroxy-2-(2,4,5-trimethoxyphenyl)-4H-chro-
men-4-one (2d) Pale yellow solid: yield 68 %; mp: 199–
201 °C. 1H NMR (CDCl3, 300 MHz): d 9.19 (bs, 1H), 8.13
(s, 1H), 7.73 (s, 1H), 7.23 (s, 1H), 6.65 (s, 1H), 3.97 (s,
3H), 3.92 (s, 3H), 3.89 (s, 3H); (CDCl3, 300 MHz, d ppm):
173.03(C=O), 158.13(C-2), 155.47(C-9), 150.86(C-20),
145.37(C-40), 144.12 (C-50), 142.21(–OH/C-3), 136.42(C-
8), 133.05(C-6), 130.20(C-10), 123.20(C-7), 121.14(C-5),
117.02 (C60), 112.32 (C-10), 97.11(C-30), 57.22, 56.15,
56.10 (OMe-C); MS (APCI); m/z 398.1 [M?H]?; HPLC:
96.06 %.
7-Fluoro-3-hydroxy-2-(2,4,5-trimethoxyphenyl)-4H-chro-
men-4-one (2i) Off white solid: yield 69 %; mp:
190–192 °C. 1H NMR (CDCl3, 300 MHz): d 9.05 (bs, 1H),
8.26 (d, J = 8.6 Hz 1H), 7.21 (d, J = 9 Hz, 1H), 7.17 (m,
1H), 7.12 (s, 1H), 6.65 (s, 1H), 3.96 (s, 3H), 3.90 (s, 3H), 3.90
(s, 3H); 13C NMR (CDCl3, 300 MHz, d ppm): 173.60(C=O),
162.71(C-2), 160.14(C-9), 155.19(C-20), 152.29(C-40),
151.19(C-50), 144.12(C-7), 141.31(–OH/C-3), 137.25(C-5),
129.23(C-10), 118.16(C-6), 113.35(C-8), 111.14 (C-60),
102.54(C-10), 95.72 (C-30), 57.30, 56.28, 56. 23 (OMe-C);
MS (APCI); m/z 347.1 [M?2H]?; HPLC: 95.33 %.
8-Chloro-3-hydroxy-4-oxo-2-(2,4,5-trimethoxyphenyl)-4H-
chromene-6-carbonitrile (2e) Off white solid: yield 57 %;
mp: 203–205 °C. 1H NMR (CDCl3, 300 MHz): d 9.27 (bs,
1H), 8.31 (s, 1H), 8.26 (s, 1H), 7.15 (s, 1H), 6.86 (s, 1H),
3.89 (s, 3H), 3.86 (s, 3H), 3.83 (s, 3H); (CDCl3, 300 MHz,
d ppm): 172.13(C=O), 157.14(C-2), 154.57(C-9), 150.66
(C-20), 146.42(C-40), 143.32(C-50), 142.31(–OH/C-3), 139.16
(C-8), 132.20(C-10), 124.15(C-5), 123.36(C-7), 119.13(CN),
117.12 (C-60), 113.16(C-6), 111.33(C-10), 97.11(C-10), 57.23,
56.25, 56.14 (OMe-C); MS (APCI); m/z 388.1 [M?1]?;
HPLC: 94.39 %.
8-Fluoro-3-hydroxy-2-(2,4,5-trimethoxyphenyl)-4H-chro-
men-4-one (2j) Off white solid: yield 65 %; mp: 189–
1
191 °. H NMR (CDCl3, 300 MHz): d 9.05 (bs, 1H), 8.04
(d, J = 8.6 Hz, 1H), 7.44 (d, J = 9 Hz, 1H), 7.33 (m, 1H),
7.15 (s, 1H), 6.66 (s, 1H), 3.96(s, 3H), 3.90 (s, 3H), 3.89 (s,
3H); 13C NMR (CDCl3, 300 MHz, d ppm): 175.60(C=O),
163.71(C-2), 160.14(C-9), 155.19(C-20), 152.29(C-40),
151.19(C-50), 146.12(C-8), 141.31(–OH/C-3), 137.25(C-
10), 129.23(C-5), 118.16(C-6), 115.35(C-7), 113.14 (C-60),
102.54(C-10), 95.72 (C-30), 57.27, 56.21, 56. 20 (OMe-C);
MS (APCI); m/z 347.1 [M?H]?; HPLC: 94.39 %.
5-Bromo-3-hydroxy-2-(2,4,5-trimethoxyphenyl)-4H-chro-
men-4-one (2f) Off white solid: yield 63 %; mp: 193–
195 °C. 1H NMR (CDCl3, 300 MHz): d 9.05 (bs, 1H), 8.14
(s, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.57 (d, J = 9 Hz 1H),
7.04 (s, 1H), 6.78 (s, 1H), 3.82 (s, 3H), 3.74 (s, 3H), 3.66 (s,
3H); 13C NMR (CDCl3, 300 MHz, d ppm): 172.49(C=O),
163.72(C-2), 164.14(C-9), 155.19(C-20), 152.29(C-40),
151.19(C-50), 143.25(C-5), 142.31(–OH/C-3), 138.12(C-7),
127. 2 3(C-10), 120.16(C-6), 115.35(C-8), 110.14 (C-60),
105.54(C-10), 99.72 (C-30), 57.17, 56.30, 56.29 (OMe-C);
MS (APCI); m/z 409.0 [M?2H]?; HPLC: 96.65 %.
6,8-Difluoro-3-hydroxy-2-(2,4,5-trimethoxyphenyl)-4H-
chromen-4-one (2k) Pale yellow solid: yield 60 %; mp:
206–208 °C. 1H NMR (CDCl3, 300 MHz): d 9.19 (bs, 1H),
7.90 (dd, 1H), 7.65 (dd, 1H), 7.10 (s, 1H), 6.85 (s, 1H),
3.88 (s, 3H), 3.80 (s, 3H), 3.72 (s, 3H); 13C NMR (CDCl3,
300 MHz, d ppm): 173.03(C=O), 158.13(C-2), 155.47(C-
9), 150.86(C-20), 145.37(C-40), 144.12(C-50), 142.21(–OH/
C-3), 141.42(C-8), 140.05(C-6), 132.20(C-10), 125.20(C-
7), 122.14(C-5), 119.02(C-60), 113.32(C-10), 97.15(C-30),
57.23, 56.15, 56.12 (OMe-C); MS (APCI); m/z 365.1
[M?H]?; HPLC: 98.57 %.
6-Bromo-3-hydroxy-8-nitro-2-(2,4,5-trimethoxyphenyl)-
4H-chromen-4-one (2g) Yellow solid: yield 70 %; mp: 197
1
–199 °C. H NMR (CDCl3, 300 MHz): d 9.27 (BS, 1H),
8.31 (s, 1H), 8.26 (s, 1H), 7.15 (s, 1H), 6.84 (s, 1H), 3.89 (s,
3H), 3.83 (s, 3H), 3.78 (s, 3H); (CDCl3, 300 MHz, d ppm):
174.03(C=O), 157.13(C-2), 155.47(C-9), 150.86(C-20),
146.37(C-40), 144.12(C-50), 14 2.21(–OH/C-3), 141.42(C-
8), 133.05(C-6), 129.20(C-10), 123.20(C-7), 121.14(C-5),
117.02 (C-60), 1 12.32 (C-10), 97.11(C-30), 57.24, 56.21,
56.18 (OMe-C); MS (APCI); m/z 453.1 [M?H]?; HPLC:
95.03 %.
5,7-Difluoro-3-hydroxy-2-(2,4,5-trimethoxyphenyl)-4H-
chromen-4-one (2l) Pale yellow solid: yield 66 %; mp:
200–202 °C. 1H NMR (CDCl3, 300 MHz): d 9.19 (bs, 1H),
7.09 (s, 1H), 6.76 (dd, 1H), 6.64 (s, 1H), 6.52 (dd, 1H), 3.96 (s,
3H), 3.88(s, 3H), 3.86(s, 3H);(CDCl3, 300 MHz, dppm):13
C
NMR(CDCl3, 300 MHz, dppm): 174.03(C=O), 159.14(C-2),
157.47(C-9), 152.86(C-20), 146.37(C-40), 145.12(C-50), 143.21
(–OH/C-3), 142.52(C-7), 141.25(C-5), 130.20(C-10), 127.20
(C-6), 124.14(C-8), 118.02(C-60), 114.32(C-10), 98.15(C-30),
57.21, 56.17, 56. 14 (OMe-C); MS (APCI); m/z 365.1 [M?1]?;
HPLC: 96.34 %.
5-Fluoro-3-hydroxy-2-(2,4,5-trimethoxyphenyl)-4H-chro-
men-4-one (2h) Brown solid: yield 71 %; mp: 186–188 °C.
1H NMR (CDCl3,300 MHz):d9.05 (bs, 1H), 7.70 (d, J = 9 Hz
3H), 7.07 (s, 1H), 6.80 (s, 1H), 3.84 (s, 3H), 3.76 (s, 3H), 3.68
(s, 3H); 13C NMR (CDCl3, 300 MHz, d ppm):172.60(C=O),
123

Med Chem Res
8-Fluoro-3-hydroxy-6-nitro-2-(2,4,5-trimethoxyphenyl)-
4H-chromen-4-one (2m) Off white solid: yield 57 %;
mp: 211–213 °C. 1H NMR (CDCl3, 300 MHz): d 9.27 (bs,
1H), 8.31 (s, 1H), 8.27(s, 1H), 7.17 (s, 1H), 6.89 (s, 1H),
3.91 (s, 3H), 3.89 (s, 3H), 3.86 (s, 3H); 13C NMR (CDCl3,
300 MHz, d ppm): 176.03(C=O), 159.13(C-2), 157.47(C-
9), 152.86(C-20), 146.37(C-40), 145.12(C-50), 143.21(–OH/
C-3), 142.42(C-6), 137.05(C-8), 130.20(C-10), 124.20(C-
7), 122.14(C-5), 118.02 (C60), 113.32 (C-10), 97.25(C-30),
57.27, 56.25, 56.21 (OMe-C); MS (APCI); m/z 392.1
[M?H]?; HPLC: 95.54 %.
1H), 6.66 (s, 1H), 3.83 (s, 3H), 3.74 (s, 3H), 3.71 (s, 3H);
13C NMR (CDCl3, 300 MHz, d ppm): 175.01(C=O),
159.13(C-2), 157.47(C-9), 150.86(C-20), 147.37(C-40),
145.12(C-50), 141.21(–OH/C-3), 131.42(C-7), 129.05(C-6),
127.17 (C/CF3), 130.20(C-10), 123.20(C-8), 121.14(C-5),
118.02(C-60), 112.32(C-10), 98.11(C-30), 57.24, 56.16,
56.12 (OMe-C); MS (APCI); m/z 397.1 [M?H]?; HPLC:
95.03 %.
3-Hydroxy-6-nitro-2-(2,4,5-trimethoxyphenyl)-4H-chromen-
4-one (2r) Yellow solid: yield 66.5 %; mp: 196–198 °C.
1H NMR (CDCl3, 300 MHz): d 9.35 (bs, 1H), 8.82 (s, 1H),
8.50 (d, J = 8.5 Hz, 1H), 7.87 (d, J = 9 Hz, 1H), 7.11
(s, 1H), 6.83 (s, 1H), 3.89 (s, 3H), 3.81 (s, 3H), 3.72 (s,
3H); 13C NMR (CDCl3, 300 MHz, d ppm): 173.23(C=O),
159.51(C-2), 158.47(C-9), 151.85(C-20), 146.37(C-40), 145.30
(C-50), 142.10(C-6), 141.21(–OH/C-3), 131.42(C-7), 130.20
(C-10), 123.20(C-8), 121.14(C-5), 119.02(C-60), 112.32(C-10),
98.20(C-30), 57.24, 56.18, 56.15 (OMe-C); MS (APCI); m/
z 374.1 [M?H]?; HPLC: 97.58 %.
8-Fluoro-3-hydroxy-4-oxo-2-(2,4,5-trimethoxyphenyl)-4H-
chromene-6-carbonitrile (2n) Brown solid: yield 64 %;
1
mp: 209–211 °C. H NMR (CDCl3, 300 MHz): d 9.27 (bs,
1H), 8.31 (s, 1H), 8.26 (s, 1H), 7.15 (s, 1H), 6.84 (s, 1H), 3.89
(s, 3H), 3.83 (s, 3H), 3.78 (s, 3H); 13C NMR (CDCl3,
300 MHz, d ppm): 172.13(C=O), 157.14(C-2), 154.57(C-9),
151.66(C-20), 146.27 (C-40), 144.32(C-50), 143.16(C-8),
142.31(–OH/C-3), 132.20(C-10), 124.15(C-5), 123.36(C-7),
120.13(CN), 117.12(C-60), 115.16(C-6), 112.33(C-10), 97.20
(C-10), 57.23, 56.24, 56.17 (OMe-C); MS (APCI); m/z 372.1
[M?H]?; HPLC: 93.67 %.
3-Hydroxy-6-(methylsulfonyl)-2-(2,4,5-trimethoxyphenyl)-
4H-chromen-4-one (2s) Off white solid: yield 66 %; mp:
220–222 °C. 1H NMR (CDCl3, 300 MHz): d 9.24 (bs, 1H),
8.58 (s, 1H), 8.20 (d, J = 9 Hz, 1H), 7.86 (d, J = 8.6 Hz,
1H), 7.10 (s, 1H), 6.83 (s, 1H), 3.86 (s, 3H), 3.82 (s, 3H),
3.78 (s, 3H); 13C NMR (CDCl3, 300 MHz, d ppm): 172.45
(C=O), 158.51(C-2), 157.47(C-9), 151.81(C-20), 146.37(C-
40), 145.30(C-50), 141.21(–OH/C3), 137.42.(C-6), 131.42
(C-7), 131.20(C-10), 123.20(C-8), 120.14(C-5), 119.02(C-
60), 112.32(C-10), 96.20(C-30), 57.25, 56.19, 56.16 (OMe-
C), 39.89 (Me/SO2); MS (APCI); m/z 407.1 [M?H]?;
HPLC: 94.95 %.
3-Hydroxy-7-methyl-2-(2,4,5-trimethoxyphenyl)-4H-chro-
men-4-one (2o) Brown solid: yield 68 %; mp: 189–191 °C.
1H NMR (CDCl3, 300 MHz): d 9.25 (bs, 1H), 8.13 (s,
J = 8 Hz, 1H), 7.31 (s, 1H),7.23 (d, J = 8.5 Hz, 1H), 7.11
(s, 1H), 6.65 (s, 1H), 3.96 (s, 3H), 3.92 (s, 3H), 3.88 (s,
3H), 2.49 (s, 3H); 13C NMR (CDCl3, 300 MHz, d ppm):
174.21(C=O), 158.10(C-2), 155.91(C-9), 151.31(C-20),
148.55(C-40), 147.37(C-50), 141.10(C-7), 140.31(–OH/C-
3), 130.21(C-5), 125.41(C-10), 120.51(C-6), 118.12(C-8),
113.33(C-60), 111.10(C-10), 98.30(C-30), 57.35, 56.53,
56.42 (OMe-C), 21.23 (Me/C); MS (APCI); m/z 343.1
[M?H]?; HPLC: 98.64 %.
3-Hydroxy-4-oxo-2-(2,4,5-trimethoxyphenyl)-4H-chromene-
8-carbonitrile (2t) Off white solid: yield 58 %; mp:
199–201 °C. 1H NMR (CDCl3, 300 MHz): d 9.05 (bs, 1H),
8.22 (d, J = 8.5 Hz, 1H), 8.06 (d, J = 9 Hz, 1H), 7.41 (m,
1H), 7.21 (s, 1H), 6.75 (s, 1H), 3.81 (s, 3H), 3.77 (s, 3H), 3.67
(s, 3H); 13C NMR (CDCl3, 300 MHz, d ppm): 172.60(C=O),
163.71(C-2), 161.14(C-9), 156.19(C-20), 153.29(C-40), 151.19
(C-50), 142.31(–OH/C-3), 130.33(C-10), 129.12(C-7), 125.25
(C-8), 117.16(C-6), 115.35(C-5), 112.14 (C-60), 107.54(C-10),
98.72 (C-30), 56.27, 56.25, 56.24 (OMe-C); MS (APCI); m/
z 372.1 [M?H]?; HPLC: 95.36 %.
3-Hydroxy-6-methyl-8-nitro-2-(2,4,5-trimethoxyphenyl)-
4H-chromen-4-one (2p) Yellow solid: yield 70 %; mp:
181–183 °C. 1H NMR (CDCl3, 300 MHz): d 9.25 (bs, 1H),
8.34 (s, 1H), 8.27 (s, 1H), 7.14 (s, 1H), 6.85 (s, 1H), 3.89 (s,
3H), 3.81 (s, 3H), 3.72 (s, 3H), 2.51 (s, 3H); 13C NMR
(CDCl3, 300 MHz, d ppm): 173.03(C=O), 157.13(C-2),
155.47(C-9), 151.86(C-20), 145.37(C-40), 143.12(C-50),
142.21(–OH/C-3), 141.42(C-8), 139.05(C-6), 129.20(C10),
123.20(C-7), 121.14(C-5), 117.02 (C-60), 114.32(C-10),
97.14(C-30), 57.21, 56.23, 56.18 (OMe-C), 21.27 (Me/C);
MS (APCI); m/z 388.1 [M?H]?; HPLC: 95.04 %.
3-Hydroxy-4-oxo-2-(2,4,5-trimethoxyphenyl)-4H-chromene-
5-carbonitrile (2u) Off white solid: yield 60 %; mp:
207–209 °C. 1H NMR (CDCl3, 300 MHz): d 9.05 (bs, 1H),
8.67 (s, 1H), 8.18 (d, J = 8.5 Hz, 1H), 7.59 (d, J = 9 Hz
1H), 7.59 (s, 1H), 6.78 (s, 1H), 3.83 (s, 3H), 3.76 (s, 3H),
3.68 (s, 3H); 13C NMR (CDCl3, 300 MHz, d ppm):
173.16(C=O), 162.20(C-2), 160.10(C-9), 156.21(C-20),
3-Hydroxy-6-(trifluoromethyl)-2-(2,4,5-trimethoxyphenyl)-
4H-chromen-4-one (2q) Off white solid: yield 66 %; mp:
216–218 °C. 1H NMR (CDCl3, 300 MHz): d 9.05 (bs, 1H),
8.59 (s, 1H), 7.85 (dd, 1H), 7.63 (d, J = 9 Hz, 1H), 7.12 (s,
123

Med Chem Res
154.29(C-40), 151.19(C-50), 142.31(–OH/C-3), 129.79(C-
10), 129.12(C-7), 126.25(C-5), 118.16(C-6), 115.35(C-8),
113.14 (C-60), 107.54(C-10), 97.70 (C-30), 56.22, 56.21,
56.19 (OMe-C); MS (APCI); m/z 372.1 [M?H]?; HPLC:
96.76 %.
flavus (MTCC 277), and Fusarium solani (MTCC 350) by
agar-well diffusion method with little modification. Normal
saline has been used to make a suspension of spores of
fungal strain, prepared as same as antibacterial assay.
Miconazole has been used as standard antifungal agent.
Acknowledgments We are grateful to the Department of Chemis-
try, Maharashtra Udayagiri Mahavidyalaya, Udgir for providing
necessary facilities and would like to thank Mr. R. B. Ingle,
Department of Microbiology, Shri Shivaji College, Akola for bio-
logical screening studies of the newly synthesized compounds.
Biological assay
Anti-inflammatory assay
Proinflammatory cytokine production by LPS in THP-1 cells
has been measured according to the method described by
Hwang in 1993. During assay, THP-1 cells were cultured in
RPMI 1640 culture medium (Gibco BRL, Pasley, UK)
containing 100 U/mL penicillin and 100 mg/mL strepto-
mycin containing 10 % fetal bovine serum (FBS, JRH). Cells
have been differentiated with phorbol myristate acetate
(PMA, Sigma). Following cell plating, the test compounds in
0.5 % DMSO have been added to each well separately and
the plate has been incubated for 30 min at 37 °C. Finally,
LPS (E. coli 0127:B8, Sigma Chemical Co., St. Louis, MO,
USA) has been added at a final concentration of 1 lg/mL in
each well. Plates have been further incubated at 37 °C for
24 h in 5 % CO2. After incubation, supernatants have been
harvested, and assayed for TNF-a and IL-6 by ELISA as
described by the manufacturer (BD Biosciences).
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