Design, synthesis, molecular modelling and biological evaluation of novel 3-(2-naphthyl)-1-phenyl-1H-pyrazole derivatives as potent antioxidants and 15-Lipoxygenase inhibitors

Article abstract of DOI:10.1080/14756366.2020.1742116

Oxidative stress is one of the main causes of significant severe diseases. The discovery of new potent antioxidants with high efficiency and low toxicity is a great demand in the field of medicinal chemistry. Herein, we report the design, synthesis molecu

Full text of DOI:10.1080/14756366.2020.1742116

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis, molecular modelling and
biological evaluation of novel 3-(2-naphthyl)-1-
phenyl-1H-pyrazole derivatives as potent
antioxidants and 15-Lipoxygenase inhibitors
Sahar A. Ali, Samir Mohamed Awad, Ahmed Mohammed Said, Shahenda
Mahgoub, Heba Taha & Naglaa Mohamed Ahmed
To cite this article: Sahar A. Ali, Samir Mohamed Awad, Ahmed Mohammed Said, Shahenda
Mahgoub, Heba Taha & Naglaa Mohamed Ahmed (2020) Design, synthesis, molecular modelling
and biological evaluation of novel 3-(2-naphthyl)-1-phenyl-1H-pyrazole derivatives as potent
antioxidants and 15-Lipoxygenase inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry,
35:1, 847-863, DOI: 10.1080/14756366.2020.1742116
To link to this article: https://doi.org/10.1080/14756366.2020.1742116
© 2020 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 27 Mar 2020.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 847–863
https://doi.org/10.1080/14756366.2020.1742116
RESEARCH PAPER
Design, synthesis, molecular modelling and biological evaluation of novel
3-(2-naphthyl)-1-phenyl-1H-pyrazole derivatives as potent antioxidants and
15-Lipoxygenase inhibitors
Sahar A. Alia, Samir Mohamed Awadb,c, Ahmed Mohammed Saidb,d, Shahenda Mahgouba, Heba Tahaa and
Naglaa Mohamed Ahmedb
b
aDepartment of Biochemistry and Molecular Biology, Faculty of Pharmacy, Helwan University, Ein-Helwan, Cairo, Egypt; Department of
c
Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Helwan University, Ein-Helwan, Cairo, Egypt; Department of Pharmacy, Al-Zahrawi
d
University College, Karbala, Iraq; Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, NY, USA
ABSTRACT
ARTICLE HISTORY
Received 18 December 2019
Revised 9 February 2020
Accepted 26 February 2020
Oxidative stress is one of the main causes of significant severe diseases. The discovery of new potent anti-
oxidants with high efficiency and low toxicity is a great demand in the field of medicinal chemistry.
Herein, we report the design, synthesis molecular modelling and biological evaluation of novel hybrids
containing pyrazole, naphthalene and pyrazoline/isoxazoline moiety. Chalcones 2a–e were synthesized
efficiently and were used as starting materials for synthesis of a variety of heterocycles. A novel series of
pyrazoline 3a–e, phenylpyrazoline 4a–e, isoxazoline 5a–e and pyrazoline carbothioamide derivatives 6a–e
were synthesized and screened for in vitro antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH),
nitric oxide (NO) and superoxide radical scavenging assay as well as 15-lipoxygenase (15-LOX) inhibition
activity. Compounds 3a, 4e, 5b, 5c, 6a, 6c, and 6e showed excellent radical scavenging activity in all
three methods in comparison with ascorbic acid and 15-LOX inhibition potency using quercetin as stand-
ard then were subjected to in vivo study. Catalase (CAT) activity, glutathione (GSH) and malondialdehyde
(MDA) levels were assayed in liver of treated rats. Compounds 5b, 5c, and 6e showed significant in vivo
antioxidant potentials compared to control group at dose of 100mg/kg B.W. Molecular docking of com-
pound 6a endorsed its proper binding at the active site pocket of the human 15-LOX which explains its
potent antioxidant activity in comparison with standard ascorbic acid.
KEYWORDS
Pyrazole; hybrids;
antioxidant activity;
scavenging activity; 15-
lipoxygenase inhibitors
GRAPHICAL ABSTRACT
1. Introduction
neutralization of ROS by antioxidants is very delicate4. Every day a
human cell is targeted by ROS, the hydroxyl radical (.OH), and
other species inducing oxidative stress5. Free radicals (atoms, mol-
ecules or ions contain an unpaired electron) are highly unstable
Oxidative stress is one of the main causes of significant severe dis-
eases, i.e. cancer, aging, atherosclerosis, hypertension, inflamma-
tion, renal disorders, liver disorders, rheumatoid arthritis,
neurological disorders, cardiovascular, autoimmune diseases and
neurodegenerative disorders such as Alzheimer’s, Huntington’s dis-
eases and Parkinson’s diseases1–3. It is caused by the human body
excessive production of reactive oxygen species (ROS) and nitro-
gen reactive species (NRS) such as hydrogen peroxides (H2O2) and
.
and very reactive species that are able to create ROS such as OH,
.
hydroperoxyl radical (HO2 ), superoxide anion (.O2ꢀ), nitric oxide
(NO), singlet oxygen (O) and H2O2 as well as nitrogen reactive
species (RNS) and reactive sulphur species (RSS). These species are
generated either internally from normal metabolic activities or
free radicals. The balance between the production and external factors, such as smoking, environmental pollutants and
CONTACT Naglaa Mohamed Ahmed
nogamoon2005@yahoo.com
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Helwan University,
P.O. Code11795, Ein-Helwan, Cairo 11795, Egypt
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

848
S. A. ALI ET AL.
Figure 1. Biologically active compounds have pyrazole ring.
radiation, that promote the production of free radicals. The main anticancer, antibiotic22 and Fezolamine V as antidepressant23
human body targets of ROS, RNS and RSS are sugars, proteins, lip- (Figure 1).
ids, DNA and RNA molecules6. High concentrations of such species
The pyrazole (1,2-diazole) has antioxidant activity and can pre-
can cause damage to the normal cell structures, embedded pro- vent oxidative stress by increasing antioxidant enzymes, such as
teins, carbohydrates, lipids, and disrupt nitrogen bases of nucleic GPx, and diminishing the lipid peroxidation process. Examples for
acids leading to the above-mentioned diseases. The human body the pharmacological effects of 1,2-diazole or its related drugs. 1,2-
creates a primary defence antioxidant mechanism for the detoxifi- Diazole was found to be effective in preventing nephrotoxicity
cation of the formed free radicals. This mechanism involves three caused by the anti-neoplastic drug cisplatin24. Edaravone VI
enzymes: superoxide dismutase (SOD), catalase (CAT) and glutathi- (Figure 2) is a novel antioxidant that has been used for patients in
one peroxidase (GPx)7. The action of these enzymes is more prom- cerebral infarction as support therapy for stroke25,26 and improves
inent in the presence of antioxidant agents. Antioxidants are ischemia/reperfusion-induced hepatic energy metabolism27.
molecules that delay and prevent oxidative damage to a target
Recently, the study of pyrazole as a pharmacophore for the
molecule. In addition, antioxidants inhibit ROS production and development of potential antioxidants has led to the synthesis of
diminish oxidative stress8. The essential defence role of antioxi- several compounds containing pyrazole core in their structures.
dants in the human body is via scavenging or regulating the pro- Among the reported synthetic pyrazoles: 3-(Pyridin-4-yl)-1H-pyra-
duction and elimination of ROS and RNS. The presence of zole-5-carboxamide chalcones VII showed potent radical scaveng-
favourable balance between ROS and antioxidants is important for ing activity (RSA) against 2,2-diphenyl-1-picrylhydrazyl (DPPH)
healthy tissues and proper physiological function. It is also well radical28, Moreover, in comparison with the standard ascorbic
known that the balance between free radicals, antioxidants and acid, 1,5-diarylpyrazoles VIII showed good DPPH RSA29. It was
co-factors can contribute to the delay of the aging process, reduce found that 3,5-diarylpyrazole IX has shown potent RSA as well.
the incidence of diseases and thus contributing to a better quality The antioxidant activity of pyrazole is attributed to the presence
of life. Therefore, the discovery of new potent antioxidants with of NH proton of the pyrazole moiety30. In addition, 3,5-diarylpyra-
.
.
2
high efficiency and low toxicity is of a great demand in the field zoline derivative X showed excellent RSA using DPPH, OH, O2
of medicinal chemistry.
and NO anion assays, compared to butylated hydroxy toluene
Pyrazole ring is an important scaffold in medicinal chemistry. (BHT7)31. Derivatives of pyrazole such as bipyrazole XI showed
Pyrazole is a five-membered heterocyclic ring that consists of good scavenging activity (19%, BHT7¼20%) in the DPPH assay at
three carbons and two adjacent nitrogen atoms. Pyrazole deriva- 10ꢀ4 M concentration32. As well as, Bis-isoxazoline XII showed
tives have received considerable attention due to their remarkable good RSA using DPPH, NO and H2O2 methods in comparison with
broad spectrum of medicinal and pharmacological activities i.e. ascorbic ascid33. Moreover, Pyrazolyl-1,2,4-oxadiazoles XIII pos-
anticancer9, antiviral10, anti-tubercular11, anti-microbial12, antimal- sessed potent DPPH RSA34. 4,5-Dihydropyrazole-1-carbothioamide
arial13, anti-inflammatory14, antihypertensive15, anti-Alzheimer’s16, derivative XIV exhibited good antioxidant activity at low concen-
antipsychotic17, and antiparkinsonian18. Various drugs that have trations (0.25 mg/mL) in DPPH method35 (Figure 2).
pyrazole ring are available in the market with diverse medicinal
activities i.e. celecoxib I as anti-inflammatory19, Crizotinib II as ous pathways such as free radical scavengers (preventive oxidants)
anticancer20, Apixaban III as anticoagulant21, Pyrazofurin IV as and as lipoxygenase inhibitors (pro-oxidative enzymes)36,37
The mechanism of action of antioxidants can be through vari-
.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
849
Figure 2. Structure of the lead antioxidant pyrazole derivatives and the designed target compounds 2–6.
15-Lipoxygenases (15-LOXes)38,39 are a unique class of non-heme inhibition with potent activity against human 15-LOX-1 in both a
iron containing enzymes that catalyse the peroxidation of polyun- cellular and an in vivo model of stroke45 (Figure 3).
In this study, we report the design, synthesis and biological
evaluation of a hybrid scaffold in which 3-naphthyl pyrazole is
substituted with pyrazoline/isoxazoline ring at position 3 to gener-
ate novel and new derivatives of 3-(2-naphthyl)-1-phenyl-1H-pyra-
saturated fatty acids such as arachidonic acid (AA) and linoleic
acid to their related hydroperoxides. In addition, 15-LOXes are
involved in various human diseases. 15-lipoxygenase-1 (15-LOX-1)
has been recently documented as a target for reduction of the
biosynthesis of eoxines, pro-inflammatory mediator40 and cancer zole (Figure 3). These novel hybrid derivatives were tested against
promoter41. Also, it was reported that 15-LOX participates in the 15-LOX enzymatic assay. Moreover, these compounds were eval-
oxidative modification of low-density lipoproteins (LDLs) that leads uated for their potential as antioxidants in DPPH, NO, and super-
to the progress of atherosclerosis42. Moreover, human 15-LOX-1 is oxide scavenging assays as well as in vivo antioxidant activity
one of the key mediators in neurodegenerative diseases such as using CAT, glutathione (GSH) and lipid peroxidation (MDA) assays.
Alzheimer’s disease43. There has been some literature work target- The results of in vitro antioxidant activity of the newly designed
ing 15-LOX-1. It was reported that 3,4,5-trisubstituted pyrazole (A) hybrids and their 15-LOX inhibitory activity would identify the
was found to work as a potent rabbit 15-LOX-1 inhibitor44
Recently, oxazole derivative (ML351) (B) showed novel 15- LOX design of 15-LOX inhibitors for the future studies. The
.
required antioxidant parameters that are most reliable in the

850
S. A. ALI ET AL.
Figure 3. Design strategy of new pyrazole hybrid compounds as 15-LOX inhibitors.
structure–activity relationship (SAR) and possible mechanisms of The reaction mixture was stirred at 70–80 ꢁC for 5–6 h. The pro-
action of these derivatives were also investigated.
gress of reaction was monitored by TLC using hexane and ethanol
(90:10). The reaction was cooled to room temperature, then
poured into cold water and a saturated solution of sodium bicar-
bonate was added to neutralise the mixture. The white solid
obtained was filtered followed by washing with water.
2. Materials and methods
2.1. Instruments
3-(3-Naphthalen-2-yl-1-phenyl-1H-pyrazole-4-yl)-1-aryl prope-
none (2a–e). A mixture of 4-substituted acetophenone (0.03 mol)
and the aldehyde 1 (0.03 mol) in 25 mL 50% alcoholic NaOH solu-
tion were stirred at room temperature for 24 h, then the solution
was cooled, poured on ice/water acidified with dil. HCl. The pro-
duced solid was filtered off, dried and crystallized from ethanol to
give compounds 2a–e.
3-(3-Naphthalen-2-yl-1-phenyl-1H-pyrazole-4-yl)-1-phenyl
propenone (2a).Yellow solid, yield 81%, m.p.158–159 ꢁC. IR (KBr)
vmax (cmꢀ1): 3150 (CH–Ar), 1695 (C¼O), 1604 (C¼N). 1H NMR
(300 MHz, DMSO-d6) d: 6.5 (d, 1H, J¼6.8 Hz, –CH¼CH–), 6.6 (d, 1H,
J¼19.1 Hz, –CH¼CH–), 6.8 (s, 1H, pyrazole), 7.1–7.9 (m, 17H,
Ar–H).13C NMR (300 MHz, DMSO-d6) d: 105.0 (pyrazole-C4), 126.0
(pyrazole-C5), 114.7–140.1 (aromatic Cs), 129. 26, 142.8 (CH¼CH),
160.0 (pyrazole-C3), 187.0 (C¼O). MS (EI): m/z: 400 [Mþ] (20%).
Anal. Calcd for C28H20N2O (400.471): C, 83.98; H, 5.03; N, 7.00;
Found: C,83. 77; H, 5.15; N, 6.93.
Melting points were determined with Electro-thermal IA 9100
apparatus (Shimadzu, Japan) and the values given were uncor-
rected. Fourier-transform infrared spectroscopy (FT-IR) spectra
were recorded as KBr pellets on a Perkin-Elmer 1650 spectropho-
tometer (USA), Faculty of Science, Cairo University, Cairo, Egypt.
Proton nuclear magnetic resonance (1HNMR) and carbon-13
nuclear magnetic resonance (13C-NMR) spectra were recorded in
dimethyl sulfoxide-d6 (DMSO-d6) on a Varian Mercury (300 MHz)
spectrometer (Varian UK) using TMS as internal standard and
chemical shifts were given as ppm (Faculty of Science, Cairo
University, Cairo, Egypt). Mass spectra were carried out using
70 eV EI Ms-QP 1000 EX (Shimadzu, Japan), Faculty of Science,
Cairo University, and Cairo, Egypt. Microanalyses were performed
on Vario, Elementar apparatus (Shimadzu, Japan), Organic
Microanalysis Unit, Faculty of Science, Cairo University, Cairo,
Egypt and the results were within the accepted range (0.40) of
the calculated values. Column Chromatography was performed on
(Merck) Silica gel 60 (particle size 0.06–0.20 mm).
1-(4-Methoxyphenyl)-3-(3-naphthalen-2-yl-1-phenyl-1H-pyra-
zole-4-yl)-propenone
(2b).
Brown
solid,
yield
85%,
m.p.187–188 ꢁC. IR (KBr) vmax (cmꢀ1): 2970 (CH-sp3), 3157 (CH–Ar),
1691 (C¼O), 1605 (C¼N). 1H NMR (300 MHz, DMSO-d6) d: 3.3 (s,
3H, OCH3), 7.0 (d, 1H, J¼6.5 Hz, –CH¼CH–), 7.4 (d, 1H, J¼18.1 Hz,
–CH¼CH–), 6.6 (s, 1H, pyrazole), 7.5-8.55 (m, 16H, Ar–H). 13C NMR
(300 MHz, DMSO-d6) d: 55.87 (OCH3), 105.21 (pyrazole-C4), 126.66
(pyrazole-C5), 113.33–145.0 (aromatic carbons), 129.30, 148.40
(CH¼CH), 161.0 (pyrazole-C3), 183.0 (C¼O). MS (EI): m/z: 430 [Mþ]
(20%). Anal. Calcd for C29H22N2O2 (430.497): C,80.91; H, 5.15; N,
6.51. Found: C,80.78; H,5.17; N,6.72.
2.2. Chemistry
3-(2-Naphthyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (1). The
titled compound 1 was synthesized according to the literature
procedure46,47. A mixture of b-acetyl naphthalene (0.03 mol) and
0.04 mol of phenyl hydrazine (0.03 mol) in absolute ethanol
(50 mL) and few drops of glacial acetic acid were heated on water
bath for 30 min. The progress of reaction was monitored by thin-
layer chromatography (TLC) using hexane and ethanol (90:10).
3-(3-Naphthalen-2-yl-1-phenyl-1H-pyrazol-4-yl)-1-p-tolyl pro-
Cooling the mixture and filtering the formed precipitate that was penone (2c). Yellow solid, yield 80%, m.p.146–147 ꢁC. IR (KBr) vmax
dried and crystallized from ethanol, a pure phenyl hydrazone was (cmꢀ1): 2975 (CH-sp3), 3160 (CH–Ar), 1696 (C¼O), 1605 (C¼N). 1H
obtained. Pyrazole-4-carbaldehyde was carried out by the applica- NMR (300 MHz, DMSO-d6) d: 2.3 (s, 3H, CH3), 6.5 (d, 1H, J¼6.7 Hz,
tion of two moles of cold solution of Vismyeir–Haack (VH) reagent –CH¼CH–), 6.7 (d, 1H, J¼18.1 Hz, –CH¼CH–), 6.8 (s, 1H, pyrazole),
(DMF-POCl3) with the phenyl hydrazone (0.01 mol) in DMF (3 mL). 7.0–7.9 (m, 16H, Ar–H). 13C NMR (300 MHz, DMSO-d6) d: 20.7 (CH3),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
851
105.0 (pyrazole-C4), 126.50 (pyrazole-C5), 112.7–142.1 (aromatic (pyrazoline-C3) 160.0 (pyrazole-C3). MS (EI): m/z: 428 [Mþ] (9.8%).
carbons), 126.26, 140.8 (CH¼CH), 163.0 (pyrazole-C3), 187.0 (C¼O). Anal. Calcd for C29H24N4 (428.53): C,81. 28; H, 5.65; N, 13.07;
MS (EI): m/z: 414 [Mþ] (17.7%). Anal. Calcd for C29H22 N2O Found: C,81.27; H,5.58; N,13.03.
5-(4-Chlorophenyl)-30-naphthalen-2-yl-10-phenyl-3,4-dihydro-
(414.49): C,84.03; H, 5.35; N, 6.76; Found: C,84.19; H,5.27; N,6.67.
1-(4-chlorophenyl)-3-(3-naphthalen-2-yl-1-phenyl-1H-pyrazol-
2H,1’H-[3,40] bipyrazole (3d). Yellow crystals, yield 75%,
4-yl)propenone (2d). Yellow solid, yield 77%, m.p.161–162 ꢁC. IR m.p.190–191 ꢁC. IR (KBr) vmax (cmꢀ1): 2979 (CH-sp3), 3173 (CH–Ar),
(KBr) vmax (cmꢀ1): 3157 (CH–Ar), 1692 (C¼O), 1655 (C¼N). H NMR 3354 (NH), 1605 (C¼N). 1H NMR (300 MHz, DMSO-d6) d: 3.2-3.5
1
(300 MHz, DMSO-d6) d: 6.4 (d, 1H, J¼6.6 Hz, –CH¼CH–), 6.8 (d, 1H, (dd, 1H, pyrazoline-C4-H), 5.20 (t, J ¼ 11.6 Hz, 1H, pyrazoline-C5-H),
J¼17.1 Hz, –CH¼CH–), 6.9 (s, 1H, pyrazole), 7.1–7.8 (m, 16H, Ar–H). 6.8 (s,1H,pyrazole), 7-7.8 (m, 16H, Ar–H), 8.01 (s, 1H,NH-pyrazoline,
13C NMR (300 MHz, DMSO-d6) d:105.5 (pyrazole-C4), 126.2 (pyra- D2O exchangeable). 13C NMR (300 MHz, DMSO-d6) d: 34.30 (pyra-
zole-C5), 115.7–145.1 (aromatic carbons), 126.2,141.1 (CH¼CH), zoline-C4), 56.57 (pyrazoline-C5), 105.0 (pyrazole-C4), 127.0 (pyra-
160.3 (pyrazole-C3), 187.0 (C¼O). MS (EI): m/z: 434 [Mþ] (20.1%), zole-C5), 117.7–147.1 (aromatic carbons), 148.50 (pyrazoline-C3),
436 (M þ 2, 6.7%). Anal. Calcd for C28H19ClN2O: (434.916): C, 77.33; 161.0
(pyrazole-C3).
(11.8%),450(M þ 2,4.1%).
MS
Anal.
(EI):
Calcd
m/z:
for
448
C28H21ClN4
[Mþ]
H, 4.40; N, 6.44; Found: C,77.29; H, 4.45; N,6.47.
1-(3,4-Dichlorophenyl)-3-(3-naphthalen-2-yl-1-phenyl-1H-pyr-
(448.95):C,74.91; H,4.71; N,12.48; Found: C,74.87; H, 4.80; N,12. 53.
azol-4-yl)propenone (2e). Yellow solid, yield 79%, m.p.168-169 ꢁC.
5-(3,4-Dichlorophenyl)-30-naphthalen-2-yl-10-phenyl-3,4-dihy-
IR (KBr) vmax (cmꢀ1): 3156 (CH–Ar), 1691 (C¼O), 1603 (C¼N). 1H dro-2H,1’H-[3,40]-bipyrazole (3e). Yellow solid, yield 77%,
NMR (300 MHz, DMSO-d6) d: 6.2 (d, 1H, J¼6.6 Hz, –CH¼CH–), 6.4 m.p.195–196 ꢁC. IR (KBr) vmax (cmꢀ1): 2973 (CH-sp3), 3175 (CH–Ar),
(d, 1H, J¼18.1 Hz, –CH¼CH–), 6.7 (s, 1H, pyrazole), 7.0–7.9 3351 (NH), 1608 (C¼N). 1H NMR (300 MHz, DMSO-d6) d: 3.4 (dd,
(m,15H,Ar–H). 13 C NMR (300 MHz, DMSO-d6) d: 105.0 (pyrazole-C4), J¼16.5, 11.1 Hz, 1H, pyrazoline-C4-H), 3.2 (dd, J¼16.4, 11.2 Hz, 1H,
126.0 (pyrazole-C5), 113.3–140.4 (aromatic carbons), 126.2,142.2 pyrazoline-C4-H), 5.23 (t, J¼11.6 Hz, 1H, pyrazoline-C5-H), 6.8 (s,
(CH¼CH), 160.2 (pyrazole-C3), 187.0 (C¼O). MS(EI): m/z: 469 [Mþ] 1H, pyrazole), 7.2–7.8 (m, 15H, Ar–H), 8.0 (s, 1H, NH-pyrazoline,
(15.3%), 471 (M þ 2, 5.1%). Anal. Calcd for C28H18Cl2N2O (469.36): D2Oexchangeable). 13C NMR (300 MHz, DMSO-d6) d: 38.30 (pyrazo-
C,71.65; H, 3.87; N, 5.97; Found: C,71.55; H, 3.85; N,5.83.
3-Naphthalen-2-yl-5-aryl,1’-phenyl-3,4-dihydro-2H,1H’-[3,4]
bipyrazole (3a–e). A solution of (2a–e) (1.0 mmol) and hydrazine
line-C4), 58.50 (pyrazoline-C5), 108.0 (pyrazole-C4), 128.0 (pyrazole-
C5), 118.7–140.1 (aromatic carbons), 148.54 (pyrazoline-C3), 158.5
(pyrazole-C3). MS (EI): m/z: 482 [Mþ] (14.5%), 484 (M þ 2, 4.8%).
hydrate 99% (1.0 mmol) in absolute ethanol (15 mL) was refluxed Anal. Calcd for C28H20Cl2N4 (483.39): C,69.57; H, 4.17; N, 11.59;
for 6-8 h. The resulting solution was concentrated, cooled, the
solid obtained was filtered off and recrystallized from ethanol to
give compounds 3a–e.
found: C,69.77; H,4.10; N,11.60.
3-Naphthalen-2-yl-5-aryl,2,1’-diphenyl-3,4-dihydro-2H,1H’-
[3,4] bipyrazole (4a–e). A solution of (2a–e) (1.0 mmol) and phe-
nyl hydrazine (1.0 mmol) in 25 mL ethanol containing 0.5 mL
piperidine was refluxed for 6–8 h. The mixture was cooled, filtered
off and recrystallized from ethanol to give compounds 4a–e.
30-Naphthalen-2-yl-2,5,10-triphenyl-3,4-dihydro-2H,1’H-
30-Naphthalen-2-yl-5,10-diphenyl-3,4-dihydro-2H,1’H-
[3,40]bipyrazole (3a). Yellow solid, yield 61%, m.p.172–173 ꢁC. IR
(KBr) vmax (cmꢀ1): 2960 (CH-sp3), 3052 (CH–Ar), 3439 (NH), 1593
(C¼N). 1H NMR (300 MHz, DMSO-d6) d: 3.10–3.88 (dd, 2H, pyrazo-
line -C4-H), 5.26 (t, J ¼ 11.5 Hz, 1H, pyrazoline-C5-H), 6.8 [3,40]bipyrazole (4a). Yellow solid, yield 68%, m.p.187–188 ꢁC. IR
(s,1H,pyrazole),7.0–8.0 (m, 17H, Ar–H), 8.3 (s, 1H, NH-pyrazoline, (KBr) vmax (cmꢀ1): 2963 (CH-sp3), 3186 (CH–Ar), 1607 (C¼N). 1H
D2O exchangeable). 13C NMR (300 MHz, DMSO-d6) d: 39.79 (pyra- NMR (300 MHz, DMSO-d6) d: 3.0–3.4 (dd, 2H, pyrazoline-C4-H), 5.21
zoline-C4), 55.87 (pyrazoline-C5), 105.0 (pyrazole-C4), 125.99 (pyra- (t, J¼11.5 Hz, 1H, pyrazoline-C5-H), 6.8 (s, 1H, pyrazole), 6.9-7.7 (m,
zole-C5), 118.68–145.05 (aromatic carbons), 150.71 (pyrazoline-C3), 22H, Ar–H). 13C NMR (300 MHz, DMSO-d6) d: 35.2 (pyrazoline-C4),
158.01 (pyrazole-C3). MS (EI): m/z:414 [Mþ] (13.7%). Anal. Calcd for 57.50 (pyrazoline-C5), 106.2 (pyrazole-C4), 129.0 (pyrazole-C5),
C28H22N4 (414.50): C,81.13; H, 5.35; N, 13.52; Found: C,81.17; 116.0-140.0 (aromatic carbons), 148.8 (pyrazoline-C3), 158.8 (pyra-
H,5.23; N,13.43.
zole-C3). MS (EI): m/z: 490 [Mþ] (9.7%). Anal.Calcd for C34H26N4
(490.6): C, 83.24; H, 5.34; N, 11.42; Found: C,83.47; H,5.33; N,11.49.
5-(4-Methoxyphenyl)-30-naphthalen-2-yl-2,10-diphenyl-3,4-
5-(4-Methoxyphenyl)-30-naphthalen-2-yl-10-phenyl-3,4-dihy-
dro-2H,1’H-[3,40] bipyrazole (3b). Brown solid, yield 67%,
m.p.177–178 ꢁC. IR (KBr) vmax (cmꢀ1): 2965 (CH-sp3), 3163 (CH–Ar), dihydro-2H,1’H-[3,40] bipyrazole (4b). Brown solid, yield 60%,
3356 (NH), 1605 (C¼N). 1H NMR (300 MHz, DMSO-d6) d: 3.22–3.40 m.p.156–157 ꢁC. IR (KBr) vmax (cmꢀ1): 2967 (CH-sp3), 3187 (CH–Ar),
1
(dd, 2H, pyrazoline-C4-H), 3.9 (s, 3H, OCH3), 5.20 (t, J ¼ 11.5 Hz, 1H, 1600 (C¼N). H NMR (300 MHz, DMSO-d6) d: 3.1–3.3 (dd, 2H, pyra-
pyrazoline-C5-H), 6.7(s, 1H, pyrazole), 6.9–7.9 (m, 16H, Ar–H), 8.0 (s, zoline-C4-H), 3.8 (s, 3H, OCH3), 5.3 (t, J¼11.5 Hz, 1H, pyrazoline-C5-
1H, NH-pyrazoline, D2O exchangeable). 13C NMR (300 MHz, DMSO- H), 6.6 (s, 1H, pyrazole), 6.8-7.6 (m, 21H, Ar–H). 13C NMR (300 MHz,
d6) d: 38.30 (pyrazoline-C4), 55.60 (pyrazoline-C5), 56.0(OCH3), DMSO-d6) d: 35.8 (pyrazoline-C4), 57.6 (pyrazoline-C5), 56.0 (OCH3),
105.0 (pyrazole-C4), 126.0 (pyrazole-C5), 112.2–141.1 (aromatic car- 106.8 (pyrazole-C4), 129.2 (pyrazole-C5), 116.0 ꢀ 140.0 (aromatic
bons), 148.54 (pyrazoline-C3), 159.0 (pyrazole-C3). MS (EI): m/z: carbons), 149.0 (pyrazoline-C3), 159.0 (pyrazole-C3). MS (EI): m/z:
444 [Mþ] (14.3%). Anal. Calcd for C29H24N4O: (444.53):C,78.36; H, 520 [Mþ] (11.3%). Anal. Calcd for C35H28N4O (520.62): C,80.74; H,
5.44; N, 12.60; Found: C,78. 33; H,5.43; N,12.70.
5.42; N, 10.76. Found: C,80.73; H,5.43; N,10.70.
3’-Naphthalen-2-yl-1’-phenyl-5-p-tolyl-3,4-dihydro-2H,
1’H
30-Naphthalen-2-yl-2,10-diphenyl-5-p-tolyl-3,4-dihydro-2H, 1’H
-[3,4’]bipyrazole (3c). Yellow crystals, yield 66%, m.p.187-188 ꢁC. -[3,40]bipyrazole (4c). Yellow solid, yield 77%, m.p.198–199 ꢁC. IR
IR (KBr) vmax (cmꢀ1): 2963 (CH-sp3), 3164 (CH–Ar), 3357 (NH), 1605 (KBr) vmax (cmꢀ1): 2923 (CH-sp3), 3052 (CH–Ar), 1595 (C¼N). 1H
(C¼N). 1H NMR (300 MHz, DMSO-d6) d: 2.5 (s, 3H, CH3), 3.20–3.51 NMR (300 MHz, DMSO-d6) d: 2.5 (s, 3H, CH3), 3.3–3.6 (dd, 2H, pyra-
(dd, 2H, pyrazoline-C4-H), 5.3 (t, J ¼ 11.5 Hz, 1H, pyrazoline-C5-H), zoline-C4-H), 5.28 (t, J¼11.5 Hz, 1H, pyrazoline-C5-H), 6.6 (s, 1H,
6.6 (s, 1H, pyrazole), 7.0-7.9 (m, 16H, Ar–H), 8.0 (s, 1H, NH-pyrazo- pyrazole), 6.9–8.2 (m, 21H, Ar–H). 13C NMR (300 MHz, DMSO-d6) d:
line, D2O exchangeable). 13C NMR (300 MHz, DMSO-d6) d: 20.7 20.3 (CH3), 39.5 (pyrazoline-C4), 55.8 (pyrazoline-C5), 107.1 (pyra-
(CH3), 36.30 (pyrazoline-C4), 57.50 (pyrazoline-C5), 105.0 (pyrazole- zole-C4), 129.5 (pyrazole-C5), 118.6-140.6 (aromatic carbons), 150.0
C4), 125.0 (pyrazole-C5), 115.5-145.1 (aromatic carbons), 148.54 (pyrazoline-C3), 159.0 (pyrazole-C3). MS(EI): m/z: 504 [Mþ] (13.5%).

852
S. A. ALI ET AL.
Anal. Calcd for C35H28N4(504.62): C,83.30; H, 5.59; N, 11.10; Found: (EI): m/z: 429 [Mþ] (11.8%). Anal. Calcd for C29H23N3O (429.51):
C,83.37; H, 6.00; N,11.01.
C,81.09; H, 5.40; N, 9.78; Found: C,81.17; H, 5.43; N,9.76.
5-(4-Chlorophenyl)-30-naphthalen-2-yl-2,10-diphenyl-3,4-dihy-
3-(4-Chlorophenyl)-5-(3-naphthalen-2-yl-1-phenyl-1H-pyrazol-
dro-2H,1’H-[3,40] bipyrazole (4d). Yellow solid, yield 71%, 4-yl)-4,5-dihydro isoxazole (5d). White solid, yield 79%,
m.p.192-193 ꢁC. IR (KBr) vmax (cmꢀ1): 2978 (CH-sp3), 3174 (CH–Ar), m.p.185–186 ꢁC. IR (KBr) vmax (cmꢀ1): 2916 (CH-sp3), 3046 (CH–Ar),
1
1
1605 (C¼N). H NMR (300 MHz, DMSO-d6) d: 3.3–3.5 (dd, 1H, pyra- 1594 (C¼N). H NMR (300 MHz, DMSO-d6) d: 3.31–3.50 (dd, 1H, iso-
zoline-C4-H), 5.27 (t, J¼11.6 Hz, 1H, pyrazoline-C5-H), 6.7 (s, 1H, xazoline-C4-H), 5.56 (t, J¼11.6 Hz, 1H, isoxazoline-C5-H), 6.60(s, 1H,
pyrazole), 7-7.8 (m, 21H, Ar–H). 13C NMR (300 MHz, DMSO-d6) pyrazole),7-8 (m, 16H, Ar–H). 13C NMR (300 MHz, DMSO-d6) d:32.5
d:35.9 (pyrazoline-C4), 57.8 (pyrazoline-C5), 106.9 (pyrazole-C4), (isoxazoline-C4), 60.8 (isoxazoline-C5), 115.6 (pyrazole-C4), 127.1
129.4 (pyrazole-C5), 116.3–141.1 (aromatic carbons), 149.6 (pyrazo- (pyrazole-C5), 117.6-140.2 (aromatic carbons), 159.7 (pyrazole-C3),
line-C3), 159.6 (pyrazole-C3). MS (EI): m/z: 524 [Mþ] (10.8%), 426 162.8(isoxazoline-C3). MS (EI): m/z: 449 [Mþ] (12.6%), 451 (M þ 2,
(M þ 2, 3.5%). Anal. Calcd for C34H25ClN4(525.04): C,77.78; H, 4.80; 4.2%). Anal. Calcd for C28H20ClN3 (449.93): C,74.74; H, 4,48; N, 9.34;
N, 10.67; Found: C,77.87; H, 4.82; N,10.53.
Found: C,74.77; H,4.43; N,9.36.
5-(3,4-Dichlorophenyl)-30-naphthalen-2-yl-2,10-diphenyl-3,4-
3-(3,4-Dichlorophenyl)-5-(3-naphthalen-2-yl-1-phenyl-1H-pyr-
dihydro-2H,1’H-[3,40]-bipyrazole (4e). Brown solid, yield 75%, azol-4-yl)-4,5-dihydro isoxazole (5e). White solid, yield 79%,
m.p.172–173 ꢁC. IR (KBr) vmax (cmꢀ1): 2974 (CH-sp3), 3176 (CH–Ar), m.p.187–189 ꢁC. IR (KBr) vmax (cmꢀ1): 2963 (CH-sp3), 3166 (CH–Ar),
1605 (C¼N). 1H NMR (300 MHz, DMSO-d6) d: 3.5 (dd, J¼16.8, 1604 (C¼N). 1H NMR (300 MHz, DMSO-d6) d: 3.69 (dd,
11.1 Hz, 1H, pyrazoline-C4-H), 3.3 (dd, J¼16.6, 11.2 Hz, 1H, pyrazo- J¼16.6,11.1 Hz, 1H, isoxazoline-C4-H), 3.82 (dd, J¼16.7, 11.2 Hz, 1H,
line-C4-H), 5.26 (t, J¼11.6 Hz, 1H, pyrazoline-C5-H), 6.8 (s, 1H, pyra- isoxazoline-C4-H), 6.06(t, J¼11.6 Hz, 1H, isoxazoline-C5-H), 6.6 (s,
zole), 7–7.8 (m, 20H, Ar–H). 13C NMR (300 MHz, DMSO-d6) d: 38.80 1H, pyrazole), 7.0-7.8 (m, 15H, Ar–H). 13C NMR (300 MHz, DMSO-d6)
(pyrazoline-C4), 58.80 (pyrazoline-C5), 108.8 (pyrazole-C4), d: 32.5 (isoxazoline-C4), 60.3 (isoxazoline-C5), 118.0 (pyrazole-C4),
128.2(pyrazole-C5), 118.8–140.4 (aromatic carbons), 148.58 (pyrazo- 128.8 (pyrazole-C5), 119.9-143.0 (aromatic carbons), 159.5 (pyra-
line-C3), 158.8 (pyrazole-C3). MS(EI): m/z: 558 [Mþ] (16.5%), 560 zole-C3), 162.0 (isoxazoline-C3). MS (EI): m/z: 484 [Mþ] (20.7%),486
(M þ 2, 5.3%). Anal. Calcd for C34H24Cl2N4 (559.48): C,72.99; H, 4.32; (M þ 2, 6.9%). Anal. Calcd for C28H19Cl2N3O (484.3): C,69.43; H,3,95;
N, 10.01; Found: C,72.96; H,4.30; N,10.10.
N,8.68. Found:C,69. 47; H,3.93; N,8.66.
5-(3-Naphthalen-2-yl-1-phenyl-1H-pyrazol-4-yl)-3-aryl-4,5-
3’-Naphthalen-2-yl-5-aryl,1’-phenyl-3,4-dihydro-1’H-
dihydro-isoxazole (5a–e).
A solution (2a–e) (1.0 mmol), and [3,4’]bipyrazolyl-2-carbo thioic acid amide (6a–e). To a solution
hydroxylamine HCl 99% (1.0 mmol) in absolute ethanol (15 mL) of chalcones 2a–e (1.6 mmol) in absolute ethanol (25 mL), semicar-
with 0.5 mL piperidine was refluxed for 8–10 h. The resulting solu- bazide hydrochloride (3.66 mmol) and piperidine (0.5 mL) were
tion was concentrated, cooled, the solid obtained was filtered off added and the solution was refluxed for 9–12 h. The reaction mix-
and recrystallized from ethanol to give compounds 5a–e.
5-(3-Naphthalen-2-yl-1-phenyl-1H-pyrazol-4-yl)-3-phenyl-4,5-
dihydro-isoxazole (5a).White solid, yield 81%, m.p.190–191 ꢁC. IR
ture was poured on ice water. The obtained solid was filtered off
and recrystallized from ethanol to give compounds 6 a–e.
30-Naphthalen-2-yl-5,10-diphenyl-3,4-dihydro-1’H-[3,40]bipyra-
(KBr) vmax (cmꢀ1): 2967 (CH-sp3), 3167 (CH–Ar), 1608 (C¼N). 1H zolyl-2-carbothioic acid amide (6a). Yellow solid, yield 65%,
NMR (300 MHz, DMSO-d6) d: 3.60-3.80 (dd, 2H, isoxazoline-C4-H), m.p.198–199 ꢁC. IR (KBr) vmax (cmꢀ1): 2966 (CH-sp3), 3166 (CH–Ar),
5.50 (t, J¼11.4 Hz, 1H, isoxazoline-C5-H), 6.5 (s, 1H, pyrazole), 3350 (NH2), 1603 (C¼N). 1H NMR (300 MHz, DMSO-d6) d: 2.9–3.3
6.8–7.6 (m, 17H, Ar–H). 13C NMR (300 MHz, DMSO-d6) d: 37.30 (iso- (dd, 2H, pyrazoline-C4-H), 5.40 (t, J¼11.7 Hz, 1H, pyrazoline-C5-H),
xazoline-C4), 56.50 (isoxazoline-C5), 114.0 (pyrazole-C4), 125.0 (pyr- 6.7(s,1H, pyrazole), 7–7.7 (m, 17H, Ar–H), 10.1 (s, 2H, NH2, D2O
azole-C5), 118.0–140.1 (aromatic carbons), 157.0 (pyrazole-C3), 160. exchangeable). 13C NMR (300 MHz, DMSO-d6) d: 45.1 (pyrazoline-
0 (isoxazoline-C3). MS (EI): m/z: 415 [Mþ] (17.7%). Anal. Calcd for C4), 55.5 (pyrazoline-C5), 117.1 (pyrazole-C4), 125.1 (pyrazole-C5),
C28H21N3O (415.48): C,80.94; H, 5.09; N, 10.11; Found: C,80.97; H, 118.5–145.0 (aromatic carbons), 155.0 (pyrazoline-C3), 157.0 (pyra-
5.13; N,10.23.
zole-C3), 180.0 (C¼S). MS (EI): m/z:473 [Mþ] (16.7%). Anal. Calcd
3-(4-Methoxyphenyl)-5-(3-naphthalen-2-yl-1-phenyl-1H-pyra-
for C29 H23N5S (473.59): C,73.55; H, 4.90; N, 14.79. Found: C,73.47;
zol-4-yl)-4,5-dihydro isoxazole (5b). Yellow solid, yield 85%, H,4.87; N,14.73.
m.p.197–198 ꢁC. IR (KBr) vmax (cmꢀ1): 2966 (CH-sp3), 3169 (CH–Ar),
5-(4-Methoxyphenyl)-30-naphthalen-2-yl-10-phenyl-3,4-dihy-
1
1605 (C¼N). H NMR (300 MHz, DMSO-d6) d: 3.61-3.90 (dd, 2H, iso- dro-1’H-[3,40] bipyrazolyl-2-carbothioic acid (6b). Yellow solid,
xazoline-C4-H), 3.5 (s, 3H, OCH3), 5.40 (t, J¼11.5 Hz,1H, isoxazoline- yield 67%, m.p.193-194 ꢁC. IR (KBr) vmax (cmꢀ1): 2956 (CH-sp3),
C5-H), 6.6 (s,1H,pyrazole), 7.1-7.9 (m, 16H, Ar–H). 13C NMR 3049 (CH–Ar), 3255 (NH2), 1597 (C¼N). 1H NMR (300 MHz, DMSO-
(300 MHz, DMSO-d6) d: 33.30 (isoxazoline-C4), 56.1 (OCH3), 60.0 d6) d: 2.8-3.03 (dd, 2H, pyrazoline-C4-H), 3.36 (s, 3H, OCH3), 5.66 (t,
(isoxazoline-C5), 115.0 (pyrazole-C4), 127.0 (pyrazole-C5), J¼11.7 Hz, 1H, pyrazoline-C5-H), 6.0 (s, 1H, pyrazole), 6.7–7.6 (m,
119.0–141.0 (aromatic carbons), 158.0 (pyrazole-C3), 162.0 (isoxa- 16H, Ar–H), 9.66 (s, 2H, NH2, D2Oexchangeable). 13C NMR
zoline-C3). MS (EI): m/z: 445 [Mþ] (18.9%). Anal. Calcd for (300 MHz, DMSO-d6) d:40.0 (pyrazoline-C4), 55.0 (pyrazoline-C5),
C29H23N3O2: (445.51): C,78.18; H, 5.20; N, 9.43; Found: C,78.27; 56.0 (OCH3), 113.9 (pyrazole-C4), 126.9 (pyrazole-C5), 118.6–148.4
H,5.14; N,9.33.
(aromatic carbons), 155.6 (pyrazoline-C3), 158.3 (pyrazole-C3),
180.0 (C¼S). MS(EI): m/z: 503 [Mþ] (12.8%). Anal. Calcd for
C30H25N5OS: (503.62): C,71.55; H, 5.00; N, 13.91. Found: C,71.47;
H,4.97; N,13.90.
5-(3-Naphthalen-2-yl-1-phenyl-1H-pyrazol-4-yl)-3-p-tolyl-4,5-
dihydroisoxazole (5c). Yellowish brown solid, yield 77%,
m.p.181–182 ꢁC. IR (KBr) vmax (cmꢀ1): 2967 (CH-sp3), 3170 (CH–Ar),
1
1600 (C¼N). H NMR (300 MHz, DMSO-d6) d: 2.5 (s, 3H, CH3), 3.66-
30-Naphthalen-2-yl-10-phenyl-5-p-tolyl-3,4-dihydro-1’H-
3.82 (dd, 2H, isoxazoline -C4-H), 5.55 (t, J¼11.4 Hz, 1H, isoxazoline- [3,40]bipyrazolyl-2-carbo thioic acid amide (6c). Yellow solid,
C5-H), 6.7 (s, 1H, pyrazole), 7.0-7.9 (m, 16H, Ar–H). 13C NMR yield 63%, m.p.184–185 ꢁC. IR (KBr) vmax (cmꢀ1): 2968 (CH-sp3),
(300 MHz, DMSO-d6) d: 20.5 (CH3), 32.30 (isoxazoline-C4), 60.6 (iso- 3161 (CH–Ar), 3307 (NH2), 1606 (C¼N). 1H NMR (300 MHz, DMSO-
xazoline-C5), 115.5 (pyrazole-C4), 127.2 (pyrazole-C5), 118.0-144.0 d6) d: 2.51(s, 3H, CH3), 3.25–3.51 (dd, 2H, pyrazoline-C4-H), 5.43 (t,
(aromatic carbons), 157.5 (pyrazole-C3), 160.6 (isoxazoline C3). MS J¼11.8 Hz, 1H, pyrazoline-C5-H), 6.7(s, 1H, pyrazole), 6.9–7.9(m,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
853
16H, Ar–H), 10.5 (s,1H, NH2, D2Oexchangeable). 13C NMR (300 MHz, different concentrations of the tested compounds (12.5, 25,
DMSO-d6) d: 20.6 (CH3), 45.5 (pyrazoline-C4), 56.6 (pyrazoline-C5), 50,100, and 200 mg/mL) were pipetted into a 96-well plate. Then,
117.5 (pyrazole-C4), 125.5 (pyrazole-C5), 118.7–144.0 (aromatic car- 50 mL of 10-mM sodium nitroprussides dissolved in phosphate-buf-
bons), 155.6 (pyrazoline-C3), 157.50 (pyrazole-C3), 180.2 (C¼S).
fered saline PBS (pH 7.4) were added to each well and the plate
was incubated for 90 min at room temperature. Next, an equal
MS(EI): m/z: 487 [Mþ] (16.3%). Anal. Calcd for C30H25N5S (487.62):
C,73.89; H, 5.17; N, 14.36. Found: C,73.87; H,5.13; N,14.43.
volume of Griess reagent (1% of sulphanilamide and 0.1% of
naphthyl ethylene diamine in 2.5% H3PO3) was added to each
well to measure the nitrite content. The absorbance of the formed
pink-coloured chromophore was measured at k 546 nm. DMSO and
ascorbic acid were used as the negative and positive control,
respectively. All tests were performed in triplicate. The percentage
of NO scavenging activity was calculated according to the follow-
ing equation:
5-(4-Chlorophenyl)-30-naphthalen-2-yl-10-phenyl-3,4-dihydro-
1’H-[3,40] bipyrazo lyl-2-carbothioic acid (6d). Yellow solid, yield
69%, m.p.186–187 ꢁC. IR (KBr) vmax (cmꢀ1): 2963 (CH-sp3), 3162
(CH–Ar), 3310 (NH2), 1606 (C¼N). 1H NMR (300 MHz, DMSO-d6) d:
2.90–3.37 (dd, 1H, pyrazoline-C4-H), 5.62 (t, J¼11.4 Hz, 1H, pyrazo-
line-C5-H), 6.6 (s, 1H, pyrazole), 7.0–7.8 (m, 16H, Ar–H), 10.1 (s, 1H,
NH2, D2O exchangeable). 13C NMR (300 MHz, DMSO-d6) d: 45.1
(pyrazoline-C4), 56.6 (pyrazoline-C5), 117.6.0 (pyrazole-C4), 125.3
(pyrazole-C5), 118.9–143.0 (aromatic carbons), 157.7 (pyrazoline-
C3), 160.0 (pyrazole-C3), 180.0 (C¼S). MS (EI): m/z: 507 [Mþ] (20%),
509 (M þ 2, 6.7%). Anal. Calcd for C29H22ClN5S (508.3): C,68.56; H,
4.36; N, 13.79. Found: C, 68.57; H,4.33; N,13.63.
% of NO scavenging ¼ ½ðAcontrol ꢀ AsampleÞ=Acontrolꢃ ꢂ 100
where Acontrol is the absorbance of the control reaction (with all
reagents except the test compound), and Asample is the absorb-
ance of the test sample. Linear regression analysis was performed
to calculate drug concentration showing 50% free radical inhib-
ition activity (IC50).
5-(3,4-Dichlorophenyl)-30-naphthalen-2-yl-10-phenyl-3,4-dihy-
dro-1’H-[3,40]bi pyrazolyl-2-carbothioic acid (6e). Yellow solid,
yield 69%, m.p. 180–181 ꢁC. IR (KBr) vmax (cmꢀ1): 2967 (CH–sp3),
3163 (CH–Ar), 3317 (NH2), 1600 (C¼N). 1H NMR (300 MHz, DMSO-
d6) d: 3.01 (dd, J¼16.1, 11.2 Hz, 1H, pyrazoline-C4-H), 3.6 (dd,
J¼16.2, 11.2 Hz, 1H, pyrazoline-C4-H), 5.25 (t, J ¼ 11.5 Hz, 1H, pyra-
zoline-C5-H), 6.8 (s, 1H, pyrazole), 7.0–7.8 (m, 15H, Ar–H), 10.2 (s,
1H, NH2, D2O exchangeable). 13C NMR (300 MHz, DMSO-d6) d: 45.3
(pyrazoline-C4), 56.3 (pyrazoline-C5), 117.3.0 (pyrazole-C4), 125.4
(pyrazole-C5), 118.1–142.0 (aromatic carbons), 155.3 (pyrazoline-
C3), 157.1 (pyrazole-C3), 180.0 (C¼S). MS (EI): m/z: 541 [Mþ]
(22.1%), 543 (M þ 2, 7.3%). Anal. Calcd for C29H21Cl2N5S
(542.48):C,64.21; H, 3.90; N,12.91. Found: C,64.27; H,3.83; N,12.83.
ꢀ.
2.3.3. Superoxide scavenging assay (O2
)
The improved pyrogallol autoxidation method was used to deter-
ꢀ.
mine O2
RSA of all synthesized compounds as previously
described51. Briefly, 50 mL of test compounds (12.5, 25, 50,100, and
200 mg/mL) was added to 2900 ml of 5-mM Tris HCl buffer (0.05 M,
pH 7.4) containing 1-mM Na2EDTA. Next, 50 mL of 60-mM pyrogal-
lol in1mM HCl had been thoroughly mixed with the mixture. The
absorbance of the reactꢀion mixture was measured at A325 nm
every 30 s for 5 min. O2 RSA was expressed by the oxidation
degree of a test group in comparison to that of the control. The
absorbance at 325 nm was measured against the Tris-HCl buffer
every 30 s for 5 min. The percentage of scavenging effect was cal-
culated using the following equation:
2.3. In vitro assays for biological antioxidant Activity
Chemicals: All chemicals required for all assays were used as ana-
lytical grade, and were purchased from Sigma-Aldrich Chemicals
Co., St. Louis, MO, USA.
ꢀ
O2 radical scavenging %
¼ ½ðDA325, control=TÞ ꢀ ðDA325nm, sample=TÞ=DA325nm, control=Tꢃ ꢂ 100
2.3.1. DPPH scavenging method
The DPPH scavenging activity of all synthesized compounds was
measured as previously described by Nahar et al.48 with some
modifications. Briefly, 100 mL of different concentrations of the
tested compounds (12.5, 25, 50, 100, and 200 mg/mL) were pipet-
ted into a 96-well plate. Then, 100 mL of 100-mM DPPH methanolic
solution were added to each well and the plate was incubated
protected from light at room temperature for 30 min. The absorb-
where DA325
mixture without the sample and DA325 nm,
mixture with the sample; T¼5 min. The experiments were per-
formed in triplicate. The IC50 value was defined as the concentra-
tion for 50% superoxide free radical inhibition and was calculated
by linear regression.
is the increase in A325 nm of the reaction
nm, control
is that for the
sample
49
ance of the solution was measured at k517 nm. Ascorbic acid was
used as the positive control while DMSO was the negative control.
The percentage of DPPH scavenging activity was calculated
according to the following equation:
2.4. In-vitro lipoxygenase inhibition activity
The assay was performed using Cayman’s lipoxygenase inhibitor
screening assay kit (Catalog No. 760700, Cayman Chemical, USA)
according to the manufacturer’s instructions. Briefly, 90 mL of 15-
LOX was pipetted into a 96-well plate. Next, 10 mL of test com-
pound at concentrations (2.5, 5.0 and 10 mM) dissolved in DMSO
were added to each well. The reaction was initiated by adding
10 mL substrate (AA) and the plate was placed on a shaker for at
least 5 minutes. Finally, 100 mL of chromogen (prepared according
to manufacturer’s instructions) was added to each well to stop
enzyme catalysis and develop the reaction. 100 mL of Assay buffer
(0.1 M Tris-HCl, pH7.4) was used in blank wells. Quercetin and
Â
Ã
% of DPPH scavenging ¼ ðAcontrol–AsampleÞ=Acontrol ꢂ 100,
where Acontrol is the absorbance of the control reaction (with all
reagents except the test compound), and Asample is the absorb-
ance of the test sample. Linear regression analysis was performed
to calculate drug concentration showing 50% free radical inhib-
ition activity (IC50). All tests were performed in triplicates.
2.3.2. No scavenging method
The NO scavenging activity of all synthesized compounds was DMSO were used as the positive control and 100% initial activity,
measured as previously described by Ho et al.50 Briefly, 50 mL of respectively. The absorbance of the solution was measured at k

854
S. A. ALI ET AL.
490–500 nm. The percentage inhibition was calculated according 2.5.3.1. CAT activity. Catalase activity in 10% liver homogenates
was determined spectrophotometrically according to Sinha AK54.
to the following equation:
The decrease in absorbance at 240 nm due to H2O2 decompos-
% inhibition ¼ ½ðIA ꢀ Ainhibitor sampleÞ=IAꢃ ꢂ 100
ition was measured and the results were expressed in U/
mg tissue.
where (IA) is the 100% initial activity and (Ainhibitor
is the
sample)
absorbance of the test sample. Dose–response curve was plotted
between % inhibition and the drug concentration. The non-linear
dose–response curve was used for calculating drug concentration
showing 50% enzyme inhibition.
2.5.3.2. Determination of reduced glutathione. Levels of glutathi-
one (GSH) in liver homogenates were assayed by the deproteiniza-
tion of tissue homogenate55. Then 200 mL supernatant was mixed
with di-potassium hydrogen phosphate buffer (pH 8) and 0.4%
5,50-dithiobis-2-nitrobenzoic acid (Ellman’s reagent). The yellow-
coloured substance formed was measured at 412 nm. The results
were expressed as GSH mg/g tissue.
2.5. In vivo biological antioxidant assays
To determine in vivo antioxidant potentials of the test compounds
which showed promising in vitro antioxidant activities, CAT activ-
ity, GSH and MDA levels were assayed in liver of treated animals.
2.5.3.3. Determination of lipid peroxide level. Lipid peroxidation
level in the liver homogenates was determined as thiobarbituric
acid reactive substances (TBARS) by measuring malondialdehyde
‘MDA’ level according to Mihara and Uchiyama56. Briefly, 0.5 mL
supernatant of tissue homogenate was mixed with 0.6% thiobarbi-
turic acid (TBA) and 1% orthophosphoric acid solution, and heated
in a boiling water bath for 45 min. The pink-coloured chromogen
formed by the reaction of TBA with MDA was extracted by n-buta-
nol and measured at 535 nm. The results were expressed as nmol/
g tissue.
2.5.1. Animals
The complete progress of the experiment was conducted using
male Wistar albino rats (200–250 g), delivered by the Institutional
Breeding House, Egypt, reared and maintained in the animal
house of the institution. The animals had free access to food and
water ad libitum and maintained in a controlled environment
under standard conditions of temperature and humidity with an
alternating 12 h light and dark cycle for about a week for acclima-
tization. The protocol of the study was approved by the Animal
Ethics Committee of the Faculty of Pharmacy, Helwan University
(ethical code number: 05A2019; date: October 2019). The study
was conducted in accordance with the EC, directive 86/609/EEC
for animal experiments.
2.6. Data presentation and statistical analysis
The data were represented as mean SEM. Significant differences
between groups were tested by using GraphPad InStat software
version 3.05 (GraphPad Inc., La Jolla, CA, USA). Appropriate graphs
were plotted when needed using GraphPad Prism version 5 for
Windows (GraphPad Inc., USA). The results were analysed using
one-way analysis of variance (ANOVA) with post hoc Scheffe’s test.
A value of p < 0.05 was considered statistically significant.
2.5.2. Acute oral toxicity study
The acute toxicity study of the selected compounds was per-
formed on albino rats according Organization for Economic Co-
operation and development guidelines-42552. The animals were
fasted overnight prior to the experiment with free access to water.
Selected drugs were administered at doses equal to and half of
Ascorbic Acid dose (50 and 100 mg/kg/p.o.), and the behavioural
change was observed up to 24 h. The selected compounds were
found to be non-toxic in the selected doses. Dose selected for
in vivo antioxidant study was 100 mg/kg B.W.
2.7. Molecular modelling procedure
The modelling experiment described in this study was performed
by using the Discovery Studio (DS) version 4.5 (Accelrys Inc., San
Diego, CA, USA) software57. The required pdb coordinates were
downloaded from the Brookhaven website (www.rcsb.org). The
hydrogen atoms were then added to both the small molecule and
the 15-LOX enzyme structure. The atom and bond types as well
as the protonation state for the small molecule and the binding
site were checked and corrected when needed. Water molecules
were deleted. This was followed by minimising the complex with
the DS force field using the default parameters. The resulted bind-
ing mode of the designed compound in bound to catalytic active
site of 15-LOX will be discussed later.
2.5.3. Animal treatment
Rats are weighed at the beginning and at the end of experiment.
Fifty-four male albino rats (n¼6) were divided into nine different
groups. Group I served as a control group and treated with the
same volume vehicle only. Group II treated with 100 mg/kg of
Ascorbic acid as standard antioxidant drug. Groups (III–IX) orally
administered 100 mg/kg of compounds 3a, 4e, 5b, 5c, 6a, 6c, 6e,
respectively, for 3 days. The animals were sacrificed by cervical dis-
location 24 h after the last dose. Sacrificing is carried out at the
same time of the day, to avoid the circadian variation in the level
of tissue GSH53. Each liver was excised, weighed, rinsed in ice-cold
normal saline and frozen for not more than 72 h await analysis of
endogenous antioxidant status (GSH levels and CAT activity) and
lipid peroxide concentrations. For performing biochemical assays,
a 10% liver homogenate in 10 mM phosphate buffer was prepared
3. Results and discussion
3.1. Chemistry
The synthesis of the target compounds (2-6) was depicted in
Schemes (1-3). The key starting derivative 3-(2-naphthyl)-1-phenyl-
1H-pyrazole-4-carbaldehyde (1) was prepared via Vilsmeier-Haack
reaction14 of a phenyl hydrazone, derived from the reaction of
b-acetyl naphthalene with phenyl hydrazine, in refluxing absolute
ethanol containing few glacial acetic acid followed by the addition
of two equivalents of dimethyl formamide and POCl3. Claisen-
Schmidt condensation58 of (1) with different aromatic ketones
R
using tissue homogeniser (Glas-ColV, Cat no.099C K6424, TERRE
HAUTE, USA).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
855
Scheme 1. Synthesis of the designed compounds 1 and 2 (a–e).
such
as
1-phenylethanone,
1-(4-methoxyphenyl)ethanone, at d 2.80–3.90 ppm and triplet at d 5.20-5.66 ppm corresponding
1-(4-methylphenyl)ethanone,
1-(4-chlorophenyl)ethanone
and
to the protons at C-4 and C-5 of the pyrazoline ring in addition to
the signals of pyrazole and other protons (CH3, OCH3, and aro-
matic Hs) . Moreover, the H NMR spectra of compounds 3 and 6
1-(3,4-dichloro phenyl)ethanone was performed in 30% ethanolic
sodium hydroxide solution at room temperature to afford the cor-
responding chalcones (2a–e), respectively, as outlined in (Scheme
1) . The formed chalcone derivatives (2a–e) was used as key inter-
mediates for synthesizing the target pyrazole-pyrazolines (3) and
(4) through 1,4-addition of hydrazine hydrate or phenyl hydrazine
to the a, b-unsaturated carbonyl system of the precursor chal-
cones 2a–e, followed by dehydration and rearrangement.
Cyclocondensation of the chalcones (2a–e) with hydrazine hydrate
or phenyl hydrazine59–61 in absolute ethanol with catalytic amount
of piperidine gave pyrazolines (3a–e) and phenylpyrazolines
(4a–e), respectively (Scheme 2). Cyclization of chalcones (2a–e)
into the corresponding isoxazolines (5a–e) was conducted by con-
densation of the chalcones with hydroxylamine hydrochloride62 in
ethanol containing a catalytic amount of piperidine to give the
target derivatives. In addition, pyrazoline-1-carboxamides (6a–e)
were prepared by base-catalysed cyclization of chalcones 2a–e
through reaction with semicarbazide HCl63 in absolute ethanol
and piperidine (Scheme 3). The reaction mechanism for formation
of carbothioamide is via the nucleophilic attack of thiosemicarba-
zide at b-carbon of the a-b unsaturated C¼O of chalcone followed
by the proton transfer and intramolecular cyclization of molecule
by the nucleophilic attack of NH2 to carbonyl carbon which is sta-
bilized by the proton transfer and further dehydration leads to
the formation of pyrazoline (Figure 4).
1
showed singlet exchangeable signal at around d 8 ppm or d
10 ppm corresponding to (NH) of the pyrazoline or (NH2) of the
carboxamide, respectively. 13C NMR showed the characteristic sig-
nals at d d 32.1-38.3 and 55.1-60.6 ppm corresponding to C-4 and
C-5 carbon of the pyrazoline ring, respectively. In addition to the
other signals for the carbons of the target compounds, The 13C
NMR spectra of compounds 2 and 6 showed the presence of sig-
nals corresponding to C¼O around at d 187 ppm and C¼S at
around d 180 ppm, respectively (cf. experimental part).
3.2. Biological antioxidant studies
This study presents the synthesis and biological evaluation of anti-
oxidant activity of compounds having pyrazole, naphthalene and
pyrazoline/isoxazoline pharmacophore. Most of LOX inhibitors
show antioxidant or free radical scavenging activities as lipoxyge-
nation occurs via a carbon centred radical64. Thus, all compounds
2–6 (a–e) were investigated for their RSA by DPPH, NO and super-
oxide assays while Ascorbic Acid (AA) was used as antioxidant ref-
erence standard. The in vitro antioxidant activities of tested
compounds were expressed as IC50 values (Table 1).
The formed new compounds (2-6) were confirmed by IR,
1HNMR, 13C NMR, mass spectroscopy and microanalysis. The IR
spectrum of compounds 2a–e exhibited characteristic bands at
3.2.1. DPPH scavenging activity
Currently, antioxidants showing DPPH scavenging activity are
receiving attention due to their role as anticancer, anti-inflamma-
tory and antiaging agents65. Therefore, the antioxidant potential
1
around 1690 cmꢀ1 for C¼O. H NMR spectra showed two doublets
1
signals at d 6.2–7.4 ppm for CH¼CH protons of chalcones. The H
NMR spectra of compounds 3-6 showed signal doublet of doublet of all novel molecules (2–6) was determined using DPPH radical

856
S. A. ALI ET AL.
Scheme 2. Synthesis of the designed compounds 3 (a–e) and 4 (a–e).
Scheme 3. Synthesis of the designed compounds 5 (a–e) and 6 (a–e).
scavenging assay in comparison with ascorbic acid (AA) as control carbothioamides possessed potent DPPH RSA. Compounds 3a, 6a,
treatment. The mechanism of radical scavenging is based on and 6c (IC50¼13.99 0.78, 20.47 1.43 and 18.98 1.73 lg/mL,
acidic H-atom transfer from the compound to the DPPH to form respectively) displayed good RSA but lower than ascorbic acid. In
DPPH-H. The results are presented in Table 1. Out of the twenty- addition, N-phenyl pyrazolyl pyrazolines 4b (IC50¼21.28 1.14 lg/
five tested pyrazole derivatives, sixteen showed moderate to mL), 4c (IC50¼24.42 0.9 lg/mL) and 4e (IC50¼ 19.56 1.06 lg/mL)
potent activity, which indicates their radical scavenging and their showed good activity in comparison to standard treatment AA.
reducing activities. The rest being less active derivatives .The pyra- The lower DPPH RSA of N-phenyl pyrazolyl pyrazolines (4a–e) and
zolyl pyrazolines (3a-d) and pyrazoline carbothioamides (6a-d) pyrazolyl isoxazolines (5a–e) prove the significant role of NH of
have potent antioxidant activities while pyrazolyl isoxazolines (5a- pyrazoline in antioxidant activity. All compounds (3–6) showed
d) have demonstrated moderate RSA. The most active compounds higher antioxidant RSA than their precursors, chalcones (2a–e)
were 3b, 3c, 3e, 6b and 6e (IC50¼11.70 0.29, 12.06 1.17, thus indicating that pyrazoline and isoxazoline rings enhance RSA
9.63 0.55, 12.02 0.63, and 9.66 0.34 lg/mL, respectively). They of these compounds. SAR studies showed that antioxidant activity
exhibited potent RSA than ascorbic acid (IC50¼ 13.67 0.97 lg/ of compounds tested by DPPH assay depends not only on the
mL). Suggesting that, the presence of free NH of pyrazoline type of heterocyclic pharmacophore but also on substituents R on
enhances the antioxidant activity by increasing their hydrogen the aromatic ring of pyrazoline/isooxazoline since antioxidant
donor capacity. Moreover, good radical scavenger property of S activity is related to electron or hydrogen donation capacity to
atom C¼S and free NH2 that act as hydrogen donor in pyrazoline DPPH. Radicals (Figure 5). Regarding heterocyclic pharmacophore,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
857
Figure 4. Proposed reaction mechanism for the formation of pyrazole-carbothioamide.
Table 1. In vitro antioxidant potential and 15-LOX inhibition activity of compounds (2–6).
a
a
a
a
Compounds
DPPH IC50 (mg/mL)
NO IC50 (mg/mL)
Superoxide IC50 (mg/mL)
15-LOX IC50 (mM)
2a
2b
2c
2d
>200
182.17 0.99
192.70 1.63
>200
>200
78.25 1.25
>200
>200
>200
>200
NDb
3.13 0.09
2.80 0.06
ND
>200
>200
2e
3a
3b
3c
118.99 1.78
13.99 0.78
11.70 0.29
12.06 1.17
>200
11.04 0.72
27.65 1.53
147.95 1.32
>200
>200
4.63 0.09
2.23 0.07
4.60 0.06
3.77 0.07
ND
50.42 1.45
118.65 1.03
>200
3d
>200
>200
3e
4a
4b
4c
9.63 0.55
34.39 1.03
21.28 1.14
24.42 0.9
>200
175.72 1.41
129.12 0.82
176.14 1.63
65.63 1.46
145.7 1.42
>200
2.53 0.07
2.53 0.09
4.00 0.06
1.83 0.07
ND
>200
>200
179.9 1.31
>200
4d
4e
5a
5b
5c
19.56 1.06
97.17 1.4
46.62 1.63
47.27 1.13
>200
57.01 1.29
37.5 1.36
75.53 1.43
38.99 1.31
>200
130.19 1.1
192.37 1.74
101.8 1.39
44.54 1.44
>200
3.53 0.07
5.53 0.07
4.37 0.09
4.23 0.07
ND
5d
5e
6a
6b
6c
6d
6e
38.44 1.28
20.47 1.43
12.02 0.63
18.98 1.73
>200
9.66 0.34
13.67 0.97
ND
96.56 1.4
36.37 0.75
198.08 1.28
23.79 0.83
148.08 1.36
71.39 1.25
37.9 1.31
ND
168.77 1.42
127.25 1.47
>200
140.17 1.52
>200
87.31 1.58
124.99 1.32
ND
3.00 0.06
1.50 0.06
1.90 0.06
2.10 0.06
1.67 0.03
1.57 0.03
2.5
Ascorbic acid
Quercetin
3.34
aIC50 values are expressed as a mean SEM of three experiments.
bNot determined.
the order of free radical scavenging activity (FRSA) were found to isoxazoline compounds was found to be: 3,4-(Cl) (3e) > 4-OCH3
2
be: (3e > 6e> 4e> 5e). Pyrazolines 3 and pyrazoline carbothioa- (3b) > 4-CH3 (3c) > H (3a) > 4-Cl (3d), in descending order.
mides 6 have higher FRSA than N-phenyl pyrazolines 4 and isoxa-
The di-halogenated compounds exhibited significant DPPH RSA
zolines 5. For compounds 3e and 6e their potent antioxidant than the corresponding chloro-substituted compounds. Pyrazole
activity is due to the presence of pyrazoline and carbothioamide derivative 3e and 6e show the most potent antioxidant activity,
moiety30,66. While the replacement of N atom by O atom gives both having 3,4 (di-Cl) substituents on phenyl ring, which is in
lower antioxidant activity as shown in isoxazolines 5. Concerning accordance with the reported results67. Presence of electron
substitution patterns of pyrazolineAQ4/isoxazoline. On the other donating groups such as OCH3 and CH3 are more beneficial than
hand, the order of antioxidant activity of pyrazoline and unsubstituted or mono chloro-substituted phenyl ring, which may

858
S. A. ALI ET AL.
Figure 5. Structure activity relationship of the pyrazole derivatives against DPPH radical scavenging assay.
be due to þ I and mesomeric effects68. This all indicate that the and 101.8 1.39 lg/mL, respectively) possessed pronouncing
physicochemical properties of the designed compound impose an superoxide
important role in the extent of its antioxidant activity. It is notable
scavenging
activity
than
ascorbic
acid
(IC50¼124.99 1.32 lg/mL). Pyrazolyl isoxazoline 5c and pyrazolyl
pyrazoline 3a were the most potent derivatives with 2.8 and 2.5
folds of ascorbic acid, respectively. In addition, pyrazolyl pyrazo-
lines 3b and 3e (IC50¼118.65 1.03 and 129.12 0.82 lg/mL,
that the calculated cLog P for these derivatives are high (more
lipophilic) compared to the standard treatment. In addition, these
derivatives are cyclized heterocyclic analogues with fewer rotat-
able bonds that make them more favourable for cellular perme-
ability compared to the standard treatment. It was found that the
more lipophilic is the compound, the more active it is as 15 LOX-
inhibitor. Also, the more electron withdrawal substitutions on the
aromatic side chain of the heterocyclic ring, the more antioxidant
activity was observed.
respectively),
pyrazoline
carbothioamide
6a
(IC50¼
127.25 1.47 lg/mL) displayed comparable O22. scavenging
potency to ascorbic acid. Moreover, pyrazoline carbothioamide 6c
(IC50 ¼ 140.17 1.52 lg/mL), N-phenylpyrazoline 4c, 4e (IC50¼
145.7 1.42 and 130.19 1.1 lg/mL, respectively) showed good
superoxide scavenging activity. SAR studies showed that most of
synthesized compounds revealed higher activity than the parent
chalcones in superoxide scavenging assay. Pyrazoline 3a, isoxazo-
line 5c, pyrazoline carbothioamide 6e and N-phenylpyrazoline 4b
rings showed a significant RSA towards superoxide radical anion.
Di-halogenated compounds displayed good superoxide RSA while,
mono halogenated ones didn’t.
3.2.2. No scavenging activity
NO assay was used to determine the scavenging power of the tar-
get compounds (2–6) to NO radical. The results are presented in
Table 1. Sodium nitroprusside in aqueous solution at physiological
pH spontaneously generates Nitrite oxide which interacts with
oxygen to produce Nitrite ions, which can be measured at 550 nm
by spectrophotometer in the presence of Griess reagent69.
Compounds 2e, 6c, 3a and 6a showed NO scavenging activity
through competing with oxygen to scavenge for the nitrite rad-
ical; higher than that of ascorbic acid (IC50¼11.04 0.72,
23.79 0.83, 27.65 1.53 and 36.37 0.75 lg/mL, respectively).
Pyrazole 2e was the most potent antioxidant derivative via reduc-
ing nitrite production with 3.4 folds that of ascorbic acid, Pyrazolyl
isoxazolines 5a, 5c (IC50¼37.5 1.36 and 38.99 1.31 lg/mL,
respectively) displayed comparable potency to ascorbic acid.
Moreover, N-phenyl pyrazolyl pyrazoline 4e displayed good anti-
oxidant activity (IC50¼57.01 1.29 lg/mL). SAR studies showed
that pyrazoline carbothioamide 6c and pyrazolyl pyrazoline 3a
exhibited higher NO scavenging activity than pyrazolyl isoxazoline
5a and N-phenyl pyrazolyl pyrazoline 4e. This confirms that pyra-
zoline and carbothioamide rings were favourable substitutions
over isoxazoline and N-phenyl pyrazoline for the antioxidant activ-
ity of the tested compounds against NO assay.
3.3. In vitro 15-lipoxygenase inhibition activity
All target compounds (2–6) were tested against Soybean 15-LOX
enzyme. The results are expressed as IC50 values (mM) as shown in
Table 1. Compounds 2b, 2c, 3a, 3e, 4a, 4c, 5e, 6a, 6b, 6c and 6d
showed potential 15-LOX inhibition activity when compared to
quercetin (IC50¼3.34 mM) as reference inhibitor. Carbothioamides
6a, 6e in which the pyrazoline ring is substituted with phenyl
moiety and 3,4-di-Cl phenyl, were the most potent compounds
(IC50¼1.50 0.06 and 1.57 0.03 mM, respectively) with 2.2 and 2.1
folds that of quercetin, respectively. Pyrazoline 3c and N-phenyl
pyrazoline 4e showed comparable potency to that of quercetin
(IC50¼ 3.77 0.07 and 3.53 0.07 mM, respectively). Moreover, pyra-
zole 2e (IC50¼4.63 0.09 mM), pyrazoline 3b (IC50¼4.60 0.06 mM),
isoxazolines 5a, 5b, and 5c (IC50¼ 5.53 0.07, 4.37 0.09 and
4.23 0.07 mM, respectively) and N-phenyl pyrazoline 4b
(IC50¼4.00 0.06 mM) displayed good 15-LOX inhibitory activity
but lower than quercetin. The results of the tested compounds
(2–6) as 15-LOX inhibitors emphasized the important role of
3-naphthylpyarazole in this enzymatic assay regardless the deriva-
tive was either a, b-unsaturated ketone 2c, pyrazoline 3a, N-phe-
nylpyrazoline 4c, isoxazoline 5e or pyrazoline carbothioamide
counterpart 6a. Those derivatives were superior to quercetin in
15-LOX inhibition. 15-LOX inhibition appeared to be (6a> 4c
>3a> 2c> 5e) to confirm the excel pyrazoline carbothioamide
3.2.3. Superoxide scavenging activity
All target compounds (2–6) were evaluated for their antioxidant
activity via superoxide sꢀc.avenging assay to estimate their
capability to scavenge O2 and so, preventing the formation
of elemental oxygen. The resulted IC50 values were measured
in lg/mL as shown in Table 1. Compounds 5c, 3a, 4b, 6e and
5b (IC50¼44.54 1.44, 50.42 1.45, 65.63 1.46, 87.31 1.58 over pyrazoline, N-phenyl pyrazoline and isoxazoline for

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
859
antioxidant activity of the tested compounds against Soybean 15- related biological implications of dietary consumption, such as
effects on antioxidant enzymes and oxidation-related metabolic
pathways. It is well known that lipid peroxidation is a complex
process which occurs as a result of the interaction between
molecular oxygen and polyunsaturated fatty acids. These free radi-
cals can cause the oxidation of biomolecules (e.g. protein, lipid,
and DNA) leading to cell injury and death70. Lipid peroxidation in
biological systems can lead to various pathological consequen-
ces71. The end products of lipid peroxidation are reactive alde-
hydes, such as MDA, which are highly toxic to cells72. In addition,
the MDA can react with biomolecules and exert cytotoxic, geno-
toxic, and neurodegenerative disorders. Since, MDA is one of the
end products of lipid peroxidation, thus the level of MDA can indi-
cate the degree of lipid peroxidation in the body. In Fact, GSH
provides the first line of body defence by scavenging ROS or by
acting as a co substrate in the GPx-catalysed reduction of H2O2
and lipid peroxides. Oxidative stress readily oxidises GSH to gluta-
thione disulphide by free radicals and ROS causing depletion of
GSH level73. Moreover, the endogenous antioxidant enzymes such
as SOD removes the superoxide anion74, while CAT catalyses the
reduction of H2O2 and protects the tissues from highly reactive
stabilization that may be produced from H2O2. Pyrazole derivatives
(3a, 4e, 5b, 5c, 6a, 6c, 6e) that showed promising in vitro antioxi-
dant activities were subjected to in vivo study. In the acute tox-
icity study, the orally administered compounds did not show toxic
effects in doses up to 100 mg/kg B.W. Oral administration of test
compounds for 3 days increased CAT activity and GSH level and
decreased the MDA concentration in the liver, which indicated
that they could enhance the antioxidant status as presented in
Table 2 and Figure 6. This validates the potent in vitro antioxidant
activity shown by these compounds. However, only compounds
(5b, 5c, and 6e) showed significant potent antioxidant activity
compared to control group at dose of 100 mg/kg. This may be
LOX enzyme. It was also noticed that di-halogenated derivatives
showed significant 15-LOX inhibition activity. This might be due
to better fitting of derivative into the catalytic pocket of 15-LOX
enzyme. In summary, compounds 3a, 4e, 5b, 5c, 6a, 6c and 6e
showed significant RSA in all three methods in comparison with
ascorbic acid and 15-LOX inhibition potency using quercetin as
standard. This suggests an important influence of EDGs (CH3,
OCH3) and di-halogen (di-Cl) in benzene ring. Regarding hetero-
cyclic pharmacophore, pyrazoline carbothioamide and pyrazoline
showed higher RSA and 15-LOX inhibition potency than N-phenyl
pyrazoline and isoxazoline and these observations should be
regarded in the future on the designed LOX inhibitors.
3.4. In vivo estimation of antioxidant activity
Measuring the in vitro antioxidant ability of the synthetic com-
pounds was not enough to estimate their antioxidant effects in
biological systems. In vivo antioxidant assays could reflect the
Table 2. In vivo antioxidant potential of compounds.
Compounds CAT (U/mg tissue) GSH (mg/g tissue) TBARS (nmol/g tissue)
Control
Ascorbic a
23.99 3.83
42.56 3.76
36.21 3.49
43.48 4.51
48.19 1.24
45.42 4.30
27.97 3.46
44.38 2.81
57.84 3.50
7.07 0.50
8.25 0.45
7.00 0.27
7.05 0.56
10.71 0.86
10.92 0.96
7.07 0.35
9.01 0.72
10.65 0.97
0.422 0.04
0.392 0.07
0.350 0.05
0.402 0.04
0.330 0.02
0.346 0.01
0.348 0.03
0.428 0.02
0.344 0.05
ꢄ
3a
4e
5b
5c
6a
6c
6e
ꢄꢄ
ꢄꢄꢄ
ꢄꢄ
ꢄ
ꢄꢄ
ꢄꢄꢄ
ꢄꢄ
CAT: catalase; GSH: reduced glutathione; TBARS: thiobarbituric acid reactive sub-
ꢄ ꢄꢄ
ꢄꢄꢄ
p < 0.05, p < 0.01, and p < 0.001
stances values are mean SEM.
as compared to control (n ¼ 6).
,
and
Figure 6. Effect of compounds (3a, 4e, 5 b, 5c, 6a, 6c, 6e) and ascorbic acid on the endogenous antioxidant status of rats. GSH: reduced glutathione; TBARS: thiobar-
ꢄ ꢄꢄ ꢄꢄꢄ
bituric acid reactive substances. Data are expressed as mean SEM% control. (n ¼ 6).
,
, and
p < 0.05, p < 0.01, and p < 0.001 compared to control group.

860
S. A. ALI ET AL.
Figure 7. Molecular modelling of 15-LOX inhibitors 6a (coloured by element), into the active binding site of human 15-LOX (PDB: 4NRE), tagging the protein residues
that coordinate with Fe3þ catalytic metal (blue ball) and that interacted with the inhibitors.
attributed to the short treatment period of the animals. The DS force field by using the default parameters. Analysis of the pro-
results of our study showed that treatment of animals by those posed binding of inhibitor 6a (Figure 7) into the catalytic binding
compounds significantly increased the level of CAT enzyme by site revealed that both naphthalene ring and the close by phenyl
about 101, 89, and 141%, respectively, compared to control group. ring on the pyrazole group was directed towards the hydrophobic
The increased level of CAT leads to break down of H2O2 and pre- cavity of the active binding pocket making hydrophobic-hydro-
vent further generation of free radicals. The increase in intracellu- phobic interactions. In addition, on the other pyrazole group, the
lar thiol-based antioxidant GSH was by about 52, 55, 51%, shown S-atom was found to be directed towards the catalytic Fe3þ of the
by compounds (5b, 5c, and 6e). The antioxidant activity may be active site and the phenyl ring is making p–cationic interactions
due to potent radical-scavenging activity of isoxazoline and carbo- with the catalytic Fe3þ of the active site as well as p–p interac-
thioamide pyrazoline.
tions with the His378 amino acid residue. It also was observed
that the terminal NH2 group is able is make a weak H-bond (3.7 Å)
with the carbonyl of the Ile 676 amino acid residue.
3.5. Molecular modelling
To investigate the orientation of the most potent compound 6a
into the active binding site pocket of the human 15-LOX (PDB:
4. Conclusions
4NRE)75 and to view the inhibitor-receptor interactions, molecular In summary, novel hybrids containing pyrazole, naphthalene and
modelling study was performed. All the modelling experiments pyrazoline/isoxazoline pharmacophore were synthesized and
described here were performed by using the DS version 4.5 investigated for their in vitro antioxidant activity using DPPH, NO
(Accelrys Inc., San Diego, CA, USA). The required pdb coordinates and Superoxide radical scavenging assays as well as 15-LOX inhib-
were downloaded from the Brookhaven website (www.rcsb.org). ition activity. One important pathway for antioxidant agents is
The hydrogen atoms were then added to both the small molecule through inhibiting lipid peroxidation that is mainly catalyzed by
and the protein. The atom and bond types as well as the proton- 15-LOX. The activity of the compound 6a was assed towards both
ation state for the small molecule and the binding site were antioxidant and anti-LOX activities. It was found that compound
checked and corrected when needed. Water molecules were 6a showed NO scavenging activity higher than that of ascorbic
deleted. This was followed by minimizing the complex with the acid. In addition, it showed potent anti-LOX activity by 2.2 folds

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
861
compared to that of quercetin. Furthermore, compounds 5a and
5c showed comparable potency to ascorbic acid. Moreover, com-
pound 4e displayed good antioxidant activity. SAR studies showed
that pyrazoline carbothioamide 6c and pyrazolyl pyrazoline 3a
exhibited higher NO scavenging activity than pyrazolyl isoxazoline
5a and N-phenyl pyrazolyl pyrazoline 4e. In addition, compounds
2c, 3a, 3e, 4a, 4c, 5e, 6a, 6b, 6c, 6d, and 6e showed potential
15-LOX inhibition activity which was almost aligned with DPPH
assay results only but was conflicted for some compounds such as
2c, 3e, 4a, 4c, 5e, 6b, and 6d with the other antioxidant assays.
Interestingly, carbothioamides 6a and 6e were the most potent
compounds with 2.2 and 2.1 folds that of quercetin, respectively,
and almost showed similar potential antioxidant activity in all
three assays. Furthermore, compounds 3a, 4e, 5b, 5c, 6a, 6c, and
6e showed significant RSA in all three in vitro assays in compari-
son with ascorbic acid along with 15-LOX inhibition potency using
quercetin as standard suggesting an important influence of EDGs
(CH3, OCH3) and di-halogen (di-Cl) on the benzene ring. Regarding
heterocyclic pharmacophore, pyrazoline carbothioamide and pyra-
zoline showed higher RSA and 15-LOX inhibition potency than N-
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active site as well as p–p interactions with the His378 amino acid
residue might be required for the potential activity of 15-LOX
inhibitor. In conclusion, the obtained results suggest that these
potent compounds may serve as lead candidates for 15-LOX inhib-
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Acknowledgements
The first author (S.A.A) and the second author (S.M.A) should both
be considered as first author for this manuscript. The design, syn-
thesis and molecular modelling parts of this manuscript were per-
formed by S.M.A, A.M.S, and N.M.A. The biology part was
performed by S.A.A, S.M and H.T. The authors would like to thank
for Mossad Sayed Mohamed (Professor of Pharmaceutical Organic
Chemistry) for his inspiration and support.
Disclosure statement
18. Jimenez HN, Liu KG, Hong SP, et al. 4-(1-Phenyl-1H-pyrazol-
4-yl)quinolines as novel, selective and brain penetrant
metabotropic glutamate receptor 4 positive allosteric modu-
lators. Bioorgan Med Chem Lett 2012;22:3235–9.
19. Hassan GS, Abou-Seri SM, Kamel G, Ali MM. Celecoxib ana-
logs bearing benzofuran moiety as cyclooxygenase-2 inhibi-
tors: design, synthesis and evaluation as potential anti-
inflammatory agents. Eur J Med Chem 2014;76:482–93.
No conflict of interest was reported by the author(s).
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