New pyridone, thioxopyridine, pyrazolopyridine and pyridine derivatives that modulate inflammatory mediators in stimulated RAW 264.7 murine macrophage

Article abstract of DOI:10.1016/j.ejmech.2009.06.023

The reaction of 2-acetyl-5,6,7,8-tetrahydronaphthalene 1 with some aldehydes was conducted in the presence of ethyl cyanoacetate and ammonium acetate, yielded the cyanopyridones 2a-c, which react with phosphorous pentasulphide to afford the corresponding

Full text of DOI:10.1016/j.ejmech.2009.06.023

European Journal of Medicinal Chemistry 44 (2009) 4547–4556
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New pyridone, thioxopyridine, pyrazolopyridine and pyridine derivatives that
modulate inflammatory mediators in stimulated RAW 264.7 murine macrophage
,
b,
*
Nehal A. Hamdya , Amira M. Gamal-Eldeen
*
a Applied Organic Chemistry Department, National Research Centre, Dokki, Cairo, Egypt
b Cancer Biology Laboratory, Center of Excellence for Advanced Sciences, National Research Center, Dokki 12622, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The reaction of 2-acetyl-5,6,7,8-tetrahydronaphthalene 1 with some aldehydes was conducted in the
presence of ethyl cyanoacetate and ammonium acetate, yielded the cyanopyridones 2a–c, which react
with phosphorous pentasulphide to afford the corresponding thioxopyridine derivatives 3a–c, respec-
tively. Compounds 2a,b were converted to 2-chloropyridine derivatives 4a,b by heating with phospho-
rous oxychloride and phosphorous pentachloride, which were fused with hydrazine hydrate and benzyl
amine to afford the corresponding pyrazolopyridine 5a,b and cyanopyridine derivatives 6a,b respec-
tively. Compounds 2a,b also afforded 3-cyanopyridinyl oxy acetic acid ethyl ester 7a,b by reaction with
ethyl bromoacetate in dry acetone in the presence of anhydrous potassium carbonate, which upon
condensation with hydrazine hydrate gave the corresponding acid hydrazide 8a,b and with benzyl amine
gave the corresponding acetamide 9a,b. We investigated the effect of those new compounds on the
macrophage growth, macrophage binding affinity to fluorescein isothiocyanate-conjugated bacterial
lipoopolysaccharide (FITC-LPS), phagocytosis of FITC-zymosan, and radical scavenging affinity against
Received 21 December 2008
Received in revised form
6 June 2009
Accepted 20 June 2009
Available online 27 June 2009
Keywords:
Pyridine
Pyridone
Thioxopyridine
Pyrazolopyridine
Anti-inflammatory
Cycloxygenase-2
5-Lipoxygenase
ꢀ
OHꢀ, ROOꢀ, and Oꢁ2 , in addition to their influence of the inflammatory mediators [nitric oxide (NO), tumor
necrosis factor-
a (TNF-a), prostaglandin E-2 (PGE-2), cycloxygenase-2 (COX-2), and 5-lipoxygenase
(5-LO)] in LPS-stimulated macrophages. The findings revealed that the derivatives 2b, 3b, 5a, 7b, 9a and
9b can be recognized as promising multi-potent anti-inflammatory agents.
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
breakdown and are beneficial by strengthening and maintaining
the defense against infection [2].
Inflammation is a physiologic process in response to tissue
damage resulting from microbial pathogen infection, chemical
irritation, and/or wounding [1]. At the very early stage of inflam-
mation, neutrophils are the first cells to migrate to the inflamma-
tory sites under the regulation of molecules produced by rapidly
responding macrophages and mast cells prestationed in tissues
[2,3]. As the inflammation progresses, various types of leukocytes,
lymphocytes, and other inflammatory cells are activated and
attracted to the inflamed site by a signaling network involving
a great number of growth factors, cytokines, and chemokines [2,3].
All cells recruited to the inflammatory site contribute to tissue
There are also mechanisms to prevent inflammation response
from lasting too long [4]. A shift from antibacterial tissue damage to
tissue repair occurs, involving both proinflammatory and anti-
inflammatory molecules [4]. Prostaglandin E2 [5], transforming
growth factor-h [6], and reactive oxygen and nitrogen intermedi-
ates [3] are among those molecules with a dual role in both
promoting and suppressing inflammation. The resolution of
inflammation also requires a rapid programmed clearance of
inflammatory cells: neighboring macrophages, dendritic cells, and
backup phagocytes do this job by inducing apoptosis and con-
ducting phagocytosis [7–9].
The phagocytosis of apoptotic cells also promotes an anti-
inflammatory response, such as enhancing the production of anti-
inflammatory mediators [10–12]. However, if inflammation
resolution is dysregulated, cellular response changes to the pattern
of chronic inflammation. In chronic inflammation, the inflammatory
foci are dominated by lymphocytes, plasma cells, and macrophages
with varying morphology [1]. Macrophages and other inflammatory
cells generate a great amount of growth factors, cytokines, and
Abbreviations: COX-2, cycloxygenase-2; FITC, fluorescein isothiocyanate; 5-LO,
ꢀ
5-lipoxygenase; LPS, bacterial lipopolysaccharide; NO, nitric oxide; Oꢁ2 , superoxide
radicals; OHꢀ, hydroxyl radicals; ORAC, oxygen radical absorbance capacity; PGE-2,
prostaglandin E-2; ROOꢀ, peroxyl radicals; TNF-
a, tumor necrosis factor-a.
* Corresponding author. Tel.: þ20106053903; fax: þ20233370931.
E-mail addresses: drnehalhamdy63@hotmail.com (N.A. Hamdy), aeldeen7@
yahoo.com (A.M. Gamal-Eldeen).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.06.023

4548
N.A. Hamdy, A.M. Gamal-Eldeen / European Journal of Medicinal Chemistry 44 (2009) 4547–4556
reactive oxygen and nitrogen species that may cause DNA damage
[2]. If the macrophages are activated persistently, they may lead to
continuous tissue damage [13]. A microenvironment constituted by
all the above elements inhabits the sustained cell proliferation
induced by continued tissue damage, thus predisposes chronic
inflammation to neoplasia [14]. Epidemiologic studies support that
chronic inflammatory diseases are frequently associated with
increased risk of cancers [1,2,13], and that the development of
cancers from inflammation might be a process driven by inflam-
matory cells as well as a variety of mediators, including cytokines,
chemokines, and enzymes, which altogether establish an inflam-
matory microenvironment [2]. Consequently, finding new anti-
inflammatory agents represents a concrete strategy in fighting not
only different inflammatory diseases but also cancer.
Synthesis of the pyridine ring system and its derivatives occupy
an important place in the realm of synthetic organic chemistry, due
to their therapeutic and pharmacological properties [14–16]. They
have emerged as integral backbones of over 7000 existing drugs
[17,18]. The pyridine ring is also an integral part of anticancer and
anti-inflammatory agents [19,20]. Tetralins (tetrahydronaph-
thalene derivatives) are of increasing interest since many of these
compounds play a vital role in the biological activities, because of
their biological potentialities; for examples as potent agonists for
D2-type receptors [21], as a treatment of Alzheimer’s disease [22],
cardiovascular diseases [23], and as a preventer of dopamine-
induced cell death [24]. On the other hand, cyanopyridone and
cyanopyridine derivatives have promising antimicrobial [25] and
anticancer activities [26]. The interest in 3-cyano-2(1H)-pyridone
and their derivatives is due to their wide range of practical uses as
medicinal compounds [27]. Recently, new pyridine carbonitriles
were reported as anti-inflammatory agents [28], and pyrazolopyr-
idine derivatives have been recently reported as anti-tumor agents
[29]. In this work, we synthesized some new heterocyclic
compounds containing pyridone, thioxopyridine, halogenated
pyridine carbonitriles, pyrazolopyridine and pyridine derivatives
and we investigated their role in the modulation of various
inflammatory mediators.
acetate in n-butanol gave the corresponding cyanopyridones 2a–c
in one pot reaction. The structure of the isolated products was
confirmed on the basis of their elemental analysis and spectral data.
For example their IR spectrum exhibited absorption bands at 3420,
3424, 3306 respectively due to NH groups, 2219, 2219, 2217
respectively due to CN groups and 1638, 1630, 1643 respectively
due to C]O groups.
Their 1H NMR spectrum showed singlet signals (D2O
exchangeable) at
addition to an aromatic multiplet in the region
d
11.2, 11.2, 12.3 respectively due to NH protons in
6.7–8.1, whereas
d
their mass spectrum showed a peak corresponding to its molecular
ion at m/z 386.3, 368.2, 365.3 (Mþ) respectively. Heating a mixture
of the obtained cyanopyridone derivatives 2a–c with phosphorous
pentasulphide in dry pyridine afforded the corresponding thio-
xopyridine 3a–c. The structures of the latter compounds were
confirmed on the basis of their elemental analysis and spectral data.
For example their IR spectrum revealed absorption bands at 1202,
1209, 1200 respectively due to C]S groups and disappearance of
C]O absorption bands also their mass spectrum showed a peaks
corresponding to their molecular ion at m/z 402.3 (Mþ), 384.4 (Mþ),
380 (Mþ ꢁ 1) respectively (Scheme 1).
On the other hand, heating compounds 2a,b with phosphorous
pentachloride and phosphorous oxychloride afforded 2-chlor-
opyridines 4a,b, which upon fused with hydrazine hydrate afforded
pyrazolopyridine derivatives 5a,b (Scheme 2). The structures of the
latter compounds were confirmed on the basis of their elemental
analysis and spectral data. The mass spectrum showed the expec-
ted molecular ion peaks Mþ at m/z 400 (100%), 382 (100%)
respectively. The IR spectrum displaying bands at 3453, 3294, 3194
and 3453, 3294, 3194 for NH2, NH respectively, C]N at 1596, 1585
respectively also the IR showed the disappearance of band of CN.
Also when compounds 4a,b fused with benzyl amine afforded the
corresponding cyanopyridine derivatives 6a,b (Scheme 2).
The structure of the latter products were established on the basis of
the appearance of an NH absorption band in the region of 3360–
3431 cmꢁ1 and a band of CN function in the region of 2205,
2202 cmꢁ1 respectively in their IR spectra, whereas their 1H NMR
spectra revealed the appearance of a doublet signal due to –CH2 in
the region 4.7 and a signal (NH, D2O exchangeable) in the region
11.2 (cf. Experimental section).
2. Results and discussion
The required 2-acetyl-5,6,7,8-tetrahydronaphthalene
1
was
Moreover, treatment of compounds 2a,b with ethyl bromoace-
tate and anhydrous potassium carbonate in dry acetone afforded, 3-
Cyanopyridinyl oxy acetic acid ethyl esters 7a,b respectively
(Scheme 3), where their structures were confirmed by the mass
prepared following the literature method [30]. Cyano condensation
reaction of compound 1 with the appropriate aromatic aldehydes
and ethyl cyanoacetate in the presence of excess ammonium
Scheme 1.

N.A. Hamdy, A.M. Gamal-Eldeen / European Journal of Medicinal Chemistry 44 (2009) 4547–4556
4549
2a-b
POCL3
P2S5
Ar
CN
N
Cl
4a-b
NH2NH2
Benzyl amine
Ar
NH2
N
Ar
CN
N
H
N
N
N
H
Ph
5a-b
6a-b
4, 5, 6
Ar
OMe
OMe
a
b
Scheme 2.
spectrum containing a peak due to the corresponding molecular
ion m/z 472.28, 454.26 (Mþ) respectively. The IR spectrum showed
absorption bands at 1745, 1749 respectively due to C]O of ester
groups, also their 1H NMR spectrum showed the protons of ethyl
ester group.
appearance of three absorption bands due to NH2, NH functions in
addition to the carbonyl absorptions and CN bands. Also their 1H
NMR spectra revealed the appearance of a signal due (NH2, D2O
exchangeable) in the region 4.3, 4.27 respectively and in the region
9.4 for NH, while treatment of 7a,b with benzyl amine gave the
corresponding amides 9a,b. Their structure were supported by
their correct analytical data and IR spectrum which showed the
The reaction of 7a,b with hydrazine hydrate in refluxing ethanol
gave the hydrazides 8a,b. Their IR spectrum showed the
2a-b
Br-CH2COOC2H5
Anhydrous potassium carbonate
Ar
N
CN
O
COOEt
7a-b
Ar
Ar
NH2NH2
Benzyl amine
CONHCH2-Ph
CN
CN
O
N
O
N
CONHNH2
9a-b
8a-b
Ar
7, 8, 9
OMe
OMe
a
b
Scheme 3.

4550
N.A. Hamdy, A.M. Gamal-Eldeen / European Journal of Medicinal Chemistry 44 (2009) 4547–4556
appearance of the absorption bands due to NH, CN, C]O. Also their
1H NMR revealed the appearance of a signal at 8.7 due to (NH2, D2O
exchangeable).
except compounds 4b, 6a, 8a, and 9b, which possessed dramatic
cytotoxicity to macrophage with IC50 values of 8.4, 4.9, 3.1, and
9.9 mM, respectively, in addition to compounds 7a and 8b, which
Macrophages are the first line of defense in innate immunity
against microbial infection. Professional phagocytes engulf and kill
microorganisms and present antigens for triggering adaptive
immune responses [31]. The rate of macrophages growth repre-
sents a controlling key in that defense system. The incubation of
macrophages with the compounds of Scheme 1 (2a–c and 3a–c) for
24 h indicated that only 2b and 3b significantly induced the cell
growth up to 4-folds of the control growth (Fig. 1A), while the rest
had moderate cytotoxic effect with IC50 values of 26.2 and 27.7
respectively (Fig. 1B and C).
mM,
These results revealed that neither of the pyridine ring nor the
tetrahydronaphthalene part is directly responsible for modulation
of the macrophage growth, but the substitution with isopropyl
phenyl in the pyridine ring of the cyanopyridone (2b) and of the
thioxopyridine (3b) led to a high induction of macrophage growth.
In contrast, the substitution with isopropyl phenyl in the chlor-
opyridine (4b) and acetamide (9b) derivatives led to a remarkable
cytotoxicity to macrophages. Similarly, the substitution with
dimethoxy phenyl group in the cyanopyridine (6a) and the
hydrazide derivatives (8a) resulted in a cytotoxic effect on macro-
of compounds were not cytotoxic (IC50 > 100
mM), except 3a, which
possessed moderate cytotoxicity (IC50 29.4 M). On the other hand,
m
all compounds of Scheme 2 (4a,b, 5a,b, and 6a,b) and Scheme 3
(7a,b, 8a,b, and 9a,b) had no cytotoxic effect on macrophage as
indicated from there IC50 values (50 to >100
mM) (Fig. 1B and C),
phages. Due to the results of the viability assay, 20 mM of
A
B
C 100
500
100
80
60
40
20
0
400
300
200
100
0
80
60
40
20
0
0
20 40
60
80 100
0
20
40
60
80 100
0
20
40
60
80 100
Concentration (µM)
Concentration (µM)
Concentration (µM)
2a
3a
2b
3b
2c
3c
4a
5b
7b
9a
7a
8b
8a
9b
4b
6a
5a
6b
10000
8000
6000
4000
2000
0
D
*
*
*
*
**
**
**
**
**
LPS 2a 2b 2c 3a 3b 3c 4a 4b 5a 5b 6a 6b 7a 7b 8a 8b 9a 9b
E
250
200
150
100
50
*
*
*
*
*
**
**
**
0
2a 2b 2c
3a 3b 3c 4a 4b 5a 5b 6a 6b 7a 7b 8a 8b 9a 9b
Fig. 1. The effect of different doses of the synthesized compounds of Scheme 1 (A), Scheme 2 (B), and Scheme 3 (C) on the viability of macrophages was measured by MTT assay.
Macrophage viability was compared with the induced proliferation by 1000 U/ml M-CSF (178% of control). The results are represented as the percentage of control untreated cells
(Mean ꢂ SD, n ¼ 4). The effect of treatment with different compounds on the FITC-LPS-binding affinity to RAW 264.7 cells (D) in comparison with untreated and FITC-LPS treated
cells. The mean parentage of the binding affinity is compared with FITC-LPS treated cells. Phagocytic activity of RAW 264.7 cells against FITC-zymosan (E) was measured fluo-
rometrically after treatment with different compounds. The results were compared to the phagocytic activity of the control untreated cells (represented as 100% activity). The
significant induction (*) and the significant inhibition (**) are indicated upon the bars (P < 0.05).

N.A. Hamdy, A.M. Gamal-Eldeen / European Journal of Medicinal Chemistry 44 (2009) 4547–4556
4551
non-cytotoxic compounds (IC50 > 100
mM) and 20% of the IC50 value
cellular changes [34]. TNF-a may initiate an inflammatory cascade
of the cytotoxic compounds were used for the rest of cellular
experiments.
consisting of other inflammatory cytokines, chemokines, growth
factors, and endothelial adhesion factors, recruiting a variety of
During phagocytosis, macrophages secrete preformed granule
constituents and newly synthesized products that play a critical
role in inflammation and tissue repair [31]. Accordingly, phagocytic
activity and macrophage reactivity against bacterial epitopes are
crucial in the assessment of macrophage function. Macrophage
defense against pathogens includes cytokine secretions like tumor
activated cells at the site of tissue damage [34]. TNF-
anticancer and procancer actions, where high-dose administration
of TNF- might destruct tumor vasculature and have necrotic effects
in tumors [34]. In contrast, TNF- has been found to be required in
chemical carcinogen-elicited skin carcinogenesis [35]. In addition,
TNF- can induce DNA damage, inhibit DNA repair [36], and act as
a growth factor for tumor cells [37]. Accordingly, the inhibition of
stimulated TNF- is an important target in prevention of cancer and
a has both
a
a
a
necrosis factor-a (TNF-a), and inflammation mediators like nitric
oxide (NO). In phagocytes, NO is produced in large quantities by the
action of inducible NO synthetase (iNOS), in addition to 5-lip-
oxygenase (5-LO) and cyclooxygenase-2 (COX-2) [32].
a
in counteracting of inflammatory diseases.
Surprisingly, the treatment of macrophages with compounds 4a,
4b, 6a, 6b, and 8b, without LPS, led to highly significant increase
(P < 0.05) in the nitrites, as an index of NO generation, and in TNF-
We examined the effect of the synthesized compounds on some
of macrophage functions including LPS-binding affinity and
phagocytosis. Fluorometric analysis of LPS-binding affinity to
macrophages was investigated using LPS-FITC (Fig. 1D). The anal-
ysis indicated that compounds 2b, 3b, 5b, and 7b significantly
enhanced the macrophages binding affinity to LPS (P < 0.05), while
compounds 4a, 4b, 6a, 6b, and 9a significantly depressed the
macrophages binding affinity to LPS (P < 0.05). In phagocytosis
experiment, compounds 2b, 2c, 3b, 5b, 7b, and 9b significantly
induced the phagocytic activity of macrophages, while compounds
4a, 6a, and 9a significantly inhibited phagocytosis. The findings of
LPS-binding and phagocytosis experiments revealed that the
substitution with isopropyl phenyl group in the pyridine ring, in
many compounds, is responsible for the enhancement of the
macrophage binding affinity to LPS and the macrophage phago-
cytic activity. In contrast, it seems that the substitution with
dimethoxy phenyl group is responsible for the inhibition of both of
LPS-binding affinity and phagocytosis.
a
secretion (Table 1). It is clear that [R-6-(5,6,7,8-tetrahydronaph-
thalen-2-yl)-nicotinonitrile}part of the chloropyridine (4a and 4b)
and the cyanopyridine (6a and 6b) derivatives, whatever is the
substitution of R, led to an induction in the secretion of TNF-a and
in the generation of NO, which may be due to an induced iNOS
expression. It can be noticed also that those derivatives additionally
depressed the macrophages binding affinity to LPS and that 4a, and
6a inhibited phagocytosis.
In LPS pre-treated macrophages, the nitrites and TNF-a level, in
the macrophage culture supernatants, were significantly gener-
ated compared with control level in untreated cells (Table 1). The
co-treatment of LPS-stimulated macrophages with compounds 2a,
2b, 3b, 5a, 5b, 7b, 8a, 9a, and 9b potentially inhibited the NO
generation (P < 0.05), while 4a, 4b, 6a, 6b, and 8b led to further
additional generation of NO more that they generate alone. On the
other hand, the co-treatment of LPS pre-treated macrophages with
compounds 2b, 3b, 5b, 7b, 9a, and 9b significantly reduced the
TNF-
a
is produced mainly by activated macrophages, and by
plays a critical role in tissue
tumor cells [33]. In inflammation, TNF-
a
secreted stimulated TNF-a (P < 0.05), as shown in Table 1.
destruction, tissue remodeling and damage recovery, and in main-
taining the reversibility of microenvironments and stimulating the
Arachidonic acids are the substrate for COX-2 to produce
prostaglandins. Interestingly, arachidonic acids can be converted
Table 1
Effect of different compounds on the levels of nitrites, TNF-
a
and PGE-2 in LPS-stimulated and un-stimulated macrophages.
Sample
Nitritesa (nmol/mg protein)
TNF-aa (pg/mg protein)
PGE2a (pg/mg protein)
LPS
Control
ꢁ
þ
ꢁ
þ
ꢁ
þ
b
8.26 ꢂ 2.58
573.29 ꢂ 44.15b
84.50 ꢂ 11.20
5800.08 ꢂ 650.18
36.50 ꢂ 4.50
3150.00 ꢂ 136.5b
2a
2b
2c
14.80 ꢂ 3.85
8.51 ꢂ 1.25
16.25 ꢂ 3.46
355.78 ꢂ 41.15c
69.56 ꢂ 9.33
91.67 ꢂ 13.13
88.44 ꢂ 15.11
3860.25 ꢂ 51.77
1255.60 ꢂ 98.16c
5100.15 ꢂ 184.98
30.35 ꢂ 5.33
40.13 ꢂ 5.74
32.63 ꢂ 6.74
3120.45 ꢂ 127.88
2105.30 ꢂ 157.67c
3210.30 ꢂ 197.52
32.55 ꢂ 4.61c
505.75 ꢂ 18.97
3ad
3b
3c
8.58 ꢂ 2.22
12.66 ꢂ 3.44
15.50 ꢂ 4.06
380.12 ꢂ 66.67
79.18 ꢂ 9.55
101.5 ꢂ 22.71
75.40 ꢂ 9.87
4652.40 ꢂ 159.94
28.66 ꢂ 3.36
41.84 ꢂ 5.16
49.82 ꢂ 4.50
3309.30 ꢂ 270.20
1843.42 ꢂ 164.25c
3005.30 ꢂ 198.85
25.58 ꢂ 3.61c
1458.16 ꢂ 171.36c
4925.35 ꢂ 211.09
480.05 ꢂ 21.84
4a
512.35 ꢂ 45.77b
766.85 ꢂ 62.34
611.68 ꢂ 59.12
810.75 ꢂ 85.55b
5716.66 ꢂ 204.08
5110.40 ꢂ 261.22
38.33 ꢂ 7.60
45.39 ꢂ 6.10
3325.87 ꢂ 310.29
2870.25 ꢂ 231.86
4bd
439.13 ꢂ 38.48b
350.05 ꢂ 27.46b
5a
5b
10.53 ꢂ 1.94
5.62 ꢂ 1.12
145.67 ꢂ 19.78c
94.88 ꢂ 11.03
105.60 ꢂ 13.05
4185.85 ꢂ 194.87
28.60 ꢂ 3.68
32.21 ꢂ 7.46
3415.23 ꢂ 332.08
2974.23 ꢂ 305.40
62.42 ꢂ 8.91c
1925.78 ꢂ 184.54c
6ad
6b
720.91 ꢂ 68.41b
915.90 ꢂ 95.22
922.50 ꢂ 86.21
2460.46 ꢂ 222.23b
5520.55 ꢂ 405.08
6325.08 ꢂ 344.16
31.29 ꢂ 9.10
48.30 ꢂ 4.87
3010.66 ꢂ 280.27
3222.55 ꢂ 194.28
614.63 ꢂ 77.28b
1890.16 ꢂ 136.84b
7ad
7bd
2.65 ꢂ 0.95
3.19 ꢂ 1.02
388.40 ꢂ 40.58
66.57 ꢂ 8.41
72.45 ꢂ 6.52
3945.11 ꢂ 256.56
22.40 ꢂ 4.84
50.54 ꢂ 8.81
2954.2 ꢂ 273.2
110.54 ꢂ 13.47c
2810.66 ꢂ 177.08c
2870.50 ꢂ 274.05
8a
1.34 ꢂ 0.57
78.54 ꢂ 9.88c
124.68 ꢂ 11.79
4924.22 ꢂ 117.23
5754.50 ꢂ 242.37
25.60 ꢂ 4.38
43.60 ꢂ 7.51
3111.94 ꢂ 265.57
3405.50 ꢂ 389.77
8bd
440.24 ꢂ 37.57b
596.40 ꢂ 47.86
1364.75 ꢂ 108.99b
9a
26.65 ꢂ 5.03
10.19 ꢂ 2.31
155.25 ꢂ 18.76c
116.8 ꢂ 14.52
69.65 ꢂ 5.19
854.16 ꢂ 73.73c
53.10 ꢂ 6.31
48.15 ꢂ 7.64
2018.96 ꢂ 140.20c
9bd
32.75 ꢂ 4.82c
615.33 ꢂ 53.77c
3260.20 ꢂ 280.42
a
The nitrites, as an index for NO generation was used by Griess assay, while TNF-
Significantly different from control untreated macrophages (P < 0.05).
Significantly different from LPS-stimulated macrophages (P < 0.05).
a and PGE-2 were measured using commercial ELISA kits.
b
c
d
These compounds are toxic, so 20% of their IC50 values were used for macrophage treatment, while the rest of the compounds were not toxic (IC50 > 100 mM), so 20 mM of
each compounds were used for macrophage treatment.

4552
N.A. Hamdy, A.M. Gamal-Eldeen / European Journal of Medicinal Chemistry 44 (2009) 4547–4556
Table 2
by another enzyme lipoxygenases (LO) to leukotrienes, which were
suggested to be another link between inflammation and cancer
[32]. The prostaglandins (PG) are biologically active derivatives of
arachidonic acid and other polyunsaturated fatty acids that are
released from membrane phospholipids by phospholipase A2 [32].
PGE2 plays a role both in normal physiology and in pathology [38].
The biological actions include inflammation, pain, tumorigenesis,
vascular regulation, neuronal functions, female reproduction,
gastric mucosal protection, and kidney function [38]. Measure-
ment of PGE-2 by commercial kit, revealed that neither of the
compounds led to a significant change in PGE-2 level in macro-
phages (P > 0.05), when treated in the absence of LPS (Table 1).
However, the treatment with LPS resulted in a dramatic significant
increase in PGE-2 level compared to untreated cells and only the
co-treatment with compounds 2b, 3b, and 9a led to a significant
inhibition in this stimulated secretion of PGE-2 (P < 0.05), as
shown in Table 1.
LOs, including 5-LO, mediate oxidation of potent carcinogens
such as benzidine, o-dianisidine, and others; this activation can be
blocked by adding the LO inhibitors, which products have inhibited
the development and progression of human cancers [39]. In
comparison with normal tissues, significantly elevated levels of LO
metabolites have been found in several types of cancer cells [39].
LO-mediated products elicit diverse biological activities needed for
neoplastic cell growth, the activation of growth factor and tran-
scription factor, the induction of oncogene, stimulation of tumor
cell adhesion, and the regulation of apoptotic cell death [39].
Agents that block LO activity may be effective in preventing cancer.
LO inhibitors have prevented carcinogen-induced lung adenomas
and rat mammary gland cancers [39]. Pharmacological agents that
specifically inhibit the LO-mediated signaling pathways are now
commercially available to treat inflammatory diseases such as
asthma, arthritis, and psoriasis [39].
COX-2 expression may be induced by a wide range of stimuli,
including lipopolysaccharide, proinflammatory cytokines, such as
IL-1 and TNF, and growth factors, such as epidermal growth factor
[32]. COX-2 is also overexpressed in various types of cancer and
involved in cellular proliferation; antiapoptotic activity, angiogen-
esis, and an increase of metastasis [32]. The products of COX-2
enzyme are prostaglandins, which are key mediators of inflamma-
tion. Various nonsteroidal anti-inflammatory drugs inhibit COX-2
activity, which reduce the risk of several cancers.
Using immunoblotting analysis, COX-2 and 5-LO enzymes were
assessed in macrophage lysates. The treatment of macrophage
with all of the compounds alone resulted in an unchangeable level
of 5-LO, while COX-2 level was significantly induced (P < 0.05) by
4a, 6a, 6b, and 8b (Table 2). In LPS-stimulated macrophages, COX-2
and 5-LO were remarkable enhanced (P < 0.05), as shown in
Table 2. The treatment of LPS-stimulated macrophages with
compounds 2b, 3b, 3c, 5a, 5b, 7b, and 9b significantly depressed
the stimulated synthesis of COX-2 (P < 0.05), similarly, compounds
2b, 3b, 3c, 5b, 7b, and 9b were recognized as potential inhibitors of
5-LO (P < 0.05), as shown in Table 2.
Effect of different compounds on the levels of COX-2 and 5-LO in LPS-stimulated and
un-stimulated macrophages.
Sample
COX-2,a % of relative intensity
5-LO,a % of relative intensity
LPS
Control
ꢁ
þ
ꢁ
þ
16.80 ꢂ 2.40
87.40 ꢂ 5.30b
21.10 ꢂ 3.10
93.60 ꢂ 9.10b
2a
2b
2c
14.60 ꢂ 2.40
21.40 ꢂ 1.20
16.30 ꢂ 1.80
76.30 ꢂ 5.60
42.70 ꢂ 4.80c
82.40 ꢂ 6.40
15.80 ꢂ 3.10
20.30 ꢂ 2.40
23.50 ꢂ 1.80
92.10 ꢂ 8.40
25.10 ꢂ 3.40c
82.30 ꢂ 7.70
3ad
3b
3c
12.30 ꢂ 1.70
18.20 ꢂ 1.40
28.10 ꢂ 3.30
72.80 ꢂ 6.60
36.70 ꢂ 5.40c
59.80 ꢂ 4.40c
18.30 ꢂ 2.20
14.20 ꢂ 2.20
23.80 ꢂ 3.20
85.40 ꢂ 7.50
31.60 ꢂ 5.20c
69.40 ꢂ 6.20c
4a
36.60 ꢂ 4.30b
88.70 ꢂ 5.20
80.10 ꢂ 8.30
25.30 ꢂ 3.40
29.50 ꢂ 1.60
84.50 ꢂ 5.40
94.60 ꢂ 5.90
4bd
28.70 ꢂ 2.50
5a
5b
15.70 ꢂ 2.60
22.60 ꢂ 3.40
63.70 ꢂ 8.20c
24.00 ꢂ 3.20
18.10 ꢂ 2.30
81.80 ꢂ 6.50
28.10 ꢂ 4.20c
43.60 ꢂ 4.80c
6ad
6b
46.30 ꢂ 6.40b
99.20 ꢂ 2.60
97.30 ꢂ 4.20
30.20 ꢂ 3.80
29.80 ꢂ 3.60
94.70 ꢂ 4.40
88.70 ꢂ 4.50
53.20 ꢂ 6.30b
7ad
7bd
27.5 ꢂ 2.60
68.70 ꢂ 6.70
21.90 ꢂ 3.30
11.90 ꢂ 2.60
79.60 ꢂ 8.40
18.70 ꢂ 3.20
31.30 ꢂ 4.30c
38.70 ꢂ 6.20c
8a
10.20 ꢂ 2.80
71.30 ꢂ 5.80
92.60 ꢂ 4.50
13.80 ꢂ 2.60
33.12 ꢂ 4.60
83.20 ꢂ 4.80
91.80 ꢂ 7.60
8bd
63.10 ꢂ 7.80b
9a
23.70 ꢂ 3.30
20.60 ꢂ 3.20
73.30 ꢂ 3.40
18.90 ꢂ 2.80
21.70 ꢂ 2.40
77.40 ꢂ 9.10
9bd
20.10 ꢂ 5.20c
61.70 ꢂ 8.60c
a
The change in COX-2 and 5-LO content were assayed by dot immunoblotting.
Significantly different from control untreated macrophages (P < 0.05).
Significantly different from LPS-stimulated macrophages (P < 0.05).
These compounds are toxic, so 20% of their IC50 values were used for macro-
b
c
d
phage treatment, while the rest of the compounds were not toxic (IC50 > 100
20 M of each compounds were used macrophage treatment.
mM), so
m
Table 3
Radical scavenging activity of different compounds against OHꢀ and ROOꢀ as
ꢀ
measured by ORAC assay, and against O2ꢁ as measured by xanthine/xanthine
oxidase assay.
Compounda ORAC-OHꢀ(units)b ORAC-ROOꢀ(units)b X/XO (O2ꢁ ) SC50
(
m
g/ml)c
ꢀ
2a
2b
2c
3a
3b
3c
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
0.54
0.92
0.21
0.85
5.41
4.2
0.41
0.75
0.35
0.90
4.32
6.13
0.61
0.92
1.92
2.45
2.95
1.34
2.01
2.36
5.12
0.81
3.56
2.22
>100
85.40
78.51
65.42
10.15
4.22
0.35
1.1
87.87
>100
>100
21.88
38.43
77.56
55.68
81.42
8.67
5.85
2.28
3.54
1.01
2.47
1.05
5.84
0.99
2.31
3.44
42.47
19.89
24.62
a
In ORAC assay, only 1
1.0 ORAC unit equals the net protection of
m
M of each compound was used.
b
b
-phycoerythrin produced by 1.0
mM
During inflammation, different types of reactive oxygen and
nitrogen species are released. Scavenging of those species
represents a defense mechanism against everlasting inflamma-
tion in inflammatory diseases and in cancer. For this reason we
screened the radical scavenging activity of the synthesized
compounds revealed that 3b, 3c, 5b, 6a, 7a, 8a, 9a, and 9b were
strong non specific scavengers of different types of radicals (OHꢀ,
Trolox.
c
SC50
: half-maximal scavenging concentration was calculated using dose
dependant curve of each compound.
3. Conclusion
Collectively, the cyanopyridone 2b induced the macrophage
growth, macrophages binding affinity to LPS, and phagocytic
activity, and it inhibited LPS-stimulated NO, TNF-a, PGE-2, 5-LO,
and COX-2. Other cyanopyridones 2a and 2c were only NO inhibitor
and phagocytosis induced, respectively. The thioxopyridine 3b
induced macrophage growth, macrophages binding affinity to LPS,
ꢀ
ROOꢀ, and Oꢁ2 ), as indicated from their high ORAC values against
ꢀ
OHꢀ and ROOꢀ and low SC50 values against Oꢁ2 (Table 3). On the
other hand, 5a was a specific strong scavenger of OHꢀ, in
comparison to trolox activity, and 7b was a relative specific
scavenger of ROOꢀ (Table 3).

N.A. Hamdy, A.M. Gamal-Eldeen / European Journal of Medicinal Chemistry 44 (2009) 4547–4556
4553
and phagocytic activity, and inhibited the LPS-stimulated NO, TNF-
, PGE-2, 5-LO, and COX-2 and it was a strong radical scavenger. The
from ethanol/DMF to afford the corresponding derivatives 2a–c as
yellow crystals, respectively.
a
other thioxopyridine 3c was a strong COX-2 and 5-LO inhibitor and
radical scavenger. The 2-chloropyridines 4a,b were found to be
4.1.1.1. 4-(3,4-Dimethoxy-phenyl)-2-oxo-6-(5,6,7,8-tetrahydronaph-
significant inducer of NO, and TNF-
a
. Compound 4a inhibited and
thalen-2-yl)-1,2-dihydro-pyridine-3-carbonitrile (2a). Yield (85%);
phagocytosis the macrophages binding affinity to LPS and induced
COX-2, while 4b was cytotoxic to macrophages and inhibited the
macrophages binding affinity to LPS. The pyrazolopyridine 5a
inhibited the LPS-stimulated NO and COX-2 and it was specific
scavenger of OHꢀ, while the other pyrazolopyridine 5b induced
phagocytosis and macrophages binding affinity to LPS and it
mp 268–270 ꢃC; IR (KBr) ( , cmꢁ1): 3420 (NH), 2933 (CH alicyclic),
n
2219 (CN), 1628 (C]O); 1H NMR (DMSO-d6):
d (ppm) 1.7 (m, 4H,
aliphatic 2CH2 of tetrahydronaphthalene), 2.7 (m, 4H, aliphatic
2CH2 of tetrahydronaphthalene), 3.8 (s, 6H, OCH3), 6.7–7.9 (m, 7H
Ar-H) and 11.2 (s, 1H, NH D2O exchangeable); MS m/z (%): 386.3
(Mþ, 100%). Anal. calcd. for C24H22N2O3: C, 74.59; H, 5.74; N, 7.25.
Found: C, 74.48; H, 5.82; N, 7.30.
inhibited the LPS-stimulated NO, TNF-
a strong radical scavenger.
a, COX-2 and 5-LO and it was
The cyanopyridine derivatives 6a,b were found to be significant
inducers of NO, TNF- , and COX-2 and inhibitors of macrophages
4.1.1.2. 4-(4-Isopropyl-phenyl)-2-oxo-6-(5,6,7,8-tetrahydronaph-
a
thalen-2-yl)-1,2-dihydro-pyridine-3-carbonitrile (2b). Yield (86%);
binding affinity to LPS. The cyanopyridine 6a, regardless its high
antioxidant activity, was cytotoxic to macrophages and it inhibited
phagocytosis. Although the 3-Cyanopyridinyl oxy acetic acid ethyl
ester 7a showed only radical scavenging affinity, 7b showed
a multi-potent agent, since it induced phagocytosis and macro-
phages binding affinity to LPS, inhibited the LPS-stimulated NO,
mp 308–310 ꢃC; IR (KBr) ( , cmꢁ1): 3424 (NH), 2934 (CH alicyclic),
n
2219 (CN), 1630 (C]O); 1H NMR (DMSO-d6):
d (ppm) 1.25 (d,
J ¼ 6.85 Hz, 6H, 2CH3), 1.75 (m, 4H, aliphatic 2CH2 of tetrahy-
dronaphthalene), 2.8 (m, 4H, aliphatic 2CH2 of tetrahydronaph-
thalene), 3.1 (m, 1H, CH), 7.43 (d, J ¼ 6.85 Hz, 2H, Ar-H), 7.65 (d,
J ¼ 6.85 Hz, 2H, Ar-H), 7.9–8.0(m, 4H Ar-H) and 11.2 (s, 1H, NH D2O
exchangeable); MS m/z (%): 368.2 (Mþ, 100%). Anal. calcd. for
C25H24N2O: C, 81.49; H, 6.57; N, 7.60. Found: C, 81.38; H, 6.60; N, 7.65.
TNF-a
, 5-LO, and COX-2, and it was a specific scavenger of ROOꢀ.
Despite the cytotoxic effect of the hydrazide derivative 8a to
macrophage, it was strong antioxidant and NO inhibitor. However,
the hydrazide derivative 8b was an inducer of NO, TNF-
2. The acetamide derivative 9a inhibited phagocytosis, the macro-
phages binding affinity to LPS, NO, TNF- , and PGE-2 and had high
a
, and COX-
4.1.1.3. 4-(1H-Indol-3-yl)-2-oxo-6-(5,6,7,8-tetrahydronaphthalen-2-yl)-
1,2-dihydro-pyridine-3-carbonitrile (2c). Yield (83%); mp 320–
a
321 ꢃC; IR (KBr) ( , cmꢁ1): 3306 (NH), 2929 (CH alicyclic), 2217 (CN),
n
antioxidant activity. On the other hand, the acetamide derivative 9b
1643 (C]O); 1H NMR (DMSO-d6):
d (ppm) 1.7 (m, 4H, aliphatic
was cytotoxic to macrophage, induced the phagocytic activity,
inhibited NO and TNF-a, COX-2, and 5-LO and had a strong anti-
2CH2 of tetrahydronaphthalene), 2.8 (m, 4H, aliphatic 2CH2 of tet-
rahydronaphthalene), 6.85–8.1 (m, 9H Ar-H), 11.97 (s, 1H, NH D2O
exchangeable) and 12.30 (s, 1H, NH D2O exchangeable); MS m/z (%):
365.3 (Mþ, 100%). Anal. calcd. for C24H19N3O: C, 78.88; H, 5.24; N,
11.50. Found: C, 78.75; H, 5.34; N, 11.55.
oxidant activity. Taken together, the derivatives cyanopyridone 2b,
thioxopyridine 3b, pyrazolopyridine 5a, 3-cyanopyridinyl oxy ace-
tic acid ethyl ester 7b and acetamide 9a and 9b can be recognized as
promising multi-potent anti-inflammatory agents, with consid-
ering the cytotoxicity of 9b.
4.1.2. General procedure for the synthesis of compounds (3a–c)
A mixture of compound 2 (0.01 mol) and P2S5 (2 g) in pyridine
(10 mL) was refluxed for 6 h. The product poured into ice-cold
dilute HCl. The separated solid was filtered off and recrystallized
from appropriate solvent to afford the corresponding thione
derivatives 3a–c, respectively.
4. Experimental section
4.1. Chemistry
All melting points are uncorrected and measured using Elec-
tro-thermal IA 9100 apparatus (Shimadzu, Tokyo, Japan). IR
spectra were recorded as potassium bromide pellets on a Perkin–
Elmer 1650 spectrophotometer (Perkin–Elmer, Norwalk, CT, USA).
1H NMR was determined on a Jeol-Ex-300 NMR spectrometer
(JEOL, Tokyo, Japan) and chemical shifts were expressed as part
4.1.2.1. 4-(3,4-Dimethoxy-phenyl)-6-(5,6,7,8-tetrahydronaphthalen-2-yl)-
2-thioxo-1,2-dihydro-pyridine-3-carbonitrile(3a). This compound
was obtained as orange crystals and recrystallized from CHCl3/
petroleum ether. Yield (70%); mp 218–220 ꢃC; IR (KBr) ( , cmꢁ1):
n
3420 (NH), 2934 (CH alicyclic), 2243 (CN), 1591 (C]N) and 1202
(C]S); 1H NMR (DMSO-d6):
d
(ppm) 1.76 (m, 4H, aliphatic 2CH2 of
per million (ppm) (
d
values) against TMS as internal reference.
tetrahydronaphthalene), 2.78 (m, 4H, aliphatic 2CH2 of tetrahy-
dronaphthalene), 3.8 (s, 6H, OCH3), 7.1–7.9 (m, 7H Ar-H) and 8.8 (s,
1H, NH D2O exchangeable); MS m/z (%): 402.3 (Mþ, 100%). Anal.
calcd. for C24H22N2O2S: C, 71.61; H, 5.51; N, 6.96; S, 7.97. Found: C,
71.53; H, 5.62; N, 6.72; 7.84.
Mass spectra were recorded on VG 2AM-3F mass spectrometer
(Thermo electron corporation, USA). Microanalyses were operated
using Mario El Mentar apparatus, Organic Microanalysis Unit, and
the results were within the accepted range (ꢂ0.20) of the calcu-
lated values. Follow up of the reactions and checking the purity of
the compounds was made by TLC on silica gel-precoated
aluminum sheets (Type 60 F254, Merck, Darmstadt, Germany).
Compounds 1 were prepared according to a reported method
[30].
4.1.2.2. 4-(4-Isopropyl-phenyl)-6-(5,6,7,8-tetrahydronaphthalen-2-yl)-
2-thioxo-1,2-dihydro-pyridine-3-carbonitrile (3b). This compound
was obtained as orange crystals and recrystallized from benzene.
Yield (72%); mp 244–246 ꢃC; IR (KBr) ( , cmꢁ1): 3444 (NH), 2928
n
(CH alicyclic), 2230 (CN), 1595 (C]N) and 1209 (C]S); 1H NMR
(DMSO-d6):
4.1.1. General procedure for the synthesis of compounds (2a–c)
A mixture of 1-(5,6,7,8-tetrahydronaphthalen-2-yl)ethanone (1)
(0.01 mol), aromatic aldehydes namely: 3,4-dimethyloxybenz-
aldehyde, p-isopropyl benzaldehyde, 3-indolaldehyde (0.01 mol),
ethyl cyanoacetate (0.01 mol) and ammonium acetate (0.08 mol) in n-
butanol (40 mL) was refluxed for 3 h. The obtained precipitate was
filtered off, washed successively with ethanol and finally recrystallized
d
(ppm) 1.25 (d, J ¼ 6.9 Hz, 6H, 2CH3), 1.75 (m, 4H,
aliphatic 2CH2 of tetrahydronaphthalene), 2.78 (m, 4H, aliphatic
2CH2 of tetrahydronaphthalene), 3.2 (m, 1H, CH), 7.08 (s, 1H, Ar-H),
7.2 (d, J ¼ 8.45 Hz, 2H, Ar-H), 7.44 (d, J ¼ 8.45 Hz, 2H, Ar-H), 7.6–
7.67(m, 3H Ar-H) and 8.0 (s, 1H, NH D2O exchangeable); MS m/z (%):
384.4 (Mþ, 100%). Anal. calcd. for C25H24N2S: C, 78.09; H, 6.29; N,
7.28; S, 8.34. Found: C, 78.15; H, 6.21; N, 7.34; S, 8.29.

4554
N.A. Hamdy, A.M. Gamal-Eldeen / European Journal of Medicinal Chemistry 44 (2009) 4547–4556
4.1.2.3. 4-[4-(1H-Indol-3-yl)-phenyl]-6-(5,6,7,8-tetrahydronaph-
thalen-2-yl)-2-thioxo-1,2-dihydro-pyridine-3-carbonitrile (3c). This
compound was obtained as yellow crystals and recrystallized from
3.0 (m, 1H, CH), 5.6 (s, 2H, NH2 D2O exchangeable), 7.12–7.88 (m, 8H
Ar-H), 12.3 (s, 1H, NH D2O exchangeable); MS m/z (%):382 (Mþ, 100).
Anal. calcd. for C25H26N4: C, 78.50; H, 6.85; N, 14.65. Found: C, 78.62; H,
6.79; N, 14.56.
CHCl3. Yield (68%); mp 273–275 ꢃC; IR (KBr) ( , cmꢁ1): 3445 (NH),
n
3248 (NH), 2923 (CH alicyclic), 2215 (CN), 1596 (C]N) and 1200
(C]S); 1H NMR (DMSO-d6):
d
(ppm) 1.75 (m, 4H, aliphatic 2CH2 of
4.1.4.3. 2-Benzylamino-4-(3,4-dimethoxy-phenyl)-6-(5,6,7,8-tetra-
tetrahydronaphthalene), 2.78 (m, 4H, aliphatic 2CH2 of tetrahy-
dronaphthalene), 7.1–8.3 (m, 9H Ar-H), 12.0 (s, 1H, NH D2O
exchangeable) and 13.7 (s, 1H, NH D2O exchangeable); MS m/z (%):
380 (Mþꢁ1, 100%), 381 (Mþ, 49%). Anal. calcd. for C24H19N3O: C,
78.74; H, 5.07; N, 9.18; S, 7.01. Found: C, 78.62; H, 5.15; N, 9.22; S, 6.93.
hydronaphthalen-2-yl)-nicotinonitrile (6a). Yield (70%); mp 160–
162 ꢃC; IR (KBr) ( , cmꢁ1): 3360 (NH), 2930 (CH alicyclic), 2205 (CN),
n
1581 (C]N); 1H NMR (DMSO-d6):
d (ppm) 1.7 (m, 4H, aliphatic 2CH2
of tetrahydronaphthalene), 2.7 (m, 4H, aliphatic 2CH2 of tetrahy-
dronaphthalene), 3.8 (s, 6H, OCH3), 4.7 (d, J ¼ 5.4 Hz, 2H, CH2), 7.1–
7.75 (m,12HAr-H) and 11.2 (s,1H, NH D2O exchangeable); MSm/z (%):
474.3 (Mþ ꢁ 1, 100%), 475.3 (Mþ, 87.5%). Anal. calcd. for C31H29N3O2:
C, 78.29; H, 6.15; N, 8.84. Found: C, 78.33; H, 6.04; N, 8.72.
4.1.3. General procedure for the synthesis of compounds (4a,b)
A suspension of compound 2a or 2b (0.01 mol), PCl5 (0.5 g) and
POCl3 (5 ml) was heated on a water bath for 3 h. The reaction
mixture was poured gradually into ice-cold water and neutralized
by dilute ammonia solution. The separated solid was filtered off and
recrystallized from ethanol/DMF to afford the corresponding
derivatives 4a,b as yellow crystals, respectively.
4.1.4.4. 2-Benzylamino-4-(4-isopropyl-phenyl)-6-(5,6,7,8-tetrahy-
dronaphthalen-2-yl)-nicotinonitrile (6b). Yield (68%); mp 132–
134 ꢃC; IR (KBr) ( , cmꢁ1): 3431 (NH), 2929 (CH alicyclic), 2202
n
(CN), 1579 (C]N); 1H NMR (DMSO-d6):
d
(ppm) 1.23 (d, J ¼ 6.85 Hz,
6H, 2CH3), 1.7 (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 2.7
(m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 3.0 (m, 1H, CH),
4.7 (d, J ¼ 5.75 Hz, 2H, CH2), 7.38 (d, J ¼ 8 Hz, 2H, Ar-H), 7.56 (d,
J ¼ 8 Hz, 2H, Ar-H), 7.9–8.1 (m, 9H Ar-H) and 11.2 (s, 1H, NH D2O
exchangeable); MS m/z (%): 456.35 (Mþ ꢁ 1, 100%), 457.3 (Mþ,
85.74%). Anal. calcd. for C31H29N3: C, 83.99; H, 6.83; N, 9.18. Found:
C, 84.08; H, 6.79; N, 9.22.
4.1.3.1. 2-Chloro-4-(3,4-dimethoxy-phenyl)-6-(5,6,7,8-tetrahy-
dronaphthalen-2-yl)-nicotinonitrile (4a). Yield (64%); mp 172–
174 ꢃC; IR (KBr) ( , cmꢁ1): 2930 (CH alicyclic), 2224 (CN), 1576
n
(C]N); 1H NMR (DMSO-d6):
d (ppm) 1.76 (m, 4H, aliphatic 2CH2 of
tetrahydronaphthalene), 2.78 (m, 4H, aliphatic 2CH2 of tetrahy-
ꢃ
dronaphthalene), 3.86 (s, 6H, 2 CH3), 7.15–8.12 (m, 7H Ar-H); MS
m/z (%): 406 (Mþ, Cl37, 33.79), 404 (Mþ, Cl35, 100). Anal. calcd. for
C24H21ClN2O2: C, 71.19; H, 5.23; Cl, 8.76; N, 6.92. Found: C, 71.32; H,
5.15; Cl, 8.84; N, 6.87.
4.1.5. General procedure for the synthesis of compounds (7a,b)
A mixture of compound 2a or 2b (0.01 mol), ethyl bromoacetate
(0.01 mol), anhydrous potassium carbonate (0.04 mol) in dry
acetone (30 mL) was refluxed for 20 h. After cooling, water was
added to the mixture and the formed solid was filtered off,
recrystallized from appropriate solvent to afford the corresponding
derivatives 7a,b, respectively.
4.1.3.2. 2-Chloro-4-(4-isopropyl-phenyl)-6-(5,6,7,8-tetrahydronaph-
thalen-2-yl)-nicotinonitrile (4b). Yield (60%); mp 182–184 ꢃC; IR
(KBr) (
NMR (DMSO-d6):
n
, cmꢁ1): 2930 (CH alicyclic), 2223 (CN), 1581 (C]N); 1H
d
(ppm) 1.25 (d, J ¼ 6.7 Hz, 6H, 2CH3), 1.76 (m, 4H,
aliphatic 2CH2 of tetrahydronaphthalene), 2.78 (m, 4H, aliphatic
2CH2 of tetrahydronaphthalene), 3.0 (m, 1H, CH), 7.19 (d, J ¼ 8.1 Hz,
1H, Ar-H), 7.47 (d, J ¼ 6.6 Hz, 2H, Ar-H), 7.70 (d, J ¼ 6.6 Hz, 2H, Ar-H),
7.9 (m, 2H Ar-H), 8.13 (s,1H, CH-pyridine ring); MS m/z (%):388 (Mþ,
Cl37, 34), 386 (Mþ, Cl35, 100). Anal. calcd. for C25H23ClN2: C, 77.61; H,
5.99; Cl, 9.16; N, 7.24. Found: C, 77.69; H, 5.89; Cl, 9.21; N, 7.10.
4.1.5.1. [3-Cyano-4-(3,4-dimethoxy-phenyl)-6-(5,6,7,8-tetrahydronaph-
thalen-2-yl)-pyridin-2-yloxy]-acetic acid ethyl ester (7a). This compound
was obtained as white crystals and recrystallized from ethanol. Yield
(69%); mp 154–156 ꢃC; IR (KBr) (
n
, cmꢁ1): 2936 (CH alicyclic), 2214
(ppm) 1.2 (t,
(CN), 1745 (C]O) and 1590 (C]N); 1H NMR (DMSO-d6):
d
3H, CH3), 1.75 (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 2.76
(m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 3.85 (s, 6H, 2 ꢃCH3),
4.2 (q, 2H, COOCH2), 5.12 (s, 2H, OCH2COO), 7.1–7.8 (m, 7H Ar-H); MS
m/z (%): 472.28 (Mþ, 100%). Anal. calcd. for C28H28N2O5: C, 71.17; H,
5.97; N, 5.93. Found: C, 71.25; H, 5.85; N, 5.87.
4.1.4. General procedure for the synthesis of compounds
(5a,b) and (6a,b)
Compound 2a or 2b (0.01 mol) was fused with hydrazine
hydrate (1 ml) or benzyl amine (1.2 ml) over sand bath for 3 h. The
reaction mixture poured into ice-cold dilute hydrochloric acid and
the separated solid was filtered off and recrystallized from ethanol
to afford the corresponding derivatives 5a,b or 6a,b, respectively.
4.1.5.2. [3-Cyano-4-(4-isopropyl-phenyl)-6-(5,6,7,8-tetrahydronaph-
thalen-2-yl)-pyridin-2-yloxy]-acetic acid ethyl ester (7b). This
compound was obtained as white crystals and recrystallized from
ethanol/DMF. Yield (71%); mp 168–170 ꢃC; 2931 (CH alicyclic), 2219
4.1.4.1. 4-(3,4-Dimethoxy-phenyl)-6-(5,6,7,8-tetrahydronaphthalen-
2-yl)-1H-pyrazolo[3,4-b]pyridin-3-ylamine (5a). Yield (69%); mp
(CN),1749 (C]O) and 1590 (C]N); 1H NMR (DMSO-d6):
d (ppm) 1.5
142–144 ꢃC; IR (KBr) ( , cmꢁ1): 3444 (NH), 3351 (NH), 3192 (NH), 2927
n
(CH alicyclic), 1596 (C]N); 1H NMR (DMSO-d6):
d (ppm) 1.74 (m, 4H,
(m, 9H, CH3of ethyl group þ 2CH3), 2.08 (m, 4H, aliphatic 2CH2 of
tetrahydronaphthalene), 3.09 (m, 4H, aliphatic 2CH2 of tetrahy-
dronaphthalene), 3.3 (m, 1H, CH), 4.52 (q, 2H, COOCH2), 5.45 (s, 2H,
OCH2COO), 7.5 (d, J ¼ 12 Hz, 1H, Ar-H), 7.79 (d, J ¼ 9 Hz, 2H, Ar-H),
8.02 (d, J ¼ 9 Hz, 2H, Ar-H), 8.13 (s, 1H, pyridine-H), 8.18–8.2 (m, 2H
Ar-H); MS m/z (%): 454.26 (Mþ, 100%). Anal. calcd. for C25H24N2O: C,
81.49; H, 6.57; N, 7.60. Found: C, 81.38; H, 6.60; N, 7.65.
aliphatic 2CH2 of tetrahydronaphthalene), 2.74 (m, 4H, aliphatic 2CH2
of tetrahydronaphthalene), 3.8 (s, 6H, OCH3), 5.6 (s, 2H, NH2 D2O
exchangeable), 7.1–7.8 (m, 7H Ar-H), 12.0 (s, 1H, NH D2O exchange-
able); MS m/z (%): 400 (Mþ, 100). Anal. calcd. for C24H24N4O2: C, 71.98;
H, 6.04; N, 13.99. Found: C, 72.03; H, 6.00; N, 13.88.
4.1.4.2. 4-(4-Isopropyl-phenyl)-6-(5,6,7,8-tetrahydronaphthalen-2-yl)-
4.1.6. General procedure for the synthesis of compounds (8a,b)
A solution of compound 7a or 7b (0.01 mol) in ethanol (50 ml)
and hydrazine hydrate (0.01 mol) was refluxed for 6 h. The sepa-
rated solid after cooling was recrystallized from appropriate solvent
to afford the corresponding hydrazides 8a,b, respectively.
1H-pyrazolo[3,4-b]pyridin-3-ylamine (5b). Yield (65%); mp 190–
192 ꢃC; IR (KBr) ( , cmꢁ1): 3453 (NH), 3294 (NH), 3194 (NH), 2925
n
(CH alicyclic), 1585 (C]N); 1H NMR (DMSO-d6):
d (ppm) 1.27
(d, J ¼ 6.9 Hz, 6H, 2CH3), 1.76 (m, 4H, aliphatic 2CH2 of tetrahy-
dronaphthalene), 2.76 (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene),

N.A. Hamdy, A.M. Gamal-Eldeen / European Journal of Medicinal Chemistry 44 (2009) 4547–4556
4555
4.1.6.1. [3-Cyano-4-(3,4-dimethoxy-phenyl)-6-(5,6,7,8-tetrahydronaph-
thalen-2-yl)-pyridin-2-yloxy]-acetic acid hydrazide (8a). This compound
was obtained as white crystals and recrystallized from AcOH. Yield
bovine serum (FBS), 2 mM L-glutamine, 100 units/ml penicillin
sodium, 100 units/ml streptomycin sulphate, and 250 ng/ml
amphotericin B. Cells were maintained sub-confluent at 37 ꢃC in
humidified air containing 5% CO2. RAW 264.7 cells were harvested
by gentle scraping. Cells were used when confluence had reached
75%. Compounds were dissolved in 10% dimethyl sulfoxide (DMSO)
supplemented with the same concentrations of antibiotics.
Compounds were tested for endotoxin, using PyrogentÒ Ultra gel
clot assay, and they were found endotoxin-free. After the viability
(70%); mp 234–236 ꢃC; IR (KBr) ( , cmꢁ1): 3442-3329 (NH2,NH), 2929
n
(CH alicyclic), 2220 (CN), 1676 (C]O) and 1589 (C]N); 1H NMR
(DMSO-d6): (ppm) 1.77 (m, 4H, aliphatic 2CH2 of tetrahydronaph-
d
thalene), 2.8 (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 3.86 (s,
6H, 2 ꢃCH3), 4.3 (s, 2H, NH2 D2O exchangeable), 4.9 (s, 2H, OCH2), 7.15–
7.91 (m, 7H Ar-H) and 9.4 (s, H, NH D2O exchangeable); MS m/z (%):
458.3(Mþ, 10.70%), 82.9 (100%). Anal. calcd. for C26H26N4O4: C, 68.11; H,
5.72; N, 12.22. Found: C, 68.22; H, 5.68; N, 12.20.
assay, for the rest of cellular experiments, 20
mM of non-cytotoxic
compounds (IC50 > 100 M) and 20% of the IC50 value of the cyto-
m
toxic compounds were used. All experiments were repeated four
times, unless mentioned, and the data were represented as mean
values. Cell culture material was obtained from Cambrex BioScience
(Copenhagen, Denmark). Chemicals were purchased from Sigma
Aldrich (VA, USA), except mentioned.
4.1.6.2. [3-Cyano-4-(4-isopropyl-phenyl)-6-(5,6,7,8-tetrahydronaph-
thalen-2-yl)-pyridin-2-yloxy]-acetic acid hydrazide (8b). This
compound was obtained as white crystals and recrystallized from
ethanol, Yield (76%); mp 190–191 ꢃC; IR (KBr) ( , cmꢁ1): 3440-3277
n
(NH2,NH), 2919 (CH alicyclic), 2215 (CN), 1665 (C]O) and 1593
(C]N); 1H NMR (DMSO-d6):
d
(ppm) 1.27 (d, J ¼ 6.9 Hz, 6H, 2CH3),
4.2.2. Viability assay
1.76 (m, 4H, aliphatic 2CH2 of tetrahydronaphthalene), 2.77 (m, 4H,
aliphatic 2CH2 of tetrahydronaphthalene), 3.0 (m, 1H, CH), 4.27 (s,
2H, NH2 D2O exchangeable), 4.98 (s, 2H, OCH2), 7.38 (d, J ¼ 8.4 Hz,
1H, Ar-H), 7.47 (d, J ¼ 8.4 Hz, 2H, Ar-H), 7.6–7.9 (m, 5H Ar-H) and 9.4
(s, 1H, NH D2O exchangeable); MS m/z (%): 440.14 (Mþ, 55%), 368.22
(Mþ ꢁ C2H5N2O, 100%). Anal. calcd. for C27H28N4O2: C, 73.61; H,
6.41; N, 12.72. Found: C, 73.61; H, 6.41; N, 12.72.
The effect of different compounds on the viability of RAW 264.7
cells was estimated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) assay. The yellow tetra-
zolium salt of MTT is reduced by mitochondrial dehydrogenases in
metabolically active cells to form insoluble purple formazan crys-
tals, which are solubilized by the addition of a detergent; acidic
isopropanol [40]. RAW 264.7 (5 ꢄ104 cells/well) were incubated for
24 h) with various concentrations of the compounds at 37 ꢃC,
before submitting to MTT assay. The relative cell viability was
expressed as the mean percentage of viable cells compared with
untreated cells. Treatment of macrophage with 1000 U/ml
recombinant macrophage colony-stimulating factor (M-CSF, Pierce,
USA) was used as positive control.
4.1.7. General procedure for the synthesis of compounds (9a,b)
A solution of compound 7a or 7b (0.01 mol) in ethanol and
benzyl amine (0.02 mol) was refluxed for 6 h. The cooled solution
was poured into ice-cold dilute hydrochloric acid. The separated
solid was filtered off and recrystallized from an appropriate solvent
to afford the corresponding derivatives 9a,b, respectively.
4.2.3. Macrophage binding to bacterial lipopolysaccharide
4.1.7.1. N-Benzyl-2-[3-cyano-4-(3,4-dimethoxy-phenyl)-6-(5,6,7,8-
tetrahydronaphthalen-2-yl)-pyridin-2-yloxy]-acetamide (9a). This
compound was obtained as white crystals and recrystallized from
The binding of fluorescein isothiocyanate-conjugated bacterial
lipopolysaccharides (LPS-FITC) to RAW 264.7 cells was monitored
according to [41]. In brief, cells (5 ꢄ105) were pre-incubated in the
absence or presence of different compounds for 15 min at 37 ꢃC in
RPMI-1640 containing 10% FBS (as a source of CD14 and LPS-
binding protein to induce binding) before incubated for 1 h with
FITC-conjugated LPS (200ng/ml). After washing with phosphate
buffered saline (PBS), the binding of FITC-LPS was analyzed by
microplate fluorometer (FluoStarOptima, BMG Labtechnologies,
AcOH. Yield (73%); mp 198–200 ꢃC; IR (KBr) ( , cmꢁ1): 3279 (NH),
n
2924.5 (CH alicyclic), 2215 (CN), 1648.5 (C]O) and 1589 (C]N); 1H
NMR (DMSO-d6): (ppm) 1.76 (m, 4H, aliphatic 2CH2 of tetrahy-
d
dronaphthalene), 2.76 (m, 4H, aliphatic 2CH2 of tetrahydronaph-
thalene), 3.85 (s, 6H, OCH3), 4.33 (d, J ¼ 6 Hz, 2H, N–CH2), 5.0 (s, 2H,
OCH2), 7.1–7.79 (m, 12H Ar-H) and 8.7 (s, 1H, NH, D2O exchange-
able); MS m/z (%): 533 (Mþ, 3%), 387 (Mþ ꢁ ph-CH2NHCOCH, 100%).
Anal. calcd. for C33H31N3O4: C, 74.28; H, 5.86; N, 7.87. Found: C,
74.35; H, 5.72; N, 7.99.
NC, USA). The fluorescence was measured at excitation
l of 485 nm
and an emission of 530 nm, and median fluorescence intensity,
l
international fluorescence unit (IFU), was determined.
4.1.7.2. N-Benzyl-2-[3-cyano-4-(4-isopropyl-phenyl)-6-(5,6,7,8-tetrahy-
dronaphthalen-2-yl)-pyridin-2-yloxy]-acetamide (9b). This compound
was obtained as white crystals and recrystallized from ethanol. Yield
4.2.4. Phagocytosis assay
We examined the phagocytic activity of macrophages using
FITC-conjugated zymosan according to [42]. RAW 264.7 cells were
plated in a cell density of 1 ꢄ105 cells/well in phenol red-free RPMI-
1640 and incubated for 24 h at 37 ꢃC in humidified air containing
5% CO2. Cells were incubated for 1 h with compounds. Twenty
microlitres of FITC-zymosan (Molecular Probes, USA) were added
and re-incubated for 1 h. After washing with medium for three
(80%); mp 188–189 ꢃC; IR (KBr) ( , cmꢁ1): 3294.6 (NH), 2922.5 (CH
n
alicyclic), 2218 (CN), 1647.5 (C]O) and 1590 (C]N); 1H NMR (DMSO-
d6):
d
(ppm) 1.26 (d, J ¼ 7.2 Hz, 6H, 2CH3), 1.75 (m, 4H, aliphatic 2CH2 of
tetrahydronaphthalene), 2.75 (m, 4H, aliphatic 2CH2 of tetrahy-
dronaphthalene), 3.0 (m, 1H, CH), 4.33 (d, J ¼ 5.7 Hz, 2H, N-CH2), 5.0 (s,
2H, OCH2), 7.09–7.78 (m, 12H, Ar-H), 7.93 (s, 1H, pyridine-H), and 8.7 (s,
1H, NH D2O exchangeable); MS m/z (%): 515 (Mþ, 5%),
369.17(Mþ ꢁ C9H9NO, 100%). Anal. calcd. for C34H33N3O2: C, 79.19; H,
6.45; N, 8.15. Found: C, 79.00; H, 6.56; N, 8.08.
times, 100
shacked. The fluorescence was measured at excitation
and an emission of 530 nm.
ml of triton X-100 (10%) were added, and vigorously
l
of 485 nm
l
4.2.5. Nitrite assay
The accumulation of nitrite, an indicator of nitric oxide (NO)
synthesis, was measured by Griess reagent [43]. RAW 264.7 were
grown in phenol red-free RPMI-1640 containing 10% FBS. In the
first experiment, cells were incubated for 24 h with compounds
without LPS, while in the second experiment; cells were incubated
4.2. Effect of synthesized compounds on inflammatory mediators
4.2.1. Cell culture
Murine raw macrophage cell line (RAW 264.7, ATCC, USA) was
routinely cultured in RPMI-1640 supplemented with 10% fetal
for 24 h with LPS (1 mg/ml) in the presence or absence of different

4556
N.A. Hamdy, A.M. Gamal-Eldeen / European Journal of Medicinal Chemistry 44 (2009) 4547–4556
compounds. Fifty microlitres of cell culture supernatant were
mixed with 50 l of Griess reagent and incubated for 10 min. The
absorbance was measured spectrophotometrically at 550 nm.
A standard curve was blotted using serial concentrations of sodium
nitrite. The nitrite content was normalized to the cellular protein
content as measured by bicinchoninic acid assay [44].
variance (ANOVA) test followed by the Tukey post hoc test was used
to detect the statistical significance. p value more than 0.05 was
considered insignificant.
m
Acknowledgements
This work was supported by the Center of Excellence for
Advanced Sciences, National Research Center, Dokki, Cairo, Egypt.
4.2.6. Determination of tumor necrosis factor-a and prostaglandin E2
RAW 264.7 cells were incubated for 24 h with compounds
without LPS, and in another experiment; cells were incubated for
References
24 h with LPS (1
compounds. TNF-
m
a
g/ml) in the presence or absence of different
and prostaglandin E2 (PGE2) were determined
[1] M. Philip, D.A. Rowley, H. Schreiber, Semin. Cancer Biol. 14 (2004) 433–439.
[2] L.M. Coussens, Z. Werb, Nature 420 (2002) 860–867.
[3] C. Nathan, Nature 420 (2002) 846–852.
[4] M.C. Maiuri, G. Tajana, T. Iuvone, Am. J. Pathol. 165 (2004) 115–126.
[5] B.D. Levy, C.B. Clish, B. Schmidt, K. Gronert, C.N. Serhan, Nat. Immunol. 2
(2001) 612–619.
in the harvested supernatants using commercial kits (Endogen Inc.,
MA, USA) and (Cayman Chemical, MI, USA), respectively, according
to the manufacturer protocols.
4.2.7. Estimation of COX-2 and 5-LO by dot immunoblotting
RAW 264.7 cells were incubated for 24 h with compounds
without LPS, and in another experiment; cells were incubated for
[6] J. Hodge-Dufour, M.W. Marino, M.R. Horton, Proc. Natl. Acad. Sci. USA 95
(1998) 13806–13811.
[7] J. Savill, A.H. Wyllie, J.E. Henson, M.J. Walport, P.M. Henson, C. Haslett, J. Clin.
Invest. 83 (1989) 865–875.
24 h with LPS (1
compounds. Lysates, extracted from macrophages, were used for
the assessment of COX-2 and 5-LO. Briefly, 20 g of isolated soluble
mg/ml) in the presence or absence of different
[8] J. Savill, V.A. Fadok, Nature 407 (2000) 784–788.
[9] J. Savill, I. Dransfield, C. Gregory, C. Haslett, Nat. Rev. Immunol. 2 (2002) 965–975.
[10] V.A. Fadok, D.L. Bratton, A. Konowal, P.W. Freed, J.Y. Westcott, P.M. Henson, J.
Clin. Invest. 101 (1998) 890–898.
m
proteins were applied at dot blotting set using nitrocellulose
membranes. Membranes were blocked and then incubated with
rabbit anti-mouse COX-2 (# ab6665) or rabbit anti-mouse 5-LO
[11] P.P.McDonald,V.A.Fadok,D.Bratton,P.M.Henson,J.Immunol.163(1999)6164–6172.
[12] M.L.N. Huynh, V.A. Fadok, P.M. Henson, J. Clin. Invest. 109 (2002) 41–50.
[13] M. Macarthur, G.L. Hold, E.M. El-Omar, Am. J. Physiol. Gastrointest. Liver
Physiol. 286 (2004) G515–G520.
[14] Henry G.D., Tetrahedron 60 (2004) 6043.
[15] M.C. Bagley, K. Chapaneri, D.W. Dale, X. Xiong, J. Bower, J. Org. Chem. 70 (2005)
1389–1396.
[16] T.L. Gilchrist, J. Chem. Soc., Perkin Trans. (2001) 2491–2497.
[17] A.H. Li, S. Moro, N. Forsyth, N. Melman, X.-D. Ji, K.A. Jacobsen, J. Med. Chem. 42
(1999) 706–721.
[18] B. Vacher, B. Bonnand, F. Funes, N. Jubault, W. Koek, M.-B. Assie, C. Cosi,
M. Kleven, J. Med. Chem. 42 (1999) 1648–1655.
[19] J.K. Son, L.X. Zhao, A. Basnet, P. Thapa, R. Karki, Y. Na, Y. Jahng, T.C. Jeong,
B.S. Jeong, C.S. Lee, E.S. Lee, Eur. J. Med. Chem. 43 (2008) 675–682.
[20] A.G. Amr, M.M. Abdulla, Bioorg. Med. Chem. 14 (2006) 4341–4352.
[21] A.K. Dutta, X. Fei, M.E.A. Reith, Bioorg. Med. Chem. Lett. 12 (2002) 619–622.
[22] P. Taddei, A. Torreggiani, G. Fini, Biopolymers 67 (2002) 289–293.
[23] H. Hatori, B. Sato, I. Sato, T. Shibata, Y. Tsurumi, K. Sakamoto, S. Takase,
H. Veda, M. Hino, T. Fujii, J. Antibiot. 57 (2004) 264–270.
[24] J.H. Song, A.J. Slot, R.W.J. Ryan, G.M. Ross, Neuropharmacology 46(2004) 984–993.
[25] A.G. Hammam, N.A. Abdel Hafez, W.H. Midura, M.Z. Mikolajczyk, Z. Natur-
forsch. 55 (2000) 417–423.
(# ab59341) and
UK).
b-actin proteins (# ab1801, Abcam, Cambridge,
After being washed, bound antibody was detected using goat
anti-rabbit antibody linked to horseradish peroxidase (# ab6721,
Abcam, Cambridge, UK) and bound complexes were detected using
O-phenylenediamine dihydrochloride (OPD). The percentage of the
relative enhanced color intensity was normalized to dot intensity
with
b-actin control. The dot photographing and analysis was
performed using gel documentation system (Biometra, Goettingen,
Germany).
4.2.8. Oxygen radical absorbance capacity
The oxygen radical absorbance capacity (ORAC) of tested
compounds against peroxyl, and hydroxyl (OHꢀ) radicals, was
[26] M. Abo-Ghalia, M.M.Z. Abdulla, A.E. Amr, Z. Naturforsch. 58 (2003) 903–912.
[27] P. Dorigo, R.M. Gaion, P. Beiiuco, D. Fraccarollo, I. Maragno, G. Bombieri,
F. Benetollo, L. Mosti, F. Orsini, J. Med. Chem. 36 (1993) 2475–2484.
[28] M.M. Ismail, Y.A. Ammar, H.S. El-Zahaby, S.I. Eisa, E.S. Barakat, Arch. Pharm.
(Weinheim) 340 (2007) 476–482.
[29] Z. Lu, G.R. Ott, R. Anand, R.Q. Liu, M.B. Covington, K. Vaddi, M. Qian,
R.C. Newton, D.D. Christ, J. Trzaskos, J.J. Duan, Bioorg. Med. Chem. Lett. 18
(2008) 1958–1962.
investigated by ORAC assay [45] as modified at [46]. b-Phycoery-
thrin was used as a radical-sensitive fluorescent indicator protein,
2,2,-azobis-(2-amidinopropane) dihydrochloride (AAPH) was used
as a peroxyl radical (ROOꢀ) initiator, and a mixture of H2O2–CuSO4
was used as a hydroxyl radical generator. A final concentration of
each compound (1 mM) was used, and the reaction was initiated by
[30] N.L. Allinger, E.S. Jones, J. Org. Chem. 27 (1962) 70–73.
[31] M. Girotti, J.H. Evans, D. Burke, C.C. Leslie, J. Biol. Chem. 279 (2004) 19113–19121.
[32] H. Lu, W. Ouyang, C. Huang, Mol. Cancer Res. 4 (2006) 221–233.
[33] R.M. Locksley, N. Killeen, M.J. Lenardo, Cell 104 (2001) 487–501.
[34] F. Balkwill, Cytokine Growth Factor Rev. 13 (2002) 135–141.
[35] C. Huang, J. Li, W.Y. Ma, Z. Dong, J. Biol. Chem. 274 (1999) 29672–29676.
[36] M. Jaiswal, N.F. LaRusso, L.J. Burgart, G.J. Gores, Cancer Res. 60 (2000) 184–190.
[37] S. Wu, C.M. Boyer, R.S. Whitaker, Cancer Res. 53 (1993) 1939–1944.
[38] B. Samuelsson, R. Morgenstern, P.J. Jakobsson, Pharmacol. Rev. 59 (2007) 207–224.
[39] V.E. Steele, C.A. Holmes, E.T. Hawk, L. Kopelovich, R.A. Lubet, J.A. Crowell,
C.C. Sigman, G.J. Kelloff, Cancer Epidemiol. Biomarkers Prev. 8 (1999) 467–483.
[40] M.B. Hansen, S.E. Nielsen, K. Berg, J. Immunol. Methods 119 (1989) 203–210.
[41] J. Carracedo, R. Ramirez, A. Martin-Malo, M. Rodriguez, P. Aljama, Nephrol.
Dial. Transplant. 17 (2002) 428–434.
[42] K.I. Kim, K.S. Shin, W.J. Jun, B.S. Hong, D.H. Shin, Biosci. Biotechnol. Biochem.
65 (2001) 2369–2377.
[43] C. Gerha¨user, K. Klimo, E. Heiss, I. Neumann, A. Gamal-Eldeen, Mutat. Res.
523–524 (2003) 163–172.
[44] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, Anal. Biochem.
150 (1985) 76–85.
addition of one of the radical generators. The decay of b-phycoer-
ythrin fluorescence was measured kinetically, at 37 ꢃC for 2 h using
a microplate fluorescence reader. One ORAC unit equals the net
protection of b-phycoerythrin produced by 1.0 mM Trolox, a known
standard antioxidant.
4.2.9. Scavenging of superoxide anion radicals
ꢀ
Superoxide anion radicals (O2ꢁ ) were generated by the oxidation
of 50
m
M hypoxanthine to uric acid by 12 mU xanthine oxidase
ꢀ
(XO), in a non cellular assay. Scavenging of Oꢁ2 was quantified by
a method based on [47] and modified by [48], which depends on
the reduction rate of nitroblue tetrazolium to dark-blue formazan.
The half-maximal scavenging concentration SC50 was calculated
from serial dilutions of each compound, and superoxide dismutase
was used as a standard inhibitor.
[45] G. Cao, R.L. Prior, Methods Enzymol. 299 (1999) 50–62.
[46] A. Gamal-Eldeen, S. Kawashty, L. Ibrahim, M. Shabana, S. Negoumy, J. Nat.
Remedies 4 (2004) 81–96.
[47] H. Ukeda, S. Maeda, T. Ishii, M. Sawamura, Anal. Biochem. 251 (1997) 206–209.
[48] C. Gerha¨user, A. Alt, E. Heiss, A. Gamal-Eldeen, K. Klimo, et al., Mol. Cancer
Ther. 1 (2002) 959–969.
4.2.10. Statistical analysis
Data were statistically analyzed using Statistical Package for
Social Scientists (SPSS) 10.00 for windows (SPSS Inc., Chicago, USA).
The student’s unpaired t-test as well as the one-way analysis of