Synthesis, biological evaluation and docking studies of novel benzopyranone congeners for their expected activity as anti-inflammatory, analgesic and antipyretic agents

Article abstract of DOI:10.1016/j.bmc.2009.05.073

8-Acetyl-7-hydroxy-4-phenyl-2H-benzopyran-2-one as starting material a number of 8-substituted derivatives (i.e., hydrazones 2a,b, imine 2c, chalcones 3, pyrazoles 4, 3-cyano-2-oxo-dihydropyridines 5, and/or 3-cyano-2-imino-dihydropyridines 6) were synthe

Full text of DOI:10.1016/j.bmc.2009.05.073

Bioorganic & Medicinal Chemistry 17 (2009) 5059–5070
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and docking studies of novel benzopyranone
congeners for their expected activity as anti-inflammatory, analgesic and
antipyretic agents
c
Amal A. M. Eissa a, Nahla A. H. Farag b, , Gamal A. H. Soliman
*
a Medicinal and Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El Aini Street, Cairo, Egypt
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Misr International University, Km 28 Cairo-Ismailia Road, PO Box 1, Heliopolis, Cairo, Egypt
c Pharmacology Department, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
8-Acetyl-7-hydroxy-4-phenyl-2H-benzopyran-2-one as starting material a number of 8-substituted
derivatives (i.e., hydrazones 2a,b, imine 2c, chalcones 3, pyrazoles 4, 3-cyano-2-oxo-dihydropyridines
5, and/or 3-cyano-2-imino-dihydropyridines 6) were synthesized and assayed for their anti-inflamma-
tory, analgesic and antipyretic activities. Compounds 3c, 4b and 4i showed significant anti-inflammatory,
analgesic and antipyretic activities. In addition, 1, 3b, 4d, 4e, 5b, 6a, 6c, 6d, 6e showed anti-inflammatory
activity, 2b, 4h, 5e exhibit analgesic activity, and 2b, 4h, 5e showed antipyretic effect. In addition, molec-
ular modeling and docking of the tested compounds into cyclooxygenase II complexed with its bound
inhibitor indomethacin (4COX) using MOLSOFT ICM 3.4-8C program was performed in order to predict the
affinity and orientation of the synthesized compounds at the active site. Also, it was found that the active
compounds 1, 4i, 6a–e interact with both Serine 530, and Tyrosine 385 amino acids which are the main
amino acids involved in the mechanism of cyclooxygenase II inhibition.
Received 28 April 2009
Revised 20 May 2009
Accepted 23 May 2009
Available online 13 June 2009
Keywords:
Benzopyranone
Coumarin
Anti-inflammatory
Analgesic
Antipyretic
Docking
The synthesis of the pyrazole-containing new compounds 4 proved a successful hit; also, the 2-imino
derivatives of 3-cyano-dihydropyridines were more successful than the 2-oxo derivatives.
According to these results, we can conclude that compounds 1, 3c, 4b, 4i, and 6c appear to be the most
interesting and seem potentially attractive as anti-inflammatory, analgesic, and antipyretic agents.
Ó 2009 Elsevier Ltd. All rights reserved.
Internal coordinate mechanics (ICM)
1. Introduction
Due to the importance of coumarin derivatives considerable
efforts have been made by several investigators, to prepare new
Several natural products with a coumarinic moiety have been
reported to have multiple biological activities, antitumor,1–3
chronic HBV/HCV,4 antibacterial and antifungal5–8 and other activ-
ities related to the benzopyranone-containing compounds.9–17 Hy-
droxy coumarins might affect the formation and scavenging of
reactive oxygen species (ROS) and influence processes involving
free radical-mediated injury, possessing antioxidant property,18
anti-inflammatory7,9–21 and analgesic7,21 activities. Moreover cou-
marin and its 7-hydroxy-derivative known as umbelliferones are
present in many plant species in nature,4 they inhibit prostaglan-
din biosynthesis, which involves fatty acid hydroperoxy intermedi-
ates and protect plants from cellular damage, infestations, trauma
and infections.
compounds bearing single substituent or more complicated
systems, including heterocyclic rings mainly at 3-, 4- and/or
7-positions. Also, literature survey reveals an excellent anti-
inflammatory activity with some compounds containing heterocy-
clic ring as pyrazole.22–28
Encouraged by these findings we thought of preparing new
derivatives 2–6 of 7-hydroxy-4-phenyl-2H-benzopyran-2-ones
substituted at C-8 with different hydrazones 2a,b and imine 2c,
chalcones 3, pyrazoles 4, 3-cyano-2-oxo-dihydropyridines 5, and/
or 3-cyano-2-imino-dihydropyridines 6 in order to screen them
for anti-inflammatory, analgesic and antipyretic activities.
Non-steroidal anti-inflammatory drugs (NSAIDs) exert their ef-
fects by inhibition of prostaglandin production. The pharmacolog-
ical target of NSAIDs is cyclooxygenase (COX, also known as
prostaglandin synthase), which catalyses the first committed step
in arachidonic acid metabolism. Two isoforms of the membrane
protein COX are known: COX-1, which is constitutively expressed
in most tissues, is responsible for the physiological production of
prostaglandins; and COX-2, which is induced by cytokines, mito-
gens and endotoxins in inflammatory cells, is responsible for the
Natural products like esculetin, fraxetin, daphnetin and other
related coumarin derivatives are recognized as inhibitors not only
of the lipooxygenase and cyclooxygenase enzymic systems, but
also of the neutrophil-dependent superoxide anion generation.
* Corresponding author. Tel.: +20 224534418/123378871; fax: +20 2 4771560.
E-mail address: nahlahassan91@yahoo.com (N.A.H. Farag).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.073

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A. A. M. Eissa et al. / Bioorg. Med. Chem. 17 (2009) 5059–5070
elevated production of prostaglandins during inflammation.29 It
was reported that crystal structures of COX enzymes with carbox-
ylic acid-containing NSAIDs shows that the inhibitors are posi-
tioned in a similar fashion with their carboxylates coordinates to
Arg-120 and their aromatic functionality projecting into the cyclo-
oxygenase active site towards Tyr-385,30,31 and diarylheterocycles
inhibitors of COX-2 bind in the cyclooxygenase active site above
Arg-120 and insert their sulfonamide or sulfone groups into a side
pocket bordered by Val-523.1–3
Molsoft36 as flexible docking program enables us to predict
favorable protein–ligand complex structures with reasonable accu-
racy and speed. The docking technique will undoubtedly continue
to play an important role in drug discovery.37
So, we docked the prepared and tested compounds into cyclox-
ygenase II (4cox) active site in order to predict their binding
modes, their binding affinities and orientation of these compounds
at the active site of the cycloxygenase II enzyme.
Recently, X-ray analysis of a cocrystal of arachidonic acid with
COX-2 revealed that its carboxylate coordinated to Serine 530
and Tyrosine 385,32 NSAIDs exert their action through their acety-
lation of Serine 530, and also hydrogen bonding by Tyrosine 385 is
proposed to stabilize the negative charge of the tetrahedral inter-
mediate that developed during Serine 530 acetylation,33 it was
demonstrated that carboxylate group of NSAI binds to COX-2
through hydrogen bonds to Tyrosine 385 and Serine 530,34 Tyro-
sine 385 and Serine 530 have a structural and functional evidence
for the importance of them in the chelation of the ligands.34
Computer docking technique plays an important role in the
drug design as well as in the mechanistic study by placing a mol-
ecule into the binding site of the target macromolecule in a non-
covalent fashion.35
2. Results and discussion
2.1. Chemistry
In this study we synthesized five new series of 7-hydroxy-4-
phenyl-2H-1-benzopyran-2-one substituted at C-8 with different
hydrazones 2a,b and imine 2c, chalcones 3, pyrazoles 4, 3-cya-
no-2-oxo-dihydropyridines 5, and/or 3-cyano-2-imino-dihydro-
pyridines 6 to be tested as anti-inflammatory, analgesic, and
antipyretic activities.
The synthesis of our target compounds 2–6 is outlined in
Scheme 1. The key intermediate 8-acetyl-7-hydroxy-4-phenyl-
2H-1-benzopyran-2-one 1 was previously prepared.38 Condensa-
tion of 1 with hydrazines39,40 or phenylene diamine gave the
C6H5
NO2 H
N
H
N
R1 =
a
NH2
O2N
,
,
HO
O
CH3
O
N
R1
2a-c
C6H5
C6H5
c
b
HO
O
O
HO
O
O
C6H5
N
O
R2
N
R2
R3
4a-i
3a-c
HO
O
O
O
CH3
R2 =
,
,
N
CH3O
C6H5
Cl
1
H
,
R3 =
,
O2N
NO2
HO
O
O
d
NH
R4
O
CN
C6H5
5a-f
O
O
HO
R4
R4 =
,
,
,
N
Cl
CH3O
OH
e
OMe
NH
N
NH
,
OMe
,MeO
CN
6a-e
Scheme 1. Reagents: (a) hydrazines/absolute ethanol; (b) aldehydes/alcoholic potassium hydroside; (c) hydrazines; (d) ethyl cyanoacetate/aldehydes/ammonium acetate;
(e) malononitrile/aldehydes/ammonium acetate.

A. A. M. Eissa et al. / Bioorg. Med. Chem. 17 (2009) 5059–5070
5061
corresponding hydrazones 2a,b and imine 2c, respectively. Mean-
while, reaction of 1 with three different aldehydes39,41–48 yielded
the corresponding chalcones 3a–c, which were further reacted
with hydrazine hydrate and/or substituted hydrazines to afford
4a–i incorporating a mono or disubstituted pyrazole ring sys-
tem.23,28,39,41,43–47 Furthermore, the 8-acetyl intermediate 1 was
reacted with ethyl cyanoacetate, ammonium acetate, and the
appropriate aldehydes in a multicomponent reaction to give 5a–f
in good yield.42,48 Also, the one pot synthesis was utilized to syn-
thesize compounds 6a–e, using malononitrile, ammonium acetate,
and the appropriate aldehydes.42,44,45,48–53
Table 1
Anti-inflammatory effect of the new synthesized compounds in doses equal to 1/10 of
their LD50 and Celecoxib (20 mg kgꢀ1) in rats (n = 5)
Groups
Paw size (mm) (mean S.E.M)
Reduction %
Control
Celecoxib
1
2a
2b
2c
3a
3b
3c
4a
4b
4c
4d
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
29.54 1.15
17.82 1.24***
23.55 1.28**
25.89 1.24
29.34 1.25
26.97 1.34
25.89 1.55
23.55 1.26**
24.40 1.53*
26.97 1.64
23.97 1.31**
26.11 1.24
24.40 1.48*
23.97 1.20**
26.21 1.25
26.97 1.36
27.83 1.16
24.50 1.35**
27.83 1.26
24.71 1.24*
27.40 1.26
27.83 1.35
28.26 1.19
27.42 1.28
24.83 1.27*
28.15 1.29
23.97 1.28**
24.72 1.27*
24.75 1.23*
—
39.67
20.27
12.35
0.67
2.2. Pharmacology
2.2.1. Statistical analysis
All rats treated with different compounds up to 150 mg kgꢀ1
were alive during the 24 h of observation. These animals did not
show visible signs of acute toxicity. LD50 of compounds 3c, 4f, 5c,
5d and 6b was 390 mg kgꢀ1. LD50 of compounds 2b, 2c, 4b, 4c, 5f
and 6e was 370 mg kgꢀ1. LD50 of compounds 3b, 4a, 4e, 4h, 4i,
5a and 6a was 330 mg kgꢀ1. LD50 of compounds 4d, 4g, 5b, 6c
and 6d was 310 mg kgꢀ1. Finally, LD50 of compounds 1, 2a, 3a
and 5e was 290 mg kgꢀ1. The tested compounds are considered
non-toxic.
8.70
12.35
20.27
17.40
8.70
18.85
11.61
17.40
18.85
11.27
8.70
5.78
17.06
5.78
16.35
7.24
5.78
2.2.2. Anti-inflammatory activity
All the final new compounds were tested for their in vivo anti-
inflammatory efficacy using carrageenan-induced paw edema
method.54
The tested compounds and the standard drug celecoxib pro-
duced significant reduction of paw size as compared to the control
group (Table 1).
The key intermediate 1 showed significant anti-inflammatory
activity. On the other hand, hydrazone 2a showed only slight
anti-inflammatory activity. As for chalcones 3a–c, compound 3b
showed very good activity (20.27% reduction) more than 3c
4.33
7.17
6a
6b
6c
6d
6e
15.94
4.70
18.85
16.31
16.21
Significant at: *P 6 0.05, **P 6 0.01, ***P 6 0.001.
Table 2
Analgesic effect (represented by number of writhes) of the new synthesized compounds in doses equal to 1/10 their LD50 and diclofenac sodium (5 mg kgꢀ1) in mice using acetic
acid-induced writhing method (n = 5)
Groups
Number of writhes
2 h
0 h
1 h
3 h
4 h
Control
Diclofenac sodium
1
42.3 2.5
41.2 2.8
42.6 2.9
41.5 2.6
42.5 2.4
42.6 3.2
42.5 2.8
42.4 3.7
42.5 2.6
42.0 2.9
42.2 3.6
41.8 2.8
42.2 3.2
41.7 3.0
42.6 2.8
42.5 2.5
42.5 3.2
42.5 3.4
42.2 2.7
42.1 3.2
42.4 3.3
42.4 3.0
42.3 2.6
42.0 2.8
42.5 3.1
42.6 2.7
42.5 2.8
42.6 2.9
42.7 2.4
42.8 2.6
10.7 2.2***
42.6 2.5
41.0 3.1
35.4 2.6
38.4 2.8
38.5 2.4
42.4 2.7
25.6 3.3***
37.8 3.2
24.8 2.8***
40.3 3.4
41.9 3.1
41.4 2.9
42.2 2.7
41.9 3.1
25.6 2.8***
26.8 2.4***
41.8 3.0
42.1 3.1
38.3 2.9
42.2 2.6
24.6 2.7***
38.3 2.7
42.3 3.1
41.2 2.5
38.5 2.8
38.5 3.1
39.5 2.9
43.7 3.2
10.2 2.0***
41.6 2.6
40.4 3.0
34.7 3.2
37.3 2.7
38.5 2.9
42.0 3.3
26.8 3.3***
37.5 3.0
25.7 2.9***
39.8 2.6
41.5 2.5
40.6 2.8
42.0 3.4
41.8 3.2
25.4 2.7***
27.5 3.2**
41.0 2.8
41.6 2.9
39.2 2.6
42.1 3.2
29.4 2.7**
38.3 2.7
42.2 2.5
41.5 2.9
37.4 3.5
38.6 3.2
39.7 3.0
45.3 3.1
13.0 1.9***
40.5 2.0
40.3 3.0
36.8 2.6
38.5 2.7
38.5 2.9
41.7 3.0
32.8 2.6**
39.8 2.3
34.5 2.7*
40.3 2.5
41.5 3.0
40.3 3.7
42.2 2.5
41.5 2.8
33.8 3.1*
34.4 3.4**
38.2 2.7
41.8 2.6
39.2 3.0
42.1 3.1
34.2 2.8*
40.5 3.0
42.0 3.1
40.3 2.7
37.6 2.7
38.5 2.9
40.5 3.3
45.8 3.3
18.2 2.0***
41.4 2.4
40.7 2.7
40.6 3.3
41.9 3.5
41.0 2.5
42.0 2.7
38.5 3.0
41.7 2.8
39.4 3.4
40.5 2.8
41.7 3.0
40.4 3.2
42.0 2.9
41.6 3.2
39.6 3.0
39.4 3.5
41.7 2.9
41.4 2.8
41.4 2.7
41.8 2.9
39.6 3.0
41.6 2.7
41.7 3.0
41.1 2.7
40.9 2.8
41.5 2.7
41.5 2.7
2a
2b
2c
3a
3b
3c
4a
4b
4c
4d
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
Significant at: *P 6 0.05, **P 6 0.01, ***P 6 0.001.

5062
A. A. M. Eissa et al. / Bioorg. Med. Chem. 17 (2009) 5059–5070
(17.40% reduction), while compound 3a had slight activity (12.35%
On the other hand, screening of substituted pyrazoles 4a–i re-
reduction).
vealed that six compounds possess anti-inflammatory activity.
Compounds
4b, 4e, 4d, 4i gave significant activity (18.85%,
18.85%, 17.40%, 17.06% reduction), respectively, while compounds
4c, and 4f showed slight activity (11.6%, 11.27% reduction).
Among 2-oxo-dihydropyridine-3-carbonitrile 5a–f, only com-
pound 5b showed moderate activity (16.35% reduction). Finally,
2-imino-dihydropyridine-3-carbonitrile 6a–e, compounds 6c, 6d,
6e, 6a had significant activity (18.85%, 16.31%, 16.21%, 15.94%
reduction), respectively
Table 2b
Analgesic effect (represented by % protection against writhing) of the new synthe-
sized compounds in doses equal to 1/10 of their LD50 and diclofenac sodium
(5 mg kgꢀ1) in mice using acetic acid-induced writhing method (n = 5)
Groups
% Protection against writhing
0 h
1 h
2 h
3 h
4 h
Control
Diclofenac sodium
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
74.02
0.00
3.75
16.70
9.85
9.41
0.0
75.24
2.34
2.58
18.35
12.44
9.41
0.94
36.94
10.71
39.09
4.78
1.65
2.63
1.40
1.64
40.23
35.29
2.84
1.18
7.54
0.70
30.49
8.80
0.70
2.58
12.00
9.38
7.02
68.44
4.92
2.81
13.41
9.62
9.41
1.65
22.82
5.23
18.24
3.58
1.65
3.35
0.93
2.35
20.47
19.05
9.47
0.71
7.54
0.70
19.14
3.57
1.17
5.39
11.52
9.62
5.15
55.82
2.81
1.17
4.47
1.64
3.52
0.94
9.41
0.71
6.63
3.58
1.18
3.11
1.40
2.11
6.82
7.29
1.18
1.66
2.35
1.41
6.38
0.95
1.88
3.52
3.76
2.58
5.15
2.2.3. Analgesic activity
2a
2b
2c
3a
3b
3c
4a
4b
4c
4d
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
All the newly synthesized compounds were subjected to testing
their analgesic activity using the acetic acid-induced writhing
method.55
Significant protection against writhing was observed in animals
treated with compounds5e, 4b, 4h, 3c, 4i where number of writhes
after 1 h were 24.6, 24.8, 25.6, 25.6, 26.8, respectively compared to
42.3 in the control group (Table 2a).
Also, the tabulated results revealed that compounds 5e, 4b, 4h,
3c, 4i and exhibited the highest percentage protection against
writhing 41.84, 41.23, 39.76, 37.41 and 36.94, respectively (Table
2b).
37.41
10.00
41.23
3.58
0.71
0.71
0.93
1.41
39.76
36.94
0.94
0.00
9.66
0.47
41.84
8.80
4.70
3.28
9.41
9.62
7.49
2.2.4. Antipyretic activity
The antipyretic activity was evaluated for the newly prepared
compounds by using the yeast-induced hyperpyrexia method.56
The tabulated results showed that compounds 2b, 3c, 4b, 4h, 4i,
5e, and 6c, significantly decrease the temperature of pyretic rats at
1, 2 and 3 h after compound administration (Table 3). The maxi-
mum mean rectal temperatures produced by brewer’s yeast in
the presence of compounds 2b and 4h were (38.05, 38.01 °C),
respectively. In addition, compounds 4i, 4b, 6c, 5e, 3c again
showed a decrease in the rectal temperature after 2 h were
6a
6b
6c
6d
6e
Table 3
Antipyretic effect of the new synthesized compounds in doses equal to 1/10 of their LD
and paracetamol (100 mg kgꢀ1) in hyperthermic rats (n = 5)
50
Groups
Rectal temperature (°C) after compounds administration
1 h
2 h
3 h
4 h
Control
Paracetamol
1
39.24 0.22
37.38 0.19***
38.91 0.25
39.11 0.22
38.42 0.22*
38.61 0.23
38.91 0.25
39.05 0.22
38.31 0.28*
38.81 0.23
38.51 0.23*
38.81 0.22
38.90 0.28
38.91 0.22
38.81 0.22
38.91 0.23
38.40 0.28*
38.54 0.22*
38.81 0.25
38.91 0.33
38.91 0.28
38.91 0.23
38.34 0.29*
38.71 0.32
38.90 0.24
38.91 0.22
38.38 0.23*
39.01 0.23
39.11 0.31
39.11 0.26
37.20 0.17***
38.91 0.26
38.81 0.22
38.05 0.27**
38.41 0.23
38.91 0.22
38.91 0.22
38.21 0.25*
38.91 0.23
38.14 0.27*
38.91 0.25
38.84 0.29
38.81 0.23
38.91 0.23
38.90 0.25
38.01 0.29**
38.10 0.28*
38.71 0.26
38.84 0.19
38.91 0.22
38.91 0.22
38.20 0.28*
38.81 0.23
38.81 0.22
39.00 0.25
38.17 0.21*
38.91 0.23
38.94 0.19
39.20 0.24
37.27 0.18***
39.00 0.28
38.91 0.15
38.34 0.19**
38.81 0.15
38.95 0.28
38.84 0.23
38.51 0.16*
38.91 0.26
38.44 0.23*
38.91 0.23
38.90 0.25
38.91 0.22
38.91 0.22
38.91 0.26
38.43 0.24*
38.42 0.25*
38.64 0.22
38.81 0.23
38.90 0.22
38.81 0.23
38.47 0.20*
38.81 0.22
38.81 0.25
38.84 0.26
38.39 0.29*
38.91 0.22
38.91 0.23
39.17 0.20
37.16 0.19***
39.06 0.23
38.91 0.22
38.93 0.19
38.90 0.23
39.11 0.12
38.91 0.23
38.86 0.19
39.15 0.28
38.87 0.21
38.91 0.26
38.91 0.29
39.05 0.25
38.94 0.23
38.91 0.28
38.88 0.21
38.91 0.22
38.91 0.22
38.91 0.23
39.11 0.25
38.91 0.22
38.91 0.22
38.91 0.23
38.91 0.28
38.91 0.22
38.89 0.21
38.94 0.26
38.91 0.29
2a
2b
2c
3a
3b
3c
4a
4b
4c
4d
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
Significant at: *P 6 0.05, **P 6 0.01, ***P 6 0.001.

A. A. M. Eissa et al. / Bioorg. Med. Chem. 17 (2009) 5059–5070
5063
(38.10, 38.14, 38.17, 38.20, 38.21 °C), respectively compared to
39.24 °C in the control group.
2.3. Molecular modeling and docking studies
To pre-asses the anti-inflammatory behavior of our benzopy-
ranone derivatives 1–6 on a structural basis,, automated docking
studies were carried out using MOLSOFT ICM 3.4-8C program,36 the
scoring functions and hydrogen bonds formed with the surround-
ing amino acids are used to predict their binding modes, their
binding affinities and orientation of these compounds at the active
site of the cyclooxygenase II enzyme. The protein–ligand complex
was constructed based on the X-ray structure (PDB entry 4cox)
cyclooxygenase II with its bound inhibitor indomethacin.22 The
scoring functions of the compounds were calculated from mini-
mized ligand protein complexes.
In order to compare the binding affinity of the newly synthe-
sized benzopyranone analogues, we docked compounds 1–6 into
the empty binding site of cyclooxygenase II (4COX), with its bound
inhibitor indomethacin, Figures 1–6 show the docking solutions
with the highest predicted binding affinity for cyclooxygenase II.
Figure 1 shows orientation of indomethacin, Figure 1b, while Fig-
ures 2–7 show orientations of compounds 1, 3c, 4b, 4i, 6c, 6e,
respectively.
Figure 2. Binding mode of compound 1 into the binding site of cycloxygenase II, it
has ICM score ꢀ66.01, and form 4 hydrogen bonds shown as white dotted lines
(Table 4), showing two hydrogen bonds between O of 2-oxo of benzopyranone
moiety withTyr-385, and Ser-530 of distances 2.15 A and 1.62 A, respectively, and
another bond between O-1 and Ser-530 of distance 2.71 A and between H of OH-7
and O of Met-522 of distance 1.66 A.
As shown from Tables 1–4 and Figures 1–7 the following results
can be drawn: indomethacin (the original ligand) reveals ICM score
of ꢀ88.93 and form four hydrogen bonds between carboxylic acid
moiety and Arg-120, and another two hydrogen bonds between
carbonyl oxygen with Ser-530, and another bond between OH of
carboxylic acid with Tyr-355 (Table 4, Fig. 1b). Compound 1 exhib-
its relatively weak binding affinity with ICM score of ꢀ66.01 but
form two hydrogen bonds between 2-oxo of benzopyranone with
Tyr-385, and Ser-530, and another bond between O-1 of benzopy-
ran-2-one with Ser-530, and between OH-7 with Met-522 (Table 4,
Fig. 2). Hydrazones 2a–b, and imine 2c possess ICM scores of
ranges from ꢀ69.70 to ꢀ85.87, and the most biologically active
anti-inflammatory is compound 2b with lowest ICM score
ꢀ85.87 and form two hydrogen bonds between OH-7 with 2-NH
of Arg-120, and O of 20-nitro phenyl group and Gly-526 (Table 4).
Chalcones 3a–c have ICM scores of ranges from ꢀ78.09 to
ꢀ86.62, and the most biologically active one is compound 3c with
Figure 3. Binding mode of compound 3c into the binding site of cycloxygenase II, it
has ICM score ꢀ80.29, and form 2 hydrogen bonds shown as white dotted lines
(Table 4), showing one hydrogen bond between O of 2-oxo of benzopyranone
moiety with S-530 and Tyr-385 of distances of 1.43 A, 2.04 A, respectively.
Figure 1. Binding mode of the original ligand Indomethacin (Imn) into its binding
site of cycloxygenase II, it has ICM score ꢀ88.93, and form 6 hydrogen bonds shown
as white dotted lines (Table 4), showing two hydrogen bonds between O (COOH), O
of (COOH) with NH-1 of Arg-120 distance 1.60 A and 2.63 A, respectively, and
another two hydrogen bonds between O (COOH), O of (COOH) with NH-2 of Arg-120
distance 2.13 A and 2.43 A, another two hydrogen bonds between O of oxo-2 and
Ser-530 of distance 1.94 A, and between O of (COOH) and Tyr-355 distance 2.43 A.
Figure 4. Binding mode of compound 4b into the binding site of cycloxygenase II, it
has ICM score ꢀ97.27 A, and form 2 hydrogen bonds shown as white dotted lines
(Table 4), showing two hydrogen bonds between O of OH-7 and H of OH-7 with Tyr-
355 of distances of 2.23 A, and 2.76 A, respectively.

5064
A. A. M. Eissa et al. / Bioorg. Med. Chem. 17 (2009) 5059–5070
Figure 7. Binding mode of compound 6e into the binding site of cycloxygenase II, it
has ICM score ꢀ79.70 A, and form 10 hydrogen bonds shown as white dotted lines
(Table 4), showing one hydrogen bond between O of 2-oxo of benzopyranone
moiety with S-530 of distance 2.14 A, and another three bonds of NH of imino group
, and two of 3-cyano group with S-353 of distances 2.70 A, 1.95 A, and 1.90 A,
respectively, and two bonds of NH of dihydropyridine ring , and NH of imino group
with Val-349 of distances 2.80 A, and 1.41 A, respectively, and two bonds of N of 4-
(3-pyridinyl) with Arg-120 of distances 1.61 A, and 2.45 A, and finally, bond with H
of OH-7with Leu-352 of distance 2.66 A.
Figure 5. Binding mode of compound 4i into the binding site of cycloxygenase II, it
has ICM score ꢀ105.59 A, and form 10 hydrogen bonds shown as white dotted lines
(Table 4), showing one hydrogen bonds between O of 2-oxo of benzopyranone
moiety with S-530 of distance 1.03 A, and two bonds of O-1 with S-530 of distances
2.77 A, and 2.29 A, and another one between N of pyrazole ring with Ser-530 of
distance 2.49 A, and two hydrogen bonds between O of OH-7 with Tyr-385, and Tyr-
348 of distances 1.77 A, and 2.16 A, respectively, and another two hydrogen bonds
between H of OH-7 with Tyr-385, and Tyr-348 of distances1.39 A, and 2.12 A,
respectively, and between O of NO2 with Val-523, and E-524 of distances 1.58 A,
and 2.47 A, respectively.
idine-3-carbonitile 5a–f have ICM scores of ranges from ꢀ78.95 to
ꢀ88.96, but the most biologically active one is compound 5b with
ICM score ꢀ78.98. Finally, 2-imino-dihydropyridine-3-carbonitile
6a–e have ICM scores of ranges from ꢀ78.08 to ꢀ89.67, the biolog-
ically activity ranges from compounds 6c, 6d, 6e, 6a where ICM
scores are ꢀ84.50, ꢀ86.96, ꢀ79.70, and ꢀ89.67, where 6c is the
most active one, it forms three hydrogen bonds, two of them be-
tween 3-cyano moiety with Ser-530, and Tyr-385, and another
bond between 20-OH with Met-522 (Table 4, Fig. 6).
3. Conclusions
In our study we focused on substitution in position 8 of 7-hy-
droxy-2H-1-benzopyran-2-one moiety. Reaction of 8-acetyl-7-hy-
droxy derivatives 1 with hydrazines, or phenylene diamine to
give hydrazone 2a,b and imine 2c, respectively, only 2a retains
anti-inflammatory activity, while 2b reveals antipyretic activity.
Reaction of 1 with different aldehydes in alcoholic potassium
hydroxide to give chalcones 3a–c, all the chalcones retain anti-
inflammatory activity in the order of 3b > 3c > 3a, and 3c reveals
also analgesic and antipyretic effects. Cyclization of chalcones
3a–c through reaction with different hydrazine derivatives to give
substituted pyrazoles 4a–i retains the anti-inflammatory effect of
4b, 4d, 4e, 4f, 4i , also, compounds 4b, 4i possess analgesic, and
antipyretic activities. Reaction of 1 with ethyl cyanoacetate, and
ammonium acetate with different aldehydes to afford the substi-
tuted 2-oxo-dihydropyridine-3-carbonitrile derivatives 5a–f, abol-
ish the anti-inflammatory activity except compound 5b which
retain the activity, and compound 5e which possesses analgesic
and antipyretic effects. Finally the reaction of 1 with malononitrile,
and ammonium acetate with different aldehydes to afford the
substituted 2-imino-dihydropyridine-3-carbonitrile derivatives
6a–e, retain the anti-inflammatory activity of all members of the
series except compound 6b Shows slight activity, and compound
6c possesses antipyretic effect.
Figure 6. Binding mode of compound 6c into the binding site of cycloxygenase II, it
has ICM score ꢀ84.50 A, and form 3 hydrogen bonds shown as white dotted lines
(Table 4), showing two hydrogen bonds between N of 3-cyano moiety with Tyr-385,
and S-530 of distance 1.68 A, and 2.00 A, respectively, and another bond of H of OH
of 4-(20-hydroxy) phenyl group with Met-522 of distances 1.55 A.
ICM score ꢀ82.21 which form two hydrogen bonds between 2-oxo
moiety with Ser-530, and Tyr-385 (Table 4, Fig. 3). Substituted pyr-
azoles 4a–i possess ICM scores of ranges from ꢀ84.58 to ꢀ105.59,
where compounds4i, 4h, 4b with ICM score ꢀ105.59, ꢀ100.02 and
ꢀ97.27, respectively, where they have significant biological activi-
ties, compound 4i is the most active one in the series, it form ten
hydrogen bonds, two bonds with O-1 of benzopyranone moiety
with Ser-530, another one between 2-oxo and Ser-530, between
N of pyrazole ring and Ser-530, four hydrogen bonds between
OH-7 and Tyr-348, and Tyr-385, and two bonds between O of nitro
group and Val-523, and E-524 (Table 4, Fig. 5). 2-Oxo-dihydropyr-
The active compounds 1, 4i, 6a–e interact with Ser-530, and
Tyr-385 which are the amino acids involved in the mechanism of
cyclooxygenase II inhibition.34

A. A. M. Eissa et al. / Bioorg. Med. Chem. 17 (2009) 5059–5070
5065
Table 4
ICM Scores of indomethacin, the compounds, and hydrogen bonds formed with amino acid residues and their lengths
Compounds
ICM scores
No. of hydrogen bonds
6
Involved group of amino acid
Atom of ligand involved
Length of hydrogen bond (A)
Indomethacin
ꢀ88.93
Y355ꢁ ꢁ ꢁHH
S530ꢁ ꢁ ꢁHG
R120ꢁ ꢁ ꢁNH
R120ꢁ ꢁ ꢁNH
R120ꢁ ꢁ ꢁNH
R120ꢁ ꢁ ꢁNH
Y385ꢁ ꢁ ꢁHH
S530ꢁ ꢁ ꢁHG
S530ꢁ ꢁ ꢁHG
M522ꢁ ꢁ ꢁO
V349ꢁ ꢁ ꢁO
OH of COOH
CO of benzoyl
CO of COOH
OH of COOH
CO of COOH
OH of COOH
2-Oxo
2.43
1.94
1.60
2.63
1.33
2.43
2.15
1.62
2.71
1.66
2.15
2.53
1.37
2.60
2.59
1.97
1.45
2.76
1.64
2.76
2.05
1.43
2.04
2.21
2.76
2.23
2.75
2.47
1.91
2.43
1.31
2.19
—
1
ꢀ66.01
4
2-Oxo
–O–
H of 7-OH
H of 7-OH
O of 7-OH
O of 7-OH
O of 7-OH
2-Oxo
2a
2b
ꢀ79.70
ꢀ85.87
1
3
R120ꢁ ꢁ ꢁ(NH)
R120ꢁ ꢁ ꢁH12
G526ꢁ ꢁ ꢁNH
S530ꢁ ꢁ ꢁHG
M522ꢁ ꢁ ꢁO
M522ꢁ ꢁ ꢁO
Y355ꢁ ꢁ ꢁHH11
R120ꢁ ꢁ ꢁNH
R120ꢁ ꢁ ꢁNH
V349ꢁ ꢁ ꢁO
2c
3a
ꢀ69.95
ꢀ86.62
3
3
H of NH2
H of NH2
O of 7-OH
O of 7-OH
O of 7-OH
H of 7-OH
2-Oxo
3b
3c
ꢀ78.09
ꢀ80.29
1
2
S530ꢁ ꢁ ꢁHG
Y38ꢁ ꢁ ꢁHH
2-Oxo
2-Oxo
4a
4b
ꢀ86.92
ꢀ97.27
1
2
S530ꢁ ꢁ ꢁHG
Y355ꢁ ꢁ ꢁHH
Y355ꢁ ꢁ ꢁOB
R120ꢁ ꢁ ꢁHH 11
Y355ꢁ ꢁ ꢁHH
Y355ꢁ ꢁ ꢁOH
L531ꢁ ꢁ ꢁNH
L531ꢁ ꢁ ꢁNH
S530ꢁ ꢁ ꢁHG
—
O of 7-OH
H of 7-OH
MO6
O of 7-OH
H of 7-OH
O of nitro group
O of nitro group
2-Oxo
—
MN1
MO4
MO5
4c
ꢀ100.17
5
4d
4e
4f
ꢀ84.58
ꢀ95.01
ꢀ95.59
1
0
4
S530ꢁ ꢁ ꢁHG
S530ꢁ ꢁ ꢁHG
S530ꢁ ꢁ ꢁHG
L531ꢁ ꢁ ꢁNH
Y355ꢁ ꢁ ꢁHH
Y355ꢁ ꢁ ꢁHH
Y355ꢁ ꢁ ꢁOH
V523ꢁ ꢁ ꢁNH
Y348ꢁ ꢁ ꢁHH
Y348ꢁ ꢁ ꢁOH
Y385ꢁ ꢁ ꢁHH
Y385ꢁ ꢁ ꢁOH
E524ꢁ ꢁ ꢁNH
S530ꢁ ꢁ ꢁNH
S530ꢁ ꢁ ꢁHG
S530ꢁ ꢁ ꢁNH
S530ꢁ ꢁ ꢁHG
S530ꢁ ꢁ ꢁHG
Y385ꢁ ꢁ ꢁOH
Y348ꢁ ꢁ ꢁOH
Y355ꢁ ꢁ ꢁHH
Y355ꢁ ꢁ ꢁOH
S530ꢁ ꢁ ꢁHG
Y355ꢁ ꢁ ꢁHH
S530ꢁ ꢁ ꢁHG
M522ꢁ ꢁ ꢁO
V523ꢁ ꢁ ꢁO
1.84
2.11
2.62
2.66
2.69
2.61
2.06
1.58
2.16
2.12
1.77
1.39
2.47
1.03
2.49
2.77
2.29
2.49
1.39
2.12
2.17
1.66
1.50
1.73
1.97
1.56
2.80
1.60
2.27
1.28
1.77
1.36
2.08
2.80
2.65
1.44
2.36
2.11
1.70
2.14
1.35
(continued on next page)
MO5
4g
4h
ꢀ89.67
1
2
O of 7-OH
O of 7-OH
H of 7-OH
O of nitro group
O of 7-OH
H of 7-OH
O of 7-OH
H of 7-OH
O of nitro group
2-Oxo
ꢀ100.02
4i
ꢀ105.59
10
N of pyrazole
–O–
–O–
5a
5b
5c
ꢀ88.95
ꢀ78.98
ꢀ85.23
3
3
4
N of 3-cyano group
H of 7-OH
H of 7-OH
H of 7-OH
O of 7-OH
N of 3-cyano group
N of 3-cyano group
N of 3-cyano group
H of 4 (2’-hydroxy phenyl)
H of 4 (20-hydroxy phenyl)
O of 7-OH
N of 3-cyano
H of 7-OH
O of 7-OH
H of 7-OH
N of 3-cyano
O of 20-methoxy
O of 60-methoxy
N of 3-cyano
N of pyridine
N of 3-cyano
O of 7-OH
N of 3-cyano
H of 7-OH
5d
5e
ꢀ86.58
ꢀ88.96
3
5
Y355ꢁ ꢁ ꢁHH
S530ꢁ ꢁ ꢁHG
Y355ꢁ ꢁ ꢁOH
Y355ꢁ ꢁ ꢁHH
Y355ꢁ ꢁ ꢁOH
S530ꢁ ꢁ ꢁHG
S530ꢁ ꢁ ꢁHG
A527ꢁ ꢁ ꢁNH
Y385ꢁ ꢁ ꢁHH
W387ꢁ ꢁ ꢁHH
S530ꢁ ꢁ ꢁHG
Y355ꢁ ꢁ ꢁHH
Y355ꢁ ꢁ ꢁHG
S530ꢁ ꢁ ꢁOH
5f
ꢀ79.41
ꢀ89.67
3
3
6a

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A. A. M. Eissa et al. / Bioorg. Med. Chem. 17 (2009) 5059–5070
Table 4 (continued)
Compounds
6b
ICM scores
No. of hydrogen bonds
Involved group of amino acid
Atom of ligand involved
Length of hydrogen bond (A)
ꢀ78.08
3
Y385ꢁ ꢁ ꢁHH
S530ꢁ ꢁ ꢁHG
S530ꢁ ꢁ ꢁHG
Y385ꢁ ꢁ ꢁHH
S530ꢁ ꢁ ꢁHG
M522ꢁ ꢁ ꢁO
S530ꢁ ꢁ ꢁHG
Y355ꢁ ꢁ ꢁHH
Y355ꢁ ꢁ ꢁOH
R120ꢁ ꢁ ꢁNH
R120ꢁ ꢁ ꢁNH
S353ꢁ ꢁ ꢁNH
S353ꢁ ꢁ ꢁHG
S353ꢁ ꢁ ꢁOG
S530ꢁ ꢁ ꢁHG
V349ꢁ ꢁ ꢁO
N of 3-cyano
NH of imino group
N of 3-cyano
N of 3-cyano
N of 3-cyano
H of 4 (20-hydroxy phenyl)
N of 3-cyano
O of 7-OH
H of 7-OH
N of 4-(3-pyridyl)
N of 4-(3-pyridyl)
N of 3-cyano
N of 3-cyano
NH of imino group
2-Oxo
NH of dihydropyridine
NH of imino group
NH of imino group
H of 7-OH
1.88
1.70
2.57
1.68
2.00
1.55
2.30
1.60
1.29
1.61
2.45
1.95
1.90
2.70
2.14
2.80
1.41
2.49
2.66
6c
6d
6e
ꢀ84.50
ꢀ86.96
ꢀ79.70
3
3
10
V349ꢁ ꢁ ꢁO
S530ꢁ ꢁ ꢁO
L352ꢁ ꢁ ꢁO
The synthesis of the pyrazole 4 containing new compounds
proved a successful hit, since most of them showed anti-inflamma-
tory, analgesic or antipyretic activities, also, concerning the
compounds containing the 3-cyano-dihydropyridine ring; the 2-
imino derivatives were more successful than the 2-oxo one.
4.2.2.2. 8-(1-(2,4-Dinitrophenylazo)
ethyl)-7-hydroxy-4-phe-
nyl-2H-1-benzopyran-2-one 2b. Yield 83%; mp 203–204 °C. IR:
3400–3300 (br) (OH, NH), 2900 (CH3), 1720 (C@O), 1620, 1590,
1550 (NH, C@N, C@C), 1515, 1340 (NO2). cmꢀ1 1H NMR (DMSO-
.
d6) d: 2.49 (s, 3H, CH3), 6.25 (s, 1H, C3–H), 6.86–8.07 (m, 10H
aromatic), 8.90 (s, 1H, NH exchangeable), 9.59 (s, 1H, OH exchange-
able). MS (m/z, %): 460 (M+, 68.2), Anal. Calcd for C23H16N4O7: C,
60.00; H, 3.50; N, 12.17. Found: C, 60.20; H, 3.60; N, 12.10.
4. Experimental
4.1. General
4.2.2.3. 8-(1-(2-Aminophenylimino) ethyl)-7-hydroxy-4-phenyl-
2H-1-benzopyran-2-one 2c. ortho-Phenylene diamine (0.011 mol)
was added to a solution of 1 (0.01 mol) in absolute ethanol (30 ml).
The reaction wasrefluxed for 16 h, and left overnightat roomtemper-
ature to separate. The precipitated product was collected, dried and
recrystallized from ethanol/chloroform.
Melting points were determined on Gallenkamp and Kofler
melting point apparatus and are uncorrected. Elemental analyses
(C, H, N) were performed by Micro Analytical Center, Faculty of Sci-
ence, Cairo University, the values were found to be within 0.4% of
the theoretical ones unless otherwise indicated. Infrared spectra
were recorded on Shimadzu IR 435 Spectrophotometer or on a
Genesis II FTIR TM, Mattson, 5225, Verona Road, Madison wi.
53711 USA, using KBr discs. 1H NMR spectra were scanned on Var-
ian 360 MHz and 90 MHz spectrometers (chemical shifts are given
in part per million (ppm) downfield from TMS). Mass spectra were
made on a Finnigan Mat 212-spectrometer (EI. 120 eV, R 1000).
Analytical thin-layer chromatography (TLC) was performed on pre-
coated silica gel plates 60-F-254 (Merck; 0.25 mm), developing
with chloroform.
Yield 73%; mp above 360 °C. IR: 3450, 3350, 3325 (OH, NH2),
2900 (br) (CH3), 1722 (C@O), 1610, 1591 (NH2, C@N,C@C) cmꢀ1
.
1H NMR (DMSO-d6) d: 2.51 (s, 3H, CH3), 6.24 (s, 1H, C3–H), 6.80–
7.80 (m, 11H aromatic), 8.08 (s, 2H, NH2 exchangeable), 9.59 (s,
1H, OH exchangeable). MS (m/z): 370 (M+, 100), Anal. Calcd for
C23H18N2O3: C, 74.58; H, 4.89; N, 7.56. Found: C, 74.50; H, 4.79;
N, 7.77.
4.2.3. General procedure for synthesis of 8-(3-(4-
Aryl)acetyloyl)-7-hydroxy-4-phenyl-2H-1-benzopyran-2-one
3a–c
4.2. Chemistry
A 30 ml ethanolic solution of the appropriate aromatic aldehyde
(0.01 mol) was added with continuous stirring and cooling to a
solution of 8-acyl-7-hydroxy-4-phenyl-2H-1-benzopyran-2-one 1
(2.8 g, 0.01 mol) in 2.5% solution of sodium hydroxide. The reaction
mixture was stirred at room temperature for 3 h and left overnight.
It was then poured onto crushed ice; the separated precipitate was
filtered, left to dry and recrystallized from ethanol/chloroform 9:1.
4.2.1. 8-Acetyl-7-hydroxy-4-phenyl-2H-1-benzopyran-2-one 1
Compound 1 was previously reported.38
4.2.2. General procedure for synthesis of 8-(1-(substituted
phenylazo) ethyl)-7-hydroxy-4-phenyl-2H-1-benzopyran-2-one
2a,b
To a solution of 1 (0.01 mol) in absolute ethanol (30 ml), phenyl
hydrazine, or 2,4-dinitrophenyl hydrazine (0.011 mol) was added.
The reaction was left to reflux for 16 h, and then left overnight at
room temperature. The precipitated product was filtered, left to
dry and crystallized from ethanol/chloroform 9:1.
4.2.3.1.
8-(3-(4-Anisoyl)acetyloyl)-7-hydroxy-4-phenyl-2H-1-
benzopyran-2-one 3a. Yield 75%; mp 134–135 °C. IR: 3329,
3276 (OH), 2895, 2836 (CH3), 1729, 1624 (C@O(s)), 1591, 1511
(C@C) cmꢀ1 1H NMR (DMSO-d6) d: 3.87 (s, 3H, OCH3), 6.18 (s,
.
1H, C3–H), 6.81–7.89 (m, 13H aromatic and CH@CH), 9.87 (s, 1H,
OH exchangeable). MS (m/z): 398 (M+, 67), Anal. Calcd for
C25H18O5: C, 75.37; H, 4.55. Found: C, 75.44; H, 4.34.
4.2.2.1. 8-(1-Phenylazo) ethyl)-7-hydroxy-4-phenyl-2H-1-benz-
opyran-2-one 2a. Yield 85%; mp 194–195 °C. IR: 3450, 3300 (OH,
NH), 1710 (C@O), 1600, 1540, 1500 (NH, C@N, C@C), 1515 cmꢀ1. 1H
NMR (CDCl3) d: 2.63 (s, 3H, CH3), 6.19 (s, 1H, C3–H), 6.86–7.74 (m,
12H aromatic), 8.10 (s, 1H, NH exchangeable), 9.80 (s, 1H, OH
exchangeable). MS (m/z): 370 (M+, 100), Anal. Calcd for C23H18N2O3:
C, 74.58; H, 4.89; N, 7.56. Found: C, 74.62; H, 4.98; N, 7.37.
4.2.3.2.
2H-1-benzopyran-2-one 3b. Yield 78%; mp 196–197 °C. IR:
3400 (OH), 1720, 1660 (C@O(s)), 1600, 1560 (C@C), 700 (Cl) cmꢀ1
1H NMR (DMSO-d6) d: 6.35 (s, 1H, C3–H), 7.09–7.61 (m, 13H,
8-(3-(4-Chlorophenyl)acetyloyl)-7-hydroxy-4-phenyl-
.

A. A. M. Eissa et al. / Bioorg. Med. Chem. 17 (2009) 5059–5070
5067
aromatic and CH@CH), 12.50 (s, 1H, OH exchangeable). MS (m/z):
402 (M+, 47), 404 (M++2, 27)), Anal. Calcd for C24H14Cl O4: C,
71.74; H, 3.51. Found: C, 71.9; H, 4.00.
4.2.4.5. 8-(5-p-Chlorophenyl-1-phenyl-4,5-dihydro-1H-pyrazol-
3-yl)-7-hydroxy-4-phenyl-2H-1-benzopyran-2-one 4e. Crystal-
lization solvent ethanol, yield 81%; mp 148–149 °C. IR: 3350
(OH), 1704 (C@O), 1598, 1493 (C@N, C@C), 690 (Cl) cmꢀ1 1H
.
NMR (CDCl3) d: 2.16, 2.60 (m, 2H, CH2 of pyrazole), 3.80
(m, 1H, CH of pyrazole), 6.17 (s, 1H, C3–H), 6.81–7.70 (m, 15H
aromatic), 12.40 (s, 1H, OH exchangeable). MS (m/z): 492 (M+,
47.9), 490 (M+ꢀ2, 100), 494 (M++2, 42.9). Anal. Calcd for
C30H21ClN2O3: C, 73.09; H, 4.29; N, 5.68. Found: C, 73.20; H,
4.50; N, 5.61.
4.2.3.3. 8-(3-(4-N,N-Dimethylaminophenyl)acetyloyl)-7-hydroxy-
4-phenyl-2H-1-benzopyran-2-one 3c. Yield 75%; mp 211–213 °C.
IR: 3319, 3275 (OH), 2863 (br) (CH3(s)), 1728, 1650 (C@O(s)), 1595,
1500 (C@C) cmꢀ1 1HNMR (CDCl3) d: 3.07 (s, 6H, 2CH3), 6.24 (s, 1H,
.
C3–H), 6.71–8.19 (m, 13H aromatic and CH@CH), 14.45 (s, 1H, OH
exchangeable). MS (m/z): 411 (M+, 41.5), Anal. Calcd for C26H21NO4:
C, 75.90; H, 5.14; N, 3.40. Found: C, 75.73; H, 4.56; N, 3.75.
4.2.4.6. 8-(5-p-Chlorophenyl-1-(2,4-dinitrophenyl)-4,5-dihydro-
1H-pyrazol-3-yl)-7-hydroxy-4-phenyl-2H-1-benzopyran-2-one
4f. Crystallization solvent acetic acid/water, yield 92%; mp
124–126 °C. IR: 3350 (OH), 1720 (C@O), 1620, 1590 (C@N,
4.2.4. General procedure for synthesis of 8-(5-aryl-1-substituted-
4,5-dihydro-1H-pyrazol-3-yl)-7-hydroxy-4-phenyl-2H-1-benzo-
pyran-2-one 4a–i
A solution of 3a–c (0.01 mol) in absolute ethanol (30 ml), was
heated, then the suitable hydrazine derivative (0.011 mol) was
added. The reaction mixture was heated for 16 h under reflux con-
ditions. It was left overnight at room temperature. The aggregated
precipitate was filtered, dried and recrystallized from suitable
solvent.
C@C), 1515, 1340 (NO2), 700 (Cl) cmꢀ1 1H NMR (DMSO-d6) d:
.
2.12, 2.52 (m, 2H, CH2 of pyrazole), 3.86 (m, 1H, CH of pyrazole),
6.19 (s, 1H, C3–H), 7.34–8.83 (m, 13H aromatic), 12.76 (s, 1H,
OH exchangeable). MS (m/z): 582.2 (M+, 37,8), 580.2 (M+ꢀ2,
100). Anal. Calcd for C30H19Cl N4O7: C, 61.81; H, 3.29; N, 9.61.
Found: C, 61.75; H, 3.53; N, 9.38.
4.2.4.7. 8-(5-p-N,N-Dimethylaminophenyl-4,5-dihydro-1H-pyra-
zol-3-yl)-7-hydroxy-4-phenyl-2H-1-benzopyran-2-one 4g. Crys-
tallization solvent ethanol/water, yield 85%; mp 140–142 °C.
IR: 3500–3200 (br) (OH, NH), 2950, 2900 (CH3(s)), 1720
4.2.4.1. 8-(5-p-Anisoyl-4,5-dihydro-1H-pyrazol-3-yl)-7-hydroxy-
4-phenyl-2H-1-benzopyran-2-one 4a. Crystallization solvent etha-
nol/water, yield 83%; mp 182–184 °C. IR: 3450–3350 (br) (OH, NH),
2900 (CH3), 1720 (C@O), 1600, 1570, 1520, 1500 (NH, C@N, C@C)
(C@O), 1615, 1570, 1530 (NH, C@N, C@C) cmꢀ1
.
1H NMR
cmꢀ1 1H NMR (DMSO-d6) d: 2.50, 2.67 (m, 2H, CH2 of pyrazole),
.
(DMSO-d6) d: 2.24, 2.51 (m, 2H, CH2 of pyrazole), 2.99 (s, 6H, 2CH3),
3.46 (m, 1H, CH of pyrazole), 6.15 (s, 1H, C3–H), 6.69–7.68 (m, 10H
aromatic), 8.49 (s, 1H, NH exchangeable), 15.00 (s, 1H, OH exchange-
able). MS (m/z): 425.25 (M+, 53.4), Anal. Calcd for C26H23N3O3: C,
73.40; H, 5.45; N, 9.88. Found: C, 73.88; H, 5.04; N, 9.71.
3.48 (m, 1H, CH of pyrazole), 3.68 (s, 3H, OCH3), 6.35 (s, 1H, C3–H),
6.58–7.80 (m, 10H aromatic), 9.60 (s, 1H, NH exchangeable), 10.90
(s, 1H, OH exchangeable). MS (m/z): 412 (M+, 100), Anal. Calcd for
C25H20N2O4: C, 72.80; H, 4.89; N, 6.79. Found: C, 72.58; H, 5.00; N,
6.80.
4.2.4.8. 8-(5-p-N,N-Dimethylaminophenyl-1-phenyl-4,5-dihydro-
1H-pyrazol-3-yl)-7-hydroxy-4-phenyl-2H-1-benzopyran-2-one
4h. Crystallization solvent ethanol, yield 98%; mp 145–147 °C. IR:
3300 (OH), 2900, 2825 (CH3(s)), 1720 (C@O), 1600, 1540, 1520 (C@N,
4.2.4.2. 8-(5-p-Anisoyl-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-
7-hydroxy-4-phenyl-2H-1-benzopyran-2-one 4b. Crystalliza-
tion solvent ethanol, yield 96%; mp 120–122 °C. IR: 3450 (OH),
2925, 2850 (CH3), 1720 (C@O), 1600, 1510 (C@N, C@C) cmꢀ1 1H
.
C@C) cmꢀ1 1H NMR (DMSO-d6) d: 2.49, 2.60 (m, 2H, CH2 of pyra-
.
NMR (DMSO-d6) d: 2.27, 2.49 (m, 2H, CH2 of pyrazole), 3.28 (m,
1H, CH of pyrazole), 3.87 (s, 3H, OCH3), 6.21 (s, 1H, C3–H), 6.83–
7.54 (m, 15H, aromatic), 15.29 (s, 1H, OH exchangeable). Anal.
Calcd for C31H24N2O4: C, 76.22; H, 4.95; N, 5.73. Found: C, 75.98;
H, 5.14; N, 5.71.
zole), 2.85 (s, 6H, 2CH3), 3.91 (m, 1H, CH of pyrazole), 6.20 (s, 1H,
C3–H), 6.64–7.53 (m, 15H aromatic), 12.10 (s, 1H, OH exchange-
able). Anal. Calcd for C32H27N3O3: C, 76.63; H, 5.43; N, 8.38. Found:
C, 76.64; H, 5.50; N, 8.59.
4.2.4.9. 8-(5-p-N,N-Dimethylaminophenyl-1-(2,4-dinitro-
phenyl)-4,5-dihydro-1H-pyrazol-3-yl)-7-hydroxy-4-phenyl-2H-
1-benzopyran-2-one 4i. Crystallization solvent dimethyl form-
amide/water, yield 98%; mp 197–199 °C. IR: 3300 (OH), 2900,
2850 (CH3(s)), 1720 (C@O), 1620, 1590 (C@N, C@C), 1510, 1330
4.2.4.3.
8-(5-p-Anisoyl-1-(2,4-dinitrophenyl)-4,5-dihydro-1H-
pyrazol-3-yl)-7-hydroxy-4-phenyl-2H-1-benzopyran-2-one
4c. Crystallization solvent dimethyl formamide/ethanol/water
6:2:2, yield 98%; mp 151–152 °C. IR: 3300 (OH), 2900, 2825
(CH3), 1720 (C@O), 1620, 1590 (C@N, C@C), 1510, 1340 (NO2)
(NO2) cmꢀ1 1H NMR (DMSO-d6) d: 2.12, 2.52 (m, 2H, CH2 of pyra-
.
cmꢀ1 1H NMR (DMSO-d6) d: 2.52, 2.74 (m, 2H, CH2 of pyrazole),
.
zole), 2.99 (s, 6H, 2CH3), 3.66 (m, 1H, CH of pyrazole), 6.18 (s, 1H,
C3–H), 6.65–7.57 (m, 13H aromatic), 11.60 (s, 1H, OH exchange-
able). Anal. Calcd for C32H25N5O7: C, 64.97; H, 4.26; N, 11.84.
Found: C, 65.14; H, 4.42; N, 10.99.
3.22 (m, 1H, CH of pyrazole), 3.79 (s, 3H, OCH3), 6.14 (s, 1H, C3–
H), 6.98–7.56 (m, 13H aromatic), 15.60 (s, 1H, OH exchangeable)..
MS (m/z): 578 (M+, 18.3), Anal. Calcd for C31H22N4O8: C, 64.36; H,
3.83; N, 9.68. Found: C, 64.45; H, 4.12; N, 9.82.
4.2.5. General procedure for synthesis of 4-(aryl)-6-(7-hydroxy-
2-oxo-4-phenyl-2H-1-benzopyran-8-yl)-2-oxo-1,2-
dihydropyridine-3-carbonitrile 5a–f
4.2.4.4. 8-(5-p-Chlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)-7-
hydroxy-4-phenyl-2H-1-benzopyran-2-one 4d. Crystallization
solvent ethanol/water, yield 53%; mp 159–160 °C. IR: 3399–3300
(br) (OH, NH), 1716 (C@O), 1603, 1489, 1480 (NH, C@N,C@C),
699 (Cl) cmꢀ1. 1H NMR (DMSO-d6) d: 2.34, 2.52 (m, 2H, CH2 of pyr-
azole), 3.81 (m, 1H, CH of pyrazole), 6.15 (s, 1H, C3–H), 7.46–7.54
(m, 10H aromatic), 9.63 (s, 1HNH exchangeable), 15.60 (s, 1H, OH
exchangeable). MS (m/z): 416 (M+, 36.8) and 418 (M++2, 24.9) Anal.
Calcd for C24H17ClN2O3: C, 69.15; H, 4.11; N, 6.72. Found: C, 69.00;
H, 3.49; N, 6.65.
To equimolar amounts of 8-acyl-7-hydroxy-4-phenyl-2H-1-
benzopyran-2-one
1
(2.8 g, 0.01 mol), ethyl cyanoacetate
(1.13 ml, 0.01 mol) and the chosen aldehyde (0.01 mol); ammo-
nium acetate (0.08 mol), was added in absolute ethanol (50 ml).
The mixture was stirred and refluxed for 8 h. After cooling, the so-
lid product that separated was gathered and crystallized from suit-
able solvent.

5068
A. A. M. Eissa et al. / Bioorg. Med. Chem. 17 (2009) 5059–5070
4.2.5.1. 4-(Anisoyl)-6-(7-hydroxy-2-oxo-4-phenyl-2H-1-benzo-
pyran-8-yl)-2-oxo-1,2-dihydropyridine-3-carbonitrile 5a. Crys-
tallization solvent ethanol, yield 75%; mp 320–321 °C. 1H NMR
(CDCl3) d: 3.84 (s, 3H, OCH3), 6.05, 6.20 (2s, 2H, C3–H, C50–H),
6.96–7.99 (m, 11H aromatic), 8.18 (s, 1H, NH exchangeable),
11.60 (s, 1H, OH exchangeable). MS (m/z): 462 (M+, 57.8) 461
(M+ꢀ1, 100). Anal. Calcd for C28H18N2O5: C, 72.72; H, 3.92; N,
6.06. Found: C, 72.41; H, 4.36; N, 5.92.
0.01 mol), ammonium acetate (0.08 mol), and the appropriate aro-
matic aldehyde (0.01 mol) in absolute ethanol (50 ml) were stirred
and refluxed for 8 h. The reaction mixture was cooled; the solid
precipitate that was formed was filtered and recrystallized from
the suitable solvent.
4.2.6.1. 4-(Anisoyl)-6-(7-hydroxy-2-oxo-4-phenyl-2H-1-benzo-
pyran-8-yl)-2-oxo-1,2-dihydropyridine-3-carbonitrile 6a. Crys-
tallization solvent acetic acid/water, yield 83%; mp 203–204 °C. IR:
3400, 3352, 2220 (OH, NH(s)), 2840 (CH3(s)), 2210 (CN), 1731
4.2.5.2. 4-(p-Chlorophenyl)-6-(7-hydroxy-2-oxo-4-phenyl-2H-1-
benzopyran-8-yl)-2-oxo-1,2-dihydropyridine-3-carbonitrile
5b. Crystallization solvent ethanol/chloroform 9:1, yield 78%; mp
above 360 °C. IR: 3471, 3345 (OH, NH), 2219 (CN), 1721, 1684
(C@O(s)), 1602, 1514, 1458 (NH, C@N, C@C), 702 (Cl) cmꢀ1. 1H NMR
(DMSO-d6) d: 6.15, 6.30 (s, 1H, C3–H, C50–H), 6.83–7.67 (m, 11H aro-
matic), 9.60 (s, 1H, NHexchangeable), 11.60 (s, 1H, OHexchangeable).
MS (m/z): 467.25 (M+, 5.0). Anal. Calcd for C27H15 Cl N2O4: C, 69.46; H,
3.24; N, 6.00. Found: C, 69.53; H, 3.37; N, 5.80.
(C@O), 1602, 1514, 1460 (NH, C@N, C@C) cmꢀ1 1H NMR (CDCL3)
.
d: 3.85 (s, 3H, OCH3), 6.19, 6.42 (2s, 2H, C3–H, C50–H), 7.12–7.64
(m, 11H aromatic), 8.00, 8.60 (2s, 2H, 2NH exchangeable), 12.70
(s, 1H, OH exchangeable). Anal. Calcd for C28H19N3O4: C, 72.88;
H, 4.15 N 9.11. Found: C, 72.49; H, 4.35; N, 8.89.
4.2.6.2. 4-(p-Chlorophenyl)-6-(7-hydroxy-2-oxo-4-phenyl-2H-
1-benzopyran-8-yl)-2-imino-1,2-dihydropyridine-3-carboni-
trile 6b. Crystallization solvent ethanol/chloroform, yield 75%;
mp 244–246 °C. IR: 3350, 3200 (OH, NH(s)), 2200 (CN), 1720
4.2.5.3. 4-(N,N-Dimethylaminophenyl)-6-(7-hydroxy-2-oxo-4-
phenyl-2H-1-benzopyran-8-yl)-2-oxo-1,2-dihydropyridine-3-
carbonitrile 5c. Crystallization solvent acetic acid/water, yield
86%; mp 275–276 °C. IR: 3350–3297 (br) (OH, NH(s)), 2909, 2866,
2828 (CH3(s)), 2206 (CN), 1704 (C@O(s)), 1611, 1567, 1522 (NH,
(C@O), 1615, 1560 (NH, C@N, C@C), 700 (Cl) cmꢀ1 1H NMR
.
(DMSO-d6) d: 6.17, 6.42 (2s, 2H, C3–H, C50–H), 7.31–7.66 (m, 11H
aromatic), 8.10, 8.79 (2s, 2H, 2NH exchangeable), 12.60 (s, 1H,
OH exchangeable) MS (m/z): 465.65 (M+, 80.27), 467.15 (M++1,
32.38). Anal. Calcd for C27H16 Cl N3O3: C, 69.61; H, 3.46; N, 9.02.
Found: C, 69.45; H, 3.59; N, 9.07.
C@N,C@C) cmꢀ1 1H NMR (DMSO-d6) d: 3.05 (2s, 6H, 2CH3), 6.30,
.
6.60 (2s, 2H, C3–H, C50–H), 6.76–8.07 (m, 11H aromatic), 9.60 (s,
1H, NH exchangeable), 12.30 (s, 1H, OH exchangeable). Anal. Calcd
for C29H21N3O4: C, 73.25; H, 4.45; N, 8.84. Found: C, 73.01; H, 4.46;
N, 8.80.
4.2.6.3. 4-(N,N-Dimethylaminophenyl)-6-(7-hydroxy-2-oxo-4-
phenyl-2H-1-benzopyran-8-yl)-2-imino-1,2-dihydropyridine-3-
carbonitrile 6c. Crystallization solvent ethanol/chloroform, yield
82%; mp 245–246 °C. IR: 3350, 3200 (OH, NH(s)), 2925 (CH3(s)),
4.2.5.4. 4-(2,4,6-Trimethoxyphenyl)-6-(7-hydroxy-2-oxo-4-phe-
nyl-2H-1-benzopyran-8-yl)-2-oxo-1,2-dihydropyridine-3-car-
bonitrile 5d. Crystallization solvent ethanol/chloroform, yield
85%; mp 265–266 °C. IR: 3437, 3305, 3189 (OH, NH(s)), 2940,
2840 (CH3(s)), 2215 (CN), 1714 (C@O(s)), 1602, 1459 (NH, C@N,
2200 (CN), 1720 (C@O), 1615, 1560, 1520 (NH, C@N, C@C) cmꢀ1
.
1H NMR (DMSO-d6) d: 3.09 (s, 6H, 2CH3), 6.44, 6.76 (2s, 2H, C3–
H, C50–H), 7.17–7.85 (m, 11H aromatic), 8.00, 8.30 (2s, 2H, 2NH
exchangeable), 10.80 (s, 1H, OH exchangeable). MS (m/z): 474.2
(M+, 0.67). Anal. Calcd for C29H22N4O3: C, 73.41; H, 4.67; N,
11.81. Found: C, 73.14; H, 4.89; N, 11.69.
C@C) cmꢀ1 1H NMR (DMSO-d6) d: 3.70, 3.81, 3.85 (3s, 9H,
.
3OCH3), 6.27, 6.36 (2s, 2H, C3–H, C50–H), 7.39–8.16 (m, 9H aro-
matic), 9.60 (s, 1H, NH exchangeable), 15.60 (s, 1H, OH exchange-
able). MS (m/z): 523.4 (M+, 36.31), Anal. Calcd for C30H22N2O7: C,
68.96; H, 4.24; N, 5.36. Found: C, 68.93; H, 4.75; N, 5.38.
4.2.6.4. 4-o-Hydroxyphenyl)-6-(7-hydroxy-2-oxo-4-phenyl-2H-
1-benzopyran-8-yl)-2-imino-1,2-dihydropyridine-3-carboni-
trile 6d. Crystallization solvent acetic acid/water, yield 79%; mp
202–203 °C. IR: 3311, 3204 (OH(s), NH(s)), 2204 (CN), 1711 (C@O),
4.2.5.5. 4-(o-Hydroxyphenyl)-6-(7-hydroxy-2-oxo-4-phenyl-2H-
1-benzopyran-8-yl)-2-oxo-1,2-dihydropyridine-3-carbonitrile
5e. Crystallization solvent acetic acid/water, yield 65%; mp above
360 °C. IR: 3431, 3318, 3277, 3210 (OH(s), NH(s)), 2210 (CN), 1705
1605, 1485 (NH, C@N, C@C) cmꢀ1 1H NMR (DMSO-d6) d6.17,
.
6.79 (2s, 2H, C3–H, C50–H), 7.40–8.00 (m, 11H aromatic), 8.20,
9.60 (2s, 2H, 2NH exchangeable), 11.60, 13.50 (2s, 2H, 2OH
exchangeable). MS (m/z): 446 (M+ꢀ1, 1.47). Anal. Calcd for
C27H17N3O4: C, 72.48; H, 3.83; N, 9.39. Found: C, 72.60; H, 4.29;
N, 9.60.
(C@O(s)), 1604, 1486 (NH, C@N, C@C) cmꢀ1 1H NMR (DMSO-d6)
.
d: 6.14, 6.19 (2s, 2H, C3–H, C50–H), 6.82–7.54 (m, 11H aromatic),
9.60 (s, 1H, NH exchangeable), 11.60 (s, 2H, 2OH exchangeable).
MS (m/z): 448 (M+, 100). Anal. Calcd for C27H16N2O5: C, 72.32; H,
3.60; N, 6.25. Found: C, 72.34; H, 3.66; N, 6.28.
4.2.6.5. 4-(3-Pyridyl)-6-(7-hydroxy-2-oxo-4-phenyl-2H-1-benzo-
pyran-8-yl)-2-imino-1,2-dihydropyridine-3-carbonitrile 6e. Crys-
tallization solvent ethanol/chloroform 9:1, yield 75%; mp 260–261 °C.
IR: 3350, 3200 (OH, NH(s)), 2200 (CN), 1720 (C@O), 1610, 1560, 1520
(NH, C@N, C@C) cmꢀ1. 1H NMR (DMSO-d6) d: 6.18, 6.79 (2s, 2H, C3–
H, C50–H), 6.97–8.05 (m, 11H aromatic), 8.20, 8.70 (2s, 2H, 2NH
exchangeable), 11.60 (s, 1H, OH exchangeable) Anal. Calcd for
C26H16N4O3, H2O: C, 69.33; H, 4.03; N, 12.44. Found: C, 68.96; H,
3.73; N, 12.36.
4.2.5.6. 4-(3-Pyridyl)-6-(7-hydroxy-2-oxo-4-phenyl-2H-1-benz-
opyran-8-yl)-2-oxo-1,2-dihydropyridine-3-carbonitrile
5f. Crystallization solvent ethanol/chloroform, yield 55%; mp
228–229 °C. IR: 3333, 3285, 3195 (OH, NH(s)), 2210 (CN), 1721
(C@O(s)), 1606, 1564 (NH, C@N, C@C) cmꢀ1 1H NMR (DMSO-d6)
.
d: 6.21, 6.46 (2s, 2H, C3–H, C50–H), 6.67–8.2 (m, 11H aromatic),
9.70 (s, 1H, NH exchangeable), 11.60 (s, 1H, OH exchangeable).
Anal. Calcd for C26H15N3O4: C, 72.05; H, 3.49; N, 9.69. Found: C,
71.50; H, 3.50; N, 9.61.
4.3. Pharmacological screening
4.2.6. General procedure for synthesis of4-(Aryl)-6-(7-hydroxy-
2-oxo-4-phenyl-2H-1-benzopyran-8-yl)-2-imino-1,2-
dihydropyridine-3-carbonitrile 6a–e
All the tested compounds were dissolved in dimethylformam-
ide (DMF) before subcutaneous injection in the tested animals.
The control group was similarly injected with DMF. The LD50 of
the newly synthesized compounds was determined.
A
mixture comprised of 8-acyl-7-hydroxy-4-phenyl-2H-1-
benzopyran-2-one (2.8 g, 0.01 mol), malononitrile (0.66 ml,
3

A. A. M. Eissa et al. / Bioorg. Med. Chem. 17 (2009) 5059–5070
5069
4.3.1. Acute toxicity and lethality (LD50) test
perature of each rat by introducing 1.5 cm of digital thermometer
in rectum, pyrexia was induced by subcutaneous injection of 1 ml
of 15% brewer’s yeast suspension in saline solution. After 18 h of
yeast injection, rats which showed a rise in temperature of at least
0.6 °C were taken for the study. One hundred and forty five, hyper-
thermic rats were divided into 29 equal groups. Rats of the 1st
group were subcutaneously injected with the vehicle and left as
control while the 2nd one was orally given paracetamol in a dose
of 100 mg kgꢀ1 (standard). The rest of the groups were injected
The acute toxicity and lethality (LD50) of the new compounds
were estimated in albino mice (25–30 g)57 In a preliminary test,
animals in groups of three, received one of 10, 50, 150, 300, or
600 mg kgꢀ1 of the tested compounds by subcutaneous injection.
Animals were observed for 24 h for signs of toxicity and number
of deaths. From the results of the first test, 150, 300, 450 and
600 mg kgꢀ1 doses of the tested compounds were administered
to fresh groups of mice. Control animals received the vehicle and
were kept under the same conditions without any treatments.
Signs of toxicity and number of deaths per dose in 24 h were re-
corded and the LD50 was calculated as the geometric mean of the
dose that resulted in 100% mortality and that which caused no
lethality at all.
with the tested compounds in doses equal to 1/10 of their LD50
.
Rectal temperature of each rat was then recorded at 1 h interval
after administration for 4 h. The antipyretic efficacy was decided
on the basis of the difference in the mean temperature between
the control and the tested compounds.
4.3.5. Statistical analysis
4.3.2. Anti-inflammatory activity
All values were expressed as mean S.E.M. Statistical signifi-
cance was determined by comparing the values of the test com-
pounds and the standards with those obtained in the presence of
the vehicle using Student’s t-test.58,59
The anti-inflammatory effect of the newly synthesized com-
pounds was evaluated in correspondence to the carrageenan-in-
duced paw edema method.54 Twenty nine groups of animals
each consisting of five rats weighing 180–200 g were selected.
The 1st group was treated with the vehicle and left as control while
the 2nd one was given celecoxib by subcutaneous injection in a
dose of 20 mg kgꢀ1 (standard). Other groups were subcutaneously
injected with the tested compounds in doses equal to 1/10 their
LD50. After 30 min, acute inflammation was induced by subplantar
injection of 0.1 ml of 1% suspension of carrageenan in the right
hind paw of all rats. Paw size was measured by wrapping a piece
of cotton thread round the paw and measuring the circumference
with a meter rule. Measurement was carried out immediately be-
fore and 3 h following carrageenan injection. Percent inhibition of
tested compounds and standard drug was calculated in compari-
son with vehicle control (100%). Carrageenan-induced hind paw
edema is the standard experimental model of acute inflammation.
Carrageenan is the phlogistic agent of choice for testing anti-
inflammatory drugs as it is not known to be antigenic and is devoid
of apparent systemic effects.
4.4. Drug modeling studies
All docking studies were performed using ‘Internal Coordinate
Mechanics (MOLSOFT ICM 3.4–8C)’. ICM docking is probably the most
accurate predictive tool of binding geometry today.36,60–62
4.4.1. Preparation of small molecule
A set of 7-hydroxy-2H-1-benzopyran-2-one analogues synthe-
sized to inhibit cyclooxygenase II was compiled by us earlier;
ChemDraw 3D structures were constructed using Chem 3D ultra
8.0 software [Molecular Modeling and Analysis; Cambridge Soft
Corporation, USA (2004)], and then they were energetically mini-
mized by using MOPAC (semi-empirical quantum mechanics), Jop
Type with 100 iterations and minimum RMS gradient of 0.01,
*
and saved as MDL MolFile ( .mol).
4.4.2. Generation of ligand and enzyme structures
The crystal structure of target protein cyclooxygenase (4cox) is
a murine COX-II complexed with indomethacin22 was retrieved
from the Protein Data Bank (http://www.rcsb.org/pdb/welcome.-
do). All bound waters ligands and cofactors were removed from
the protein. The amino acids of the binding site where defined
using data in pdbsum (http://www.ebi.ac.uk/thoronton-srv/dat-
abases/pdbsum/.
4.3.3. Analgesic activity
The analgesic validation of tested compounds was evaluated
using the acetic acid-induced writhing method55 Mice (25–30 g)
were assigned into 29 groups, each containing five animals. The
1st group was kept as a control (received the vehicle) while the
2nd one was subcutaneously injected with diclofenac sodium in
a dose of 5 mg kgꢀ1 (standard). The other groups were injected
with the tested compounds in doses equal to 1/10 of their LD50
.
Writhing was induced 30 min later, by intraperitoneal injection
of 0.1 ml of 0.6% acetic acid. Numbers of writhes (abdominal con-
tractions) in all animals were counted for 30 min, immediately
after acetic acid injection (0 time) and hourly after administration
for 4 h. Analgesic activity was expressed as the percentage protec-
tion against writhing produced by the new compounds allover the
experimental period comparing between control and those pre-
treated with the new compounds using the ratio:
4.4.3. Docking using MOLSOFT ICM 3.4-8C program
1- Convert our PDB file into an ICM object: This conversion
involves addition of hydrogen bonds, assignment of atoms
types, and charges from the residue templates.
2- To perform ICM small molecule docking:
a) Setup docking project:
1) Set project name:
% reduction
2) Setup the receptor:
3) Review and adjust binding site:
4) Make receptor maps:
Writhes mean at ð0Þ time ꢀ Writhes mean at ðtÞ time
¼
ꢂ 100
Writhes mean at ð0Þ time
The abdominal constriction response induced by acetic acid is a
sensitive procedure to establish peripherally acting analgesics. This
response is thought to involve local peritoneal receptors.
b) Start docking simulation:
3- Display the result:
ICM stochastic global optimization algorithm attempts to find
the global minimum of the energy function that include five grid
potentials describing interaction of the flexible ligand with the
receptor and internal conformational energy of the ligand, during
4.3.4. Antipyretic activity
The antipyretic activity of the newly synthesized compounds
was screened in male rats (180–200 g), in consistent to the
yeast-induced hyperpyrexia method.56 After measuring rectal tem-

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