European Journal of Medicinal Chemistry 145 (2018) 594e605

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Design, synthesis, biological evaluation and docking studies of new

3-(4,5-dihydro-1H-pyrazol/isoxazol-5-yl)-2-phenyl-1H-indole
derivatives as potent antioxidants and 15-lipoxygenase inhibitors
Haydi Saher ElBordiny a, Mostafa Mahmoud El-Miligy b, Shaymaa Emam Kassab a, *,
Hoda Daabees a, Waleed Ali Mohamed Ali c, Soad Abdelhamid Mohamed El-Hawash b
a
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Damanhour University, Damanhour, El-Buhaira, 22516, Egypt
b
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria, 21526, Egypt
c
Biochemistry Department, General Hospital, Cairo, 11753, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: New candidates of 3-(4,5-dihydro-1H-pyrazol/isoxazol-5-yl)-2-phenyl-1H-indole derivatives (4e7) were

Received 31 October 2017 designed combining the pyrazoline/isoxazoline heterocycles and 2-phenylindole to explore its potential
Received in revised form as 15-lipoxygenase (15-LOX) inhibitors. The design of the new derivatives was based on utilizing the
22 December 2017
antioxidant properties of pyrazoline, 2-phenylindole and the good 15-LOX inhibition properties of
Accepted 8 January 2018
Available online 10 January 2018
indolylpyrazoline. The derivatives were synthesized adopting simple and laboratory friendly reaction
conditions to give the target compounds in quantitative yields. The resulting indolylpyrazolines/iso-
xazolines were evaluated as antioxidants against 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO)
and superoxide dismutase (SOD); indolylpyrazoline (4b) was the most potent antioxidant against SOD
assay (IC50 ¼ 1.78 mM) to be superior to ascorbic by 2 folds. Consistently, (4b) was the most potent in-
hibitor when tested against Soybean 15-LOX (IC50 ¼ 3.84 mM) excelling quercetin as standard inhibitor by
1.8 folds. Some of the new derivatives were docked into the active binding site of human 15-LOX (PDB
entry 4NRE) emphasizing the most potent derivative (4b) and the least potent one (4c). Docking solu-
tions of compounds (4b), (4c), (5b) and (6c) revealed that (4b) was the only compound that got stabilized
into the catalytic pocket of enzyme by p-cation interaction with the catalytic Feþ and formation of one
hydrogen bond with Ile 676 amino acid. Other derivatives including the least potent one variably got
stabilized into the active binding pocket by p-cation interaction with the catalytic Feþ but failed to form
hydrogen bond with Ile 676. For the future optimization of the generated inhibitors, (i) antioxidant
activity against SOD, (ii) the inhibitor stabilization by p-cation interaction with the catalytic Feþ3 and (iii)
formation of hydrogen bond with Ile 676 should be regarded.
© 2018 Elsevier Masson SAS. All rights reserved.

1. Introduction isoforms of LOXs; 5-LOX, 12-LOX and 15-LOX were found to be

Lipoxygenases (LOXs) constitute a family of dioxygenases, that
catalyze dioxygenation of 1,4-cis,cis-pentadiene-containing poly-
unsaturated fatty acids such as arachidonic (AA) and linoleic acids
to related hydroperoxides [1,2]. They are class of non-heme iron
containing enzyme found in both plant and animals. Three main
LOXs including 5, 12 and 15-lipoxygenases have been distinguished
based on the peroxidation site of AA [3]. Different mammalian
* Corresponding author.
E-mail addresses: shaymaakassab@yahoo.com, shaymaa.kassab@pharm.dmu.
edu.eg (S.E. Kassab).
involved in the etiology and progression of various human diseases
that attracted the attention to the design and emerge of new drug
candidates to work as potential lipoxygenase inhibitors [4]. Inter-
estingly, it has been proved that 15-LOX participated in cardiovas-
cular complications, astherosclerosis as it has a role in the oxidative
modification of low-density lipoproteins (LDL) [5]. Moreover, 15-
LOX has been implicated in the pathophysiology of neurodegen-
erative diseases like Alzheimer's disease [6], progression of certain
cancer cell types [7e9], and formation of pro-inflammatory and
thrombotic mediators that result from oxidation of arachidonic and
linoleic acids [10,11]. Thus, 15-LOX is definitely a considerable
medicinal target for therapeutic intervention [12].

https://doi.org/10.1016/j.ejmech.2018.01.026
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.
 H.S. ElBordiny et al. / European Journal of Medicinal Chemistry 145 (2018) 594e605
Regarding the crystal structure of different LOX members
whether prokaryotic, plant, fungal, or vertebrate/mammalian, all
have the same catalytic domain that contains the non-heme iron
atom with conserved amino acids [13e17]. More careful investi-
gation about the sequence identity of different LOXs; those are from
plant origin and those are mammalian revealed that the highest
sequence identity between LOXs of both lies in the area of catalytic
domain [18,19]. These important findings made the researchers
confidently depend on the Soybean LOX which is more accessible
than human LOX for designing, testing, characterizing, defining the
inhibition mechanism and discovery of small molecule enzyme
inhibitors [20].
Most of publications that reported elaboration of new small
molecule 15-LOX inhibitors adopted two strategeies [21e25] in the
molecular design to alter the enzyme activity pathways as the
following: (i) antioxidants to interfer with the redox cycle of 15-
LOX and (ii) iron-chelators to stop the enzyme catalysis via coor-
dination with the metal.
In pursuing the antioxidant potential of various heterocycles, we
have noticed that some interesting pyrazolines solely (Fig. 1-II) and/
or in conjunction to other heterocycles exhibited potential anti-
oxidant activity [26e28]. On the other hand, 2-phenylindole de-
rivatives (Fig. 1-I) showed promising antioxidant activity in
comparison to melatonin when tested against 2,2-diphenyl-1-
picrylhydrazyl (DPPH), superoxide radical scavenging and ferric
ions reducing antioxidant (FRAP) assays [29,30]. Interestingly, a
report confirmed the good to moderate 15-LOX inhibition activity
of some indolylpyrazoline derivatives (Fig. 1-III) and emphasized
the importance of pyrazoline ring for the LOX inhibition activity
that was lost when it got aromatized [31].
Accordingly, we describe herein the design of a hybrid scaffold
in which 2-phenylindole is substituted with pyrazoline ring at
position 3 to ultimately formulate new candidates of 3-(4,5-
dihydro-1H-pyrazol-5-yl)-2-phenyl-1H-indole derivatives (Fig. 1-
target 15-LOX inhibitors) that combines the beneficial effects of
Fig. 1. Rational design of the target molecular hybrid of 15-LOX inhibitors.
595
both 2-phenylindole and pyrazoline as antioxidants to utilize the
good LOX inhibition activity of indolylpyrazoline for investigation
of the new hybrids as potent small molecule inhibitors against
Soybean 15-LOX enzymatic assay. Isoxazoline ring have been cho-
sen to represent isosteric replacement of pyrazoline ring of the new
hybrid structures in a way to examine the role of such isostere in
the potential activity of new derivatives against the target enzyme.
Moreover, it is planned to evaluate the potential of the generated
target inhbitors as antioxidants against DPPH, NO, and SOD assays.
Testing the antioxidant activity of the new candidates against
multiple antioxidant assays is targeted aiming at characterizing the
antioxidant performance of the compounds that to us, is as
important as 15-LOX inhibition activity. Because this would help us
identify the antioxidant parameter that is most reliable in the
emerge of redox 15-LOX inhibitors for the present study and for the
future studies as well.
2. Results and discussion
2.1. Chemistry
In the present study, the key intermediate (3) that would be
utilized in synthesizing the target indolylpyrazolines (4) and
indolylisoxazolines (5); wlas prepared as shown in Scheme 1. 2-
phenylindole-3-carboxaldehyde (1) was prepared as reported
previously adopting Vilsmeier-Haack reaction [32]. 4-/3-
acetylpyridines and 2-acetylthiophene were reacted with 2-
phenyl-1H-indole-3-carboxaldehyde (1) by fusion in the presence
of catalytic amount of piperidine [33] to afford the corresponding
chalcones (3a-c) in very quantitative yields. All trials to synthesize
the N-acetylchalcone counterparts of (3a-c) from 1-acetyl-2-
phenyl-1H-indole-3-carboxaldehyde (2) [34] adopting the same
reaction conditions varying the reaction time, temperature, and/or
relative amount of the reagents were unsuccessful and deacetyla-
tion occurred to eventually afford the N-deacetylchalcones (3a-c).
596 H.S. ElBordiny et al. / European Journal of Medicinal Chemistry 145 (2018) 594e605

Scheme 1. Synthesis of indoly-3-pyrazolines (4) and indoly-3-isoxazolines (5).

This was confirmed by the m.p, IR and 1H-NMR of the products. to obtain the corresponding indolyl-3-(1-acetyl/propionylpyrazo-
Cyclization of chalcones (3a-c) into the corresponding indo- line) [38] failed to achieve the target products but it gave 2-
lylpyrazolines (4a-c) was conducted by treating the chalcones with phenylindole.
slight excessive equivalent amount of hydrazine hydrate in Compounds (6) and (7) were confirmed by IR and 1H-NMR, 13C-
refluxing ethanol [35,36] to give the target derivatives in quanti- NMR, Mass spectroscopy and Microanalysis.
tative yields. While indolylisoxazolines (5a-c) were synthesized by
reacting the respective chalcones (3a-c) with 1.4 equivalent 2.2. Antioxidant activity
amount of hydroxylamine hydrochloride in refluxing ethanol con-
taining catalytic amount of glacial acetic acid [37]. The resulting
compounds (3), (4), and (5) were confirmed by IR and 1H-NMR, 13C-
NMR, Mass spectroscopy and Microanalysis.
The preparation of the second-line of target compounds,
indolyl-3-(1-acylpyrazolines) (6) and indolyl-3-(1-
methylpyrazoline) (7) as shown in Scheme 2 was achieved by
adopting a procedure in which the indolyl-3-pyrazolines (4a,b)
were subjected to acetyl chloride or benzoyl chloride or methyl
iodide while cooling the starting indolylpyrazoline solution in dry
acetone in ice-bath; in the presence of triethylamine (TEA) [36]. (5) (4, 6, 7)
After complete addition of acyl chloride or methyl iodide, the re-
action temperature was raised to room temperature and the reac-
tion mixture was left stirring until judged complete to give the
respective indolyl-3-(1-acylpyrazolines) (6a-d) and indolyl-3-(1- 2.2.1. DPPH scavenging activity
methylpyrazoline) (7). It is important to report that refluxing a DPPH assay is used to determine the antioxidant potential of the
solution of indolylpyrazoline (4a) in either acetic or propionic acid new candidates (4e7) implementing Nahar et al.'s method [39]. The
 H.S. ElBordiny et al. / European Journal of Medicinal Chemistry 145 (2018) 594e605
Scheme 2. Synthesis of indoly-3-acylpyrazolines (6) and indoly-3-(1-methylpyrazoline) (7).
data as shown in Table 1 revealed the potential antioxidant activity
of indolylpyrazolines (4a-c) and the modest antioxidant property of
the isoxazoline counterparts (5a-c) when compared to that of the
reference standard ascorbic acid (IC50 ¼ 23.40 mg/mL). Indolylpyr-
azoline (4b) was the most potent antioxidant derivative
(IC50 ¼ 13.61 mg/mL) even with 1.7 folds that of ascorbic acid. In
addition, indolyl-3-(1-acetylpyrazoline) derivative (6c)
(IC50 ¼ 37.97 mg/mL) and indolyl-3-(1-methylpyrazoline) derivative
(7) (IC50 ¼ 33.10 mg/mL) displayed good antioxidant activity but
lower than ascorbic acid. The lower antioxidant activity of indoly-
lisoxazolines (5a-c), N-acylated indolylpyrazolines (6a-d) and N-
methyl indolylpyrazoline (7) made us infer that the free pyrazoline-
NH might play a significant role in the redox reaction process.
2.2.2. NO scavenging activity
NO assay is used to estimate the scavenging power of the target
compounds (4e7) to NO. The results recorded in Table 1 showed
that indolylpyrazolines (4a-c) displayed higher NO scavenging
potential than the indolylisoxazoline counterparts (5a-c) when
compared to the ascorbic acid as a reference standard. Indolylpyr-
azoline (4a) was the most potent NO scavenger (IC50 ¼ 20.99 mg/
mL) with 1.2 folds that of ascorbic acid (IC50 ¼ 24.50 mg/mL).
Additionally, indolylpyrazolines (6a,c) showed comparable NO
scavenging activity (IC50 ¼ 29.14, and 29.80 mg/mL respectively) to
ascorbic acid. The significant lower antioxidant activity of indoly-
lisoxazolines (5a-c) confirms that isoxazoline ring was not favour-
able substitution over pyrazoline ring for the antioxidant potaential
of the tested compounds against NO assay.
2.2.3. SOD inhibition activity
New target compounds (4e7) were evaluated against SOD
enzyme to examine their antioxidant potencies and the corre-
sponding capability to prevent the generation of elemental oxygen.
The resulted IC50 values measured in mM as shown in Table 1.
Ascorbic acid was used as reference standard for the enzymatic
assay.
Based on the data listed in Table 1, the tested compounds (4b),
(5a), and (6c) showed SOD inhibition activity higher than that of
597
ascorbic acid (IC50 ¼ 1.78, 1.87 and 2.57 mM respectively). Indo-
lylpyrazoline (4b) was the most potent derivative (IC50 ¼ 1.78 mM)
with 2 folds that of ascorbic acid (IC50 ¼ 3.54 mM). In addition,
indolylpyrazoline (4a) (IC50 ¼ 3.24 mM) exhibited comparable po-
tency to ascorbic acid and indolylisoxazoline (5b) (IC50 ¼ 3.54 mM)
was equipotent to ascorbic acid. The rest of compounds displayed
good SOD inhibition activities. The resulted activities of the tested
compounds (4e7) as antioxidants against SOD emphasized the
important role of 2-phenylindole in the antioxidant activity in this
enzymatic assay regardless the derivative was either indolylpyr-
azoline (4b) or indolylisoxazoline (5b) or pyrazoline-N-substituted
counterpart (6c). Those dertivatives were even superior to ascorbic
acid in the antioxidant potential. But the contribution of pyrazoline
and isoxazoline rings in controlling the activity of the generated
hybrids couldn't be neglected. So, the lower activity upon removal
of isoxazoline and pyrazoline rings for compounds (4b), (5a) and
(6c) is highly expected according to the IC50 values reported for the
compounds. This is because the antioxidant activity appeared to be
(4b) > (5a) > (6c) to confirm the excel of indolylpyrazoline over
both indolylisoxazoline and N-acetylindolylpyrazoline and infer
the role of pyrazoline and isoxazoline in discriminating the po-
tential antioxidant properties among potent compounds.
2.3. In vitro 15-lipoxygenase inhibition activity
New target inhibitors (4e7) were tested against Soybean 15-LOX
enzyme and the results were expressed as IC50 values measured in
mM as shown in Table 1. Compounds 4a,b; 5a,b; and 6b,c showed
potential 15-LOX inhibition activity at concentration lower than
that of the standard inhibitor quercetin (IC50 ¼ 6.87 mM). Indo-
lylpyrazoline (4b) in which the pyrazoline ring is substituted with
3-pyridyl moiety, was the most potent compound (IC50 ¼ 3.84 mM)
that gave potency 1.8 times that of quercetin. Among the tested
indolylisoxazolines (5a-c), only derivatives (5a,b) showed potential
activity against the target enzyme. On contrary, 2-thienyl de-
rivatives of both indolylpyrazoline (4c) (IC50 ¼ 9.41 mM) and indo-
lylisoxazoline (5c) (IC50 ¼ 7.51 mM) gave lower potency when
compared to 4-pyridyl derivatives (4a) (IC50 ¼ 6.54 mM); (5a)
598 H.S. ElBordiny et al. / European Journal of Medicinal Chemistry 145 (2018) 594e605

Table 1
15-LOX inhibition activity and antioxidant potential of compounds (4e7).

Compound R Ar Soybean 15-LOX IC50a (mM) ±SEM DPPH IC50b (mg/mL) ±SD NO IC50c (mg/mL) ±SD SOD IC50d (mM) ±SEM

4a H 06.54 ± 0.032 15.83 ± 0.63 20.99 ± 2.50 3.24 ± 0.083

4b H 03.84 ± 0.091 13.61 ± 0.52 35.01 ± 1.64 01.78 ± 0.125

4c H 09.41 ± 0.021 18.30 ± 0.913 24.04 ± 1.82 06.51 ± 0.030

5a e 04.89 ± 0.092 59.94 ± 0.44 70.50 ± 0.54 01.87 ± 0.116

5b e 06.71 ± 0.023 60.70 ± 1.214 116.70 ± 1.98 03.54 ± 0.111

5c e 07.51 ± 0.037 117.5 ± 1.92 233.40 ± 2.44 04.26 ± 0.081

6a CH3CO 08.74 ± 0.051 98.54 ± 0.71 29.140 ± 0.93 05.47 ± 0.055

6b C6H5CO 05.14 ± 0.062 107.32 ± 3.00 52.54 ± 1.23 04.27 ± 0.061

6c CH3CO 04.56 ± 0.053 37.97 ± 0.84 29.80 ± 0.72 02.57 ± 0.077

6d C6H5CO 07.23 ± 0.055 48.21 ± 1.11 50.33 ± 0.29 04.62 ± 0.074

7 CH3 09.32 ± 0.027 33.10 ± 0.66 37.10 ± 0.85 05.41 ± 0.050

Quercetin e e 06.87 ± 0.046 NDe ND ND

Ascorbic acid e e ND 23.40 ± 1.18 24.50 ± 3.94 03.54 ± 0.091
a
IC50 values are expressed as a mean ± SEM of three experiments in mM.
b
IC50 of DPPH values are expressed as a mean ± SD of three experiments in mg/mL.
c
IC50 of NO values are expressed as a mean ± SD of three experiments in mg/mL.
d
IC50 of SOD values are expressed as a mean value ± SEM of three experiments in mM.
e
Not determined.

(IC50 ¼ 4.89 mM) and 3-pyridyl derivatives (4b) (IC50 ¼ 3.84 mM);
(5b) (IC50 ¼ 6.71 mM). It was also noticed that substitution of pyr-
azoline ring at position 1 with methyl, or acetyl, or benzoyl didn't
significantly improve the potency of the corresponding non-
substituted derivative (4a) and reduced the potency of derivatives
(4b,c). This might be attributed to the role of the larger volume of 3/
4-pyridyl at position 3 of pyrazoline/isoxazoline ring in imparting
better fitting of the small molecule inhibitor into the catalytic
pocket of target enzyme. The data recorded for the tested com-
pounds shows that aryl substitution at positin 3 of pyrazoline/iso-
xazoline ring might be having a key role in improving the potency
of the new inhibitors for futur optimization of the compounds.
In summary, the non-substituted pyrazoline ring played a sig-
nificant role in letting the target compounds perform from excel-
lent to good antioxidant activity against DPPH, NO and SOD assays.
Interestingly, the antioxidant potency values of the tested com-
pounds (4e7) against only SOD assay almost went aligned with the
15-LOX inhibition potency values for the same compounds but it
went conflicted for some compounds like (4c), (5a-c), and (6a) with
the other antioxidant assays. Thus, the SOD as a type of antioxidant
assays is that should be regarded in the future studies on the
generated LOX inhibitors.
2.4. Docking study
In order to investigate the orientation of the most potent com-
pound (4b) into the active binding site pocket of the human 15-LOX
(PDB entry 4NRE) [40] and to view the inhibitor-receptor in-
teractions, docking study was performed using (MOLSOFT ICM
3.4e8C) software. Analysis of the docking solution of (4b) (Fig. 2A)
into the active binding pocket revealed that the benzene ring of
indole inhibitor was laid into the hydrophobic cavity of the active
binding pocket with pyrazoline ring and stabilized by the p-cation
interaction with the catalytic Feþ3. Only one hydrogen bond was
formed between the amino acid Ile 676 and pyrazoline-NH.
The docking solution of the lowest potent candidate; 3-
thienylindolylpyrazoline (4c) (IC50 ¼ 9.41 mM) confirmed the
implication of p-cation interaction with the catalytic Feþ3 in
inhibiting the target enzyme by both the non-substituted pyrazo-
line-NH and indole-benzene ring. This is because the orientation of
(4c) into the active binding pocket (Fig. 2B) showed the difficulty of
the inhibitor to get stabilized by p-cation interaction with the
 H.S. ElBordiny et al. / European Journal of Medicinal Chemistry 145 (2018) 594e605 599

Fig. 2. Docking solutions of 15-LOX inhibitors A) 4b, B) 4c, C) 5b, D) 6c (colored by element), into the active binding site of human 15-LOX (PDB entry 4NRE), tagging the protein
residues that coordinate with Fe3þ catalytic metal (light green ball) and that interacted with the inhibitors. Hydrogen bonds are represented as white dots. Hydrogen bond distance
is indicated by numbers in Å. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

catalytic metal specifically via pyrazoline-NH that almost directed
away but it was possible via indole-benzene ring. Moreover, the
inability of the compound to form hydrogen bond with Ile 676 the
same way as indolylpyrazoline (4b).
Considering the docking solution of the most potent inhibitor
(4b) as a reference simulation to the inhibitor-receptor interaction
pattern to which the other inhibitors' docking solutions are
compared, indolylisoxazoline (5b) (Fig. 2C) was chosen as a direct
counterpart to (4b) to be docked into the active binding pocket of
15-LOX. This is an attempt to realize the structural requirements
among the new candidates to perform potent inhibition to the
target enzyme. The docking solution and the orientation of indo-
lylisoxazoline (5b) into the binding pocket showed the possibility
of the compound to get stabilized by p-cation interaction with the
catalytic metal via both isoxazolineeoxygen and indole-benzene
ring without any possible formation of hydrogen bond with Ile
676 due to absence of hydrogen bond donor NH group. Interest-
ingly, the binding mode of (5b) combined with the binding mode of
(4c) indicated that the formation of hydrogen bond with amino acid
Ile 676 via either hydrogen bond donor or acceptor might be
essential for potent inhibition of the target enzyme. Thus, the lower
potency of all the candidates other than (4b) might be attributed to
the inability of the compounds to form hydrogen bond with Ile 676.
All the other candidates generated in this study were docked
into the active binding pocket and the docking solutions revealed
that all of them were variably stabilized by p-cation interaction but
failed to form hydrogen bond with Ile 676. Moreover, the docking
solution of indolyl-3-(1-acetylpyrazoline) derivative (6c) (Fig. 2D)
provided us with a convenient justification to the decrease in the
potency of 15-LOX inhibition (IC50 ¼ 4.56 mM) when compared to
the none-substituted indolylpyrazoline counterpart (4b)
(IC50 ¼ 3.84 mM). The N-COCH3 enabled the compound (6c) to get
stabilized by p-cation interaction with the catalytic metal via
carbonyl-oxygen but failed via indole ring. Also, absence of
hydrogen bond donor of pyrazoline-NH made the compound un-
able to interact with Ile 676 via formation of hydrogen.
3. Conclusion
The present study introduced new candidates of indolylpyr-
azolines and indolylisoxazolines in which 2-phenylindole was
combined with pyrazoline/isoxazoline heterocycles to get
benefited of both as antioxidants and the good LOX inhibition ac-
tivity of indolylpyrazolines. The 15-LOX inhibition potential of the
new hybrids was verified upon testing the designed compounds
against 15-LOX enzyme. Among the target compounds tested
against 15-LOX, indolylpyrazoline (4b) was the most potent in-
hibitor and showed 1.8 times more potency than quercetin
600 H.S. ElBordiny et al. / European Journal of Medicinal Chemistry 145 (2018) 594e605
standard inhibitor. The same compound exhibited the most anti-
oxidant potential when tested against SOD with 2 times more po-
tency than ascorbic acid as standard antioxidant to infer that SOD
was the most reliable antioxidant parameter to depend on in the
future studies when compared with the data obtained from other
antioxidant parameters. Docking solutions of the most potent
candidate (4b), the less potent candidates (4c), (5b) and (6c)
revealed that stabilization of the ligand inhibitor via p-cation
interaction with the catalytic Feþ3 and formation of hydrogen bond
with the amino acid Ile 676 might be required for potential inter-
ference with the catalytic process of the target enzyme. Conclu-
sively, the study of the new hybrids generated as potential 15-LOX
inhibitors coupled with the significant antioxidant activity high-
lights some important fundamentals that should be regarded in the
future studies for further optimization of the generated candidates:
i) SOD inhibition activity is the most reliable parameter for testing
the antioxidant activity of the inhibitors ii) Ligand inhibitor stabi-
lization by p-cation interaction with the catalytic Feþ3 and iii)
formation of hydrogen bond with Ile 676 amino acid contribute to
the potential activity of the inhibitors.
4. Experimental
4.1. Chemistry
Melting points were determined on digital Gallen-Kamp MFB-
595 instrument using open capillary tubes and are uncorrected. IR
spectra were recorded as potassium bromide discs on Schimadzu
FT-IR 440 spectrometer. 1H-NMR spectra were recorded on Bruker
spectrophotometer at 300 MHz in DMSO-d6, Varian Mercury VX at
400 MHz in DMSO-d6; values (d) are given in parts per million
(ppm) downfield from tetramethylsilane (TMS) as internal refer-
ence standard. 13C-NMR spectra were recorded using the same
spectrophotometers that used for recording 1H NMR. Mass spectra
were recorded on Shimadzu GCMS/QP-2010 plus (70eV) mass
spectrometer. The elemental analyses were performed at the
Microanalytical Center, Cairo University, Cairo, Egypt. Reactions
were followed up by thin layer chromatography (TLC) using Merck
Silica gel/TLC cards with fluorescent indicator UV254, the spots
were visualized using Spectroline E series dual wavelength UV
lamp at l ¼ 254 nm.
4.1.1. General procedure for (E)-3-(2-Phenyl-1H-indol-3-yl)-1-
(aryl) prop-2-en-1-ones (3a-c)
A mixture of 2-phenyl-1H-indole-3-carboxaldehyde (1)
(1.0 mmol, 0.22 g) and 3- or 4-acetylpyridine (1.0 mmol, 0.1 mL) or
2-acetylthiophen (1.3 mmol) and piperidine (0.2 mL) was heated
for 1 h, then ethanol (1.5 mL), glacial acetic acid and water (1:1)
were added dropwise to the resulting red solution until first
appearance cloudiness. The resulting product was filtered off,
washed with water and recrystallized from ethanol. In case of 2-
thienyl derivative of chalcone, the product obtained was sticky
mass, triturated with a mixture of petroleum ether and diethyl
ether to afford yellow precipitate, then recrystallized from ethanol.
4.1.2. (E)-3-(2-Phenyl-1H-indol-3-yl)-1-(pyridin-4-yl) prop-2-en-
1-one (3a)
Yield: 85%. Orange crystals; m.p.: 278e280  C. IR (KBr) ʋmax/
cm1: 3015 (NH), 1645 (CO), 1H-NMR (DMSO-d6, 400 MHz):
d 7.28e7.31 (m, 2H, indole-C5,6-H), 7.49e7.56 (m, 2H, indole-C4-H,-
CH¼CH-C¼O), 7.58e7.63 (m, 5H, 2-phenyl-C2,3,4,5,6-H), 7.92 (d,
J ¼ 5.0 Hz, 2H, pyridine-C3,5-H), 8.05 (d, J ¼ 15.45 Hz, 1H, -CH¼CH-
C¼O), 8.20 (d, J ¼ 7.00 Hz, 1H, indole-C4-H), 8.78 (d, J ¼ 5.0 Hz, 2H,
pyridine-C2,6-H), 12.34 (s, 1H, NH, D2O exchangeable). 13C-NMR
(DMSO-d6, 100 MHz): d 109.44 (indole-C3), 112.35 (indole-C7),
115.54 (indole-C5), 121.26 (indole-C6), 121.50 (pyridine-C3,5), 121.88
(indole-C4), 123.45 (2-phenyl-C4), 125.71 (indole-C2), 128.99 (2-
phenyl-C2,6), 129.41 (C¼C-C¼O), 129.75 (2-phenyl-C3,5), 130.70
(indole-C3a), 137.00 (2-phenyl-C1), 140.58 (C¼C-C¼O), 144.70
(indole-C7a),146.20 (pyridine-C4), 150.62 (pyridine-C2,6), 188.56
(CO). MS EIMS: m/z (calcd) 324.13 (found) 323.90 (Mþ). Anal.
Calcd for C22H16N2O: C, 81.46; H, 4.97; N, 8.64. Found: C, 81.63; H,
5.15; N 8.79.
4.1.3. (E)-3-(2-Phenyl-1H-indol-3-yl)-1-(pyridin-3-yl) prop-2-en-
1-one (3b)
Yield: 90%. Yellow crystals; m.p.: 224e225  C, IR (KBr) ʋmax/
cm1: 3265 (NH), 1645 (CO), 1H-NMR (DMSO-d6, 400 MHz):
d 7.31e7.78 (m, 10H, indole-C4,5,6,7eH, 2-phenyl-C2,3,4,5,6-H,
CH¼CHCO), 8.09 (d, J ¼ 15.00 Hz, 1H, CH¼CH-CO), 8.21 (d,
J ¼ 8.00 Hz, 1H, pyridine-C6-H), 8.28 (d, J ¼ 7.00 Hz, 1H, pyridine-C4-
H), 8.79 (m, 1H, pyridine-C5-H), 9.26 (s, 1H, pyridine-C2-H), 12.3 (s,
1H, NH, D2O exchangeable). 13C-NMR (DMSO-d6, 100 MHz):
d 109.39 (indole-C3), 112.23 (indole-C7), 115.92 (indole-C5), 121.37
(indole-C6), 121.78 (indole-C4), 123.34 (indole-C2), 123.83 (indole-
C3a), 125.73 (C¼C-CO), 128.99 (2-phenyl-C2,6), 129.34 (2-phenyl-C4),
129.73 (2-phenyl-C3,5), 130.81 (pyridine-C3), 133.63 (2-phenyl-C1),
135.68 (C4-pyridine), 136.92 (indole-C7a), 139.58 (C5-pyridine),
145.71 (C¼C-CO), 149.25 (pyridine-C2), 152.67 (pyridine-C6), 187.89
(CO). MS EIMS: m/z (calcd) 324.13 (found) 323.98 (Mþ). Anal.
Calcd for C22H16N2O: C, 81.46; H, 4.97; N 8.64. Found: C, 81.58; H,
4.79; N, 8.49.
4.1.4. (E)-3-(2-Phenyl-1H-indol-3 -yl)-1-(thiophen-2-yl) prop-2-
en-1-one (3c)
Yield: 70%. Orange crystals; m.p.: 254e255  C. IR (KBr) ʋmax/
1
cm : 3215 (NH), 1626 (CO), 1H-NMR (DMSO-d6, 400 MHz):
d 7.22e7.31 (m, 3H, indole-C5,6eH, 2-phenyl-C4-H),7.49 (d,
J ¼ 7.50 Hz, 1H, C7-indole-H), 7.55e7.64 (m, 5H, C4,5-thiophen-H, 2-
phenyl- C3,5-H, -CH¼CH-CO), 7.78 (d, J ¼ 8Hz, 2H, 2-phenyl C-2,6-H),
8.02 (d, J ¼ 15.00 Hz, 1H, -CH¼CH-CO,), 8.20e8.26 (m, 2H, indole-
C4-H, thiophen-C3-H), 12.35 (s, 1H, NH, D2O exchangeable). 13C-
NMR (DMSO-d6, 100 MHz): d 112.02 (indole-C3), 116.06 (indole-C7),
121.07 (indole-C5), 121.65 (indole-C6), 122.45 (indole-C4), 123.27
(indole-C2), 123.71 (indole-C3a), 125.77 (2-phenyl-C2,6), 128.76
(C¼C-CO), 128.98 (2-phenyl-C4), 129.25 (2-phenyl-C3,5), 129.69
(thiophen-C4), 129.85 (thiophen-C3), 129.91 (2-phenyl-C1), 132.18
(thiophen-C5), 134.22 (indole-C7a), 135.91 (thiophen-C2), 137.85
(C¼C-CO), 185.51 (CO). MS EIMS: m/z (calcd) 329.09 (found) 329.22
(Mþ). Anal. Calcd for C21H15NOS: C,76.57; H, 4.59; N, 4.25. Found:
C, 76.38; H, 4.77; N, 4.56.
4.1.5. General procedure for 2-Phenyl-3-(3-aryl-4,5-dihydro-1H-
pyrazol-5-yl)-1H-indoles (4a-c)
A solution (3a-c) (1.0 mmol), and hydrazine hydrate 99%
(1.1 mmol, 0.06 g) in absolute ethanol (10 mL) was refluxed for
8e10 h. The resulting solution was concentrated, left to cool, the
solid obtained was filtered off and recrystallized from the appro-
priate solvent.
4.1.6. 2-Phenyl-3-[3-(pyridin-4-yl)-4,5-dihydro-1H-pyrazol-5-yl]-
1H-indole (4a)
Yield: 60.0%. Recrystallized from ethanol; pale yellow crystals;
m.p.: 209e210  C. IR (KBr) ʋmax/cm1: 3359 (NH). 1H-NMR (DMSO-
d6, 300 MHz): d 11.35 (s, 1H, NH, D2O exchangeable), 8.55 (d,
J ¼ 4.7 Hz, 2H, pyridine-C2,6eH), 8.01 (s, 1H, NH-pyrazoline, D2O
exchangeable), 7.63 (d, J ¼ 7.7 Hz, 2H, 2-phenyl-C2,6eH), 7.54 (dd,
J ¼ 11.5, 5.4 Hz, 4H, indole-C4,7, pyridine-C3,5eH), 7.42 (dd, J ¼ 12.6,
6.0 Hz, 3H, 2-phenyl-C3,4,5-H), 7.10 (t, J ¼ 7.5 Hz, 1H, indole-C6-H),
6.92 (t, J ¼ 7.4 Hz, 1H, indole-C5-H), 5.26 (t, J ¼ 11.6 Hz, 1H,
 H.S. ElBordiny et al. / European Journal of Medicinal Chemistry 145 (2018) 594e605
pyrazoline-C5eH), 3.50e3.41 (m, 1H, pyrazoline-C4eH), 3.20 (dd,
J ¼ 16.6, 11.1 Hz, 1H, pyrazoline-C4eH). 13C-NMR (DMSO-d6,
75 MHz): d 37.59 (pyrazoline-C4), 56.85 (pyrazoline-C5), 111.630
(indole-C3), 111.50 (indole-C7), 118.80 (indole-C4), 119.27 (indole-
C5), 120.00 (indole-C6), 121.56 (indole-C2), 126.15 (indole-C3a),
127.85 (2-phenyl-C4), 128.68 (pyridine-C2,6), 128.83 (2-phenyl-C2,6),
132.26 (2-phenyl-C3,5), 135.92 (2-phenyl-C1), 136.45 (indole-C7a),
140.51 (pyridine-C1), 145.44 (pyrazoline-C3), 149.83 (pyridine-C3,5).
MS EIMS: m/z (calcd) 338.15 (found) 337.95 (Mþ). Anal. Calcd for
C22H18N4: C, 78.08; H, 5.36; N, 16.56. Found: C, 78.27; H, 5.54; N,
16.37.
4.1.7. 2-Phenyl-3-[3-(pyridin-3-yl)-4,5-dihydro-1H-pyrazol-5-yl]-
1H-indole (4b)
Yield 57.0%. Recrystallized from ethanol; yellow powder; m.p.
179e180  C. (KBr) ʋmax/cm1: 3056, 3318 (NH). 1H-NMR (DMSO-
d6, 300 MHz): d 11.33 (s, 1H, NH, D2O exchangeable), 8.86 (s, 1H,
pyridine-C2-H), 8.51 (d, J ¼ 2.2 Hz, 1H, pyridine-C4-H), 8.04 (d,
J ¼ 8.0 Hz, 1H, pyridine-C6-H), 7.68 (s, 1H, NH-pyrazoline, D2O
exchangeable), 7.64 (d, J ¼ 7.4 Hz, 2H, indole-C4,7-H), 7.52 (dd,
J ¼ 15.2, 7.7 Hz, 3H, pyridine-C5-H, 2-phenyl-C2,6eH), 7.46e7.37 (m,
3H, 2-phenyl-C3,4,5eH), 7.10 (t, J ¼ 7.5 Hz, 1H, indole-C6-H), 6.93 (t,
J ¼ 7.5 Hz, 1H, indole-C5-H), 5.22 (t, J ¼ 11.3 Hz, 1H, pyrazoline-C5-
H), 3.47 (dd, J ¼ 16.3, 11.9 Hz, 1H, pyrazoline-C4-H), 3.24 (dd,
J ¼ 16.6, 11.4 Hz, 1H, pyrazoline-C4-H). 13C-NMR (DMSO-d6,
75 MHz): d 38.20 (pyrazoline-C4), 56.57 (pyrazoline-C5), 111.45
(indole-C3), 111.68 (indol-C7), 118.72 (indole-C4), 120.22 (indole-C5),
121.52 (indole-C6), 123.66 (indole-C2), 127.81 (indol-C3a), 128.67
(pyridine-C2,6), 128.83 (2-phenyl-C2,6), 129.36 (2-phenyl-C4), 132.14
(2-phenyl-C3,5), 135.90 (2-phenyl-C1), 136.46 (indol-C7a), 145.94
(pyridine-C1), 146.48 (pyridine-C3,5), 148.54 (pyrazoline-C3). MS
EIMS: m/z (calcd) 338.15 (found) 338.44 (Mþ). Anal. Calcd for
C22H18N4: C, 78.08; H, 5.36; N, 16.56. Found: C, 78.28; H, 5.54; N,
16.73.
4.1.8. 2-Phenyl-3-[3-(thiophen-2-yl)-4, 5-dihydro-1H-pyrazol-5-
yl]-1H-indole (4c)
Yield: 55.0%. Recrystallized from benzene; mustard yellow
powder; m.p. 124e125  C. IR (KBr) ʋmax/cm1: 3388, 3213 (NH). 1H-
NMR (DMSO-d6, 400 MHz): d 11.29 (s, 1H, NH, D2O exchangeable),
7.59 (s,1H, NH-pyrazoline), 7.52 (m, 3H, 2-phenyl-C3,4,5-H), 7.43 (m,
5H, indole-C4,7-H, thiophen-C5eH, 2-phenyl-C2,6-H), 7.07 (m, 3H,
thiophen-C3eH, indole-C5,6-H), 6.96 (m, 1H, indole-C5-H), 5.24 (t,
J ¼ 11.4 Hz, 1H, pyrazoline-C5-H), 3.40 (dd, J ¼ 16.4, 11.5 Hz, 1H,
pyrazoline-C4-H), 3.20 (dd, J ¼ 16.4, 11.6 Hz, 1H, pyrazoline-C4-H),
MS EIMS: m/z (calcd) 343.11 (found) 343.32 (Mþ). Anal. Calcd for
C21H17N3S: C,73.44; H, 4.99; N, 12.24. Found: C, 73.62; H, 4.78; N,
12.43.
4.1.9. General procedure for 5-(2-Phenyl-1H-indol-3-yl)-3-(aryl)-4,
5-dihydroisoxazoles (5a-c)
A solution of 3a-c (1.0 mmol) and hydroxylamine hydrochloride
(1.4 mmol, 0.1 g) and a catalytic amount of glacical acetic acid
(0.5 mL) in absolute ethanol (10 mL), was heated under reflux from
8 to 10 h until the reaction was judged complete, the reaction
mixture left to cool down then poured onto crushed ice. The pre-
cipitate obtained was filtered off, washed with water and recrys-
tallized from ethanol.
4.1.10. 5-(2-Phenyl-1H-indol-3-yl)-3-(pyridin-4-yl)-4,5-
dihydroisoxazole(5a)
Yield: 81%. Off-white powder. m.p.: 224e225  C. IR (KBr) ʋmax/
1
cm : 3201 (NH), 1H-NMR (DMSO-d6, 300 MHz): d 11.61 (s, 1H, NH,
D2O exchangeable), 8.71 (d, J ¼ 4.4 Hz, 2H, C2,6-pyridine-H), 7.71 (d,
J ¼ 4.7 Hz, 2H, C3,5-pyridineeH), 7.66e7.51 (m, 4H, indole-C4-H, 2-
601
phenyl-C2,4,6), 7.46 (t, J ¼ 8.1 Hz, 2H, indole-C5,6-H), 7.33 (d,
J ¼ 7.7 Hz, 1H, indole-C7-H), 7.15 (t, J ¼ 7.5 Hz, 1H, 2-phenyl-C3-H),
6.99 (t, J ¼ 7.4 Hz, 1H, 2-phenyl-C5-H), 6.08 (t, J ¼ 10.9 Hz, 1H, iso-
xazoline-C5eH), 3.89 (dd, J ¼ 17.3, 11.8 Hz, 1H, isoxazoline-C4eH),
3.66 (dd, J ¼ 17.4, 10.2 Hz, 1H, isoxazoline-C4eH). 13C-NMR (DMSO-
d6, 75 MHz): d 78.78 (isoxazoline-C5), 109.00 (indole-C3), 112.42
(indole-C7), 119.61 (indole-C4), 120.16 (indole-C5), 121.20 (indole-
C6), 122.54 (indole-C2), 126.0 (indole-C3a), 128.90 (pyridine-C3,5),
129.4 (2-phenyl-C2,6), 132.13 (2-phenyl-C1), 136.9 (2-phenyl-C3,5),
137.40 (indole-C7a), 138.17 (pyridine-C4), 150.87 (pyridine-C2,6),
156.24 (isoxazoline-C3). MS EIMS: m/z (calcd) 339.14 (found)
339.00 (Mþ). Anal. Calcd for C22H17N3O: C, 77.86; H, 5.05; N, 12.38.
Found: C, 78.06; H, 4.90; N, 12.56.
4.1.11. 5-(2-Phenyl-1H-indol-3-yl)-3-(pyridin-3-yl)-4,5-
dihydroisoxazole (5b)
Yield: 70%. Buff powder; m.p.119e120  C. IR (KBr) ʋmax/cm1:
3386 (NH),1H-NMR (DMSO-d6, 300 MHz): d 11.58 (s, 1H, NH, D2O
exchangeable), 8.94 (s, 1H, pyridine-C2-H), 8.68 (d, J ¼ 4.7 Hz, 1H,
pyridine-C4-H), 8.18 (d, J ¼ 7.9 Hz, 1H, pyridine-C6-H), 7.65e7.50 (m,
5H, pyridine-C5-H, indole-C4-H, 2-phenyl-C3,4,5-H), 7.51e7.41 (m,
2H, 2-phenyl-C2,6-H), 7.37 (d, J ¼ 8.0 Hz, 1H, indole-C7-H), 7.15 (t,
J ¼ 7.5 Hz, 1H, indole-C6-H), 6.99 (t, J ¼ 7.5 Hz, 1H, indole-C5-H), 6.03
(t, J ¼ 10.9 Hz, 1H, isoxazoline-C5-H), 3.92 (dd, J ¼ 17.2, 11.6 Hz, 1H,
isoxazoline-C4eH), 3.69 (dd, J ¼ 17.4, 10.4 Hz, 1H, isoxazoline-
C4eH). 13C-NMR (DMSO-d6, 75 MHz): d 77.68 (isoxazoline-C5),
109.39 (indole-C3), 111.72 (indole-C7), 119.17 (indole-C4), 119.51
(indole-C5) 121.93 (pyridine-C5), 124.02 (indole-C6), 125.82 (indole-
C2), 128.27 (2-phenyl-C2,6), 128.84 (indole-C3a), 131.65 (2-phenyl-
C4), 133.84 (2-phenyl-C3,5), 136.38 (2-phenyl-C1, pyridine-C3),
137.49 (indole-C7a, pyridine-C4), 147.82 (pyridine-C2), 152.10 (pyri-
dine-C6), 155.30 (isoxazoline-C3). MS EIMS: m/z (calcd) 339.14
(found) 339.29 (Mþ). Anal. Calcd for C22H17N3O: C,77.86; H, 5.05;
N,12.38. Found: C, 78.04; H, 5.19; N, 12.20.
4.1.12. 5-(2-Phenyl-1H-indol-3-yl)-3-(thien-2-yl)-4,5-
dihydroisoxazole(5c)
Yield 80%. Yellow powder; m.p.188e190  C. IR (KBr) ʋmax/cm1:
3345 (NH). 1H-NMR (DMSO-d6, 400 MHz): d 12.59 (s, 1H, NH, D2O
exchangeable), 7.98 (d, J ¼ 7.5 Hz, 1H, thiophen-C3-H), 7.78 (d,
J ¼ 8.0 Hz, 2H, 2-phenyl-C-2,6-H), 7.48e7.66 (m, 6H, thiophen-C4,5-
H, indole-C4,7-H, 2-phenyl- C3,5-H), 7.27e7.37 (m, 2H, indole-C6eH,
2-phenyl-C4-H), 7.10e7.11 (m, 1H, indole-C5-H), 5.93 (t, J ¼ 11.0 Hz,
1H, isoxazoline-C5-H), 3.90 (dd, J ¼ 17.5,11.0 Hz, 1H, isoxazoline-C4-
H), 3.70 (dd, J ¼ 17.5, 11.3 Hz, 1H, isoxazoline-C4-H). MS EIMS: m/z
(calcd) 344.10 (found) 344.35 (Mþ). Anal. Calcd for C21H16N2OS: C,
73.23; H, 4.68; N, 8.13. Found: C, 73.50; H, 4.79; N, 8.27.
4.1.13. General procedure for synthesis of 1-[5-(2-Phenyl-1H-indol-
3-yl)-3-(pyridin-3-/4-yl)-4,5-dihydro-1H-pyrazol-1-yl]ethan-1-
ones (6a,c) and Phenyl [5-(2-phenyl-1H-indol-3-yl)-3-(pyridin-3-/
4-yl)-4,5-dihydro-1H-pyrazol-1-yl]methanones (6b,d)
A solution of indolylpyrazolines 4a,b (1.0 mmol) and TEA
(1.0 mmol, 0.1 g) in dry acetone (15 mL) was cooled to 0  C in an ice
bath with stirring for 30 min, then acetyl- or benzoyl chloride
(1.0 mmol) was added dropwise. After complete addition, the re-
action mixture was stirred for 6 h at room temperature until the
reaction judged complete. The resulting solution was poured onto
ice-cold water and the separated solid was filtered off, washed with
water and then recrystallized from ethanol.
4.1.14. 1-[5-(2-Phenyl-1H-indol-3-yl)-3-(pyridin-4-yl)-4,5-
dihydro-1H-pyrazol-1-yl]ethan-1-one (6a)
Yield: 55%. Pale yellow powder; m.p. 184e185  C. IR (KBr) ʋmax/
cm1: 3390 (NH), 1678 (CO).1H-NMR (DMSO-d6, 300 MHz): d 11.43
602 H.S. ElBordiny et al. / European Journal of Medicinal Chemistry 145 (2018) 594e605
(s, 1H, NH, D2O exchangeable), 8.80 (d, J ¼ 5.0 Hz, 2H, pyridine-
C2,6eH), 8.03 (d, J ¼ 5.0 Hz, 2H, pyridine-C3,5eH), 7.80 (d, J ¼ 7.5 Hz,
2H, 2-phenyl-C2,6-H), 7.06e7.57 (m, 6H, indole-C4,6,7-H, 2-phenyl-
C3,4,5eH), 6.88e6.93 (m,1H, indole-C5-H), 5.24 (dd, J ¼ 12.0,
12.60 Hz, 1H, pyrazoline-C5-H), 3.90 (dd, J ¼ 18.0, 12.0 Hz, 1H, pyr-
azoline-C4-H), 3.30 (dd, J ¼ 18.0, 12.40 Hz, 1H, pyrazoline-C4-H),
2.27 (s, 3H, CH3). 13C-NMR (DMSO-d6, 75 MHz): d 22.34 (CH3CO),
41.30 (pyrazoline-C4), 55.40 (pyrazoline-C5), 111.80 (indole-C3),
112.36 (indole-C7), 118.20 (indole-C4), 120.0 (indole-C5), 122.90
(indole-C6), 123.15 (pyridine-C3,5), 125.65 (indole-C2), 128.52
(indole-C3a), 129.26 (2-phenyl-C2,6), 129.49 (2-phenyl-C3,5), 132.80
(2-phenyl-C4), 135.90 (2-phenyl-C1), 136.79 (indole-C7a), 144.90
(pyridine-C2,6), 145.10 (pyridine-C1), 151.37 (pyrazoline-C3), 169.15
(C¼O). MS EIMS: m/z (calcd) 380.16 (found) 380.42 (Mþ). Anal.
Calcd for C24H20N4O: C,75.77; H, 5.30; N,14.73. Found: C,76.04; H,
5.48; N, 14.94.
4.1.15. Phenyl[5-(2-phenyl-1H-indol-3-yl)-3-(pyridin-4-yl)-4,5-
dihydro-1H-pyrazol-1-yl]methanone (6b)
Yield 65%. White crystals; m.p.: 199e200  C. IR (KBr) ʋmax/
cm1: 3401 (NH), 1636 (CO). 1H-NMR (DMSO-d6, 300 MHz): d 11.46
(s, 1H, NH, D2O exchangeable), 8.69 (d, J ¼ 4.6 Hz, 2H, pyridine-
C2,6eH), 7.92 (d, J ¼ 7.4 Hz, 2H, benzoyl-C2,6-H), 7.76 (d, J ¼ 6.9 Hz,
2H, 2-phenyl-C2,6-H), 7.66 (d, J ¼ 4.7 Hz, 2H, pyridine-C3,5eH), 7.58
(t, J ¼ 7.5 Hz, 2H, benzoyl-C3,5-H), 7.53e7.38 (m, 5H, 2-phenyl-C3,4,5-
H, benzoyl-C4-H, indole-C4-H), 7.29 (d, J ¼ 7.9 Hz, 1H, indole-C7-H),
7.09 (t, J ¼ 7.5 Hz, 1H, indole-C6-H), 6.95 (t, J ¼ 7.4 Hz, 1H, indole-C5-
H), 6.17 (dd, J ¼ 12.2, 6.4 Hz, 1H, pyrazoline-C5-H), 3.98 (dd, J ¼ 18.2,
12.5 Hz, 1H, pyrazoline-C4-H), 3.29 (m, 1H, pyrazoline-C4-H). 13C-
NMR (DMSO-d6, 75 MHz): d 45.35 (pyrazoline-C4), 55.09 (pyrazo-
line-C5), 111.30 (indole-C3), 111.81 (indole-C7), 117.72 (indole-C4),
119.46 (indole-C5), 120.52 (indole-C6), 121.58 (pyridine-C3,5), 125.31
(indole-C2), 127.76 (2-phenyl-C2,6), 127.92 (indole-C3a), 128.71
(benzoyl-C2,6), 128.91 (benzoyl-C3,5), 129.27 (2-phenyl-C3,5), 130.82
(2-phenyl-C4), 132.40 (benzoyl-C4), 134.56 (2-phenyl-C1), 135.46
(indole-C7a), 136.32 (benzoyl-C1), 138.21 (pyridine-C5), 150.36
(pyridine-C2,6), 153.61 (pyrazoline-C3), 166.10 (CO). MS EIMS: m/z
(calcd) 442.18 (found) 442.25 (Mþ). Anal. Calcd for C29H22N4O: C,
78.71; H, 5.01; N, 12.66. Found: C, 78.96; H, 5.22; N,12.89.
4.1.16. 1-[5-(2-Phenyl-1H-indol-3-yl)-3-(pyridin-3-yl)-4,5-
dihydro-1H-pyrazol-1-yl]ethan-1-one (6c)
Yield 60%. White powder; m.p.124e125  C. IR (KBr) ʋmax/cm1:
3427 (NH), 1656 (CO). 1H-NMR (DMSO-d6, 300 MHz): d 11.40 (s, 1H,
NH, D2O exchangeable), 8.99 (s, 1H, pyridine-C2-H), 8.66 (d,
J ¼ 4.3 Hz, 1H, pyridine-C6eH), 8.22 (d, J ¼ 7.8 Hz, 1H, pyridine-
C4eH), 7.87 (d, J ¼ 7.5 Hz, 2H, indole-C4,7-H), 7.54 (t, J ¼ 7.10 Hz, 3H,
2-phenyl-C3,4,5-H), 7.42 (dd, J ¼ 15.7, 7.7 Hz, 2H, indole-C6-H, pyri-
dine-C5), 7.21e6.97 (m, 2H, 2-phenyl-C2,6-H), 6.90 (t, J ¼ 7.6 Hz, 1H,
indole-C5-H), 5.90 (dd, J ¼ 12.5, 5.7 Hz, 1H, pyrazoline-C5-H), 3.94
(dd, J ¼ 18.5, 12.7 Hz, 1H, pyrazoline-C4-H), 3.26 (m, 1H, pyrazoline-
C4-H), 2.24 (s, 3H, CH3CO). 13C-NMR (DMSO-d6, 75 MHz): 21.76
(CH3CO), 48.52 (pyrazoline-C4), 53.49 (pyrazoline-C5), 111.68
(indole-C3), 111.87 (indole-C7), 117.81 (indole-C4), 119.29 (indole-
C5), 121.45 (indole-C6), 123.91 (pyridine-C3), 125.16 (pyridine-C5),
127.21 (indole-C2), 127.79 (indole-C3a), 128.61 (2-phenyl-C2,6),
128.92 (2-phenyl-C3,5), 132.42 (2-phenyl-C4), 133.64 (2-phenyl-C1),
134.99 (indole-C7a), 136.26 (pyridine-C4), 147.57 (pyridine-C2),
150.82 (pyrazol-C3), 152.18 (pyridine-C6), 167.81 (CO). MS EIMS:
(calcd) m/z 380.16 (found) 380.33 (Mþ). Anal. Calcd for
C24H20N4O: C,75.77; H, 5.30; N,14.73. Found: C,76.01; H, 5.54; N,
14.94.
4.1.17. Phenyl[5-(2-phenyl-1H-indol-3-yl)-3-(pyridin-3-yl)-4,5-
dihydro-1H-pyrazol-1-yl]methanone (6d)
Yield 65%. Yellowish brown powder; m.p.189e190  C. IR (KBr)
ʋmax/cm1: 3268 (NH), 1699 (CO). 1H-NMR (DMSO-d6, 300 MHz):
11.39 (s,1H, NH, D2O exchangeable), 8.92 (s,1H, pyridine-C2-H), 8.66
(d, J ¼ 5.0 Hz, 1H, pyridine-C6-H), 8.13 (d, J ¼ 7.3 Hz,1H, pyridine-C4-
H), 7.90 (t, J ¼ 7.0 Hz, 1H, pyridine-C5-H), 7.70 (d, J ¼ 7.0 Hz, 1H,
indole-C4-H), 7.31e7.57 (m, 11H, indole-C7-H, 2-phenyl-C2,3,4,5,6-H,
benzoyl-C2,3,4,5,6), 7.10 (m,1H, indole-C6-H), 6.90 (m,1H, indole-C5-
H), 6.18 (dd, J ¼ 11.7, 6.3 Hz, 1H, pyrazoline-C5-H), 4.0 (dd, J ¼ 17.0,
11.7 Hz, 1H, pyrazoline-C4-H), 3.41 (m, 1H, pyrazoline-C4-H). 13C-
NMR (DMSO-d6, 75 MHz): 49.06 (pyrazoline-C4), 55.20 (pyrazo-
line-C5), 111.98 (indole-C3), 112.25 (indole-C7), 118.34 (indole-C4),
119.95 (indole-C5), 122.07 (indole-C6), 125.85 (indole-C2), 128.21
(pyridine-C5),128.39 (pyridine-C1), 129.02 (indole-C3a), 129.21 (2-
phenyl-C2,6), 129.41 (benzoyl-C2,6), 129.71 (benzoyl-C3,5), 129.79
(2-phenyl-C3,5), 131.2 (2-phenyl-C4), 132.94 (benzoyl-C4), 133.31 (2-
phenyl-C1), 134.25 (indole-C7a), 135.89 (benzoyl-C1), 148.19 (pyri-
dine-C2), 151.43 (pyridine-C6), 153.81 (pyrazoline-C3), 166.35 (CO).
MS EIMS: m/z (calcd) 442.18 (found) 441.87 (Mþ). Anal. Calcd for
C29H22N4O: C,78.71; H, 5.01; N, 12.66. Found C,78.97; H, 5.08; N,
12.94.
4.1.18. 3-[1-Methyl-3-(pyridin-4-yl)-4,5-dihydro-1H-pyrazol-5-yl]-
2-phenyl-1H-indole (7)
A solution of indolylpyrazoline 4a (1.0 mmol, 0.3 g) and TEA
(1 mmol, 0.1 g) in dry acetone (15 mL) was cooled in an ice bath
with stirring for 30 min and then methyl iodide (1 mmol, 0.14 g)
was added dropwise. The stirring was continued for 9 h at room
temperature, then the reaction mixture was poured onto ice-cold
water, the solid separated was filtered off, washed with water
and recrystallized from ethanol.
Yield 50%. Dark orange powder; m.p.145e146  C. IR (KBr) ʋmax/
cm1: 3208 (NH), 1H-NMR (DMSO-d6, 300 MHz): 11.48 (s, NH, D2O
exchangeable), 8.75 (d, J ¼ 6.9 Hz, 1H, indole-C4eH), 8.67 (d,
J ¼ 4.5 Hz, 2H, pyridine-C2,6eH), 8.55 (d, J ¼ 8.0 Hz, 1H, indole-
C7eH), 7.96 (d, J ¼ 4.2 Hz, 2H, pyridine-C3,5eH), 7.38e7.65 (m, 5H,
2-phenyl-C2,3,4,5,6-H), 7.1e7.22 (m, 1H, indole-C5eH), 6.94e6.97
(m,1H, indole-C6eH), 5.5e5.6 (m, 1H, pyrazoline-C5-H), 4.20 (s, 3H,
N-CH3), 3.45e3.6 (m, 1H, pyrazoline-C4-H), 3.15e3.22 (m, 1H, pyr-
azoline-C4-H). 13C-NMR (DMSO-d6, 100 MHz): 31.15 (CH3), 46.15
(pyrazoline-C4), 57.35 (pyrazoline-C5), 112.03 (indole-C3), 112.82
(indole-C7), 119.32 (indole-C4), 119.79 (indole-C5), 120.53 (indole-
C6), 122.08 (indole-C2), 126.18 (indole-C3a), 128.37 (2-phenyl-C4),
129.35 (pyridine-C2,6), 129.89 (phenyl-C2,6), 132.77 (2-phenyl-C3,5),
136.44 (2-phenyl-C1), 136.97 (indole-C7a), 141.01 (pyridine-C1),
145.97 (pyrazoline-C3), 150.35 (pyridine-C3,5). MS EIMS: (calc)
352.17 (found) 352.28 (Mþ). Anal. Calcd for C23H20N4: C,78.38; H,
5.72; N,15.90. Found: C,78.56; H, 5.94; N, 16.13.
4.2. Antioxidant activity
4.2.1. DPPH method
The DPPH scavenging activity of tested compounds was
measured according to the method described by Nahar et al. [39]
with some modifications. Briefly, 100 mL of different concentrations
of the tested compounds (12.5, 25, 50, 100 and 200 mg/ml) were
pipetted into a 96-well flat-bottomed plate. Next, 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 absorbance of the solution was measured at l 517 nm
[41]. Ascorbic acid and DMSO were used as the positive control and
blank respectively. The percentage of DPPH scavenging activity was
calculated according to the following equation:
 H.S. ElBordiny et al. / European Journal of Medicinal Chemistry 145 (2018) 594e605
% of DPPH scavenging ¼ [(A blank eA sample) / A blank]  100
Where A blank is the absorbance of the control reaction (containing
all reagents except the test compound), and A sample is the
absorbance of the test sample.
Dose-response curve was plotted between %free radical scav-
enging activity and the drug concentration. Linear regression
analysis was carried out for calculating drug concentration showing
50% free radical inhibition activity (IC50). All tests were undertaken
on three replicates and the results were averaged.
4.2.2. NO scavenging method
The nitric oxide (NO) scavenging activity of tested compounds
was measured according to the method described by Ho et al. [42].
In this method 50 mL of different concentrations of the tested
compounds (12.5, 25, 50,100 and 200 mg/ml) were pipetted into a 96-
well flat-bottomed plate. Next, 50 mL of 10 mM sodium nitroprusside
dissolved in Phosphate buffered saline PBS (pH 7.4) were added to
each well and the plate was incubated at room temperature for
90 min. Finally, an equal volume of Griess reagent (1% of sulphani-
lamide and 0.1% of naphthyl ethylene diamine in 2.5% H3PO3) was
added to each well to measure the nitrite content [41]. A pink-colored
chromophore was formed in diffused light. The absorbance of the
solution was measured at l 546 nm. Ascorbic acid and DMSO were
used as the positive control and blank respectively. All tests were
undertaken on three replicates and the results were averaged.
The percentage of NO scavenging activity was calculated ac-
cording to the following equation:
% of NO scavenging ¼ [(A blank -A sample) / A blank]  100
Where A blank is the absorbance of the control reaction (containing
all reagents except the test compound), and A sample is the
absorbance of the test sample.
Dose-response curve was plotted between % free radical scav-
enging activity and the drug concentration. Linear regression
analysis was carried out for calculating drug concentration showing
50% free radical inhibition activity (IC50).
4.2.3. SOD assay
The assay was performed at room temperature using superoxide
Dismutase assay Kit (Catalog#7500-100-k). In this method, in a
disposable cuvette, the following component were added in order:
60 mL of reaction buffer, 7.5 mL xanthine solution, 30 mL of Nitroblue
Tetrazolium (NBT) solution, test compound at concentrations
(2.5 mM, 5.0 mM and 10 mM), 10 mL Xanthine Oxidase (XOD) solution,
read the absorbance at 550 nm, start a timer or stopwatch and record
the absorbance reading every 30 s for 5 min [43]. The first time will
be at 30 s and the final time point will be at 5 min 30 secconds.
1. Determine the rate of increase in absorbance units (A) per
minute for the negative control and for the test sample(s):
A550 @ 5 min:30 sec:  A550 @30 sec:
D A550n =minute ¼
5min
2. Determine the % inhibition for the test sample(s):
½ðDA550n =minuteÞnegative control  ðDA550n =minuteÞtest
%Inhibition ¼
ðDA550 =minuteÞnegative control
603
Dose-response curve was plotted between % inhibition of the
rate of increase of absorbance at 550 nm due to the reduction of
NBT to NBT-diformazan by the superoxide radical and the drug
concentration. The non-linear dose-response curve was used for
calculating drug concentration showing 50% free radical inhibition
activity (IC50).
4.3. In-vitro lipoxygenase inhibition activity
The assay was performed using Abnova Lipoxygenase inhibitor
screening assay kit (Catalog#KA1329). In this method [44], 90 mL of
soybean LOX was pipetted into a 96-well flat-bottomed plate. Next,
10 mL of test compound at concentrations (2.5 mM, 5.0 mM and
10 mM) dissolved in DMSO were added to each well. The reaction
was initiated by adding 10 mL substrate (arachidonic acid or linoleic
acid) and the plate was placed on a shaker for at least five minutes.
Finally, an equal volume 100 mL of chromogen was added to each
well to stop enzyme catalysis and develop the reaction. The
absorbance of the solution was measured at l 490e500 nm. Quer-
cetin, assay buffer and DMSO were used as the positive control,
blank and 100% initial activity respectively.
The percentage inhibition was calculated according to the
following equation:
% inhibition ¼ [(IA -A sample) / IA]  100
Where IA is the 100% initial activity (containing all reagents except
the test compound), and A sample is the 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
activity (IC50).
4.4. Docking study
All docking studies were performed using ‘Internal Coordinate
Mechanics (MOLSOFT ICM 3.4e8C)’. ICM docking is probably one of
the most accurate and predictive tools of binding geometry today
[45e48].
4.4.1. Preparation of small molecule
Regarding the synthesized compounds (4b), (4c), (5b) and (6c)
are to be docked as 15-LOX inhibitors; ChemDraw 3D structures
were constructed using Chem 3D ultra 15.1 software [Molecular
Modeling and Analysis; Cambridge Soft Corporation, USA (2015)],
and then they were energetically minimized by using MOPAC
(semi-empirical quantum mechanics), Jop Type with 100 iterations
and minimum RMS gradient of 0.01, and saved as MDL MolFile
(*.mol).
4.4.2. Generation of enzyme structure
The crystal structure of target protein (15-LOX) is a human
isoform of lipoxygenase in complex with ligand substrate (arach-
idonate) was retrieved from the Protein Data Bank (http://www.
rcsb.org/pdb/welcome.- do). All bound water, substrate and co-
factors were removed from the protein. The amino acids of the
 100
604 H.S. ElBordiny et al. / European Journal of Medicinal Chemistry 145 (2018) 594e605
binding site where defined using data in pdbsum (http://www.ebi.
ac.uk/thoronton-srv/databases/pdbsum/).
4.4.3. Docking using MOLSOFT ICM 3.4e8C program
1. Convert our PDB file into an ICM object: This conversion in-
volves addition of hydrogen bonds, assignment of atoms types,
and charges from the residue templates.
2. To perform ICM small molecule docking:
a) Setup docking project:
1) Set project name:
2) Setup the receptor:
3) Review and adjust binding site:
4) Make receptor maps:
b) Start docking simulation:
3. Display the result:
ICM stochastic global optimization algorithm attempts to find
the global minimum of the energy function that include five grid
potentials describing interaction of the flexible ligand with the
receptor and internal conformational energy of the ligand, during
this process a stack of alternative low energy conformations is
saved. The binding pocket of the substrate co-crystallized with the
enzyme that was chosen for docking the inhibitors into it.
References
[1] A.R. Brash, Lipoxygenases: occurrence, functions, catalysis, and acquisition of
substrate, J. Biol. Chem. 274 (1999) 23679e23682.
[2] E. Pontiki, D. Hadjipavlou-Litina, Lipoxygenases (LOs): an heterogenous family
of lipid peroxidizing enzymes implicated in cell differentiation, inflammation,
asthma, carcinogenesis, atherogenesis-an interesting target for the develop-
ment of promising drugs, Curr. Enzym. Inhib. 1 (2005) 309e327.
[3] J.S. Larsen, E.P. Acosta, Leukotriene-receptor antagonists and 5-lipoxygenase
inhibitors in asthma, Ann. Pharmacother. 27 (1993) 898e903.
[4] G.P. Pidgeon, J. Lysaght, S. Krishnamoorthy, J.V. Reynolds, K. O'Byrne, D. Nie,
K.V. Honn, Lipoxygenase metabolism: roles in tumor progression and survival,
Canc. Metastasis Rev. 26 (2007) 503e524.
[5] L. Zhao, C.D. Funk, Lipoxygenase pathways in atherogenesis, Trends Car-
diovasc. Med. 14 (2004) 191e195.
[6] D. Pratico  , V. Zhukareva, Y. Yao, K. Uryu, C.D. Funk, J.A. Lawson,
J.Q. Trojanowski, V.M.Y. Lee, 12/15-Lipoxygenase is increased in Alzheimer's
disease: possible involvement in brain oxidative stress, Am. J. Pathol. 164
(2004) 1655e1662.
[7] D. Nie, K.V. Honn, Cyclooxygenase, lipoxygenase and tumor angiogenesis,
Cellular and Molecular Life Sciences CMLS 59 (2002) 799e807.
[8] U. Kelavkar, W. Glasgow, T.E. Eling, The effect of 15-lipoxygenase-1 expression
on cancer cells, Curr. Urol. Rep. 3 (2002) 207e214.
[9] U.P. Kelavkar, C. Cohen, H. Kamitani, T.E. Eling, K.F. Badr, Concordant induction
of 15-lipoxygenase-1 and mutant p53 expression in human prostate adeno-
carcinoma: correlation with Gleason staging, Carcinogenesis 21 (2000)
1777e1787.
[10] C. Sultana, Y. Shen, V. Rattan, V.K. Kalra, Lipoxygenase metabolites induced
expression of adhesion molecules and transendothelial migration of
monocyte-like HL-60 cells is linked to protein kinase C activation, J. Cell.
Physiol. 167 (1996) 477e487.
[11] B.N. Setty, M.H. Werner, Y.A. Hannun, M.J. Stuart, 15-Hydroxyeicosatetraenoic
acid-mediated potentiation of thrombin-induced platelet functions occurs via
enhanced production of phosphoinositide-derived second messengers-sn-
1,2-diacylglycerol and inositol-1,4,5-trisphosphate, Blood 80 (1992)
2765e2773.
[12] T. Schewe, 15-lipoxygenase-1: a prooxidant enzyme, Biol. Chem. 383 (2002)
365e374.
[13] J.C. Boyington, B.J. Gaffney, L.M. Amzel, The three-dimensional structure of an
arachidonic acid 15-lipoxygenase, Science 260 (1993) 1482e1486.
[14] A. Garreta, S.P. Val-Moraes, Q. Garcia-Fernandez, M. Busquets, C. Juan,
A. Oliver, A. Ortiz, B.J. Gaffney, I. Fita, A. Manresa, X. Carpena, Structure and
interaction with phospholipids of a prokaryotic lipoxygenase from Pseudo-
monas aeruginosa, Faseb. J. 27 (2013) 4811e4821.
[15] N.C. Gilbert, S.G. Bartlett, M.T. Waight, D.B. Neau, W.E. Boeglin, A.R. Brash,
M.E. Newcomer, The structure of human 5-lipoxygenase, Science 331 (2011)
217e219.
[16] S.A. Gillmor, A. Villasenor, R. Fletterick, E. Sigal, M.F. Browner, The structure of
mammalian 15-lipoxygenase reveals similarity to the lipases and the de-
terminants of substrate specificity, Nat. Struct. Biol. 4 (1997) 1003e1009.
[17] S. Xu, T.C. Mueser, L.J. Marnett, M.O. Funk Jr., Crystal structure of 12-
lipoxygenase catalytic-domain-inhibitor complex identifies a substrate-
binding channel for catalysis, Structure 20 (2012) 1490e1497.
[18] W. Minor, J. Steczko, B. Stec, Z. Otwinowski, J.T. Bolin, R. Walter, B. Axelrod,
Crystal structure of soybean lipoxygenase L-1 at 1.4 A resolution, Biochem-
istry 35 (1996) 10687e10701.
[19] S.T. Prigge, J.C. Boyington, M. Faig, K.S. Doctor, B.J. Gaffney, L.M. Amzel,
Structure and mechanism of lipoxygenases, Biochimie 79 (1997) 629e636.
[20] E. Skrzypczak-Jankun, K. Zhou, N.P. McCabe, S.H. Selman, J. Jankun, Structure
of curcumin in complex with lipoxygenase and its significance in cancer, Int. J.
Mol. Med. 12 (2003) 17e24.
[21] M. Mahdavi, M.S. Shirazi, R. Taherkhani, M. Saeedi, E. Alipour, F.H. Moghadam,
A. Moradi, H. Nadri, S. Emami, L. Firoozpour, A. Shafiee, A. Foroumadi, Syn-
thesis, biological evaluation and docking study of 3-aroyl-1-(4-
sulfamoylphenyl)thiourea derivatives as 15-lipoxygenase inhibitors, Eur. J.
Med. Chem. 82 (2014) 308e313.
[22] M.B. Tehrani, S. Emami, M. Asadi, M. Saeedi, M. Mirzahekmati, S.M. Ebrahimi,
M. Mahdavi, H. Nadri, A. Moradi, F.H. Moghadam, S. Farzipour, M. Vosooghi,
A. Foroumadi, A. Shafiee, Imidazo[2,1-b]thiazole derivatives as new inhibitors
of 15-lipoxygenase, Eur. J. Med. Chem. 87 (2014) 759e764.
[23] M. Roussaki, K. Zelianaios, E. Kavetsou, S. Hamilakis, D. Hadjipavlou-Litina,
C. Kontogiorgis, T. Liargkova, A. Detsi, Structural modifications of coumarin
derivatives: Determination of antioxidant and lipoxygenase (LOX) inhibitory
activity, Bioorg. Med. Chem. 22 (2014) 6586e6594.
[24] S. Dianat, S. Moghimi, M. Mahdavi, H. Nadri, A. Moradi, L. Firoozpour,
S. Emami, A. Mouradzadegun, A. Shafiee, A. Foroumadi, Quinoline-based
imidazole-fused heterocycles as new inhibitors of 15-lipoxygenase, J. Enzym.
Inhib. Med. Chem. 31 (2016) 205e209.
[25] A. Saeed, S.U. Khan, P.A. Mahesar, P.A. Channar, G. Shabir, J. Iqbal, Substituted
(E)-2-(2-benzylidenehydrazinyl)-4-methylthiazole-5-carboxylates as dual
inhibitors of 15-lipoxygenase & carbonic anhydrase II: synthesis, biochemical
evaluation and docking studies, Biochem. Biophy. Res. Commun. 482 (2017)
176e181.
[26] S. Ono, K. Okazaki, M. Sakurai, Y. Inoue, Density functional study of the radical
reactions of 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186): implication for
the biological function of MCI-186 as a highly potent antioxidative radical
scavenger, J. Phys. Chem. 101 (1997) 3769e3775.
[27] T. Taj, R.R. Kamble, S.B. Marganakop, Facile synthesis of pyrazoline derivatized
amides viasydnone ring cleavage, J. Heterocycl. Chem. 51 (2014) E323eE328.
[28] P.C. Jagadish, N. Soni, A. Verma, Design, synthesis, and in vitro antioxidant
activity of 1,3,5-trisubstituted-2-pyrazolines derivatives, J. Chem. 2013 (2013)
6.
[29] S. Suzen, P. Bozkaya, T. Coban, D. Nebiogu, Investigation of the in vitro anti-
oxidant behaviour of some 2-phenylindole derivatives: discussion on possible
antioxidant mechanisms and comparison with melatonin, J. Enzym. Inhib.
Med. Chem. 21 (2006) 405e411.
[30] S. Suzen, N. Das-Evcimen, P. Varol, M. Sarıkaya, Preliminary evaluation of rat
kidney aldose reductase inhibitory activity of 2-phenylindole derivatives:
affiliation to antioxidant activity, Med. Chem. Res. 16 (2007) 112e118.
[31] M.V. Reddy, V.K. Billa, V.R. Pallela, M.R. Mallireddigari, R. Boominathan,
J.L. Gabriel, E.P. Reddy, Design, synthesis, and biological evaluation of 1-(4-
sulfamylphenyl)-3-trifluoromethyl-5-indolyl pyrazolines as cyclooxygenase-
2 (COX-2) and lipoxygenase (LOX) inhibitors, Bioorg. Med. Chem. 16 (2008)
3907e3916.
[32] A.I. Vogel, B.S. Furniss, A.I. Vogel, Vogel's Textbook of Practical Organic
Chemistry, fifth ed., Longman Scientific & Technical; Wiley, London New York,
1989.
[33] R.C. Blume, H.G. Lindwall, 2-phenylindole-3-aldehyde and certain of its
condensation products, J. Org. Chem. 11 (1946) 185e188.
[34] A. Bourlot, E. Desarbre, J. Merour, A convenient synthesis of 1, 2-dihydro-3H-
indol-3-ones and 1, 2-dihydro-2H-indol-2-ones by Baeyer-Villiger oxidation,
Synthesis 1994 (1994) 411e416.
[35] V.D. Joshi, M.D. Kshirsagar, S. Singhal, Synthesis and antimicrobial activities of
various pyrazolines from chalcones, International J. ChemTech Res. 4 (2012)
971e975.
[36] M.A. Abdel-Sayed, S.M. Bayomi, M.A. El-Sherbeny, N.I. Abdel-Aziz, K.E. ElTahir,
G.S. Shehatou, A.A. Abdel-Aziz, Synthesis, anti-inflammatory, analgesic, COX-
1/2 inhibition activities and molecular docking study of pyrazoline de-
rivatives, Bioorg. Med. Chem. 24 (2016) 2032e2042.
[37] P.O. Patil, S.B. Bari, Synthesis and antidepressant activity of some new 5-(1H-
Indol-3-yl)-3-(substituted aryl)-4,5-dihydroisoxazoline derivatives, J. Chem.
2013 (2013) 7.
[38] S. Viveka, Dinesha, P. Shama, G.K. Nagaraja, S. Ballav, S. Kerkar, Design and
synthesis of some new pyrazolyl-pyrazolines as potential anti-inflammatory,
analgesic and antibacterial agents, Eur. J. Med. Chem. 101 (2015) 442e451.
[39] L. Nahar, W.R. Russell, M. Middleton, M. Shoeb, S.D. Sarker, Antioxidant
phenylacetic acid derivatives from the seeds of Ilex aquifolium, Acta Phar-
maceutica 55 (2005) 187e193.
[40] M.J. Kobe, D.B. Neau, C.E. Mitchell, S.G. Bartlett, M.E. Newcomer, The structure
of human 15-lipoxygenase-2 with a substrate mimic, J. Biol. Chem. 289 (2014)
8562e8569.
[41] A. Padmaja, C. Rajasekhar, A. Muralikrishna, V. Padmavathi, Synthesis and
antioxidant activity of oxazolyl/thiazolylsulfonylmethyl pyrazoles and iso-
xazoles, Eur. J. Med. Chem. 46 (2011) 5034e5038.
[42] S.-C. Ho, Y.-L. Tang, S.-M. Lin, Y.-F. Liew, Evaluation of peroxynitrite-
scavenging capacities of several commonly used fresh spices, Food Chem.
 H.S. ElBordiny et al. / European Journal of Medicinal Chemistry 145 (2018) 594e605
119 (2010) 1102e1107.
[43] D. Hadjipavlou-Litina, A. Samadi, M. Unzeta, J. Marco-Contelles, Analysis of the
antioxidant properties of differently substituted 2- and 3-indolyl carbohy-
drazides and related derivatives, Eur. J. Med. Chem. 63 (2013) 670e674.
[44] P. Plackova, M. Sala, M. Smidkova, M. Dejmek, H. Hrebabecky, R. Nencka,
H.J. Thibaut, J. Neyts, H. Mertlikova-Kaiserova, 9-Norbornyl-6-chloropurine
(NCP) induces cell death through GSH depletion-associated ER stress and
mitochondrial dysfunction, Free Radical Biology & Med. 97 (2016) 223e235.
[45] Y.A. Arnautova, R. Abagyan, M. Totrov, All-atom internal coordinate me-
chanics (ICM) force field for hexopyranoses and glycoproteins, J. Chem. Theor.
605
Comput. 11 (2015) 2167e2186.
[46] A. Guspiel, J. Ziemska, A. Czescik, R. Kawecki, J. Solecka, Intracellular antiox-
idant activity of a streptomyces Sp. 8812 secondary metabolite, 6,7-
dihydroxy-3,4-dihydroisoqino- line-3-carboxylic acid, and its synthetic de-
rivatives, Acta Poloniae Pharmaceutica 73 (2016) 645e651.
[47] T. James, M.L. Hsieh, L. Knipling, D. Hinton, Determining the architecture of a
protein-DNA complex by combining FeBABE cleavage analyses, 3-D printed
structures, and the ICM Molsoft Program, Meth. Mol. Biol. 1334 (2015) 29e40.
[48] R. Abagyan, M. Totrov, High-throughput docking for lead generation, Curr.
Opin. Chem. Biol. 5 (2001) 375e382.