3-Formylchromones: Potential antiinflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2010.05.065

The synthesis and characterization of 3-formylchromone (1) and its derivatives 2-24 and evaluation of their potential antiinflammatory activities is reported here. These compounds were characterized by ¹H NMR, EI MS, IR, and UV spectroscopic techniques and elemental analysis. The synthesized compounds were evaluated by using various in vitro and in vivo assay models for antiinflammatory activity and their effects were compared with known standard drug such as aspirin and indomethacin. Among all tested compounds, 1, 2, 5, 6, 9, 14, 16-19, 21-23, showed promising antiinflammatory activities. The results and SAR has been discussed in this report. The synthesis and potential antiinflammatory activities of 3-formylchromone (1) and its derivatives 2-24 is reported here. The synthesized compounds were evaluated by using various in vitro and in vivo assay models for antiinflammatory activity.

Full text of DOI:10.1016/j.ejmech.2010.05.065

European Journal of Medicinal Chemistry 45 (2010) 4058e4064
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
3-Formylchromones: Potential antiinflammatory agents
,
a
a
a
b
Khalid M. Khan a , Nida Ambreen , Uzma Rasool Mughal , Saima Jalil , Shahnaz Perveen ,
*
M. Iqbal Choudhary a
a H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
b PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and characterization of 3-formylchromone (1) and its derivatives 2e24 and evaluation of
their potential antiinflammatory activities is reported here. These compounds were characterized by 1H
NMR, EI MS, IR, and UV spectroscopic techniques and elemental analysis. The synthesized compounds
were evaluated by using various in vitro and in vivo assay models for antiinflammatory activity and their
effects were compared with known standard drug such as aspirin and indomethacin. Among all tested
compounds, 1, 2, 5, 6, 9, 14, 16e19, 21e23, showed promising antiinflammatory activities.
The results and SAR has been discussed in this report.
Received 22 February 2010
Received in revised form
4 May 2010
Accepted 29 May 2010
Available online 8 June 2010
Keywords:
Ó 2010 Elsevier Masson SAS. All rights reserved.
3-Formylchromones
Antiinflammatory activity
Respiratory burst
Neutrophils
COX
LOX inhibition
1. Introduction
activation of membrane-based, phagocyte-specific enzyme, NADPH
oxidase [5].
Inflammation is a defensive response of body, which induces
physiological adaptations to minimize tissue damage and to
remove the pathogenic infections. Such mechanisms involve
a complex series of cellular and modular events including dilation
of arterioles, venules and capillaries with increased vascular
permeability and exudation of fluids containing plasma proteins as
well as migration of leukocytes into the inflammatory area. A
chronic inflammation is however an important contributory factor
in morbidity and mortality. Such inflammatory disorders include
rheumatoid arthritis, osteoarthritis, inflammatory bowl diseases,
retinitis, multiple sclerosis, psoriasis and atherosclerosis [1].
Inflammation is characterized by the immediate infiltration of
key defense cells. Initially neutrophils respond to inflammation,
followed by monocytes and lymphocytes [2]. Neutrophils play a key
role in inflammatory response by neutralizing the adverse effects of
external inflammatory stimuli e.g., chemical, antigens, infections
etc. They stored specialized proteins in their granules, which are
secreted on their activation. Neutrophils also generate toxic oxygen
metabolites including reactive oxygen species (ROS) by various
pathways [3,4]. ROS are formed subsequent to the assembly and
Several methods are available to measure the superoxide
production by NADPH oxidase of phagocyte cells [5]. WST-1 used to
measure the superoxide production by neutrophils which were
activated by opsonized Zymosan A, which induces phagocytic
activation of neutrophils. This technique is more sensitive and
reliable over other available techniques and can be adopted in
microplate format. WST-1 is reduced by NADPH oxidase via
superoxides. It is a very useful assay for measuring the neutrophils
functions and antiinflammatory activity of the target compounds
[6,7].
In addition to NADPH oxidase, other cellular enzymes are also
involved in the inflammatory processes. For example, cyclo-
xygenases (COX) catalyze the conversion of arachidonic acid into
prostaglandins and thromboxane [8e15]. Lipoxygenase (LOX) is
another enzyme which has been reported to induce inflammation
via the catalysis of arachidonic acid to produce LTs (Leukotriens).
These enzymes are, therefore, important targets for the discovery of
mechanism-based drugs for the treatment of a variety of inflam-
matory disorders such as bronchial asthma, inflammation, cancer,
and autoimmune diseases. More recently, dual inhibitors of COX
and LOX are found to be significantly important for the treatment of
inflammatory disorders [9].
Traditionally, the standard treatment for most inflammatory
disorders, such as rheumatoid arthritis, is the use of NSAIDs e.g.,
* Corresponding author. Tel.: þ92 21 34824910; fax: þ92 21 34819018.
E-mail addresses: hassaan2@super.net.pk, khalid.khan@iccs.edu (K.M. Khan).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.065

K.M. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 4058e4064
4059
aspirin. However, the main side effect of aspirin is gastric ulcera-
tion. Therefore, there is a need to discover new and novel inhibitors
against these enzymes. Most of the heterocyclic bioactive
compounds are used as a source of pharmacophores. For example,
a number of chromones and their derivatives have been reported to
possess wide spectrum of useful properties of biological as well as
pharmacological importance [16]. Chromone derivatives exhibit
various biological activities including antimycobacterial, antifungal,
anticonvalescent, and antimicrobial mushroom tyrosinase inhibi-
tion activities [17e21]. These pharmacological activities have been
the major interest behind the syntheses of the chromones and their
derivatives. In addition, the importance of lead molecules for the
discovery of new synthetic drugs, several chemical structures are
used as a starting point for chemical modifications in order to
improve potency, selectivity or pharmacokinetic parameters. In
drug discovery and drug designing program of our research
group, we are working on various classes of compounds in search
of potential lead molecules [22e24]. We earlier reported the
thymidine phosphorylase inhibitory activities of Schiff bases of
3-formylchromones 2e5, 7, 8, 10, 11, 14e16 and 22 which discloses
the excellent thymidine phosphorylase inhibitory activities of
compounds 2 and 10 [25]. We also reported the antibacterial and
antifungal activities of compounds 1e4, 6, 8, 11, 12, 15e19, 21e23
[26]. To the best of our knowledge only the compound 1 previously
reported as an antiinflammatory agent in some other modified
assays [27], therefore 3-formylchromone (1) is an important and
well-studied compound serves as the starting materials for more
potent antiinflammatory compounds. Therefore, in view of our
aforementioned reports we also screened compounds 2e24 for
their antiinflammatory potential. This study provides a solid base
for further research on these types of compounds.
superoxide scavenging activity using xanthineexanthine oxidase
system, cyclooxygenase (COX-1, COX-2) and lipoxygenase inhibi-
tory activities and in vivo carrageenan-induced rat paw edema
study.
Initially, all of the compounds 1e24 were screened for their
respiratory burst inhibitory activity. The derivatives 2,16, 21, and 23
exhibited a significant activity in this assay (data not shown). Other
derivatives showed a moderate to weak activity. All the derivatives
1e24 were also tested for the superoxides scavenging activity and
most of them were found to be active in xanthine/xanthine oxidase
assay system.
For the evaluation of the enzymes related to the antiin-
flammatory activity, we selected COX-1 and -2, and LOX. The
selection of the compounds for further studies was based on the
preliminary studies; the compounds showed very weak studies in
primary studies were not further screened. Compounds (4, 7, 13, 16,
17, and 23) were screened to measure their inhibitory potential
against these enzymes. Based on in vitro antiinflammatory assay,
compound 23 was selected for the study in vivo inflammatory
carrageenan-induced rat paw edema model. This derivative was
found to be most potent candidate in the most of the in vitro
studies. The results are summarized below:
2.2.1. In vitro respiratory burst inhibitory activity
In vitro respiratory burst inhibitory activity of synthetic Schiff
bases was evaluated by using the modified method of Berridge et al.
[5]. Results, presented in Table 1, show that compound 23 is the
most active compound with an IC50 value 40.210 ꢁ 1.76
Compound 16 was found to be the second most active compound
with an IC50 55.036 ꢁ 6.17 M. Compound 21 demonstrated an
activity with an IC50 value 85.540 ꢁ 0.072 M.
mM.
m
m
2.2.2. Xanthine/xanthine oxidase superoxides scavenging activity
This is a mechanism-based model for the comparison of the
activity of the compounds in the cell-based assay and the cell-free
system. The results of this assay are shown in Table 2. Compounds
2. Results and discussion
2.1. Chemistry
2, 3, 16,19, and 21 showed IC50 values in potent range (<5
below 5 M always considered as potent range. Other compounds 1,
5, 7,18,16, 22, and 23 also appear to be significantly active (less than
30 M). The remaining derivatives 4, 6, 14 and 17 were found to be
mM) since
3-Formylchromone (1) was prepared by the VilsmeiereHaack
synthesis (Scheme 1) [28]. Derivatives of 3-formylchromone (1)
were prepared by a condensation reaction of 3-formylchromones
with a variety of aromatic, aliphatic amine and hyrazides in ethanol
(Scheme 2).
m
m
only weak active in this assay.
The preparation of derivatives was carried out by stirring the
mixture of 3-formylchromone (1, 1 mmol) with substituted
aromatic, aliphatic amines and hydrazides (1 mmol) in ethanol for
2e4 h, at 50e100 ꢀC. The progress of reaction was monitored by
TLC. The resulting products were recrystallized from ethanol and on
drying to afford the desired compounds.
The structures of the synthesized compounds were determined
by using spectroscopic techniques such as 1H NMR, EI MS, IR and
UV. Elemental analysis results were also found to be satisfactory
and found within the limit.
2.2.3. Enzyme inhibition activity
Some of the selected compounds 4, 7, 16, 17, and 23 were
screened for the inhibitory potential against the cyclooxygenases
(COX-1 and -2). The results, shown in Table 3, indicate that
compounds 16 and 17 were moderate to weakly active against both
the enzymes. However, compounds 4 and 23 were very weakly
active against only COX-1 and COX-2 (approximate 50% at
w1000 mM), respectively.
The compounds 4, 16, 17, and 23 were also screened for the
lipoxygenase (LOX) enzyme to measure their inhibitory potential.
Results shown in Table 4 indicate that none of the compounds
appear to inhibit LOX activity, rather all compounds enhanced LOX
enzyme activity.
2.2. Biological evaluation
Several biological assays were used for the study antiin-
flammatory potential of 3-formylchromone (1) and its derivatives
2e24 of which include in vitro respiratory burst inhibitory activity,
2.2.4. In vivo antiinflammatory study on carrageenan-induced rat
paws edema
Derivative 23 was further evaluated for in vivo antiinflammatory
activity, because of its -potent in vitro activities in other assays.
Compound 23 showed a promising activity in the in vivo model,
comparable to the standard drugs, aspirin and indomethacin. This
compound showed activity against carrageenan-induced inflam-
mation in a dose and time dependent manners. After the carra-
geenan injection at the lowest dose (1 mg/kg), the compound 23
OH
CH3
O
O
Dry DMF, POCl3
- H2O
CHO
O
1
Scheme 1. Synthesis of 3-formylchromone (1).

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K.M. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 4058e4064
O
O
+
H
RNH2
CH NR
O
1
O
O
2-24
Compds.
No.
R
Compds.
No.
R
Compds.
No.
R
H3CO
H3C
O
Cl
2
10
18
HN
Cl
Cl
N
OCH3
O
3
11
19
(CH2)5CH3
-HN
O
HOOC
O
C
4
5
12
13
20
21
HN
N
H3C
H3C
H3CO
O
HN
C
NO2
NO2
CH3
H3CO
6
14
22
O
NO2
-H
N
CH3
CH3
HO
7
8
15
16
23
24
O
N
-HN
O
C
H3C
O2N
O
HN
(CH2)7CH3
-HN
O
-(CH2)2-
9
17
HN
S
CH3
O
Scheme 2. Synthesis of chromone schiff bases 2e24.
showed a low inhibitory activity. Highest activity was observed
after the second hour of the inflammation (53%). Compound 23 at
a dose of 10 mg/kg showed a 7.82% inhibition during the first hour,
which reached to 67% after the fourth hour. At the lower dose of
5 mg/kg, activity remains same during the first to fourth hours.
However, it was interesting to note that the activity persisted at
both the doses. In conclusion, compound 23 was found to possess
a significant in vivo antiinflammatory activity (Table 5 and 6).
Compound 23 found to be the most active compound with a lowest
M. In addition, compounds 2, 5, 16, and 21
were also exhibited more potent activities than indomethacin and
aspirin. Compounds 1, 5, 6, 9, 14, and 22 exhibited better activities
than aspirin, however, these compounds exhibited moderate
IC50 ¼ 40.210 ꢁ 1.76
m
activities when compared with indomethacin. Compound
5
exhibited a respiratory burst inhibitory activity. Compound 23,
a butanohydrzide derivative of 1, was found to be the most potent
compound in the series. The difference in inhibitory activity of
compounds 16 and 23 may be explained on the basis of substitution
on carbonyl moiety. Compound 16 carries a phenyl, whereas
compound 23 has a propyl group at the carbonyl residue. The
phenyl is an electron withdrawing group, whereas propyl is an
electron donating group. The difference in substitution pattern
suggests that carbonyl part of hydrazide on chromone moiety
with an electron donating group enhances the respiratory burst
inhibitory activity. It appears that compound 21 which is a para-
nitrohydrazide exhibited less activity than compound 16, perhaps
3. Discussion
Synthetic 3-formylchromone (1) and its derivatives 2e24 were
synthesized and assayed for their antiinflammatory activities.
Results from respiratory burst inhibitory assay demonstrated that
compounds 2e24 have
(IC50 ¼ 40.210 ꢁ 1.76e478.39 ꢁ 10.36
known antiinflammatory compounds, indomethacin and aspirin
(IC50 values 246.35 ꢁ 12.36 and 518.52 ꢁ 3.68 M, respectively).
a
varying degree of activity
mM), which compared with
m

K.M. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 4058e4064
4061
Table 1
Table 3
Respiratory burst inhibitory activity of the synthetic chromone hydrazides deriva-
Cyclooxygenase inhibition activity of the synthetic chromone hydrazide derivatives
at a concentration of 1000 M.
tives at a concentration of 500
m
M using activated human neutrophils.
m
Compound
% Inhibition at 500
m
M
IC50 (
m
M)
Compound
COX-1 Inhibition IC50 (
mM)
COX-2 Inhibition IC50 (mM)
1
2
3
4
5
6
7
8
80.47
76.37
71.37
þ2.41*
78.53
52.59
92.75
45.17
399.99 ꢁ 16.92
98.215 ꢁ 32.85
>500
7
4
17
16
>1000
1000
150.45 ꢁ 4.25
405.96 ꢁ 4.12
>1000
þ 25.43*
þ 65.72*
203.69 ꢁ 2.65
802.32 ꢁ 1.63
1000
>1000
0.67 ꢁ 0.98
>500
130.98 ꢁ 33.98
23
500
Aspirina
750.41 ꢁ 0.54
400.85 ꢁ 5.23
>500
>500
>1000
>1000
Indomethacineb
0.05 ꢁ 0.47
a
Enhancing activity rather inhibitory.
Standard inhibitors for cyclooxygenase activity.
9
10
11
b
23.72
16.79
12
>500
13
14
15
16
17
18
21
23
e
>500
>500
399.357 ꢁ 10.76
55.036 ꢁ 6.17
e
Compounds 4, 7, 8, 10, 13, 16, and 23 were also screened for their
84.17
80.86
91.55
þ 2.41*
24.48
96.22
76.55
COX-1 and -2 inhibitory activities. Compounds 4 and 16 showed
a weak activity against both the isoforms. None of the compounds
(13, 16, 17, and 23) were found to be lipoxygenase inhibitory rather
they have enhanced the lipoxygenase activity.
>1000
85.540 ꢁ 0.072
40.210 ꢁ 1.76
288.72 ꢁ 36.51
518.5 ꢁ 3.68
246.35 ꢁ 12.36
The in vitro data suggests that compound 23 has the potential to
be a potent antiinflammatory compound, therefore, it was further
evaluated for its antiinflammatory activity in an in vivo system.
During the first hour after the carrageenan injection, compound 23
at 10 mg/kg dose inhibited the inflammation only by 7.82% which
reached up to 67% after the 4 h of treatment. At the lower dose of
5 mg/kg, activity remains the same during the first to fourth hours
(67%). At the lowest dose (1 mg/kg), it showed a reduced inhibitory
activity (42%). Highest activity was observed after the second hour
of the inflammation (53%). The data indicate that the compound 23
exhibited a very potent activity in the in vivo model, comparable
to the known antiinflammatory compounds, aspirin and
indomethacin.
22
Aspirina
Indomethacineb
a
Enhancing activity rather inhibitory.
Standard inhibitor for respiratory burst assay.
b
due to the same reasons. Similarly compound 2, a Schiff base with
dicholorophenyl moiety, also showed less activity than
compound 16.
Compound 2e24 were screened in cell-free system to measure
their superoxide scavenging activity. Most of the compounds found
to be potent scavengers of superoxides, produced by the enzymatic
system xanthine/xanthine oxidase. In this assay, compounds 2, 3,
a
4. Conclusion
16, 19, and 21 showed IC50 values less than 5 mM, which is a potent
range. In addition, compounds 1, 5, 7, 18, 16, 22, and 23 were also
found to be potent in inhibiting superoxide production at IC50 less
than 30 mM. The other compounds 4, 6, 14 and 17 either demon-
strated a weak activity or found to be inactive in this assay. The
results of this assay conferred that the possible mechanism of
action of the active compounds was their scavenging potential
against free radicals rather than inhibition of the enzyme involved
in the respiratory burst assay.
In conclusion, in vitro and in vivo studies demonstrated that
compound 23, which is butanohydrzide derivative of 3-for-
mylchromone, has potent antiinflammatory activities. This
compound has a potential to be further evaluated as a novel drug to
reduce inflammation.
4.1. In vitro antiinflammatory assays
4.1.1. Material and methods
Table 2
Heparinized human blood was obtained from local blood bank.
WST-1 was purchased from Dojindo Laboratories (Japan), Ficoll
Paque was purchased from Pharmacia Amersham (Uppsala
Sweden). Deionized water, which was prepared by using a Milli-
pore system, was used during the entire assay procedure. The
colorimetric COX (ovine) inhibitor screening kit (Cat No. 760111)
was from Cayman Chemicals, Ann Arbor, USA). Buttermilk xanthine
Superoxide scavenging activity of the synthetic chromone hydrazides derivatives at
a concentration of 1000
mM using xanthineexanthine oxidase system.
Compound
% Inhibition at 1000
m
M
IC50 (mM)
1
2
3
4
5
6
7
14
15
16
17
18
19
21
22
70.22
78.96
97.25
32.62
85.69
65.95
70.63
þ 8.47*
78.32
99.36
11.85
e
21.36 ꢁ 2.36
1.68 ꢁ 0.58
0.274 ꢁ 0.04
>1000
17.64 ꢁ 1.44
116.12 ꢁ 3.69
18.59 ꢁ 1.85
Inactive
29.19 ꢁ 12.41
0.439 ꢁ 0.07
>1000
13.95 ꢁ 5.67
4.63 ꢁ 0.69
1.00 ꢁ 0.20
20.00 ꢁ 10.43
72.77 ꢁ 12.56
>1000
oxidase (EC 1.2.3.2), xanthine, baicalein, and l-carrageenan, were
Table 4
Lipoxygenase inhibition activity of the synthetic chromone hydrazides derivatives at
a concentration of 1000
mM.
Compound
% Inhibition at 1000 mM
93.61
94.25
e
17
13
16
þ137.80*
þ47.5*
þ89.65*
þ80.78*
23
86.25
23
Aspirina
Baicleina
22.5ꢁ0.26
Indomethacineb
>1000
*Enhancing activity rather inhibitory.
a
Enhancing activity rather inhibitory.
Standard inhibitor for superoxide scavenging activity.
a
Standard inhibitor for lipooxyganase activity.
b

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K.M. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 4058e4064
Table 5
Amherst, USA). All the reactions were carried out in five replicates
to reduce the variability of the values.
Effect of compound 23 on the carrageenan-induced paw volume in rats with
different time intervals against various ranges of the dosage of compound.
Dose (mg/kg)
Paw volume (mL)
4.1.4. Cell viability assay
Cytotoxicity of the active compounds were measured by using
freshly isolated human neutrophils, as described by Berridge et al.
and Ishiyama et al. Freshly isolated human neutrophils
(1 ꢂ 107 cells/mL) were incubated in a total reaction volume of
0e1 h
0e2 h
0e3 h
0e4 h
Control
Aspirin
0.25 ꢁ 0.03
0.11 ꢁ 0.09
0.03 ꢁ 0.03
0.40 ꢁ 0.10
0.42 ꢁ 0.04
0.35 ꢁ 0.09
0.20 ꢁ 0.12
0.59 ꢁ 0.03
0.59 ꢁ 0.05
0.47 ꢁ 0.16
0.33 ꢁ 0.07
0.56 ꢁ 0.24
0.5 ꢁ 0.04
0.47 ꢁ 0.17
0.31 ꢁ 0.10
0.59 ꢁ 0.19
10
5
1
200
(200e12.5
30 min of incubation with a moderate shaking in a water bath at
37 ꢀC, WST-1 (250
M) was added and the plate was re-incubated
with shaking for 3 h. Absorbance was measured at 450 nm with
microplate reader (SpectraMax 340, Molecular Devices).
mL containing test compound with the various concentrations
(23)
10
5
1
0.23 ꢁ 0.05
0.14 ꢁ 0.06
0.15 ꢁ 0.06
0.27 ꢁ 0.07
0.15 ꢁ 0.11
0.19 ꢁ 0.05
0.26 ꢁ 0.07
0.23 ꢁ 0.15
0.36 ꢁ 0.14
0.16 ꢁ 0.14
0.16 ꢁ 0.09
0.29 ꢁ 0.13
mM). The absorbance was pre-read at 450 nm. After
m
a
Percentage cell viability was calculated by using the following
formula:
purchased from Aldrich Chem. Co. Dextran, zymosan A, enzyme
lipoxygenase (1.13.11.12 type I-B), arachidonic acid, sodium citrate
and all other reagents were purchased from Sigma (St. Louis, MO,
USA). Plathysmometer was of (Model 76 014, Ugo, Japan).
%Cell Viability ¼ fðOD test compound
ꢂ100=OD controlÞ ꢃ 100g
4.1.2. Animals
4.1.5. Cyclooxygenses inhibition assay
Wistar rats (180e210 g) of either sex were obtained from the
animal house facility of the International Center for Chemical and
Biological Sciences, University of Karachi. The animals were kept
under standardized conditions i.e. temperature 22 ꢁ 2 ꢀC, humidity
60% ꢁ 4%, 12/12 h light and dark cycles. Animals were kept on free
excess of water and normal diet.
The assay was performed as per manufacturer’s instruction
(Cayman Chemicals, Ann Arbor, USA). The activity was assayed
colorimetrically by monitoring the appearance of oxidized N, N, N0,
N0-tetramethyl-p-phenylenediamine (TMPD) at 590 nm. Inhibition
of COX activity by a variety of selective inhibitors was calculated
based on their IC50 values. Five replicates of each experiment were
performed.
4.1.3. Assay for respiratory burst inhibitory activity
Respiratory burst inhibitory activity of synthetic Schiff’s bases
were determine by using the modified method of Berridge et al.
Briefly, 15e20 mL heparinized fresh human volunteer blood was
used to isolate neutrophils, as described elsewhere (Siddiqui et. al.).
The cells were re-suspended with MHS at a concentration of
1 ꢂ 106 cells/mL. Freshly prepared human serum (preferably AB
blood group) was mixed with zymosan A (20 mg/mL in 1:1PBS).
Tube was shaken vigorously with vortex mixer and kept in
a shaking water bath at 37 ꢀC for 20 min. The tube was then
centrifuged at 3000 rpm for 10 min at room temperature, pallets
were re-suspended and kept at ꢃ20 ꢀC until used. All the test
4.1.6. Lipoxygense inhibition assay
Lipoxygenase inhibitory activity was measured by slightly
modifying the spectrophotometric method developed by Tappel
et al. [29]. In a total volume of 200 mL, test compound, and lip-
oxygenase solutions were mixed with sodium phosphate (100 mM)
buffer (pH 8.0) and incubated for 10 min at 25 ꢀC. The reaction was
then initiated by the addition of 10 mL linoleic acid solution and the
change of absorbance at 234 nm was measured for next 10 min due
to formation of (9Z, 11E)-(13S)-13- hydroperoxyoctadeca-9, 11-
dienoate. Baicalein (Aldrich Chem. Co.) was used as a positive
control in the assay as described by Ellis and Luscombe [30]. The
IC50 values were then calculated by using the Ez-Fit 5.0.
compounds were prepared as stock at a concentration of 500 mM in
5% DMSO with MHS. Aspirin and indomethacin were used as
standard antiinflammatory agents. Cells (1 ꢂ 106 cells/mL) were
4.1.7. Platelet aggregation assay
incubated with various concentrations of test samples and 250 mM
Venous blood was mixed with sodium citrate (3.8%) solution in
9:1 ratio, in order to prevent coagulation. The whole blood was
centrifuged at 1000 rpm for 15 min at 37 ꢀC to obtain platelet rich
plasma (PRP). The upper part of the plasma which was rich in
platelets was taken. The remaining plasma was again centrifuged
for 10 min at 37 ꢀC to obtain platelet poor plasma (used as blank).
This plasma hardly contained any platelet.
of WST-1 in a total volume 200
mL in 96-well plates. Pre-read
absorbance was taken at 450 nm on microplate reader (SpectraMax
340, Molecular Devices). After 30 min of incubation at 37 ꢀC, cells
were induced to generate reactive oxygen species by adding
opsonized zymosan (10 mL/well); and kinetic absorbance was taken
for 30 min. At the end of the reaction, endpoint reading was taken.
IC50 was calculated by using Ez-fit 5.0 (Perrella Scientific Inc.,
Aggregation was measured by using a Dual Channel Lumi-
aggregometer with 230
Various concentrations (200e10
aspirin, (10 L), were dissolved in DMSO (10% with normal saline)
and incubated at for 1 min before challenged with platelet aggre-
gation agonist (Arachidonic acid). Aggregation was recorded for 4
additional minutes, so the total exposure time of the platelets to
inhibitors was 5 min. Percent inhibitions were expressed in the
dose-response curves between the % aggregation and the time in
minutes.
m
L aliquots of PRP in small glass cuvette.
mM) of test compounds and
Table 6
m
Percentage inhibition of different concentrations of compound 23 at different time
intervals on carrageenan-induced rat paw edema model.
Dose (mg/kg)
% Edema inhibition
1 h
2 h
3 h
4 h
Control
Aspirin
e
10
5
e
e
e
e
64.23
89.82
25.26
52.06
72.45
19.17
51.37
66.21
42.38
1.43
32.60
28.80
1
4.1.8. In vitro superoxide scavenging assay by using
xanthine/xanthine oxidase system
(23)
10
5
1
7.82
41.50
39.80
36.31
64.48
53.97
55.37
61.87
38.84
67.17
67.33
42.17
In a total 200
mL reaction volumes, various concentrations of the
test samples were tested with xanthine oxidase (0.011 units/well).

K.M. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 4058e4064
4063
Plate was pre-incubated for 10 min at room temperature. WST-1
(15 M) was added and pre-read absorbance was recorded at
450 nm. Continues kinetics analysis was initiated by adding
xanthine (20 L) and the kinetic absorbance was read for 30 min. At
the end of reaction, endpoint absorbance was recorded. Each
experiment was the mean of triplicates.
5.6. 3-{[(3-Methylphenyl) imino] methyl}-4H-chromen-4-one (7)
m
Yield ¼ 60%; mp ¼ 116 ꢀC; Rf ¼ 0.46 (EtOAc:Hex, 3:7) [25].
5.7. 3-[Bis(tert-butylamino)methyl]-4H-chromen-4-one (8)
Yield ¼ 46%; mp ¼ 138 ꢀC; Rf ¼ 0.45 (EtOAc:Hex, 5:5) [25].
m
4.1.9. In vivo carrageenan-induced rat paw edema
antiinflammatory activity measurement
5.8. 3-{[(2-{[(4-Oxo-4H-chromen-3-yl)methylidene]amino}ethyl)
In vivo antiinflammatory activity was measured by using
modified method of Winter et al. [16]. Various doses of the test
compounds (0.5, 1, 5, and 10 mg/mL/kg body weight) were injected
intra-peritoneally (i.p.). After 30 min, edema was induced by
injecting 0.05 mL of 0.1% carrageenan (dissolved in 0.9% saline) in
the sub-planter region of the right paw of each rat. Control group of
animals received only vehicle (10% DMSO with 0.9% saline). Paw
volume was measured by using plathysmometer after 30 min, and
1, 2, 3, and 4 h, after the carrageenan injection. 0 h measurement
was taken prior to the i.p. injection of test compounds. The
percentages of the inhibition in the treated animals versus control
group were calculated by using following formula:
imino] methyl}-4H-chromen-4-one (9)
Yield ¼ 23%; mp ¼ 188 ꢀC; Rf ¼ 0.35 (EtOAc:Hex, 3:7) [25].
5.9. 3-{[(3-Methoxy-4-methylphenyl) imino] methyl}
-4H-chromen-4-one (10)
Yield ¼ 72%; mp ¼ 86 ꢀC; Rf ¼ 0.42 (EtOAc:Hex, 3:7) [25].
5.10. 3-{[(3-Methoxyphenyl)imino]methyl}-4H-chromen-4-one (11)
Yield ¼ 43%; mp ¼ 178 ꢀC; Rf ¼ 0.35 (EtOAc:Hex, 5:5) [25].
5.11. 3-[(Phenylimino) methyl]-4H-chromen-4-one (12)
% inhibition ¼ ðVT ꢃ VC=VCÞ ꢂ 100
Where, VT ¼ Volume of paw for test sample and VC ¼ Volume of
Yield ¼ 69%; mp ¼ 125 ꢀC; Rf ¼ 0.52 (EtOAc:Hex, 3:70) [25].
paw for control group
5.12. 3-{[(2-Methoxy-5-nitrophenyl) imino] methyl}-4H-chromen-
4-one (13)
5. Experimental
Yield ¼ 78%; mp ¼ 266 ꢀC; Rf ¼ 0.38 (EtOAc:Hex, 3:7) [25].
Melting points were determined on a Büchi 434 melting point
apparatus and were uncorrected. NMR was performed on a Bruker
AM 300, 400 and 500 MHz, respectively. CHN analysis was per-
formed on a Carlo Erba Strumentazion-Mod-1106, Italy. Ultraviolet
(UV) spectra were recorded on PerkineElmer Lambda-5 UV/VIS
spectrometer in MEOH. Infrared (IR) spectra were recorded on
JASCO IR-A-302 Spectrometer as KBr (disc). Electron impact mass
spectra (EIMS) were recorded on a Finnigan MAT-311A, Germany.
Thin layer chromatography (TLC) was performed on pre-coated
silica gel glass plates (Kieselgel 60, 254, E. Merck, Germany).
Chromatograms were visualized by UV at 254 and 365 nm or by
iodine vapors.
5.13. 3-{[(2-Methoxy-4-nitrophenyl) imino] methyl}-4H-chromen-
4-one (14)
Yield ¼ 63%; mp ¼ 268 ꢀC; Rf ¼ 0.46 (EtOAc:Hex, 3:7) [25].
5.14. 3-{[(3-Hydroxy-2-pyridinyl)imino]methyl}-4H-chromen-
4-one (15)
Yield ¼ 62%; mp ¼ 144 ꢀC; Rf ¼ 0.5 (EtOAc:Hex, 3:7) [25].
5.15. N0-[(4-Oxo-4H-chromen-3-yl) methylidene]
benzohydrazide (16)
5.1. 4-Oxo-4H-chromene-3-carbaldehyde (1)
Yield ¼ 58%; mp ¼ 201 ꢀC; Rf ¼ 0.42 (EtOAc:Hex, 3:7) [26].
Yield: 82%; mp ¼ 142 ꢀC; Rf: 0.56 (EtOAc: Hex, 3:7) [25].
5.16. 4-Methyl-N0-[(4-Oxo-4H-chromen-3yl) methylidene]
benzenesulfonohydrazide (17)
5.2. 3-{[(2-Benzoyl-4-chlorophenyl) imino] methyl}-4H-chromen-
4-one (3)
Yield ¼ 56%; mp ¼ 208 ꢀC; Rf ¼ 0.41 (EtOAc:Hex, 3:7) [26].
Yield ¼ 76%; mp ¼ 138 ꢀC; Rf ¼ 0.52 (EtOAc:Hex, 3:7) [25].
5.17. N0-[(4-Oxo-4H-chromen-3-yl) methylene]
isonicotinohydrazide (18)
5.3. 2-{[(4-Oxo-4H-chromen-3-yl) methylidene] amino}benzoic
acid (4)
Yield ¼ 56%; mp ¼ 278 ꢀC; Rf ¼ 0.45 (EtOAc:Hex, 3:7); UV
Yield ¼ 64%; mp ¼ 96 ꢀC; Rf ¼ 0.42 (EtOAc:Hex, 3:7) [25].
(MeOH); lmax 206.1 (log
3
¼ 4.2) nm; IR (KBr): ymax 3448 (NeH),
3050 (Ar CH), 2924 (CH3), 2862 (CH2), 1702 (C]O) cmꢃ1
;
1H NMR
(400 MHz, CD3OD)
d
: 10.45 (s, 1H NeH), 8.80 (d, J5,6 ¼ 8.1 Hz, 1H, H-
5.4. 3-{[(2,6-Dimethylphenyl)imino]methyl}-4H-chromen-4-one (5)
Yield ¼ 53%; mp ¼ 84 ꢀC; Rf ¼ 0.61(EtOAc:Hex, 3:7) [25].
0
0
0
0
5), 8.62 (s, 1H, CH]N), 8.22 (s,1H, H-2), 7.83 (d, J3 ,2 ¼ J5 ,6 ¼ 6.4 Hz,
2H, H-30,50), 7.69 (d, J2 ,3 ¼ J6 ,5 ¼ 6.4 Hz, 2H, H-20, 60), 7.47 (td,
J7,5 ¼ 1.5, J7,6 ¼ J7,8 ¼ 8.2 Hz, 1H, H-7), 7.0 (d, J8,7 ¼ 8.2 Hz, 1H, H-8),
6.91 (t, J6,5 ¼ J6,7 ¼ 8.2 Hz, 1H, H-6); EIMS: m/z (rel. abund. %) 292
(Mþ, 12), 188 (80), 172 (3.2), 120 (100); Anal. Calc. for C13H11N3O3:
(293): [Found: C, 65.52; H, 3.77; N, 14.34. Requires: C, 65.53; H,
3.78; N, 14.33].
0
0
0
0
5.5. 3-{[(3, 5-Dimethylphenyl)imino]methyl}-4H-chromen-4-one (6)
Yield ¼ 72%; mp ¼ 184 ꢀC; Rf ¼ 0.51 (EtOAc:Hex, 3: 7) [26].

4064
K.M. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 4058e4064
5.18. N0-[(4-Oxo-4H-chromen-3-yl) methylene]
heptanohydrazide (19)
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(1995) 189.
Yield ¼ 78%; mp ¼ 201 ꢀC; Rf ¼ 0.52 (EtOAc:Hex, 5:5) [26].
[9] C. Charlier, C. Michaux, Dual inhibition of cyclooxygenase-2 (COX-2) and 5-
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5.19. N0-[(4-Oxo-4H-chromen-3-yl)methylidene]
nicotinohydrazide (20)
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5.20. N-Hydroxy-N-oxo-4-({2-[(4-oxo-4H-chromen-3-yl)
methylidene]hydrazino}carbonyl)benzenaminium (21)
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(1996) 1518.
[13] D. Steinhilber, A target for antiinflammatory drug revisited. Curr. Med. Chem.
6 (1999) 71.
Yield ¼ 74%; mp ¼ 208 ꢀC; Rf ¼ 0.43 (EtOAc:Hex, 3:7) [26].
5.21. N0-[(4-Oxo-4H-chromen-3-yl) methylidene]
propanohydrazide (22)
[14] D. Nie, K.V. Honn, Cyclooxygenase, lipoxygenase and tumor angiogenesis. Cell.
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[15] G.P. Ellis, D.K. Luscombe, Progress in Medicinal Chemistry, vol. 29, Elsevier
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[16] G.P. Ellis, in: A. Weissberger, E.C. Taylor (Eds.), Chromenes, Chromanones, and
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5.22. N0-[(4-Oxo-4H-chromen-3-yl) methylidene]
butanohydrazide (23)
Yield ¼ 61%; mp ¼ 197 ꢀC; Rf ¼ 0.48 (EtOAc:Hex, 2:8) [26].
5.23. N0-[(4-Oxo-4H-chromen-3-yl)methylidene]
nonanohydrazide (24)
[19] G.R. Singh, N.K. Singh, M.P. Girdhar, S. Ishar, A versatile route to 2-alkyl-/
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inase inhibition activity of some chromones. Chem. Pharm. Bull. 50 (2002)
309.
Yield ¼ 46%; mp ¼ 278 ꢀC; Rf ¼ 0.44 (EtOAc:Hex, 3:7) [26].
Acknowledgements
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I. Choudhary, 1,3,4-Oxadiazole-2(3H)-thione and its analogues: a new class of
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Synthesis and lishmenicidal activity of 2,3,4-substituted-5-imidazolon. J. Enzym.
Inhib. Med. Chem. (2009).
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3-formylchromone as thymidine phosphorylase inhibitors. Biorg. Med. Chem.
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Schiff bases of 3-formylchromones as phytotoxic, antibacterial, and antifungal
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This work was financially supported by the Higher Education
Commission (HEC) Pakistan under the National Research Support
Program for Universities. The help of the Fatimid Foundation is
highly acknowledged for providing fresh healthy blood for this
research study.
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