Pyrazoles and pyrazolines as anti-inflammatory agents

Article abstract of DOI:10.3390/molecules26113439

The five-membered heterocyclic group of pyrazoles/pyrazolines plays important role in drug discovery. Pyrazoles and pyrazolines present a wide range of biological activities. The synthesis of the pyrazolines and pyrazole derivatives was accomplished via t

Full text of DOI:10.3390/molecules26113439

molecules
Article
Pyrazoles and Pyrazolines as Anti-Inflammatory Agents
Martha Mantzanidou, Eleni Pontiki and Dimitra Hadjipavlou-Litina *
Department of Pharmaceutical Chemistry, School of Pharmacy, Faculty of Health Sciences, Aristotle University of
Thessaloniki, 54124 Thessaloniki, Greece; martha19mantza@gmail.com (M.M.); epontiki@pharm.auth.gr (E.P.)
* Correspondence: hadjipav@pharm.auth.gr; Tel.: +30-231-099-7627
Abstract: The five-membered heterocyclic group of pyrazoles/pyrazolines plays important role in
drug discovery. Pyrazoles and pyrazolines present a wide range of biological activities. The synthesis
of the pyrazolines and pyrazole derivatives was accomplished via the condensation of the appropriate
substituted aldehydes and acetophenones, suitable chalcones and hydrazine hydrate in absolute
ethanol in the presence of drops of glacial acetic acid. The compounds are obtained in good yields
68–99% and their structure was confirmed using IR, 1H-NMR, 13C-NMR and elemental analysis.
The novel derivatives were studied in vitro for their antioxidant, anti-lipid peroxidation (AAPH)
activities and inhibitory activity of lipoxygenase. Both classes strongly inhibit lipid peroxidation.
Compound 2g was the most potent lipoxygenase inhibitor (IC50 = 80 µM). The inhibition of the
carrageenin-induced paw edema (CPE) and nociception was also determined, with compounds 2d
and 2e being the most potent. Compound 2e inhibited nociception higher than 2d. Pyrazoline 2d was
found to be active in a preliminary test, for the investigation of anti-adjuvant-induced disease (AID)
activity. Pyrazoline derivatives were found to be more potent than pyrazoles. Docking studies of the
most potent LOX inhibitor 2g highlight hydrophobic interactions with VAL126, PHE143, VAL520
and LYS526 and a halogen bond between the chlorine atom and ARG182.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Keywords: pyrazolines; pyrazoles; antioxidant activities; anti-inflammatory activities; lipoxygenase
inhibition; analgesic activity; anti-arthritis; docking study
Citation: Mantzanidou, M.; Pontiki,
E.; Hadjipavlou-Litina, D. Pyrazoles
and Pyrazolines as
Anti-Inflammatory Agents. Molecules
2021, 26, 3439. https://doi.org/
10.3390/molecules26113439
1. Introduction
Pyrazoles constitute a principal heterocyclic family containing two nitrogen atoms in
Academic Editor: Brullo Chiara
their five-membered heterocyclic ring [
agrochemical and pharmacological properties [
1
] exhibiting a wide range of chemical, biological,
]. Pyrazole is a versatile lead molecule; its
2
Received: 10 May 2021
Accepted: 3 June 2021
Published: 5 June 2021
derivatives are reported to possess innumerable biological activities such as anti-microbial,
anti-fungal, anti-tubercular, anti-inflammatory, anti-convulsant, anticancer, anti-viral, an-
giotensin converting enzyme (ACE) inhibitory, neuroprotective, cholecystokinin-1 receptor
antagonist, and estrogen receptor (ER) ligand activity [3]. Since 1883, when Knorr, L. [4]
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with regard to jurisdictional claims in
published maps and institutional affil-
iations.
gave the generic name “pyrazole” to the above class of compounds synthesizing the first
pyrazolin-5-one (3-methyl-1-phenyl-2-pyrazolin-5-one), many papers have reported the
antipyretic, anti-inflammatory and analgesic activity of several pyrazoles, pyrazolin-3-ones
and pyrazolidine-3,5-diones [5–9]. Many of these derivatives have been clinically applied
as non- steroidal anti-inflammatory agents, such as anti-pyrine (2,3-dimethyl-1-phenyl-
3-pyrazolin-5-one) or phenazone (analgesic and antipyretic), metamizole or dipyrone
(analgesic and antipyretic), phenylbutazone (anti-inflammatory and antipyretic), aminopy-
rine or aminophenazone (anti-inflammatory, antipyretic and analgesic), sulfinpyrazone
(chronic gout) and oxyphenbutazone (antipyretic, analgesic, anti-inflammatory, mild urico-
suric) [10].
It is well known that non-steroidal anti-inflammatory drugs (NSAIDs) are important
therapeutic agents for the treatment of various inflammatory disorders. The pharma-
cological activity is based on: (a) the suppression of prostaglandin biosynthesis from
arachidonic acid via the inhibition of cyclooxygenases (COXs) and thromboxane synthase
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© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
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Molecules 2021, 26, 3439
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with a different degree of selectivity, and (b) the biotransformation of arachidonic acid
via 5-lipoxygenase (5-LOX) to potent mediators of inflammation leukotrienes (LTs) and
prostaglandins (PGs) [11–13]. COX enzymes exist in two isoforms: COX-1 (constitutive) ex-
pressed in most tissues and COX-2 (inducible) induced at sites of inflammation. Currently
used NSAIDs exert their activity via the inhibition of both isoforms including major side
effects at the gastrointestinal and renal level [14] due to their inhibition of COX-1-mediated
physiological prostaglandins.
Commercially available pyrazole moiety examples as potent COX-2 inhibitors are Cele-
coxib [15], Ramifenazone [16], Lonazolac (NSAID) [17] and Rimonabant [18] (Figure 1). The
search for safer NSAIDs continues with the failure of anticipated “ideal” anti-inflammatory
drugs, the coxibs, on long term usage [19]. Design and synthesis of NSAIDs with a
potential for clinical use with less adverse side effects captured the heed of chemists
and pharmacists.
Figure 1. Structure of drugs bearing the pyrazole moiety.
Moreover, chalcones have played a crucial part in the development of heterocyclic
compounds, and they have been used extensively in organic synthesis for the synthe-
sis of several bioactive compounds. A classical synthesis of these compounds involves
the base-catalyzed Claisen–Schmidt reaction of substituted ketones and aldehydes to
give
for various biological activities such as anti-inflammatory, antimicrobial, antifungal, an-
tioxidant and anticancer [20 21]. Thus, they can be used as intermediates undergoing a
subsequent reaction with hydrazine hydrate affording pyrazoles/pyrazolines. It has been
α, β-unsaturated ketones. Chalcones represent an important scaffold responsible
,
reported that pyrazolines possess analgesic, anti-inflammatory [22
activities [25–27].
–24] and antimicrobial
It is well known that, during inflammation, free radicals are produced, leading to
peroxides and other reactive oxygen species [28]. Several researchers [29 33] have reported
–
the implication of reactive oxygen species (ROS), e.g., hydroxyl radical, superoxide anion
and hydrogen peroxide, in disorders associated with oxidative stress (e.g., coronary artery
disease, inflammatory injury, cancer and cardiovascular diseases).
Based on these observations and in continuation with our work related to the synthesis
of anti-inflammatory agents, we now describe the synthesis and the in vitro evaluation
of a number of novel pyrazole and pyrazoline derivatives as antioxidants, lipoxygenase
inhibitors and in vivo as anti-inflammatory and analgesic agents influencing adjuvant-
induced arthritis.

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2. Results and Discussion
2.1. Chemistry
The synthesis of the pyrazolines and pyrazole derivatives was accomplished via the
condensation of the appropriate substituted aldehydes, suitable chalcones and hydrazine
hydrate in absolute ethanol in the presence of drops of glacial acetic acid, as presented in
Scheme 1 [34]. Chalcones as starting materials were successfully synthesized via Claisen–
Italics Schmidt condensation using 15% KOH from the corresponding aldehydes with
acetophenone in methanol (Scheme 1) [35].
Scheme 1. Synthesis of the novel derivatives.
Products 2a
final products were recrystallized from ethanol, acetone or preparative TLC while the
chalcones were recrystallized from methanol. Structurally, compounds 2a 2b 2d and 2e
are pyrazolines, whereas 2c 2f 2g 2h are acetyl-substituted pyrazoles. Compound 2a
–2h (Table 1) were obtained in satisfactory yields (68–99%). The pure
,
,
,
,
,
has been previously reported [36]. IR spectra for pyrazolines and pyrazole−d1erivatives
revealed the presence of a N-N bond at 1500–1510 cm−1, N-H at 3220–3400 cm and C=N
1
at 1660–1680 cm−1. H-NMR, 13C-NMR and elemental analysis were used for the confir-
mation of the synthesized compounds’ structures. The physical data of the synthesized
compounds are given in detail in the Experimental Section.
2.2. Physicochemical Studies
2.2.1. Determination of Lipophilicity
Lipophilicity is the key physicochemical parameter of a bioactive molecule, linking
solubility, ligand-target binding interactions and membrane permeability with absorption,
distribution, bioavailability, metabolism and elimination (ADME) and toxicological effects,
crucial for its biological activity. The reverse phase thin layer chromatography (RPTLC)
method was used for the experimental determination of the lipophilic character of the
synthesized compounds as RM values (Table 2) [37]. Lipophilicity was also theoretically
calculated as clog P values, using the CLOGP Program of Biobyte Corp. [38] and as
LPSP-lipophilicity values through Spartan v.5.1.3. (Wavefunction Inc., Irvine, CA, USA).
According to the calculated clog P values, as well as LPSP, the most lipophilic compounds

Molecules 2021, 26, 3439
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were the compounds 2c
,
2d
,
2e and 2g. This observation was supported by the RM values
(with the exception of 2g).
Table 1. Substituted pyrazoles and pyrazoline derivatives.
Compd
2a
Template
Ar1
Ar2
2b
2c
2d
2e
2f
2g
2h
Table 2. Theoretical calculation of the properties associated with energy and charge distribution with the program Spartan
v.5.1.3. Lipophilicity values: experimental RM% (RM values are the average of at least five measurements; SD: standard
deviation < 10%). Theoretically calculated clog P values calculated using the C-QSAR Program, Biobyte.
(Å3)
(MV)
E(HOMO) E(LUMO)
∆E(HOMO-LUMO)
SM2
(kcal/mol)
Surface
(Å2)
Dipole
(D)
clog P
(C-QSAR)
a
Compd.
LPSP RM (±SD) a
(eV)
(eV)
(eV)
2a
2b
2c
2d
2e
2f
−7.65
−7.67
−8.44
−7.84
−8.08
−7.73
−7.78
−8.46
2.15
2.64
2.39
2.86
2.78
2.48
2.07
2.23
9.80
10.31
10.83
10.70
10.86
10.21
9.85
−6.29
−11.04
−25.72
−8.24
321.20
329.46
450.99
419.96
434.69
349.38
382.80
412.08
318.74
320.31
439.09
410.67
422.90
340.28
376.11
403.67
2.66
5.84
2.44
1.57
4.27
2.32
1.44
2.38
4.19
3.36
4.84
5.63
5.96
2.22
4.17
3.64
0.22 ± 0.2
0.23 ± 0.03
0.68 ± 0.05
0.72 ± 0.06
0.87 ± 0.07
−0.07 ± 0.01
−0.59 ± 0.05
0.41 ± 0.02
4.77
4.30
7.40
6.15
6.69
4.97
6.89
6.78
−10.96
−12.79
−7.37
2g
2h
10.69
−7.27
a
SD standard deviation < 10%.
Attempts to correlate clog P and RM values as well as RM values with LPSP values
resulted to the following Equations (1) and (2).
clog P (C-QSAR) = 2.714 (±2.658) RM + 4.515 (±1.474)
n = 6, r = 0.817, r2 = 0.668, q2 = 0.222, s = 0.786, F1,4 = 8.03, α = 0.05
(1)
(2)
RM = 0.241 (±0.093) LPSP − 0.588 (±0.413)
n = 7, r = 0.948, r2 = 0.898, q2 = 0.851, s = 0.117, F1,5 = 43.84, α = 0.01
From our results (Table 2), it can be concluded that RM values could be used as a suc-
cessful relative measure of the overall lipophilic/hydrophilic properties of these molecules.

Molecules 2021, 26, 3439
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2.2.2. Theoretical Calculation of Physicochemical Properties
The physicochemical properties were determined with the program Spartan v.5.1.3.
(Wavefunction Inc., Irvine, CA, USA) in the conformation of minimum energy (Table 2).
2.3. Biological Evaluation
Reactive oxygen species and free radicals can be formed either from normal essential
metabolic processes or external sources e.g., smoking, chemicals etc. [39]. They can be
derived either from enzymatic (phagocytosis, prostaglandin synthesis, P-450) or non-
enzymatic reactions (ionizing reactions, reaction of oxygen with organic compounds) [40].
Free radicals can be highly toxic, attacking macromolecules [41] leading to homeostatic
disruption and cell damage, thus detoxification is of absolute necessity. Antioxidants
(glutathione, ubiquinol, vitamin E, vitamin C etc) can delay or inhibit cellular damage
due to their free radical scavenging activities and can terminate chain reactions before
damaging vital molecules.
In this study, the novel derivatives were evaluated: (i) in vitro for their antioxi-
dant activities and inhibition of soybean lipoxygenase, and (ii) in vivo for their anti-
inflammatory activities using the carrageenin-induced edema, and for their analgesic
activity, anti-nociception applying the writhing test and for the induction of adjuvant-
induced disease (AID).
The antioxidant profile of the studied derivatives was determined through two dif-
ferent methods: (i) by measuring the scavenging ability by donating a hydrogen or an
electron on a free radical, and (ii) by generating a free radical from an antioxidant system.
The in vitro antioxidant activity was measured in terms of: (a) the interaction with the
stable free radical DPPH; (b) the ABTS+ radical cation reduction-decolorization ability; and
(c) anti-lipid peroxidation (AAPH). Factors such as solubility or steric hindrance seemed
to be important, and influenced the experimental conditions.
The novel derivatives were studied for their interaction with the stable 2,2-diphenyl-1-
picrylhydrazyl free radical (DPPH) at concentrations 50 µM, 100 µM, 200 µM and after 20
and 60 min (Table 3) [42]. This assay is based on the reduction of the DPPH by transferring
an electron from the antioxidant. Nordihydroguaretic acid (NDGA) was used as a reference
compound [43]. In general, the compounds present low or medium activity. However,
it seems that the interaction is dependent on the concentration and on the reaction time.
From this point of view, it can be concluded that the acetyl pyrazolyl derivatives 2c and 2f
seem to present the highest scavenging activities. An increase in concentration favors the
activity. On the contrary, time does not seem to affect the activity apart from compounds 2a
and 2d at 100 µM. As for compounds 2a, 2b, 2c and 2f, activity seems to be time dependent
at 200 µM. The acetyl pyrazole is more potent than the corresponding 2d pyrazoline
derivative. The presence of a chlorine group in 2e pyrazoline leads to an activity increase
(compared to 2d) in concentration 200
than 2b at 200 µM.
µM. The acetyl derivative 2f presents better activity
Table 3. Interaction with the stable radical 1,1-diphenyl-picrylhydrazyl (DPPH), In vitro lipoxygenase (LOX) inhibitory
activity at 100 µM (LOX%).
RA% 50 µM
20 min
RA% 50 µM
60 min
RA% 100 µM
RA% 100 µM
RA% 200 µM
RA% 200 µM
LOX
Compd.
20 min
60 min
20 min
60 min
% Inhibition at 100 µM
2a
2b
2c
2d
2e
2f
2g
2h
NDGA
15.0
19.3
17.1
6.3
9.5
21.0
9.0
20.0
25.0
22.4
9.5
13.0
24.7
12.7
9.5
22.9
26.1
41.9
19.4
14.8
34.7
11.2
7.8
32.0
33.6
44.0
37.4
17.8
38.8
13.6
9.6
37.2
34.9
49.4
9.4
25.6
50.1
22.5
17.2
94
49.3
44.4
59.2
14.9
30.1
59.5
27.7
20.9
96
35
17
42
13
16
3
60 (IC50 = 80 µM)
26
93 (0.45 µM)
6.7
81
83
87
93

Molecules 2021, 26, 3439
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0
For the lipid peroxidation study, 2,2 -azobis(2-amidinopropane) hydrochloride (AAPH)
was used for the generation of peroxyl radicals. The generation of the conjugated diene
hydroperoxide derived from the oxidation of sodium linoleate in an aqueous solution was
recorded at 234 nm [43,44]. The compounds presented high anti-lipid peroxidation activity
(78–100%) (Table 4). Pyrazolines 2a and 2b present lower anti-lipid peroxidation activity
(89% and 78% respectively). All the others are almost equally potent. Lipophilicity does
not seem to influence this activity.
Table 4. % Anti-lipid peroxidation (AAPH), decolorization activity ABTS+ % assays. In vivo anti-
inflammatory activity (CPE%).
AAPH%
100 µM
ABTS+ %
100 µM
Compd.
CPE a
%
2a
2b
2c
2d
2e
2f
2g
2h
Trolox
89
78
100
96
98
95
100
97
93
-
no
no
no
15
30
no
no
no
91
-
no
27.0 *
38.0 *
63.0 **
56.0 **
30.0 *
33.0 *
16.0 *
-
Indomethacin
47 **
a
* p < 0.01, ** p < 0.05. % of reduction in the rat paw edema (CPE%) induced by carrageenin at the dose of
0.0057 mmol/Kg/body weight. No: no action under the experimental conditions.
Pyrazolines 2d and 2e which are the most lipophilic compounds, showing antioxidant
activity using the ABTS radical cation (ABTS•+) generated through potassium persulfate
by oxidation with no participation of an intermediary radical. This reduction is completed
by adding electron-donating antioxidants [43]. It seems that lipophilicity influences the
activity, since both present high lipophilicity values. The compounds presented low to
moderate antioxidant activity with most potent compound, 2e, attributed to the presence
of an electron acceptor substituent p-Cl at the molecule (Table 4).
Lipoxygenase (LOX) is the key enzyme implicated in membrane lipid peroxidation
by forming hydroperoxides, thus it is considered a target for inflammatory diseases. LOX
inhibitors may act either as radical scavengers or inhibitors of free radical production,
since lipoxygenation occurs via a carbon centered radical [45]. LOX inhibitors bearing an
antioxidant profile could be expected to offer protection in inflammatory conditions, and
lead to potentially effective drugs. For the in vitro study, soybean lipoxygenase was used,
based on the homology to mammalian lipoxygenase [46,47]. It has been found that, under
the experimental conditions, all the synthesized derivatives inhibited soybean lipoxygenase
(13–60%) apart from compound 2f. Compound 2g is the most potent among the synthesized
derivatives. For the most promising compound, 2g, IC50 value was calculated.
In an attempt to determine the type of LOX inhibition (competitive or non-competitive)
for the most potent compound, 2g, the study was conducted varying the concentration of
the substrate, sodium linoleate (LLA) and keeping stable the enzyme’s and compound’s
2g concentration. From the results, it can be concluded that 2g LOX inhibition remained
steadily strong, compared to the reference compound nordihydroguaeretic acid NDGA
(Table 5). This underlines a competitive inhibition. In competitive inhibition, the in-
hibitor “competes” with the substrate at the binding site of the enzyme, and high substrate
concentrations can break down the enzyme-inhibitor complex.

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Table 5. In vitro assay for the determination of the type of inhibition of lipoxygenase (LOX).
2g-LOX
(% Inhibition 100 µM)
NDGA-LOX
(% Inhibition 100 µM)
LLA *-C
50 µM
100 µM
200 µM
89.6
-
-
39.3
36.2
6.6
* Linoleic acid sodium salt concentration.
The carrageenin-induced rat paw edema assay was used, as a model of acute in-
flammation [48]. Inflammation induced by carrageenan is acute, non-immune, well-
researched, and highly reproducible, and is described as a biphasic event. Cardinal signs of
inflammation—edema, hyperalgesia, and erythema, are developed immediately following
subcutaneous injection, resulting from the action of pro-inflammatory agents: bradykinin,
histamine, tachykinins, complement reactive oxygen and nitrogen species. It is known that
NSAIDs act during the second phase of prostaglandin release, presenting weak activity in
the first phase of histamine and serotonin release.
Pyrazolines 2d and 2e presented the highest anti-inflammatory activity among the
synthesized derivatives, exhibiting even higher activity compared to indomethacin used as
a reference compound. Compounds were administered intraperitoneally in order to ensure
systemic response via fast bioavailability, while carrageenin’s intradermal administration,
as inflammatory agent, aimed at a local response. Compound 2a was proven to be inactive
and 2h exhibited low activity. Compounds 2b, 2c, 2f and 2g presented medium activity.
Lipophilicity seems to be important, since 2d and 2e are the most lipophilic derivatives
within the set. The presence of a p-Cl at the benzyl ring slightly reduces the activity
(2e < 2d). It seems that the acetyl-substituted pyrazoles are less active.
Correlation of the in vivo anti-inflammatory activity (CPE%) with LPSP (lipophilicity
values calculated from Spartan v.5.1.3), showed that lipophilicity governs the in vivo anti-
inflammatory activity (Equation (3)).
log (CPE %) = 0.183 (±0.130) LPSP + 0.706 (±0.611)
(3)
n = 6, r = 0.890, r2 = 0.793, q2 = 0.475, s = 0.111, F1,4 = 15.35, α = 0.05
Compounds 2d and 2e, presenting the highest anti-inflammatory activity, were ex-
amined for their analgesic activity as peripheral nociception using the writhing test. The
acetic acid-induced writhing test is a quick, simple and reproducible method, despite the
fact that it lacks specialization. Pyrazoline 2e proved to be more effective than pyrazoline
2d (Table 6).
Table 6. In vivo analgesic activities of 2d and 2e, % inhibition of writhing responses (Writhing
inhibition%).
Compound
Writhing Inhibition (%) a
2d
2e
Aspirin
54.2 *
66.1 *
77 **
* p < 0.01, ** p < 0.05; a Dose of the administered 0.0057 mmol/kg body weight.
Free radicals are particularly important in the inflammatory process [49–51]. ROS
produced by phagocytes have been connected with the induction of inflammation and
tissue damage. However, recently, ROS are also implicated in the regulation of inflamma-
tion and protection from autoimmunity. Evidence for the latter comes from association
of ROS-deficiency with severe chronic inflammation in animal models and human pa-
tients in an ever-growing number of pathologic conditions, such as arthritis, lupus and
neurodegenerative diseases.
Since it has been reported that anti-inflammatory drugs may also be effective in the
prevention of free radical mediated damage [52], it is therefore to be considered that the

Molecules 2021, 26, 3439
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action of some anti-inflammatory agents may be due to their antioxidant and free radical
scavenging properties.
Taking into consideration the above, our biological findings as a whole show an
agreement between anti-inflammatory and antioxidant activity for compounds 2c
, 2d,
2e 2g. They could indicate that organic peroxy-radicals, such as lipoperoxy-radicals,
,
can be scavenged by these compounds, and this may be implicated in the mechanism
of their anti-inflammatory ability. In the literature are referred, antioxidants possessing
anti-inflammatory activity [52].
Compound 2d exerts an effect greater than that of indomethacin, which has potent
anti-inflammatory activity. The inhibition observed by compound 2d was greater than
that of indomethacin. Furthermore, the antioxidant-anti lipid peroxidation activities of
compounds 2d and 2e are in correlation to their anti-inflammatory and analgesic activity.
Furthermore, it has been claimed that compounds acting as antioxidants could act as
cyclooxygenase and/or lipoxygenase inhibitors [53].
Thus, the above tested compound 2d appeared to be an effective agent not only
on acute, but also on chronic inflammation of arthritis, and it is possibly effective in
autoimmune diseases in correlation to its antioxidant activity. Rats treated with compound
2d either did not develop or developed very mild arthritis, and simultaneously indicated
anti-inflammatory activity.
Pyrazoline 2d was selected, since it was found to be the most active of these com-
pounds in a preliminary test, for the investigation of anti-adjuvant-induced disease (AID)
activity. Adjuvant-induced disease (AID) is a good experimental model for rheumatoid
arthritis, and is often used in testing agents for anti-inflammatory activity [54]. The com-
pound was administered intraperitoneally on the 1st day, i.e., the day of the administration
of Freund’s adjuvant (FA), and once every other day for the following 24 days. Adjuvant
arthritis was developed about 14 days post-FA administration. Arthritic scores, body
weight loss and in vivo zoxazolamine metabolism impairment (expressed as the duration
of the induced paralysis) were recorded and found to be significantly reduced in compari-
son to the AID rat controls, which were treated only with the liquid vehicle. From these
results it can be concluded that the examined pyrazolines possess preventive activity. The
effect of the tested compound on inflammation is shown in Table 7 and the time course
of adjuvant arthritis development expressed as arthritic score is shown in Figure 2. In the
same experimental model, Indomethacin (IMA) was used as a reference compound.
Table 7. Assessment of the preventive action of 2d on the adjuvant-induced disease (AID). Manifestations (body weight
change, liver weight change, zoxazolamine paralysis) in comparison to indomethacin (IMA), used as a reference compound.
AID Rats-Controls
Treated only with the
Liquid Vehicle
Absolute Controls, Normal
Animals Treated only with
the Liquid Vehicle
Examined Parameters
AID Rats Treated
with IMA
AID Rats Treated with 2d
(mean ± SD)
Percent change in body
weight (g ± SD)
6 ± 0.3 **
7 ± 0.6 *
8.3 ± 0.2 *
6.3 ± 0.4 *
217 ± 17 **
3 ± 0.1 *
7.8 ± 0.4 *
284 ± 19 *
14 ± 2 *
8.7 ± 0.5 **
156 ± 18 *
Percent change in liver
weight (g ± SD)
Zoxazolamine paralysis
(minutes ± SD)
227 ± 17 *
* p < 0.01 (Student’s Test), ** p < 0.005 (Student’s Test).
2.4. Computational Studies–Docking Simulation Soybean Lipoxygenase
Molecular Modeling of the Synthesized Derivatives in Soybean LOX
In silico docking studies have been performed for all the synthesized derivatives, and
their docking scores, hydrophobic interactions, hydrogen bonds, π-cation interactions and
halogen bonds of the synthesized derivatives with different residues are given in Table S1
as Supplementary Material. Figure S1 presents the preferred docking poses of pyrazoles
(
2a [pink], 2b [blue], 2d [purple] and 2e [cyan]) bound to soybean lipoxygenase (LOX-1)
and Figure S2 presents the preferred docking poses of pyrazolines (2c [light sea green],

Molecules 2021, 26, 3439
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2f [light purple], 2g [light green] and 2h [peach pink]) bound to soybean lipoxygenase
(LOX-1) (Supplementary Material).
Figure 2. Effect of the compound 2d and IMA on the onset and severity of arthritis in AID of rats.
The favored docking position of the most active derivative 2g is shown in Figure 3.
Compound 2g has an AutoDockVina score of
−
10.3 kcal/mol binding to soybean LOX
(PDB code: 3PZW). It is well known that one-to-one correlation is difficult to reach between
the obtained results from the in vitro inhibition of soybean lipoxygenase that represent
experimental values and docking scores that are based on algorithms and scoring function
calculations. Docking describes the preferred orientation of the ligand bound to the protein.
It seems that the novel compounds interact with the SLOX through allosteric interactions.
Compound 2g presents hydrophobic interactions with VAL126, PHE143, VAL520 and
LYS526, and a halogen bond between the chlorine atom and ARG182. It is well known
that most LOX inhibitors act as antioxidants or by scavenging free radicals [43], oxidizing
the enzyme via a carbon-centered radical on a lipid chain. It is possible that compound 2g
exerts its activity by extending into the hydrophobic domain and blocking the substrates to
the binding site, and thus preventing oxidation.

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Figure 3. Preferred docking pose of 2g (depicted in cyan) bound to soybean lipoxygenase (LOX-1).
3. Experimental Section
3.1. Materials and Instruments
All chemicals, solvents, chemical and biochemical reagents were of analytical grade
and purchased from commercial sources (Fluka, Alfa and Sigma-Aldrich, Merck). Soybean
lipoxygenase, sodium linoleate, arachidonic acid (AA), 2,2-azobis (2-amidinopropane
(ABTS), 2,2-azinobis-2-methyl-propanimidamine HCl (AAPH) were obtained from Sigma
Chemical, Co. Nordihydroguaretic acid (NDGA), 1,1-diphenyl-2-picrylhydrazyl (DPPH),
salycilic acid (SA), acetylsalicylic acid (ASA), 6-Hydroxy-2,5,7,8-tetramethyl-chroman-
2-carboxylic acid (Trolox), Freund’s Adjuvant: Mycobacterium Butyricum (Difco-0640-
33) were purchased from the Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All
starting materials were obtained from commercial sources (Merck, Merck KGaA, Darmstadt,
Germany, Fluka Sigma-Aldrich Laborchemikalien GmbH, Hannover, Germany, Alfa Aesar,
Karlsruhe, Germany and Sigma, St. Louis, MO, USA) and used without further purification.
Melting points (uncorrected) were determined on a MEL-Temp II (Lab. Devices,
Holliston, MA, USA). For the in vitro tests, UV-Vis spectra were obtained on a Perkin-Elmer
554 double beam spectrophotometer. Infrared spectra (film as Nujol mulls or KBr pellets)
were recorded with Perkin-Elmer 597 spectrophotometer (Perkin-Elmer Corporation Ltd.,
Lane Beaconsfield, Bucks, UK).
The 1H Nucleic Magnetic Resonance (NMR) spectra were recorded at 300 MHz on
a Bruker AM-300 spectrometer (Bruker AnalytischeMesstechnik GmbH, Rheinstetten,
Germany) in CDCl3 or DMSO using tetramethylsilane as an internal standard unless
otherwise stated. 13C-NMR spectra were obtained at 75.5 MHz on a Bruker AM-300
spectrometer in CDCl3 or DMSO solutions with tetramethylsilane as internal reference,
unless otherwise stated. Chemical shifts are expressed in_(ppm) and coupling constants J
in Hz. Mass spectra were determined on a LC-MS 2010 EV Shimadzu (Shimadzu, Kiyoto,
Japan), using MeOH as solvent. Elemental analyses for C and H gave values acceptably
close to the theoretical values ( 0.4%) in a Perkin-Elmer 240B CHN analyzer (Perkin-Elmer
±
Corporation Ltd., Lane Beaconsfield, Bucks, UK). Reactions were monitored by thin layer
chromatography on 5554 F254 Silica gel/TLC cards (Merck and FlukaChemie GmbH Buchs,
Steinheim, Switzerland). For preparative thin layer chromatography (prep TLC) Silica gel
60 F254, plates 2 mm, Merck KGaA ICH078057 were used.

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3.2. Chemistry General Procedure
3.2.1. Synthesis of Chalcones
A Claisen–Schmidt condensation was performed between acetophenone and the
suitable substituted aryl aldehyde at a molar ratio of 1:1 in methanol [35]. Five milliliters
(3 mL) of aqueous KOH (15%) was added and the mixture was stirred at room temperature
for 24 h. Reaction completion was monitored by TLC. The pH was adjusted to acidic via
the addition of three milliliters (3 mL) of CH3COOH. The mixture was cooled down with
ice and the formed precipitate was filtered and washed with methanol.
All the synthesized chalcones have been already reported in the literature [35
except the following ones:
,55,56]
(E)-3-(4-((4-bromobenzyl)oxy)phenyl)-1-phenylprop-2-en-1-one (1d). Aurora Fine Chemicals
LLCUSA, Austria. Yield: 50%; Rf = 0.8 (CH2Cl2); m.p. 45–47 ◦C; IR (Nujol) (cm−1): (C=O)
1680; 1H-NMR (CDCl3):
δ, (ppm) 5.07 (2H, s), 6.97–8.02 (15H, m, aromatics and -CH=CH-);
13C-NMR (CDCl3): 69.4, 76.6, 77.4, 115.3, 120.2, 128.4, 128.4, 128.6, 129.0, 130.2, 131.8, 132.5,
135.5, 144.5, 160.5, 187.3; Anal. C, H, N. Calcd % (C22H17O2Br): C: 67.19, H: 4.36; Found %
(C22H17O2Br): C: 66.79; H: 4.36.
(E)-3-(4-((4-bromobenzyl)oxy)phenyl)-1-(4-chlorophenyl)prop-2-en-1-one (1e). Yield: 100%; Rf = 0.95
◦
(CH2Cl2); m.p. 175 C; IR (Nujol) (cm−1): (C=O) 1700; 1H-NMR (CDCl3):
δ, (ppm) 5.10 (2H, s),
6.97–7.96 (12H, m, aromatics and -CH=CH-); 13C-NMR (CDCl3): 40.0, 69.4, 76.6, 77.4, 115.3,
119.5, 122.1., 128.0, 128.9, 129.0, 129.8, 130.3, 131.8, 135.4, 145.0, 160.6, 189.2; Anal. C, H, N.
Calcd% (C22H16O2BrCl): C: 61.78, H: 3.77; Found% (C22H16O2BrCl): C: 61.57; H: 3.86.
3.2.2. Synthesis of Pyrazolines and Pyrazole Derivatives
In a round bottom flask, the appropriate substituted chalcone (0.01 mol) and hydrazine
hydrate (99%, 0.02 mol) were dissolved in absolute ethanol while adding drops of glacial
acetic acid. The solution was stirred and heated under reflux for 8–13 h. Reaction comple-
tion was monitored by TLC. The excess of the solvent was removed under reduced pressure
and the remaining residue was treated with a mixture of ice/cold water. The solid/semi-
solid residue was filtered. The pure final products were obtained via recrystallization from
the suitable solvents, e.g., ethanol 95◦ or acetone or preparative TLC.
5-(naphthalen-1-yl)-3-phenyl-4,5-dihydro-1H-pyrazole (2a) [36]. Yield: 77%; Rf = 0.7 (CH2Cl2);
m.p. 45–47 ◦C; IR (Nujol) (cm−1): 3346, 1688, 1600, 1560; UV (abs. C2H5OH):
λ
max = 330,
1
280, 206,
ε
= 4600, 1100, 75,400; H-NMR (DMSO-d , CDCl ):
δ, (ppm) 2.07–2.16 (d,
max
6
3
1H, -NH-pyrazoline), 3.00–3.29 (dd, 1H, CH2), 3.66–3.74 (dd, 1H, CH2), 5.66–5.73 (t, 1H,
−
CH-pyrazoline), 7.34–8.27 (m, 12H, aromatics); 13C-NMR (DMSO-d6, CDCl3): 41.0, 47.1,
123.2, 123.5, 125.6, 126.0, 126.6, 127.2, 128.89, 128.96, 128.66, 129.15, 129.28, 130.2, 131.0,
133.5, 138.1, 152.1. Anal. C, H, N. Calcd % (C19H15N2): C: 84.10, H: 5.57; N: 10.32; Found%
(C19H15N2): C: 84.45; H: 5.88, N: 10.20.
5-(3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)-1H-indole (2b). Yield: 99%; Rf = 0.5 (CH2Cl2);
m.p. 96–98 ◦C; IR (Nujol) (cm−1): 3220–3470, 1680, 1520; UV (abs. C2H5OH):
255.5,
zoline), 3.06–3.14 (dd, 1H, CH2), 3.41–3.55 (dd, 1H, CH2), 5.00–5.06 (t, 1H, CH), 6.51–6.67 (m
λ
= 325,
max
ε δ, (ppm) 2.15–2.25 (d, 1H, NH pyra-
max = 5100, 13,760; 1H-NMR (DMSO-d6, CDCl3):
,
1H, -CHβ = indole), 6.79–6.88 (br, 1H, -CH(6)-indole), 7.16–7.87 (m, 7H, aromatics), 8.34 (s,
1H, -NH-indole); 13C-NMR (DMSO-d6, CDCl3): 41.8, 58.7, 102.9, 111.9, 118, 118.6, 125.3,
127.5, 129.0, 129.3, 130.2, 131.0, 133.0, 134.5, 135.0, 137.0, 151.0; Anal. C, H, N. Calcd %
(C17H13N3Cl): C: 69.50, H: 4.45; N: 14.25; Found% (C17H13N3Cl): C: 69.46; H: 4.54, N: 14.39.
1-(5-(4-((4-bromobenzyl)oxy)phenyl)-3-(thiophen-2-yl)-1H-pyrazol-1-yl)ethan-1-one (2c). Yield:
◦
68%; Rf = 0.8 (EtOH:PE, 1:2); m.p. 47–48 C; IR (Nujol) (cm−1): 3150, 1720, 1510; UV
1
(abs. C2H5OH):
d6, CDCl3):
λ
max
= 330, 270, 233.5,
ε
= 6600, 17,000, 15,100; H-NMR (DMSO-
max
δ
, (ppm) 2.23–2.31 (m, 3H, -CH3), 5.04 (s, 2H, -OCH2-), 6.68 (s, 1H, -CH =
pyrazole), 6.97–7.62 (m, 11H, aromatics); 13C-NMR (DMSO-d6, CDCl3): 2.7, 69.7, 103.0,
104.8, 114.0, 115.7, 123.0, 124.4, 125.0, 126.0, 127.3, 128.0, 129.0, 129.4, 130, 132, 132.2, 139.0,

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140.0, 167.0; Anal. C, H, N. Calcd% (C22H17N2O2SBr): C: 58.28, H: 3.78; N: 6.18; Found %
(C22H17N2O2SBr): C: 58.55; H: 4.18, N: 6.08.
5-(4-((4-bromobenzyl)oxy)phenyl)-3-phenyl-4,5-dihydro-1H-pyrazole (2d). Yield: 94%; Rf = 0.6
(CH2Cl2); m.p. 98–100 C; IR (Nujol) (cm−1): 3360, 1660, 1517; UV (abs. C2H5OH):
◦
λ
max = 354, 262, 235, ε δ, (ppm)
max = 23,020, 16,800, 21,200; 1H-NMR (DMSO-d6, CDCl3):
2.06–2.09 (d, 1H, NH), 2.98–3.07 (dd, 1H, -CH-), 3.40–3.49 (dd, 1H, -CH-), 5.10 (s, 2H, -CH2-),
6.89–6.92 (d, 2H, aromatics), 7.26–7.55 (m, 11H, aromatics); 13C-NMR (DMSO-d6, CDCl3):
2.7, 69.7, 103.0, 104.8, 114.0, 115.7, 123.0, 124.4, 125.0, 126.0, 127.3, 128.0, 129.0, 129.4, 130.0,
132.0, 132.2, 139.0, 140.0, 167.0; Anal. C, H, N. Calcd% (C22H19N2OBr): C: 64.87, H: 4.70; N:
6.88; Found% (C22H19N2OBr): C: 64.53; H: 4.69, N: 6.82.
5-(4-((4-bromobenzyl)oxy)phenyl)-3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazole (2e). Yield: 72%;
◦
Rf = 0.7 (CH2Cl2); m.p. 108–110 C; IR (Nujol) (cm−1): 3320, 1660, 1510; UV (abs. C2H5OH):
1
λ
max
= 330, 263.5, 233,
ε
= 10,000, 21,560, 15,600; H-NMR (DMSO-d6, CDCl3):
δ,
max
(ppm) 2.06–2.09 (d, 1H, NH), 2.95–3.03 (dd, 1H, -CH2-pyrazoline), 3.37–3.46 (dd, 1H, CH3
pyrazoline), 4.85–4.92 (t, 1H, -CH-), 5.01 (s, 2H, -OCH2-), 6.90–6.93(d, 2H, aromatics),
7.26–7.60 (m, 10H, aromatics); 13C-NMR (DMSO-d6, CDCl3): 269.2, 99.0, 114.0, 114.8, 122.3,
127.0, 128.7, 128.9, 129.1, 129.3, 129.7, 130.2, 130.5, 130.7, 131.0, 131.6, 131.9, 135.0, 137.5,
149.8, 151.0, 165.6; Anal. C, H, N. Calcd% (C22H17N2OBrCl): C: 59.95, H: 3.89; N: 6.39;
Found% (C22H17N2OBrCl): C: 60.02; H: 3.79, N: 6.03.
1-(5-(1H-indol-5-yl)-3-phenyl-1H-pyrazol-1-yl)ethan-1-one (2f). Yield: 83%; Rf = 0.8 (CH2Cl2);
m.p. 65–66 ◦C; IR (Nujol) (cm−1): 3380–3200, 1720, 1500; UV (abs. C2H5OH):
λmax = 254,
ε
max = 15,000; 1H-NMR (DMSO-d6, CDCl3):
δ, (ppm) 2.39 (s, 3H, -CH3), 6.68 (s, 1H, –CH
= pyrazole), 7.36–7.97 (m, 11H, aromatics and NH indole); 13C-NMR (DMSO-d6, CDCl3):
21.7, 102.0, 104.0, 111.9, 118.0, 119,.0 124.0, 126.0, 126.9, 127.6, 128.7, 129, 129.9, 133.2, 136.0,
144.0, 145.0, 147.0, 166.1; Anal. C, H, N. Calcd % (C19H15N3O): C: 75.73, H: 5.02; N: 13.94;
Found % (C19H15N3O): C: 75.89; H: 5.40, N: 13.80.
1-(3-(4-chlorophenyl)-5-(naphthalen-1-yl)-1H-pyrazol-1-yl)ethan-1-one (2g). Yield: 94%; Rf = 0.8
◦
(CH2Cl2); m.p. 53–55 C; IR (Nujol) (cm−1): 3100, 1680, 1560; UV (abs. C2H5OH):
1
λmax = 280, 247.5,
ε
= 11,600, 14,340; H-NMR (DMSO-d6, CDCl3):
δ, (ppm) 2.59 (s,
max
3H, -CH3), 6.80 (s, 1H, -CH = pyrazole), 7.41–8.02 (m, 11H, aromatics); 13C-NMR (DMSO-
d6, CDCl3): 23.0, 104.0, 124.4, 125.9, 126.7, 127.3, 127.7, 128.3, 128.9, 129.3, 129.8, 130.0, 130.8,
131.0, 131.8, 132.0, 134.0, 143.0, 145.0, 147.0, 169.8; Anal. C, H, N. Calcd % (C21H15N2OCl):
C: 72.73, H: 4.36; N: 8.08; Found % (C21H15N2OCl): C: 72.35; H: 4.45, N: 8.12.
1-(5-(3-phenoxyphenyl)-3-phenyl-1H-pyrazol-1-yl)ethan-1-one (2h).Yield: 71%; Rf = 0.7 (CH2Cl2);
m.p. 44–46 ◦C; IR (Nujol) (cm−1): 3100, 1718, 1680, 1500; UV (abs. C2H5OH):
λ
max = 241,
= 26,180; 1H-NMR (DMSO-d , CDCl ):
δ, (ppm) 2.78 (s, 3H, -CH3), 6.82 (s, 1H, –CH =
6 3
ε
max
pyrazole), 6.99–7.15 (m, 3H, aromatics), 7.33–7.71 (m, 11H, aromatics); 13C-NMR (DMSO-d6,
CDCl3): 29.0, 100.4, 110.2, 116.0, 118.6, 119.0, 120.4, 123.5, 125.5, 128.5, 128.8, 129.8, 130.3,
140.4, 157.8, 176.9; Anal. C, H, N. Calcd% (C23H18N2O2): C: 77.95, H: 5.12; N: 7.90; Found%
(C23H18N2O2): C: 78.05; H: 5.28, N: 8.28.
3.3. Physicochemical Studies
Experimental Determination of Lipophilicity as RM Values
Lipophilicity as RM values was determined through reversed phase TLC (RP-TLC)
on silica gel plates impregnated with 5% (v/v) liquid paraffin in light petroleum ether.
Methanol/water mixture (70/30, v/v) was ◦used a mobile phase. Closed chromatography
tanks saturated with the mobile phase at 24 C were used for the development of the plates
while the spots were detected under UV light. For the determination of RM values, five
individual measurements of Rf values were recorded, and the RM values were derived
from the equation RM = log [(1/Rf) − 1] [42,43] (Table 2).

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3.4. Biological Assays
3.4.1. Biological In Vitro Assays
A stock solution (1% DMSO in the appropriate buffer with the tested compound
diluted under sonication) was prepared for the in vitro assays from which several dilutions
were made with the appropriate buffer. The in vitro experiments were performed at least in
triplicate, and the standard deviation of absorbance was less than 10% of the mean.
Determination of the Reducing Activity of the Stable Radical 1,1-Diphenyl-Picrylhydrazyl
(DPPH)
A stock solution (10 mM) of the novel derivatives in DMSO was prepared and an
equal volume of it was added to a solution of DPPH in absolute ethanol to reach final
concentrations 50, 100 µM and 200 µM. The absorbance was recorded at 517 nm, after 20
and 60 min at room temperature (Table 3) [42,43].
Inhibition of Linoleic Acid Lipid Peroxidation
AAPH was used as free radical initiator of alkylperoxyl radicals. The ability of the
compounds to prevent the oxidation of linoleic acid sodium salt from alkylperoxyl radicals
was recorded at 234 nm. Trolox was used as reference (93%) (Table 4) [43,44].
ABTS+ -Decolorization Assay for Antioxidant Activity
The ABTS radical cation (ABTS+•) was generated by mixing ABTS stock solution
in water (7 mM) with potassium persulfate (2.45 mM) and left in the dark at room tem-
perature for 12–16 h before use. The results are recorded immediately after the mixing
solutions at 734 nm. The results were compared to the appropriate standard inhibitor
Trolox (Table 4) [44].
Soybean Lipoxygenase Inhibition Study
The stock solutions of the compounds were incubated with sodium linoleate (0.1 mM
and 0.2 mL of soybean lipoxygenase solution (1/9
10−4 w/v in saline) at room temper-
ature. In vitro test was performed as previously published [42 44]. The test follows our
previously published methods [42 44] The production of 13-hydroperoxylinoleic acid was
)
×
–
–
recorded at 234 nm using using NDGA as reference compound (Table 3, Table 5).
3.4.2. Biological In Vivo Assays
The animals (Fisher 344 rats), were housed under standard conditions, and received a
diet of commercial food pellets and water ad libitum during the maintenance, but were
entirely fasted during the experiment period. Both sexes were used, while pregnant
females were excluded. Each group was composed of 6–15 animals. Our studies were in
accordance with recognized guidelines on animal experimentation (guidelines for the care
and use of laboratory animals published by the Greek Government 160/1991, based on
EU regulations 86/609). Rats were kept in the Centre of the School of Veterinary Medicine
(EL54 BIO42), Aristotle University of Thessaloniki, which is registered by the official
state veterinary authorities (presidential degree 56/2013, in harmony with the European
Directive 2010/63/EEC). The experimental protocols were approved by the Animal Ethics
Committee of the Prefecture of Central Macedonia.
Inhibition of the Carrageenin-Induced Edema
Carrageenin in water as an intradermal injection of 0.1 mL 2% was used for the
induction of the edema in the right hind paw of the rats, according to the previously
reported method [44]. Animals were divided in five-membered groups and the tested
compounds were suspended in water (0.0057 mmol/kg body weight), with a few drops
of Tween 80, ground in a mortar before administered intraperitoneally simultaneously.
The rats were euthanized 3.5 h after carrageenin injection. Comparison of the change in
paw weight with that in control animals (treated with water) was expressed as a percent

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inhibition of the oedema CPE% values using indomethacin (IMA) as a reference compound.
Values CPE% are the mean from two different experiments with a standard error of the
mean less than 10% (Table 5). Student’s T test was applied.
Anti-Nociception-Writhing Test
For the study of the analgesic activity of the tested compounds, pain is provoked
with acetic acid. Animals were divided in three five-membered groups, according to our
reported method [44]. After treatment with the tested compounds (0.0057 mmol/kg body
weight; intraperitoneally) in the first group, the second group was used as a positive control
(aspirin; 0.0057 mmol/kg body weight i.p.) and the third group served as the control in
which saline was administered (negative control). After 30 min, 0.6% acetic acid (1 mL/kg
body weight) was injected i.p. The number of writhes was recorded for each animal every
5 min during a subsequent 30 min period (Table 6).
Induction of Adjuvant-Induced Disease (AID)
Rats were divided into four groups (five-membered groups of animals were used):
groups one, two and three were injected intradermally [4] with 0.1 mL Freund’s adjuvant
(FA) into the base of the tails of the animals; group three was the FA control rats; group
four was injected with saline, used as an absolute control. Compound 2d (group 1) and
indomethacin (group 2) were injected i.p. once every other day for the following 24 days in
a dose of 0.00057 mmol/mL/kg body weight (Table 7). Adjuvant arthritis was developed
circa 14 days post-FA administration. Arthritic score was measured every 2 days from
the commencement (14th day) day. Arthritic score was assessed on the 24th day. For
quantification of arthritis (arthritic score); a single point was assigned for each wrist or
ankle area and an additional point was given for each involved phalangeal joint, up to a
maximum of five points per extremity [54,57,58].
On the 24th day and at least 12 h after the last injection, animals were administered
zoxazolamine i.p. 10 mg/1 mL/g, as an aqueous suspension with a few drops of Tween 80,
and the duration of the paralysis were assessed (Table 7) (Figure 2). The experiment was
conducted in duplicate.
3.5. Computational Methods. Molecular Docking Studies on Soybean Lipoxygenase
For the docking studies, soybean lipoxygenase (PDB code: 3PZW) was used, and the
visualization was accomplished through UCSF Chimera [59]. The protein was prepared:
water molecules were removed, missing residues were added with Modeller [60], hydrogen
atoms and AMBER99SB-ILDN charges were added, and the charge on iron was set to +2.0,
with no restraint applied to the iron atom and the ligands. OpenBabel was used to generate
and minimize ligand 3D coordinates using the MMFF94 force field [61]. Ligand topologies
and parameters were generated by ACPYPE (AnteChamberPYthon Parser interfacE) [62
]
using Antechamber [63]. Energy minimizations were carried out using the AMBER99SB-
ILDN force field [64] with GROMACS 4.6. Docking was performed with AutoDockVina
(1.1.2) [65] applying a grid box of size 100 Å, 70 Å, 70 Å in X, Y, Z dimensions. The
generation of docking input files and the analysis of the docking results was accomplished
by UCSF-Chimera. Docking was carried out with an exhaustiveness value of 10 and a
maximum output of 20 docking modes.
4. Conclusions
The synthesized compounds present antioxidant and anti-inflammatory activities,
scavenging of free radicals, and inhibition of lipid peroxidation.
In the DPPH assay, the novel derivatives showed medium antioxidant activity with
small differences dependent on time and concentration. Only compounds 2d and 2e
moderately reduced the ABTS radical cation (ABTS•+) at 100
µM, while all compounds
highly inhibited lipid peroxidation. Lipophilicity plays a significant role.

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Compound 2g presents the best lipoxygenase inhibition within the data test, in a
competitive mode. Docking studies reveal that 2g possibly interacts in an allosteric mode,
presenting hydrophobic interactions with VAL126, PHE143, VAL520 and LYS526 and a
halogen bond between the chlorine atom and ARG182.
Pyrazolines 2d and 2e seem to be the best anti-inflammatory agents. Simultaneously,
they present satisfactory analgesic activity, whereas 2d diminishes the severity and the
onset of adjuvant-induced arthritis. Thus, it can be considered to be a lead compound with
a multifunctional profile.
Supplementary Materials: Table S1: Docking scores. Hydrophobic interactions, hydrogen bonds,
π-cation interactions and halogen bonds of the synthesized derivatives with different residues,
Figure S1: Preferred docking poses of pyrazoles (2a [pink], 2b [blue], 2d [purple] and 2e [cyan])
bound to soybean lipoxygenase (LOX-1), Figure S2: Preferred docking poses of pyrazolines (2c [light
sea green], 2f [light purple], 2g [light green] and 2h [peach pink]) bound to soybean lipoxygenase
(LOX-1).
Author Contributions: M.M. synthesized and biologically evaluated the new compounds as a part
of her Master’s thesis; D.H.-L. designed and supervised the research and contributed to the writing,
design, synthesis, biological evaluation and analysis of the data; E.P. contributed to the writing,
docking studies and analysis of data. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Details are given in the biological session.
Informed Consent Statement: Not applicable, no studies were applied on humans.
Data Availability Statement: All the data of this research is included in this paper.
Acknowledgments: E. Pontiki would like to thank A. Patsilinakos from the Department of Chemistry
and Drug Technologies, “Sapienza” University of Rome, Italy. We are thankful to A. Leo and M.
Medlin as well as Biobyte for free access to the platform.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds (2a–2h) are available from the authors.
Abbreviations
AA: Arachidonic acid; AAPH: 2,20-azinobis(2-amidinopropane) hydrochloride; ACE: angiotensin
converting enzyme; ADME: Absorption, Distribution, Bioavailability, Metabolism and Elimination;
AID: Adjuvant-induced disease; clog P: theoretical calculated lipophilicity; COX: cycloxygenase;
CPE: carrageenin-induced rat paw edema; DPPH: 2,2-diphenyl-1-picrylhydrazyl free radical; ER:
estrogen receptor; LOX: Lipoxygenase; LPSP: lipophilicity values through Spartan v.5.1.3.; LTs: leu-
cotrienes; NDGA: nordihydroguaeretic acid; NSAIDs: non-steroidal anti-inflammatory drugs; PGs:
prostaglandins; QSAR: Quantitative Structure Activity Relationships; ROS: Reactive Oxygen Species;
RPTLC: Reverse Phase Thin Layer Chromatography.
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