Synthesis and biological evaluation of novel pyrazoline derivatives as anti-inflammatory and antioxidant agents

Article abstract of DOI:10.1007/s12272-012-0606-9

A series of novel 5-aryl-3-cyclopropyl-4,5-dihydropyrazole derivatives 2a-p were synthesized via cyclization of chalcones 1a-h with thiosemicarbazide or semicarbazide HCl and evaluated as anti-inflammatory/antioxidant agents. The structures were confirmed

Full text of DOI:10.1007/s12272-012-0606-9

Arch Pharm Res Vol 35, No 6, 995-1002, 2012
DOI 10.1007/s12272-012-0606-9
Synthesis and Biological Evaluation of Novel Pyrazoline Derivatives
as Anti-inflammatory and Antioxidant Agents
Nadia A. Khalil1, Eman M. Ahmed1, Hala B. El-Nassan1, Osama K. Ahmed2, and Ahmed M. Al-Abd3
1Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt, 2Biochemistry Department, Faculty
3
of Agriculture, Cairo University, Cairo, Egypt, and Department of Pharmacology, Medical Division, National Research
Center, Dokki, Cairo, Egypt
(Received October 9, 2011/Revised January 15, 2012/Accepted January 25, 2012)
A series of novel 5-aryl-3-cyclopropyl-4,5-dihydropyrazole derivatives 2a
-p were synthesized
via cyclization of chalcones 1a with thiosemicarbazide or semicarbazide HCl and evaluated
-
h
as anti-inflammatory/antioxidant agents. The structures were confirmed by elemental analy-
ses and spectral data. The free radical scavenging activity toward superoxide was determined.
Their effect on hepatocytes viability and nitric oxide (NO) production in LPS-stimulated mac-
rophages was also determined. The results showed that compounds 2e and 2n demonstrated
the highest free-radical scavenging and anti-inflammatory activities, thus can be useful in the
prevention of oxidative stress and inflammation-related disorders.
Key words: Pyrazoline, Anti-inflammatory, Antioxidant, Free-radical scavenging
important role in inflammatory conditions by interact-
ing with pro-inflammatory cytokines. The over-produced
INTRODUCTION
Oxidation and inflammation are two major factors in- pro-inflammatory cytokines may lead to inflammation,
volved in the progression of a wide variety of patholo- enhance systemic inflammatory stress and also pro-
gical conditions including cardiovascular diseases mote the deterioration of cardiac and/ or renal dysfunc-
(Leopold and Loscalzo, 2009), inflammatory conditions tions (Drimal et al., 2008). In this respect, disease pro-
(Halliwell, 1994), atherosclerosis (Cook and Samman, gression can be retarded or suppressed by administer-
1996), neurodegeneration, aging (Halliwell, 2001), and ing protective compounds such as free radical scaven-
cancer (Halliwell et al., 2000). Free radicals, especially gers (Papas, 1999). This had attracted a great deal of
reactive oxygen species (ROS) are generated either research interest in therapeutic antioxidant-based drug
exogenously from oxidative injuries related to pollu- formulations. Thus, the development of synthetic com-
tion, radiation and food constituents (Foote, 1976) or pounds capable of scavenging free radicals has been of
endogenously in the human body from the metabolic great importance.
reactions, in addition to oxidative stress (Nohl et al.,
Recently, the ∆
2-pyrazoline derivatives have raised a
2005). Overproduction of these species, such as hydroxyl great interest because of their multiple pharmacolo-
radical (•OH), hydrogen peroxide (H2O2), superoxide gical applications such as antioxidant (Dora Carrión
anions (O2•−), nitric oxide (NO•), nitrosonium (NO+), et al., 2004; Ohyama et al., 2006), anti-inflammatory
and nitroxyl anion (NO−), as well as peroxylnitrite, (Rathish et al., 2009; Fioravant et al., 2010), anti-
may contribute to the immunopathological phenomena microbial (Abdel-Wahab et al., 2009), antidepressant
related to oxidative stress (Awah et al., 2010). Fur- (Kaplanciki et al., 2010), anticancer (Shaharyar et al.,
thermore, it has been established that ROS play an 2010), and anti-Alzheimer agents (Gökhan-Kelekci et
al., 2007). However, little data have been published on
the mechanism of action of pyrazoline derivatives as
antioxidant or anti-inflammatory agents (Dora Carrión
et al., 2004).
Correspondence to: Eman M. Ahmed, Organic Chemistry Depart-
ment, Faculty of Pharmacy, Cairo University, Cairo, Egypt
Tel: 201005270276
E-mail: dr_eman2001@hotmail.com
In the present work, a novel series of 5-aryl-3-cyclo-
995

996
N. A. Khalil et al.
Scheme 1. General synthetic pathways of derivatives 2a-p
propyl-4,5-dihydro-1
and 5-aryl-3-cyclopropyl-4,5-dihydro-1
carboxamides 2i were synthesized and evaluated as
anti-inflammatory/ antioxidant agents.
MATERIALS AND METHODS
Chemistry
H
-pyrazole-1-carbothioamides 2a
-
General procedure for the synthesis of 5-aryl-
3-cyclopropyl-4,5-dihydro-1H-pyrazole-1-car-
bothioamides (2a-h) and 5-aryl-3-cyclopropyl-
4,5-dihydro-1H-pyrazole-1-carboxamides (2i-p)
h
H-pyrazole-1-
-p
To a solution of an appropriate chalcone 1a-h (0.01
mol) and thiosemicarbazide or semicarbazide HCl
(0.012 mol) in ethanol (25 mL), was added a solution
of sodium hydroxide (1 g, 0.025 mol) in water (5 mL),
Melting points were determined on Griffin appar- then the mixture was heated under reflux for 8 h. The
atus and the values given are uncorrected. IR spectra products were poured into crushed ice and the separ-
were determined on Shimadzu IR 435 spectropho- ated solid was filtered, dried and crystallized from
tometer (KBr, cm−1). 1H-NMR spectra were carried out ethanol.
using a Varian Gemini 200 MHz Spectrophotometer
and Varian Mercury-300 (300 MHz) Spectrophoto- 3-Cyclopropyl-5-phenyl-4,5-dihydro-1
H-pyrazole-
meter using TMS as internal standard. Chemical shift 1-carbothioamide (2a)
values are recorded in ppm on
Center, Cairo University, Egypt. Mass spectra were 3143 (C-H aromatic), 2900, 2850 (C-H aliphatic), 1593
δ
scale, Microanalytical Yield 85%; mp 142-143oC; IR (cm−1): 3387, 3259 (NH2),
1
recorded on a GCMP-QP1000 EX Mass spectrometer, (C=N), 1350 (C=S); H-NMR (200 MHz, DMSO-d6):
Microanalytical Center, Cairo University, Egypt. Elem- (ppm) 0.82-0.90 (m, 4H, 2CH2), 1.72-1.88 (m, 1H, CH),
ental analyses were carried out at the Microanalytical 2.39 (dd, 1H, Ha), 3.48 (dd, 1H, Hb, Jab = 18 Hz, Jbx
δ
=
Center, Cairo University, Egypt. Progress of the reac- 10.8 Hz), 5.75 (dd, 1H, Hx), 7.04-7.34 (m, 5H, Ar-H),
tions was monitored using TLC sheets precoated with 7.39 (br s, 1H, NH, D2O exchangeable), 7.73 (s, 1H,
UV fluorescent silica gel Merck 60F 254 using acetone/ NH, D2O exchangeable); Anal. Calcd for C13H15N3S: C,
benzene (1: 9) and were visualized using UV lamp.
63.64; H, 6.16; N, 17.13; Found: C, 63.85; H, 6.44; N,
All chemicals were obtained from Aldrich, Fluka, or 17.05.
Merck chemicals.
1-Cyclopropyl-3-phenyl-2-propen-1-ones (1a
prepared according to reported procedure (Osman et pyrazole-1-carbothioamide (2b)
al., 2003).
Yield 82%; mp 153-155ºC; IR(cm−1): 3437, 3248 (NH2),
-h) were 5-(2-Bromophenyl)-3-cyclopropyl-4,5-dihydro-1H-

Pyrazoline Anti-inflammatory and Antioxidant Agents
997
3147 (C-H aromatic), 2950, 2855 (C-H aliphatic), 1593 3-Cyclopropyl-5-(4-
N
,
N
-dimethylaminophenyl)-4,
(C=N), 1361 (C=S); 1H-NMR (200 MHz, DMSO-d6 ):
δ
5-dihydro-1H-pyrazole-1-carbothioamide (2f)
(ppm) 0.84-0.87 (m, 4H, 2CH2), 1.73-1.85 (m, 1H, CH), Yield 93%, mp 159-160ºC; IR (cm−1): 3410, 3251 (NH2),
2.32 (dd, 1H, Ha, Jab = 17.9 Hz, Jax = 3.3 Hz), 3.46 (dd, 3147 (C-H aromatic), 2900, 2812 (C-H aliphatic), 1600
1H, Hb, Jab = 17.9 Hz, Jbx = 11.4 Hz), 5.88 (dd, 1H, Hx, (C=N), 1361 (C=S); 1H-NMR (200 MHz, DMSO-d6 ):
δ
Jax = 3.3 Hz, Jbx = 11.4 Hz), 6.82-8.49 (m, 4H, Ar-H), (ppm) 0.85-0.92 (m, 4H, 2CH2), 2.48-2.53 (m, 1H, CH),
7.86 (br s, 1H, NH, D2O exchangeable), 8.10 (br s, 1H, 2.95 (s, 6H, 2CH3), 2.97 (dd, 1H, Ha), 3.47 (dd, 1H, Hb),
NH, D2O exchangeable); Anal. Calcd for C13H14BrN3S: 6.67 (m, 1H, Hx), 7.47-7.59 (m, 4H, Ar-H), 7.75 (br s,
C, 48.16; H, 4.35; N, 12.96; Found: C, 48.50; H, 4.25; 1H, NH, D2O exchangeable), 7.98 (br s, 1H, NH, D2O
N, 13.05.
exchangeable); Anal. Calcd for C15H20N4S: C, 62.47; H,
6.99; N, 19.43; Found: C, 62.32; H, 7.25; N, 19.75.
5-(4-Bromophenyl)-3-cyclopropyl-4,5-dihydro-1
H-
pyrazole-1-carbothioamide (2c)
3-Cyclopropyl-5-(4-fluorophenyl)-4,5-dihydro-1H-
Yield 87%; mp 200-201ºC; IR(cm−1): 3379, 3259 (NH2), pyrazole-1-carbothioamide (2g)
3143 (C-H aromatic), 2955, 2835 (C-H aliphatic), 1593 Yield 75%; mp 195-196ºC; IR (cm−1): 3383, 3255 (NH2),
1
(C=N), 1363 (C=S); H-NMR (300 MHz, DMSO-d6):
δ
3143 (C-H aromatic), 2900, 2812 (C-H aliphatic), 1593
(ppm) 0.80-0.90 (m, 4H, 2CH2), 1.77-1.83 (m, 1H, CH), (C=N), 1361 (C=S); 1H-NMR (200 MHz, DMSO-d6):
δ
2.45 (dd, 1H, Ha, Jab = 18.75 Hz, Jax = 3.3 Hz), 3.45 (dd, (ppm) 0.85-0.93 (m, 4H, 2CH2), 1.78-1.95 (m, 1H, CH),
1H, Hb, Jab = 18.75 Hz, Jbx = 11.4 Hz), 5.68 (dd, 1H, Hx, 2.45 (dd, 1H, Ha, Jab = 18 Hz, Jax = 3 Hz), 3.44 (dd, 1H,
Jax = 3.3 Hz, Jbx = 11.4 Hz), 7.01 (d, 2H, Ar-H,
Hz), 7.38 (br s, 1H, NH, D2O exchangeable), 7.48 (d, 3 Hz, Jbx = 11.2 Hz), 7.12-7.16 (m, 4H, Ar-H), 7.44 (s,
2H, Ar-H, = 8.1 Hz), 7.75 (br s, 1H, NH, D2O ex- 1H, NH, D2O exchangeable), 7.79 (s, 1H, NH, D2O
changeable); Anal. Calcd for C13H14BrN3S: C, 48.16; exchangeable); MS (EI) m/z (% rel. Int.): 263 (M+,
J = 8.1 Hb, Jab = 18 Hz, Jbx = 11.2 Hz), 5.73 (dd, 1H, Hx, Jax =
J
H, 4.35; N, 12.96; Found: C, 48.35; H, 4.58; N, 12.88.
1.66), 60 (CSNH2, 100); Anal. Calcd for C13H14FN3S:
C, 59.29; H, 5.36; N, 15.96; Found: C, 59.45; H, 5.12;
N, 15.78.
5-(2-Chlorophenyl)-3-cyclopropyl-4,5-dihydro-1
H-
pyrazole-1-carbothioamide (2d)
Yield 72%; mp 148-149ºC; IR (cm−1): 3417, 3250 (NH2), 3-Cyclopropyl-5-(4-methoxyphenyl)-4,5-dihydro-
3145 (C-H aromatic), 2916, 2850 (C-H aliphatic), 1589
1
H
-pyrazole-1-carbothioamide (2h)
Yield 89%; mp 164-165oC; IR (cm−1): 3379, 3251 (NH2),
(ppm) 0.78-0.89 (m, 4H, 2CH2), 1.76-1.81 (m, 1H, CH), 3143 (C-H aromatic), 2951, 2831 (C-H aliphatic), 1593
2.35 (dd, 1H, Ha, Jab = 18 Hz, Jax = 3.3 Hz), 3.46 (dd, (C=N), 1361 (C=S); 1H-NMR (200 MHz, DMSO-d6):
1
(C=N), 1365 (C=S); H-NMR (300 MHz, DMSO-d6 ):
δ
δ
1H, Hb, Jab = 18 Hz, Jbx = 11.4 Hz), 5.92 (dd, 1H, Hx, (ppm) 1.22-1.38 (m, 4H, 2CH2), 2.14-2.16 (m, 1H, CH),
Jax = 3.3 Hz, Jbx = 11.4 Hz), 6.86-7.48 (m, 4H, Ar-H), 2.89 (dd, 1H, Ha, Jab = 17.9 Hz, Jax = 3 Hz), 3.67 (dd,
7.82 (br s, 1H, NH, D2O exchangeable), 8.10 (br s, 1H, 1H, Hb, Jab = 17.9 Hz, Jbx = 11 Hz), 4.17 (s, 3H, OCH3),
NH, D2O exchangeable); Anal. Calcd for C13H14ClN3S: 6.21 (dd, 1H, Hx, Jax = 3 Hz, Jbx = 11 Hz), 7.26 (d, 2H,
C, 55.81; H, 5.04; N, 15.02; Found: C, 55.65; H, 5.28; Ar-H,
J = 8.4 Hz), 7.45 (d, 2H, Ar-H, J = 8.4 Hz), 7.40
N, 14.85.
(s, 1H, NH, D2O exchangeable), 7.79 (s, 1H, NH, D2O
exchangeable); MS (EI) m/z (% rel. Int.): 275 (M+,
60.82), 56 (100); Anal. Calcd for C14H17N3OS: C, 61.06;
H, 6.22; N, 15.26; Found: C, 61.35; H, 6.12; N, 15.55.
5-(4-Chlorophenyl)-3-cyclopropyl-4,5-dihydro-1
H-
pyrazole-1-carbothioamide (2e)
Yield 88%; mp 189-190ºC; IR (cm−1): 3381, 3257 (NH2),
3145 (C-H aromatic), 2900, 2850 (C-H aliphatic), 1593 3-Cyclopropyl-5-phenyl-4,5-dihydro-1
(C=N), 1363 (C=S); 1H-NMR (200 MHz, DMSO-d6 ):
1-carboxamide (2i)
(ppm) 0.82-0.90 (m, 4H, 2CH2), 1.72-1.88 (m, 1H, CH), Yield 84%; mp 184-185oC; IR (cm−1): 3460, 3282 (NH2),
2.40 (dd, 1H, Ha), 3.47 (dd, 1H, Hb, Jab = 18.5 Hz, Jbx 3190 (C-H aromatic), 2935, 2862 (C-H aliphatic), 1651
11.0 Hz), 5.69 (dd, 1H, Hx), 7.05 (d, 2H, Ar-H,
= 8.2 (C=O), 1597 (C=N); 1H-NMR (200 MHz, DMSO-d6 ):
Hz), 7.34 (d, 2H, Ar-H, = 8.2 Hz), 7.43 (br s, 1H, NH, (ppm) 0.75-0.83 (m, 4H, 2CH2), 1.70-1.83 (m, 1H, CH),
H-pyrazole-
δ
=
J
δ
J
D2O exchangeable), 7.78 (br s, 1H, NH, D2O 2.43 (dd, 1H, Ha, Jab = 17.9 Hz, Jax = 5.3 Hz), 3.20-3.29
exchangeable); MS (EI) m/z (% rel. Int.): 281 (M+2, (m, 1H, Hb), 5.18 (dd, 1H, Hx, Jax = 5.3 Hz, Jbx = 11.8
21.68), 279 (M+, 66.58), 60 (CSNH2, 100). Anal. Calcd Hz), 6.50 (s, 2H, NH2, D2O exchangeable), 7.10-7.85
for C13H14ClN3S: C, 55.81; H, 5.04; N, 15.02; Found: C, (m, 5H, Ar-H); Anal. Calcd for C13H15N3O: C, 68.10; H,
55.45; H, 5.25; N, 15.34.
6.59; N, 18.33; Found: C, 68.35; H, 6.22; N, 18.60.

998
N. A. Khalil et al.
5-(2-Bromophenyl)-3-cyclopropyl-4,5-dihydro-1
pyrazole-1-carboxamide (2j)
H-
(C=O), 1604(C=N); 1H-NMR (300 MHz, DMSO-d6):
(ppm) 0.85-0.90 (m, 4H, 2CH2), 1.77-1.84 (m, 1H, CH),
δ
Yield 78%, mp 214-215ºC; IR (KBr): 3471, 3298 (NH2), 2.67 (dd, 1H, Ha), 2.93 (s, 6H, 2CH3); 3.15 (dd, 1H, Hb),
3159 (C-H aromatic), 2931, 2850 (C-H aliphatic), 1654 5.42 (dd, 1H, Hx), 6.28 (s, 2H, NH2, D2O exchangeable),
(C=O), 1600 (C=N); 1H-NMR (200 MHz, DMSO-d6 ):
δ
6.67 (dd, 2H, Ar-H), 7.47 (dd, 2H, Ar-H); MS (EI) m/z (%
(ppm) 0.72-0.85 (m, 4H, 2CH2), 1.72-1.80 (m, 1H, CH), rel. Int.): 272 (M+, 35.02). Anal. Calcd for C15H20N4O: C,
2.42 (dd, 1H, Ha), 3.15 (dd, 1H, Hb), 5.45 (dd, 1H, Hx), 66.15; H, 7.40; N, 20.57; Found: C, 66.35; H, 7.58; N,
6.59 (s, 2H, NH2, D2O exchangeable), 7.23-8.20 (m, 20.22.
4H, Ar-H); Anal. Calcd for C13H14BrN3O: C, 50.67; H,
4.58; N, 13.64; Found: C, 50.35; H, 4.70; N, 13.77.
3-Cyclopropyl-5-(4-fluorophenyl)-4,5-dihydro-1
H-
pyrazole-1-carboxamide (2o)
5-(4-Bromophenyl)-3-cyclopropyl-4,5-dihydro-1
H
-
Yield 72%; mp 214-215ºC; IR (KBr): 3460, 3282 (NH2),
3163 (C-H aromatic), 2989, 2850 (C-H aliphatic), 1647
pyrazole-1-carboxamide (2k)
Yield 80%, mp 159-160ºC; IR (KBr): 3464, 3437 (NH2), (C=O), 1604 (C=N); 1H-NMR (200 MHz, DMSO-d6):
δ
3155 (C-H aromatic), 2950, 2855 (C-H aliphatic), 1666 (ppm) 0.78-0.90 (m, 4H, 2CH2), 1.72-1.76 (m, 1H, CH),
(C=O), 1585 (C=N); 1H-NMR (300 MHz, DMSO-d6):
2.44 (dd, 1H, Ha), 3.46 (dd, 1H, Hb), 5.20 (dd, 1H, Hx),
(ppm) 0.74-0.82 (m, 4H, 2CH2), 1.70-1.82 (m, 1H, CH), 6.54 (s, 2H, NH2, D2O exchangeable), 7.23 (dd, 2H, Ar-
δ
2.43 (dd, 1H, Ha), 3.27 (dd, 1H, Hb), 5.19 (dd, 1H, Hx), H,
J = 8.6 Hz), 7.80 (dd, 2H, Ar-H, J = 8.6 Hz); MS
6.49 (s, 2H, NH2, D2O exchangeable), 7.06-7.80 (m, (EI) m/z (% rel. Int.): 247 (M+, 15.4); Anal. Calcd for
4H, Ar-H); MS (EI) m/z (% rel. Int.): 309 (M+2, 46.34), C13H14FN3O: C, 63.15; H, 5.71; N, 16.99; Found: C,
307 (M+, 48.36), 265 (81.30), 263 (78.60); Anal. Calcd 63.10; H, 5.55; N, 17.23.
for C13H14BrN3O: C, 50.67; H, 4.58; N, 13.64; Found:
C, 50.33; H, 4.84; N, 13.42.
3-Cyclopropyl-5-(4-methoxyphenyl)-4,5-dihydro-
1H-pyrazole-1-carboxamide (2p)
5-(2-Chlorophenyl)-3-cyclopropyl-4,5-dihydro-1
H
-
Yield 86%; mp 194-195ºC; IR (KBr): 3452, 3282 (NH2),
3163 (C-H aromatic), 2954, 2858 (C-H aliphatic), 1647
pyrazole-1-carboxamide (2l)
Yield 68%; mp 219-220ºC; IR (KBr): 3468, 3278 (NH2), (C=O), 1604 (C=N); 1H-NMR (200 MHz, DMSO-d6):
δ
3159 (C-H aromatic), 2931, 2854 (C-H aliphatic), 1658 (ppm) 0.78-0.90 (m, 4H, 2CH2), 1.72-1.76 (m, 1H, CH),
(C=O), 1600 (C=N); 1H-NMR (300 MHz, DMSO-d6):
2.62 (dd, 1H, Ha), 3.25 (dd, 1H, Hb), 3.78 (s, 3H, OCH3),
(ppm) 0.72-0.81 (m, 4H, 2CH2), 1.70-1.75 (m, 1H, CH), 5.35 (dd, 1H, Hx), 6.41 (s, 2H, NH2, D2O exchangeable);
δ
2.36 (dd, 1H, Ha), 3.43 (dd, 1H, Hb), 5.40 (dd, 1H, Hx), 6.96 (dd, 2H, Ar-H,
J = 8.8 Hz), 7.67 (dd, 2H, Ar-H, J
6.54 (s, 2H, NH2, D2O exchangeable), 7.73-8.23 (m, = 8.8 Hz); MS (EI) m/z (% rel. Int.): 259 (M+, 28.11),
4H, Ar-H); MS (EI) m/z (% rel. Int.): 265 (M+2, 1.29), 215 (M-44, 100); Anal. Calcd for C14H17N3O2: C, 64.85;
263 (M+, 2.94), 228 (6.70), 219 (6.74); Anal. Calcd for H, 6.61; N, 16.20; Found: C, 64.50; H, 6.75; N, 16.42.
C13H14ClN3O: C, 59.21; H, 5.35; N, 15.93; Found: C,
59.52; H, 5.10; N, 16.15.
Pharmacological and biochemical tests
Superoxide radical scavenging assay
5-(4-Chlorophenyl)-3-cyclopropyl-4,5-dihydro-1
pyrazole-1-carboxamide (2m)
H-
The assay was based on the capacity of compounds
to inhibit formazan formation by scavenging the
Yield 85%; mp 171-172ºC; IR (KBr): 3302, 3276 (NH2), superoxide radicals generated in riboflavin-light-NBT
3105 (C-H aromatic), 2970, 2850 (C-H aliphatic), 1639 system (Beauchamp and Fridovich, 1971). Each 3 mL
1
(C=O), 1597 (C=N); H-NMR (300 MHz, CDCl3):
δ
reaction mixture contained 50 mM sodium phosphate
(ppm) 0.74-0.94 (m, 4H, 2CH2), 1.71-1.80 (m, 1H, CH), buffer (pH 7.6), 20 mg riboflavin, and 12 mM EDTA,
2.51 (dd, 1H, Ha), 3.27 (dd, 1H, Hb), 5.27 (s, 2H, NH2, 0.1 mg NBT and 1 mL sample solution. The reaction
D2O exchangeable), 5.30 (dd, 1H, Hx), 7.12 (d, 2H, Ar- was started by illuminating the reaction mixture with
H,
J = 8.4 Hz), 7.31 (d, 2H, Ar-H, J = 8.4 Hz); Anal. different concentrations of the sample (25-100 mg/mL)
Calcd for C13H14ClN3O: C, 59.21; H, 5.35; N, 15.93; for 90 sec. Immediately after illumination, the absor-
Found: C, 59.50; H, 5.15; N, 15.85.
bance was measured at 590 nm. The entire reaction
assembly was enclosed in a box lined with aluminum
3-Cyclopropyl-5-(4-N,N-dimethylaminophenyl)-4, foil. Identical tubes with the reaction mixture were
5-dihydro-1 -pyrazole-1-carboxamide (2n)
kept in the dark and served as blanks. The percentage
H
Yield 90%, mp 209-210ºC; IR (KBr): 3464, 3290 (NH2), inhibition of superoxide anion generation was calcu-
3167 (C-H aromatic), 2927, 2854 (C-H aliphatic), 1643 lated. The antioxidant activity of the synthesized com-

Pyrazoline Anti-inflammatory and Antioxidant Agents
999
pounds was measured in terms of superoxide radical MTT (0.5 mg/mL, 100
scavenging activity at different concentrations (10, 50 for 4 h, the medium was removed and 100
µ
L) was added. After incubation
µL of acidic
and 100 g/mL) (Table I). isopropanol (0.08 N HCl) was added to dissolve the
µ
formazan crystals. The absorbance was determined spec-
trophotometrically at 570 nm. Viability was defined as
In vitro anti-inflammatory activity
Macrophage cells were cultured in phenol red free the ratio (expressed as a percentage) of absorbance of
Dulbecco’s modified Eagle’s medium (DMEM) contain- treated cells to untreated cells that served as control
ing 50 units/mL penicillin, 50 mg/mL streptomycin, 44 (Table III, Fig. 1).
mM sodium bicarbonate and 10% fetal bovine serum
at 37oC in humidied air containing 5% CO2. Macro-
phage cells were plated in 1 mL of the aforementioned
RESULTS AND DISCUSSION
media in 24 well plates, cultured for 2 days to approxi-
mately 1 × 106 cells/well) then treated with 10
g/mL 1a
LPS and the test compounds at different concentra- condensation of cyclopropyl methyl ketone and the
tions (10, 50 and 100 g/mL). In all cases, cells were appropriate substituted aromatic aldehydes in ethanol
washed, and the fresh complete media was added before containing sodium hydroxide as a catalyst (Osman et
the indicated treatments. Stock solutions of each com- al. 2003). Cyclization of 1a with thiosemicarbazide
pound were prepared in DMSO so that the nal concen- or semicarbazide HCl afforded 2a and 2i , respect-
tration of DMSO did not exceed 0.5%. Six hours after ively.
LPS-treatment, the stable end products of L-arginine- The structures of the new compounds 2a
dependent NO synthesis, nitrate and nitrite were confirmed by elemental analyses and spectral data.
measured in the cell culture medium using Griess The IR spectra of the carbothioamide derivatives 2a
As shown in Scheme 1, the intermediate chalcones
µ
-h
have been synthesized by Claisen-Schmidt
µ
-h
-h
-p
-p were
-h
reaction, (Green et al., 1982) (Table II).
revealed a forked band in the region of 3437-3248 cm−1
corresponding to NH2 group as well as a band at 1365-
1350 cm−1 corresponding to C=S group. While, the IR
Hepatocyte viability assay
Liver cell culture preparation for viability test spectra of the carboxamide derivatives 2i-p demon-
The effect of the synthesized compounds on hepatocyte strated NH2 forked band at 3471-3276 cm−1 and a
viability were assessed in cultured cells (Moldeus et band in the region of 1666-1639 cm−1 corresponding to
al., 1978). Cells were grown in Dulbecco’s modified the carbonyl group. All new compounds, 2a-p displayed
Eagle’s medium (DMEM, Gibco, Invitrogen Corpora- absorption bands in the region around 1600 cm−1 corr-
tion) with glucose content of 4.5 g/L and supplemented esponding to C=N stretching because of ring closure.
with the following: inactivated fetal calf serum 10% (v/ Moreover, the formation of 2-pyrazoline ring was con-
1
v), non-essential amino acids (1%), glutamine (1%), firmed by the appearance of ABX system in H-NMR
penicillin (100 U/mL) and streptomycin (10 mg/mL). due to geminal-vicinal coupling between the two un-
The medium was adjusted at pH 7.4 and maintained equivalent protons of the methylene group Ha and Hb
in a humidified atmosphere containing CO2 (5%) at at C-4 and a methine proton Hx at C-5. The proton Ha
37oC. At 70-80% confluence, cells were trypsinized, which appeared as doublet of doublets around
δ 2.32-
centrifuged (250 × g for 5 min at 4oC), resuspended in 2.97 ppm is the proton trans to Hx and geminal to Hb.
fresh medium and plated in microtiter wells (2 × 104 The cis proton, Hb, vicinal to Hx appeared as doublet
cells/well). After attachment, they were incubated for of doublets in the range of
24 h at 37oC in serum-free medium containing differ- shifted proton around
5.18-6.67 ppm of methylene
ent concentrations of the test compounds (10, 50 and residue has been shown to be coupled with the vicinal
100 g/mL). methine proton (Hx), indicating the presence of trans
configuration. Besides, the amino protons appeared as
two exchangeable signals at 7.38-8.10 ppm in com-
The metabolic competence in viable cells was relied pounds 2a and as an exchangeable broad singlet
on the conversion of yellow MTT to the purple formazan signal at 6.28-6.59 ppm in compounds 2i . The
δ 3.15-3.48 ppm. The upfield
δ
µ
-
Methylthiazolyl tetrazolium (MTT) assay
δ
-h
δ
-p
derivative by mitochondrial succinate dehydrogenase mass spectrum of compound 2g showed the molecular
(Heras et al., 2001). The incubated cells (2 × 104 cells/ ion peak at m/z 263 and the base peak at m/z 60
well) in serum-free medium were treated with different corresponding to CSNH2. However, the mass spectra
concentrations of test compounds (10, 50 and 100 µg/ of compounds 2h, 2o and 2p displayed molecular ion
mL) for 24 h at 37oC, washed with phosphate buffered peaks at m/z 275, 247, and 259, respectively. In
saline and incubated in serum-free medium to which addition, the mass spectra of compounds 2k and 2l

1000
N. A. Khalil et al.
showed characteristic M+2 peaks. Besides, a peak sence of ‘S’ atom which was reported to act as good
corresponding to M-CONH2 appeared in the mass radical scavenger (Sankaran et al., 2010). The highest
spectrum of compounds 2k, 2l and 2p. The synthetic activity was observed with the carbothioamide derivative
routes for the preparation of the new compounds 2a
are outlined in Scheme 1.
-p
2e and the carboxamide derivative 2n compared to
rutin as a reference standard (Table I). The pyrazoline
derivatives exhibited their anti-oxidative behavior in
the riboflavin-NBT system assay and no significant
effect in the NO-based cell assay. Superoxide-derived
Biochemical assay
Superoxide radical scavenging activity
Superoxide anions are precursors to active free radi- damage of NMT signal, would be potentially studied
cals that have potential of reacting with the biological in the future.
macromolecules and thereby inducing tissue damage
(Halliwell and Gutteridge, 1984). Moreover, superoxide
has been observed to initiate lipid peroxidation either
In vitro anti-inflammatory activity
The anti-inflammatory activity of the synthesized
directly or through transformation into more reactive compounds was studied in vitro for their inhibitory
species such as hydroxyl radical (Wickens, 2001). effects on chemical mediators release (LPS-induced
Rutin is a flavonoid with strong anti-oxidant activity NO production) from macrophages (Table II). Activated
which has been used herein as positive control anti- macrophages produce large amounts of chemical medi-
oxidant for comparison (Mitrovic
´
et al., 2011).
ators that indicate inflammation. Nitric oxide (NO), a
The superoxide-scavenging activity (%) of test com- bioactive free radical, is one of these critical mediators
pounds was calculated as follows:
which is produced by inducible NO synthase (iNOS) in
inflamed macrophages when stimulated by lipopoly-
saccharide (LPS). Excessive production of NO is indi-
cated both in chronic and acute inflammation. In fact,
NO production induced by LPS through iNOS induction
may reflect the degree of inflammation and provide a
The superoxide-scabenging activity (%)
absorbancecontrol–absorbancesample
----------------------------------------------------------------------------------------------
=
×100
absorbancecontrol
The results revealed a significant concentration- measure for assessing the effect of the test compounds
dependent free radical scavenging activity of all test on the inflammatory process. The data in Table II re-
compounds. Generally, most of the carbothioamide presented NO content six hours after LPS-treatment.
derivatives displayed higher activity than the corres- The stable end products of L-arginine-dependent NO
ponding carboxamide derivatives may be due to pre- synthesis, nitrate and nitrite were measured in the
Table II. Effect of test compounds on NO content after six
hour treatment with LPS
Table I. Superoxide radical scavenging activity expressed
as % inhibition
NO content (
0.2
5.58 ± 0.13
µ
M/106 cells)
0.4
5.58 ± 0.06
Superoxide scavenging activity %
Compd No.
Compd No.
Control
0.04
µ
M
µ
M
µM
0.04
µM
0.2
µM
0.4 µM
5.62 ± 0.08
Rutin
70.40 ± 2.49 90.32 ± 0.68 97.82 ± 0.37
(Standard)
LPS (10
µ
g/mL) 77.49 ± 0.16 73.49 ± 0.65 72.99 ± 0.40
52.18 ± 0.78 45.13 ± 0.12 40.80 ± 0.57
59.44 ± 0.48 52.07 ± 1.05 47.29 ± 0.31
48.76 ± 0.47 40.40 ± 0.43 34.94 ± 0.20
60.41 ± 0.78 56.93 ± 0.41 53.72 ± 0.23
43.28 ± 0.40 39.11 ± 0.55 30.71 ± 0.40
51.28 ± 0.49 44.77 ± 0.11 44.59 ± 0.26
45.57 ± 0.41 37.03 ± 0.11 29.98 ± 0.06
66.64 ± 0.47 55.37 ± 0.38 51.54 ± 0.36
64.38 ± 0.84 54.45 ± 0.44 50.53 ± 0.43
58.50 ± 0.33 47.43 ± 0.43 48.51 ± 0.33
52.53 ± 0.60 46.69 ± 0.50 45.00 ± 0.82
55.47 ± 1.07 49.68 ± 0.35 44.54 ± 0.38
40.40 ± 0.94 36.15 ± 0.37 29.65 ± 0.29
62.42 ± 0.93 54.66 ± 0.37 50.39 ± 0.31
2a
2b
2c
2d
2e
2f
2g
2i
2j
2k
2l
2m
2n
2p
59.20 ± 1.36 80.03 ± 0.77 87.43 ± 0.77
30.06 ± 0.49 50.27 ± 0.54 60.23 ± 0.74
59.04 ± 0.85 81.34 ± 0.45 87.54 ± 0.69
39.92 ± 0.65 57.76 ± 0.34 65.57 ± 0.64
68.26 ± 0.69 90.24 ± 0.74 94.03 ± 0.45
59.26 ± 1.06 81.56 ± 0.35 88.19 ± 0.70
59.90 ± 1.10 83.46 ± 0.60 91.69 ± 0.62
26.55 ± 0.49 48.46 ± 0.49 56.52 ± 0.58
33.11 ± 0.91 57.11 ± 0.16 64.59 ± 0.49
37.40 ± 0.67 59.60 ± 0.41 67.69 ± 0.55
59.75 ± 1.00 81.29 ± 0.49 89.63 ± 0.60
46.08 ± 0.91 67.29 ± 0.65 75.37 ± 0.59
68.23 ± 0.74 87.48 ± 0.62 95.52 ± 0.65
39.56 ± 0.53 58.16 ± 0.49 68.39 ± 0.62
2a
2b
2c
2d
2e
2f
2g
2i
2j
2k
2l
2m
2n
2p
Data are presented as mean ± S.D., n = 3.
Data are presented as mean ± S.D., n = 3.

Pyrazoline Anti-inflammatory and Antioxidant Agents
1001
cell culture medium of different compounds. All test these compounds at the recommended doses against
compounds exhibited reduction of NO level. The lowest the control rats. Further data will be published on the
NO content was observed with the carbothioamide activity and mode of action of compounds 2e and 2n
derivatives 2e, 2g and the carboxamide derivative 2n as antioxidant and anti-inflammatory agents.
compared to other compounds.
REFERENCES
Hepatocyte viability assay
Abdel-Wahab, B. F., Abdel-Aziz, H. A., and Ahmed, E. M.,
Synthesis and antimicrobial evaluation of 1-(benzofuran-
2-yl)-4-nitro-3-arylbutan-1-ones and 3-(benzofuran-2-yl)-4,
5-dihydro-5-aryl-1-[4-(aryl)-1,3-thiazol-2-yl]-1H-pyrazoles.
Eur. J. Med. Chem., 44, 2632-2635 (2009).
Awah, F. M., Uzoegwu, P. N., Oyugi, J. O., Rutherford, J.,
Ifeonu, P., Yao, X.-J., Fowke, K. R., and Eze, M. O., Free
radical scavenging activity and immunomodulatory effect
The effect of the synthesized compounds on hepato-
cyte viability was assessed in cultured cells using
methylthiazolyl tetrazolium (MTT) assay (Heras et
al., 2001). Compound 2d displayed the lowest toxicity
on cell viability in all tested doses. However, compounds
2e and 2n that have shown the most potent super-
oxide scavenging activity and the lowest NO content,
were found to exhibit moderate toxicity on hepatocyte
viability. On the other hand, the highest toxicity on
cell viability, was observed with compounds 2i at a
of Stachytarpheta angustifolia leaf extract. Food Chem.
,
119, 1409-1416 (2010).
Beauchamp, C. and Fridovich, I., Superoxide dismutase: im-
proved assays and an assay applicable to acrylamide gels.
Anal. Biochem., 44, 276-287 (1971).
Cook, N. C. and Samman, S., Flavonoids-Chemistry, meta-
bolism, cardioprotective effects, and dietary sources. J.
Nutr. Biochem., 7, 66-76 (1996).
Dora Carrión, M., Camacho, M. E., Leon, J., Escames, G.,
Tapias, V., Castroviejo, D. A., Galloa, M. A., and Espinosaa,
A., Synthesis and iNOS/ nNOS inhibitory activities of new
benzoylpyrazoline derivatives. Tetrahedron, 60, 4051-4069
(2004).
Drimal, J., Knezl, V., Navarova, J., Nedelcevova, J., Paulovicova,
E., Sotnikova, R., Snirc, V., and Drimal, D., Role of inflam-
matory cytokines and chemoattractants in rat model of
streptozotocin-induced diabetic heart failure. Endocr. Regul.,
42, 129-135 (2008).
Fioravant, R., Bolasco, A., Manna, F., Rossi, F., Orallo, F.,
Alacro, S., and Cirilli, R., Synthesis and biological evaluation
of N-substituted-3,5-diphenyl-2-pyrazoline derivatives as
cyclooxygenase (COX-2) inhibitors. Eur. J. Med. Chem.,
45, 6135-6138 (2010).
Foote, C. S., Photosensitized oxidation and singlet oxygen:
consequences in biological systems. Free Radical Biol., 2,
85-133 (1976).
Gökhan-Kelekci, N., Yabanoglu, S., Küpeli, E., Salgýn, U.,
Özgen, Ö., Ucar, G., Yesilada, E., Kendi, E., Yesilada, A.,
and Bilg, A. A., A new therapeutic approach in Alzheimer
disease: Some novel pyrazole derivatives as dual MAO-B
inhibitors and antiinflammatory analgesics. Bioorg. Med.
Chem., 15, 5775-5786 (2007).
Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L.,
Wishnok, J. S., and Tannenbaum, S. R., Analysis of nitrate,
nitrite, and [15N]nitrate in biological fluids. Anal. Biochem.
126, 131-138 (1982).
concentration of 10
g/mL and 2a at a concentration of 100
III).
µ
g/mL, 2l at a concentration of 50
µ
µg/mL (Table
In summary, in vitro assays revealed that the test
compounds exhibited a significant concentration-de-
pendent free radical scavenging activity as well as sig-
nificant reduction of nitric oxide level. The carbothio-
amide derivatives showed higher radical scavenging
activity than the corresponding carboxamide derivatives
especially compounds 2e and 2n, which contain chloro
or N,N-dimethyl substituents in the para position of
the phenyl ring. Liver and kidney toxicity tests of the
most active compounds, 2e and 2n, showed that these
compounds have no significant toxicity on liver and
kidney tissues. The results reflected the safe usage of
Table III. Effect of the test compounds on liver cell viability
Liver cell viability (%)
Compd No.
0.04
µM
0.2
µM
0.4 µM
100
100
100
Control
2a
2b
2c
2d
2e
2f
2g
2i
2j
2k
2l
90.22
93.55
92.55
95.34
91.35
93.87
94.86
90.08
94.21
91.23
90.97
94.21
91.50
94.87
88.17
90.55
88.94
92.51
88.07
89.65
91.73
87.92
92.56
87.08
86.85
92.62
86.92
91.00
76.62
82.18
80.54
87.54
78.51
82.50
83.54
78.51
79.95
80.95
80.77
84.64
81.66
84.34
Halliwell, B. and Gutteridge, J. M. C., Oxygen toxicology,
oxygen radicals, transition metals and disease. Biochem.
J., 219, 1-14 (1984).
2m
2n
2p
Halliwell, B., Free radicals, antioxidants, and human disease:

1002
N. A. Khalil et al.
curiosity, cause, or consequence? Lancet, 344, 721-724 (1994).
of reactive oxygen species by mitochondria. Biochem.
Pharmacol., 69, 719-723 (2005).
Halliwell, B., Zhao, K., and Whiteman, M., The gastrointestinal
tract: a major site of antioxidant action? Free Radical
Res., 33, 819-830 (2000).
Halliwell, B., Role of Free Radicals in the Neurodegenera-
tive Diseases: Therapeutic Implications for Antioxidant
Treatment. Drug Aging, 18, 685-716 (2001).
Ohyama, H. R., Kimata, A., Suzuki, T., and Miyata, N.,
Hydroxyl radical scavenging by edaravone derivatives:
Efficient scavenging by 3-methyl-1-(pyridin-2-yl)-5-pyra-
zolone with an intramolecular base. Bioorg. Med. Chem.
Lett., 16, 5939-5942 (2006).
Heras, B. D. L., Abad, M. J., Silvan, A. M., Pascual, R., Bermejo,
P., and Rodriguez, B., Effects of six diterpenes on macro-
phage eicosanoid biosynthesis. Life Sci., 70, 269-278 (2001).
Kaplanciki, Z. A., Özdemir, A., Turan-Zitouni, G., Altintop,
M. D., and Can, Ö. D., New pyrazoline derivatives and their
antidepressant activity. Eur. J. Med. Chem., 45, 4383-4387
(2010).
Osman, A. N., El-Gendy, A. A., Kandeel, M. M., Ahmad, E.
M., and Hussein, M. M. M., Synthesis and antimicrobial
activity of certain organic compounds produced by appli-
cation and of michael addition. Bull. Fac. Pharm. Cairo
Univ., 41, 59-68 (2003).
Papas, A. M., Diet and antioxidant status. Food Chem.
Toxicol., 37, 999-1007 (1999).
Kawai, H., Nakai, H., Suga, M., Yuki, S., Watanabe, T., and
Saito, K. I., Effects of a novel free radical scavenger, MCl-
186, on ischemic brain damage in the rat distal middle
cerebral artery occlusion model. J. Pharmacol. Exp. Ther.,
281, 921-927 (1997).
Rathish, I. G., Javed, K., Ahmad, S., Bano, S., Alam, M. S.,
Pillai, K. K., Singh, S., and Bagchi, V., Synthesis and anti-
inflammatory activity of some new 1,3,5-trisubstituted
pyrazolines bearing benzene sulfonamide. Bioorg. Med.
Chem. Lett., 19, 255-258 (2009).
Leopold, J. A. and Loscalzo, J., Oxidative risk for athero-
thrombotic cardiovascular disease. Biol. Med., 47, 1673-
1706 (2009).
Sankaran, M., Kumarasamy, C., Chokkalingam, U., and
Mohan, P. S., Synthesis, antioxidant and toxicological study
of novel pyrimido quinoline derivatives from 4-hydroxy-3-
acyl quinolin-2-one. Bioorg. Med. Chem. Lett., 20, 7147-
7151 (2010).
Shaharyar, M., Abdullah, M. M., Bakht, M. A., and Majeed,
J., Pyrazoline bearing benzimidazoles: Search for anti-
cancer agent. Eur. J. Med. Chem., 45, 114-119 (2010).
Wickens, A. P., Aging and the free radical theory. Respir.
Physiol., 128, 379-391 (2001).
Mitrovic
M., Radojevic
Curcic, M., and Markovic
´
, T., Stamenkovic
, I., Stefanovic
, S., Antioxidant, antimicrobial
´
, S., Cvetkovic
´
, V., Tošic
´
, S., Stankovic
´,
´
´, O., Comic
´, L., Dac
˘ic´, D.,
˘
´
´
and antiproliferative activities of five lichen species. Int.
J. Mol. Sci., 12, 5428-5448 (2011).
Moldeus, P., Hogberg, J., and Orrenius, S., Isolation and use
of liver cells. Method Enzymol., 52, 60-71 (1978).
Nohl, H., Gille, L., and Staniek, K., Intracellular generation