Design, synthesis, and molecular docking of novel 3,5-disubstituted-1,3,4-oxadiazole derivatives as iNOS inhibitors

Article abstract of DOI:10.1002/ardp.202000469

To obtain new anti-inflammatory agents, recent studies have aimed to replace the carboxylate functionality of nonsteroidal anti-inflammatory drugs with less acidic heterocyclic bioisosteres like 1,3,4-oxadiazole to protect the gastric mucosa from free carboxylate moieties. In view of these observations, we designed and synthesized a series of 3,5-disubstituted-1,3,4-oxadiazole derivatives as inhibitors of prostaglandin E₂ (PGE₂) and NO production with an improved activity profile. As initial screening, and to examine the anti-inflammatory activities of the compounds, the inhibitions of the productions of lipopolysaccharide-induced NO and PGE₂ in RAW 264.7 macrophages were evaluated. The biological assays showed that, compared with indomethacin, compounds 5a, 5g, and 5h significantly inhibited NO production with 12.61 ± 1.16, 12.61 ± 1.16, and 18.95 ± 3.57 μM, respectively. Consequently, the three compounds were evaluated for their in vivo anti-inflammatory activities. Compounds 5a, 5g, and 5h showed a potent anti-inflammatory activity profile almost equivalent to indomethacin at the same dose in the carrageenan-induced paw edema test. Moreover, the treatment with 40 mg/kg of 5h produced significant anti-inflammatory activity data. Furthermore, docking studies were performed to reveal possible interactions with the inducible nitric oxide synthase enzyme. Docking results were able to rationalize the biological activity data of the studied inhibitors. In summary, our data suggest that compound 5h is identified as a promising candidate for further anti-inflammatory drug development with an extended safety profile.

Full text of DOI:10.1002/ardp.202000469

Received: 11 December 2020
DOI: 10.1002/ardp.202000469
Revised: 13 April 2021
Accepted: 16 April 2021
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F U L L P A P E R
Design, synthesis, and molecular docking of novel
3,5‐disubstituted‐1,3,4‐oxadiazole derivatives as
iNOS inhibitors
Meric Koksal1
Enise E. Gurdal1,3
Melda Ozgurbuz6
| Ayca Dedeoglu‐Erdogan1
| Marwa Bader1,2
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| Wolfgang Sippl3
| Hande Sipahi4
| Rengin Reis4,5
| Turgay Celik6
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1Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Yeditepe University,
Istanbul, Turkey
Abstract
To obtain new anti‐inflammatory agents, recent studies have aimed to replace
the carboxylate functionality of nonsteroidal anti‐inflammatory drugs with less
acidic heterocyclic bioisosteres like 1,3,4‐oxadiazole to protect the gastric
mucosa from free carboxylate moieties. In view of these observations, we
designed and synthesized a series of 3,5‐disubstituted‐1,3,4‐oxadiazole deriva-
tives as inhibitors of prostaglandin E2 (PGE2) and NO production with an im-
proved activity profile. As initial screening, and to examine the anti‐inflammatory
activities of the compounds, the inhibitions of the productions of
lipopolysaccharide‐induced NO and PGE2 in RAW 264.7 macrophages were
evaluated. The biological assays showed that, compared with indomethacin,
compounds 5a, 5g, and 5h significantly inhibited NO production with
12.61 1.16, 12.61 1.16, and 18.95 3.57 µM, respectively. Consequently, the
three compounds were evaluated for their in vivo anti‐inflammatory activities.
Compounds 5a, 5g, and 5h showed a potent anti‐inflammatory activity profile
almost equivalent to indomethacin at the same dose in the carrageenan‐induced
paw edema test. Moreover, the treatment with 40 mg/kg of 5h produced
significant anti‐inflammatory activity data. Furthermore, docking studies were
performed to reveal possible interactions with the inducible nitric oxide synthase
enzyme. Docking results were able to rationalize the biological activity data of
the studied inhibitors. In summary, our data suggest that compound 5h is
2Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Omar Al‐Mukhtar
University, Al Bayda, Libya
3Department of Pharmaceutical Chemistry
and Clinical Pharmacy, Martin Luther
University Halle‐Wittenberg, Halle (Saale),
Germany
4Department of Pharmaceutical Toxicology,
Faculty of Pharmacy, Yeditepe University,
Istanbul, Turkey
5Department of Pharmaceutical Toxicology,
Faculty of Pharmacy, Acibadem University,
Istanbul, Turkey
6Department of Pharmacology, Faculty of
Pharmacy, Yeditepe University, Istanbul,
Turkey
Correspondence
Meric Koksal, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Yeditepe
University, Atasehir, 34755 Istanbul, Turkey.
Email: merickoksal@yeditepe.edu.tr
identified as
a promising candidate for further anti‐inflammatory drug
development with an extended safety profile.
K E Y W O R D S
1,3,4‐oxadiazole, anti‐inflammatory, docking study, nitric oxide, PGE2
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Arch Pharm. 2021;e2000469.
wileyonlinelibrary.com/journal/ardp
© 2021 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.202000469

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1
INTRODUCTION
introduction of 1,3,4‐oxadiazole moiety and arylpiperazine/piper-
idine pharmacophore to its structure enhances its biological
activity. In our previous study, the anti‐inflammatory activity and
gastric ulcerogenic potential of the 5‐(3,4‐dichlorophenyl)‐3‐[(4‐
substitutedpiperazin‐1‐yl)methyl]‐1,3,4‐oxadiaxole‐2(3H)‐thiones
derivatives were investigated. These results showed that these 3,5‐
disubstituted‐1,3,4‐oxadiazole‐2(3H)‐thione derivatives showed the
most prominent anti‐inflammatory activity with low gastric ulcera-
tion incidence than the reference drug indomethacin.[20] In view of
these observations and in continuation to our previous
studies,[20‐22] we have designed and synthesized a series of novel 3,
5‐disubstituted‐1,3,4‐oxadiazole derivatives as inhibitors of PGE2
and NO production with an improved activity profile. All the syn-
thesized and structurally confirmed compounds were screened in
terms of their ability to inhibit the production of NO and PGE2 with
LPS‐induced RAW 264.7 macrophages cells. In addition, in vivo anti‐
inflammatory assay was performed for three active compounds that
have significant inhibitions in in vitro assays. Docking studies were
carried out to rationalize the protein–ligand interactions between
the target iNOS and the developed inhibitors.
Inflammation is a protective physiological response of the immune
system to infectious agents. Nevertheless, the inflammatory re-
sponse may cause considerable damage to the cell, because microbial
factors such as lipopolysaccharide (LPS) stimulate proinflammatory
cytokines including inducible nitric oxide synthase (iNOS) and cy-
clooxygenases. Thus, as an inflammation response, a large amount of
prostaglandin E2 (PGE2) and nitric oxide (NO) is produced in the
immune cell.[1,2] Due to their potent vasodilator properties, NO and
PGE2 may cause classic inflammation symptoms like edema and
redness. In addition, reacting with the superoxide anion, NO can be
converted to peroxynitrite, which is an oxidizing molecule related to
cellular damage.[3‐5] By acting on neurons, PGE2 also improves the
systemic response toward inflammation including pain and hy-
persensitivity.[6‐9] For this reason, as well as anti‐inflammatory ac-
tivity, PGE2 is also an essential target for analgesic activity. However,
as PGE2 was found to be an immunosuppressant, it may contribute to
the progression of inflammation and lead to severe diseases like
cancer.[10] So, the importance or inhibition of PGE2 synthesis has
been increased in inflammatory and malign diseases such as cancer
and rheumatoid arthritis.
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In biological condition, NO is produced from L‐arginine by nitric
oxide synthase (NOS) in the presence of NADPH, molecular oxygen,
and other cofactors. There are three isoforms of NOS in mammalian
tissues named according to their activity or the tissue type in which
they were first described.[11] Two of the isoforms, neuronal NOS and
endothelial NOS, are constitutively expressed in mammalian cells
and synthesize NO in response to an increase in intracellular Ca2+
levels for nerve function and blood pressure regulation, respectively.
The third isoform, iNOS, activity is independent of the level of cal-
cium in the cell and could be activated by the stimulation of cyto-
kines or bacterial LPS. In addition, the production of NO by iNOS
lasts much longer than from the other isoforms of NOS and tends to
be in much higher and cytotoxic concentrations in the cell that can
mediate inflammation and an innate immune response.[11‐13]
2
RESULTS AND DISCUSSION
Chemistry
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2.1
Scheme 1 depicts the synthesis pathway of targeted 3,5‐disubstituted‐
1,3,4‐oxadiazole‐2‐thione derivatives (5a–h). The synthetic pathway
was started with Fischer esterification of salicylic acid (1) and the re-
sulting ester (2) was converted to acid hydrazide in the presence of
hydrazine hydrate (3). The produced hydrazide group was cyclized with
carbon disulfide and potassium hydroxide to obtain 5‐(2‐
hydroxyphenyl)‐1,3,4‐oxadiazole‐2(3H)‐thione ring (4).[22] For the last
step, by using the Mannich reaction procedure, piperidine or piperazine
derivatives and oxadiazole ring were linked with methylene chain
(5a–h). The obtained compounds were purified by recrystallization with
ethanol or salt formation.
Over the past several decades, many scientists have focused on
the discovery of new nonsteroidal anti‐inflammatory drugs (NSAIDs)
with improved therapeutic profile and less side effects.[14,15] Mole-
cular and conceptual models of inflammation have kept on evolving
to bring new insight to generate novel therapeutic approaches for
inflammatory diseases. Management of NO and PGE2 production has
become the current research strategy to develop new anti‐
Structural properties of purified compounds are given in Table 1.
The newly synthesized compounds were characterized by infrared
(IR), 1H‐nuclear magnetic resonance (NMR), 13C‐NMR, and elemental
analysis. All of the compounds gave satisfactory analytical and
spectroscopic data, which were in full accordance with their depicted
structure. In general, the IR spectrum of all compounds exhibited
characteristics peaks of free hydroxyl groups at 3320–3473 cm−1, at
1610–1630 cm−1 for the C═N group, and 1435–1490 cm−1 for the
C═S group. In the 1H‐NMR spectra, the hydroxyl groups of synthe-
sized compounds appeared at δ 10.51–10.66. All the aromatic pro-
tons were obtained in the range of δ 7.00–7.65. The hydrogens of
methylene bridge (N–CH2–N) of the compounds appeared at δ
5.00–5.07, and the piperazine or piperidine protons were observed
in the range of δ 1.58–3.48. The 13C‐NMR spectra represent the
signal of thione (C═S) group at δ 176.90–177.03 and the aromatic
protons in the range of δ 108.80–157.98. The carbon of the
inflammatory drugs. In
a chemical manner, to obtain new
anti‐inflammatory agents, recent studies have aimed to replace the
carboxylate functionality of NSAIDs with many types of less acidic
heterocyclic bioisosteres like thiazole,[16] pyrazole,[17] and ox-
adiazole[18] to protect the gastric mucosa from free carboxylate
moiety. During recent years, the compounds containing 1,3,4‐
oxadiazole core have been documented as remarkable anti‐
inflammatory agents.[19] Although salicylic acid and its properties are
well known since decades and this compound is rather a weak anti‐
inflammatory agent, the results showed in this study indicate that

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SCHEME 1 Synthetic pathway of the
3,5‐disubstituted‐1,3,4‐oxadiazole
derivatives (5a–h)
TABLE 1 Structural properties of the synthesized compounds
X
N
R
N
N
S
O
OH
Compounds
Molecular formula
MW (g/mol)
mp (°C)
Yield (%)
–X
CH2
CH
CH
C
–R
5a
5b
5c
5d
5e
5f
3‐Methyl
C15H19N3O2S.H2O
C15H19N3O3S
323.41
321.39
419.92
445.96
428.93
392.47
368.45
369.44
>300
169.1
166.2
251.1
154.7
183.8
172
40
72
43
42
53
52
42
59
4‐Hydroxymethyl
4‐Phenyl
C20H21N3O2S
4‐Hydroxy‐4‐phenyl
4‐Acetyl‐4‐phenyl
4‐Cyano‐4‐phenyl
4‐Phenyl
C20H21N3O3S.HCl
C22H23N3O3S.HCl
C21H20N4O2S.HCl
C19H20N4O2S
C
C
5g
5h
N
N
4‐(2‐Pyridyl)
C18H19N5O2S
147.4
Abbreviations: MW, molecular weight, mp, melting point.
methylene bridge (N–CH2–N) was at δ 69.62–70.56. The 1H‐NMR,
13C‐NMR, and elemental analysis results of the compounds are given
in the Supporting Information.
by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide
(MTT) assay. The assay results of synthesized compounds that show
data recorded for these doses will not be due to cytotoxicity against
RAW 264.7 cells are summarized in Table 2. The results indicated
that compounds did not inhibit 50% of RAW 264.7 macrophages
growth up to 100 μM except for 5d. For compound 5d, IC50 was
found to be 37.92 9.10 μM, and therefore nitrite assay was per-
formed at 25 μM, which was determined to be a safe dose (Table 2).
Indomethacin, known as a powerful anti‐inflammatory agent, was
used as the reference drug for comparisons in all biological studies.
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2.2
Pharmacology
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2.2.1
In vitro anti‐inflammatory activity
Cell cytotoxicity
To examine the anti‐inflammatory activities of the target com-
pounds, the inhibitions of the productions of LPS‐induced NO and
PGE2 in RAW 264.7 macrophages were evaluated. The cytotoxic
effects of compounds on RAW 264.7 macrophages were examined
NO and PGE2 production inhibition assays
As an initial screening, we tested the inhibitory capacity of
synthesized compounds against NO production at their nontoxic

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TABLE 2 Cytotoxicity, nitrite, and prostaglandin E2 (PGE2) levels in lipopolysaccharide (LPS)‐stimulated RAW 264.7 cells treated with
compounds
Entry
Dose (µM)
Cytotoxicity (IC50 values) (µM SD)
Nitrite level (µM SD)
0.17 0.049
PGE2 production (pg/ml)
200.97 79.16
Control
Control + LPS
5a
39.42 2.03
4157.77 402.71
50
‐
25.30 3.66*
12.61 1.16**
‐
100
>100
3754.05 53.50
5b
5c
5d
5e
5f
100
100
25
>100
29.05 2.32
34.23 0.18
35.13 0.38
31.37 1.79
24.38 0.54*
3918.12 163.06
3677.78 25.52
>100
37.92 9.10
>100
100
100
3854.27 46.72
3702.56 52.50
>100
5g
50
‐
36.30 0.51
‐
100
>100
11.06 1.34**
3975.56 220.50
5h
50
‐
37.99 2.90
18.95 3.57*
‐
100
>100
3670.08 22.55
Indomethacin
100
>100
19.08 1.34*
266.67 15.28**
Note: The significant differences between groups and control + LPS were defined with *p < .05 and **p < .01.
doses in LPS‐induced RAW 264.7 cells, a murine macrophage cell
line that is commonly used in screening for the identification of
anti‐inflammatory compounds.[23,24] As given in Table 2, com-
pounds 5a–h expressed varying inhibitory activities against LPS‐
induced NO productions at tested concentrations. Interestingly,
the compounds possessing piperazine ring exhibited higher NO
production inhibition than reference indomethacin, whereas the
others with piperidine ring except compound 5a showed weak
activities. More specifically, compound 5g showed the most potent
inhibitory activity against NO production with 72.01 3.97%,
which is lower than the reference value, whereas the piperidine
analog of this derivative almost did not cause any inhibition.
Moreover, the compounds carrying disubstitution on the fourth
position of piperidine did not exhibit significant activities at the
tested concentration. On the basis of the in vitro anti‐
inflammatory model results illustrated in Table 2, the three
compounds (5a, 5g, and 5h) exerted higher NO inhibition capacity
as compared with the reference drug in 100 μM dose. In addition,
when 5a is tested at 50 μM, dose‐dependent nitrite inhibition can
be seen with this compound.
Inhibitor activities of all synthesized compounds were ad-
ditionally tested with a PGE2 assay kit technique. According to PGE2
test results, the compounds lead to a slight decrease in PGE2 levels,
which was statistically insignificant (Table 2). It is noticeable that the
compounds are more active as inhibitors of NO production rather
than PGE2 production. According to in vitro biological assays, com-
pounds 5a, 5g, and 5h that exhibited the most potent inhibitory
activities at 100 µM were selected for in vivo studies to identify the
lead compound of this group. Furthermore, the favorable in vitro
anti‐inflammatory activities of the compounds led us to molecular
docking to evaluate the probable interactions of these active com-
pounds with iNOS protein.
FIGURE 1 The effects of 5a (a), 5g (b), and 5h (c) on carrageenan‐induced paw edema in mice (n = 6). Data are the mean SEM. (a) p ≤ .05,
compared with vehicle (two‐way analysis of variance [ANOVA] followed by Tukey's post‐hoc test). (b) p ≤ .05, compared with indomethacin
(positive standard) (one‐way ANOVA followed by Tukey's post‐hoc test)

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FIGURE 2 Docking poses of 5h (a) and developed inhibitors (b) on inducible nitric oxide synthase enzyme (PDB ID: 1R35)
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2.2.2
In vivo anti‐inflammatory activity
after edema induction at the first, third, and fourth hours (Figure 1c). As
suggested above, the results of the acute inflammatory model indicated
that compound 5h has a potential as an anti‐inflammatory agent against
inflammatory edema.
To evaluate the in vivo anti‐inflammatory potency of selected deriva-
tives, 5a, 5g, and 5h, carrageenan‐induced paw edema test as an acute
inflammation animal model was performed.[25] The anti‐inflammatory
effects of compounds are demonstrated in Figure 1, separately. Oral
administrations of compounds 5a, 5g, and 5h both at 10‐ and 40‐mg/kg
doses exhibited a significant decrease in paw edema. As a positive con-
trol, indomethacin showed an inhibitory effect on edema development
induction at the third and fourth hours. Treatment with compounds 5a
and 5g in two applied doses yielded a similar inhibitory activity with a
dose of 10 mg/kg indomethacin and also suppressed edema formation
after edema induction at the third and fourth hours (Figure 1a,b). Al-
though, the treatment with 10 mg/kg dose of 5h showed similiar activity
profile such as 5a and 5g, 40 mg/kg dose oral administration of the
compound exhibited significantly more inhibitory activity than reference
indomethacin. Moreover, it suppressed significantly edema formation
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2.3
Molecular docking studies
iNOS can be activated by cytokines or LPS, resulting in a great
amount of NO secretion to cause inflammation and an innate im-
mune response independent of Ca2+ [26]
To evaluate the possible
.
interactions of the developed compounds, docking studies were
performed with iNOS (PDB ID: 1R35[27]). Binding to iNOS requires
the interaction with Glu377 that corresponds to Glu371 in murine
iNOS that was used for the current biological study. Docking studies
revealed a common binding mode of the developed compounds. In
particular, the inhibitors form an H‐bond between its phenol and a
glutamate residue, a salt bridge between the protonated piperidine/
piperazine, and a propionate group of protoporphyrin. In addition, an
H‐bond between the thioxo sulfur and Asp376 carboxylate was ob-
served as a common interaction. The binding mode of the active
inhibitor 5h is depicted in Figure 2 as an example to represent the
series. Equatorial conformation of bulky substituents at the
piperidine was found to be more favorable in the docking studies.
Accordingly, corresponding docking scores of all compounds are
given in Table 3.
TABLE 3 Docking scores of compounds (PDB ID: 1R35)
Compound
Docking score
−6.426
5a_R
5a_S
−6.718
5b
−6.758
5c
−6.615
5d
−6.675
5e
−6.948
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3
CONCLUSION
5f
−6.957
In conclusion, a series of 3,5‐disubstituted‐1,3,4‐oxadiazole‐2‐thione
derivatives containing piperidine and piperazine fragment was syn-
thesized. According to in vitro activity result, several compounds
were displayed as potent inhibitors of NO production rather than
inhibitors of PGE2 release. Among them, compounds 5a, 5g, and 5h
5g
−6.938
5h
−6.724
Cocrystallized ligand
−5.304 (RMSD: 0.150)
Abbreviation: RMSD, root mean square deviation.

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showed the highest activities as promising NO production inhibitors in
LPS‐induced RAW 264.7 macrophages with low cytotoxicity. These
three compounds were selected for in vivo anti‐inflammatory activity
assay and molecular docking studies were performed on all compounds
to evaluate their possible interactions with the iNOS active site.
Docking studies demonstrated that the anti‐inflammatory activities of
the compounds are reasonable due to the fulfilling of necessary inter-
actions with the iNOS enzyme, that is, hydrogen bonding to Glu371 and
Asp376 and ionic interaction with protoporphyrin. According to in vivo
acute inflammatory models, 5h is a potential anti‐inflammatory agent
against inflammatory edema. Therefore, compound 5h can be utilized as
a promising lead compound for further development of new and safe
anti‐inflammatory agents.
4.1.3
General procedure for the synthesis of 5‐(2‐
hydroxyphenyl)‐1,3,4‐oxadiazole‐2(3H)‐thione (4)
A mixture of salicylic acid hydrazide 3 (0.05 mol, 7.5 g), potassium
hydroxide (0.05 mol, 3 g), carbon disulfide (0.17 mol, 10 ml), and
ethanol (70 ml) was stirred and refluxed for 12 h. The solvent was
removed in vacuo and the residue was acidified with hydrochloric
acid (2 mol/l). The resulting precipitate was filtered, washed with
water, and recrystallized from ethanol.[21]
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4.1.4
General procedure for the synthesis of 5‐(2‐
hydroxyphenyl)‐3‐substituted‐1,3,4‐oxadiazole‐2(3H)‐
thiones (5a–h)
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4
EXPERIMENTAL
Chemistry
Formaldehyde solution 37% (0.02 mol, 1.5 ml) was added to a stirred
solution of 5‐(2‐hydroxyphenyl)‐1,3,4‐oxadiazole‐2(3H)‐thione 4 (4 g,
0.02 mol) in absolute ethanol (40 ml). An ethanolic solution (10 ml) of
the appropriate amine (0.02 mol) was added portion‐wise to the re-
action mixture, stirred for 3 h at room temperature, and left over-
night in a refrigerator. The crude products were filtered, washed
with water, dried, and crystallized from appropriate solvents.[30]
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4.1
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4.1.1
General
All chemicals and reagents used in the present study were of ana-
lytical grade. The substituted piperazine and piperidine derivatives
were ordered from chemical company Aldrich. The reactions were
monitored by thin‐layer chromatography on Merck precoated silica
GF254 plates. Melting points were determined by using a Mettler
Toledo FP62 capillary melting point apparatus (Mettler‐Toledo) and
were uncorrected. Infrared spectra were recorded on a Perkin‐Elmer
Spectrum One series FT‐IR apparatus (version 5.0.1) (Perkin El-
mer), using potassium bromide pellets, and the frequencies were
expressed in cm−1. The 1H‐ and 13C‐NMR spectra (see the Supporting
Information) were recorded with a Varian Mercury‐400 FT‐NMR
spectrometer (Varian), using tetramethylsilane as the internal re-
ference, with dimethylsulfoxide (DMSO) as a solvent, the chemical
shifts were reported in parts per million (ppm), and coupling con-
stants (J) were given in hertz (Hz). Elemental analyses were per-
formed on LECO 932 CHNS instrument (Leco‐932).
5‐(2‐Hydroxyphenyl)‐3‐[(3‐methylpiperidin‐1‐yl)methyl]‐1,3,4‐
oxadiazole‐2(3H)‐thione (5a)[21]
FT‐IR (KBr, cm−1); 3473 (OH), 2950 (CH aromatic), 2811 (CH ali-
phatic), 1625 (C═N), 1572 (C═C), 1490 (C═S), and 1235 (C–N).
1H‐NMR (DMSO, ppm); 0.80 (d, 3H, –CH3, J = 6.4 Hz), 1.59–1.40 (m,
4H, H4 + H5), 2.08 (t, 1H, piperidine H3, J = 10.8 Hz), 2.39 (t, 2H, pi-
peridine H6, J = 12 Hz), 2.96 (d, 2H, piperidine H3, J = 10.8 Hz), 5.04
(s, 2H, N–CH2–N), 6.96 (t, 1H, 2‐hydroxyphenyl H5, J = 7.6 Hz), 7.02
(d, 1H, 2‐hydroxyphenyl H6, J = 8 Hz), 7.40–7.04 (m, 1H,
2‐hydroxyphenyl H4), 7.62 (dd, 1H, 2‐hydroxyphenyl H3, J = 8 Hz,
Jʹ = 2 Hz), and 10.54 (bs, 1H, OH). 13C‐NMR (DMSO, ppm); 40.05
(piperidine, C3 + C5), 49.96 (piperidine, C2 + C6), 70.49 (N–CH2–N),
108.99 (2‐hydroxyphenyl C5), 116.94 (2‐hydroxyphenyl C4), 119.40
(2‐hydroxyphenyl C6), 128.79 (2‐hydroxyphenyl C3), 133.40
(2‐hydroxyphenyl C1), 156.28 (2‐hydroxyphenyl C2), 157.98 (ox-
adiazole C5), and 177.01 (oxadiazole C═S). Analytical calculated for
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Information.
C15H19N3O2S.H2O; Cal. C, 54.16; H, 4.31; N, 13.30; S, 7.61. Found: C,
54.10; H, 4.31; N, 13.43; S, 7.60.
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4.1.2
General procedure for the synthesis of
3,4‐dichlorobenzohydrazide (3)
5‐(2‐Hydroxyphenyl)‐3‐[(4‐hydroxymethylpiperidin‐1‐yl)methyl]‐
1,3,4‐oxadiazole‐2(3H)‐thione (5b)
A solution of 2‐hydroxybenzoic acid (salicylic acid) 1 (0.05 mol, 6.9 g),
methanol (20 ml), and a catalytic amount of conc. H2SO4 was re-
fluxed for 3 h. The reaction mixture was cooled and extracted with
dichloromethane to obtain the pure methyl ester 2.[28] The solution of
methyl ester of salicylic acid (0.1 mol, 15.2 g) and 80% hydrazine hydrate
(30 ml) in absolute ethanol (80 ml) was refluxed for 12–14 h. The excess
solvent was distilled off and the concentrated solution was poured into
ice water. The solid separated was filtered, washed, and dried; the crude
product was purified by recrystallization from ethanol.[29]
FT‐IR (KBr, cm−1); 3469 (OH), 2938 (CH aromatic), 2810 (CH ali-
phatic), 1610 (C═N), 1573 (C═C), 1436 (C═S), and 1254 (C–N).
1H‐NMR (DMSO, ppm); 1.14–1.01 (m, 2H, H5), 1.26–1.21 (m, 1H, H4),
1.61 (d, 2H, piperidine H3, J = 11.2 Hz), 2.42 (t, 2H, piperidine H6,
J = 11.6 Hz), 3.02 (d, 2H, –CH2OH, J = 11.2 Hz), 3.21 (t, 2H, piperidine
H2, J = 5.6 Hz), 4.39 (t, 1H, OH), 5.00 (s, 2H, N–CH2–N), 6.98–6.94
(m, 1H, 2‐hydroxyphenyl H5), 7.02 (d, 1H, 2‐hydroxyphenyl H6,
J = 8.4 Hz), 7.43–7.39 (m, 1H, 2‐hydroxyphenyl H4), 7.62 (dd, 1H,
2‐hydroxyphenyl H3, J = 7.8 Hz, Jʹ = 1.6 Hz), and 10.55 (bs, 1H, OH).

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13C‐NMR (DMSO, ppm); 28.60 (piperidine, C3 + C5), 37.53 (piper-
idine, C4), 50.03 (piperidine, C2 + C6), 70.56 (N–CH2–N), 109.04
(2‐hydroxyphenyl C5), 117.02 (2‐hydroxyphenyl C4), 119.45
(2‐hydroxyphenyl C6), 128.39 (2‐hydroxyphenyl C3), 133.51
(2‐hydroxyphenyl C1), 156.35 (2‐hydroxyphenyl C2), 157.35 (ox-
adiazole C5), and 177.03 (oxadiazole C═S). Analytical calculated for
13C‐NMR (DMSO, ppm); 25.47 (CH3) 32.11 (piperidine, C3 + C5),
47.40 (piperidine, C4), 53.50 (piperidine, C2 + C6), 69.98 (N–CH2–N),
108.86 (2‐hydroxyphenyl C5), 117.03 (2‐hydroxyphenyl C4), 119.40
(2‐hydroxyphenyl C6), 126.22 (phenyl C4), 126.97 (phenyl C2 + C6),
128.75 (phenyl C3 + C5), 129.02 (2‐hydroxyphenyl C3), 133.63
(2‐hydroxyphenyl C1), 141.26 (phenyl, C1), 156.36 (2‐hydroxyphenyl
C2), 157.83 (oxadiazole C5), 176.92 (oxadiazole C═S), and 208.91
(C═O). Analytical calculated for C22H23N3O3S.HCl; Cal. C, 65.37; H,
5.76; N, 11.44; S, 8.73. Found: C, 64.64; H, 5.49; N, 11.44; S, 8.73.
C
15H19N3O3S.H2O; Cal. C, 56.06; H, 5.96; N, 13.07; S, 9.98. Found: C,
55.98; H, 6.29; N, 13.02; S, 9.90.
5‐(2‐Hydroxyphenyl)‐3‐[(4‐phenylpiperidin‐1‐yl)methyl]‐1,3,4‐
oxadiazole‐2(3H)‐thione (5c)
5‐(2‐Hydroxyphenyl)‐3‐[(4‐cyano‐4‐phenylpiperidin‐1‐yl)methyl]‐
1,3,4‐oxadiazole‐2(3H)‐thione (5f)
FT‐IR (KBr, cm−1); 3469 (OH), 2959 (CH aromatic), 2817 (CH
aliphatic), 1625 (C═N), 1592 (C═C), 1489 (C═S), and 1253 (C–N).
1H‐NMR (DMSO, ppm); 10.52 (bs, 1H, OH), 7.66 (dd,
1H, 2‐hydroxyphenyl H3, J = 8 Hz, Jʹ = 2 Hz), 7.46–7.41 (m,
1H, 2‐hydroxyphenyl H4), 7.25–7.13 (m, 5H, phenyl), 7.04 (dd, 1H,
2‐hydroxyphenyl H6, J = 8.4 Hz, Jʹ = 0.8 Hz), 6,99–6.95 (m, 1H,
2‐hydroxyphenyl H5), 5.07 (s, 2H, N–CH2–N), 3.14 (d, 2H, piperidine
H2, J = 12 Hz), 2.60 (t, 2H, piperidine H3, J = 10.4 Hz), 2.46–2.40 (m, 1H,
piperidine H4), 1.73 (d, 2H, piperidine H6, J = 10.8 Hz), and 1.67–1.58 (m,
2H, piperidine H5). 13C‐NMR (DMSO, ppm); 32.84 (piperidine, C3 + C5)
40.87 (piperidine, C4), 50.57 (piperidine, C2 + C6), 70.33 (N–CH2–N),
108.96 (2‐hydroxyphenyl C5), 117.00 (2‐hydroxyphenyl C4), 119.41
(2‐hydroxyphenyl C6), 125.92 (phenyl C4), 126.57 (phenyl C2 + C6),
128.22 (phenyl C3 + C5), 129.02 (2‐hydroxyphenyl C3), 133.57
(2‐hydroxyphenyl C1), 145.98 (phenyl, C1), 156.28 (2‐hydroxyphenyl C2),
157.85 (oxadiazole C5), and 176.96 (oxadiazole C═S). Analytical calcu-
lated for C20H21N3O2S; Cal. C, 65.37; H, 5.76; N, 11.44; S, 8.73. Found: C,
64.64; H, 5.49; N, 11.44; S, 8.73.
FT‐IR (KBr, cm−1); 3363 (OH), 2952 (CH aromatic), 2832 (CH aliphatic),
2240 (C≡N), 1625 (C═N), 1597 (C═C), 1490 (C═S), and 1253 (C–N).
1H‐NMR (DMSO, ppm); 2.06–1.99 (m, 2H, piperidine H5), 2.15 (d, 2H,
piperidine H6, J = 12.8 Hz), 2.85–2.83 (m, 2H, piperidine H3), 3.25
(d, 2H, piperidine H2, J = 12.4 Hz), 5.13 (s, 2H, N–CH2–N), 7.00 (t, 1H,
2‐hydroxyphenyl H5, J = 7.6 Hz), 7.09 (d, 1H, 2‐hydroxyphenyl H6,
J = 8.4 Hz), 7.54–7.35 (m, 5H, phenyl), 7.65 (dd, 1H, 2‐hydroxyphenyl H3,
J = 7.6 Hz, Jʹ = 1.6 Hz), and 10.61 (bs, 1H, OH). 13C‐NMR (DMSO, ppm);
35.12 (piperidine, C3 + C5), 41.29 (piperidine, C4), 47.52 (piperidine,
C2 + C6), 69.62 (N–CH2–N), 108.81 (2‐hydroxyphenyl C5), 117.06
(2‐hydroxyphenyl C4), 119.43 (2‐hydroxyphenyl C6), 121.60 (phenyl C4),
125.52 (phenyl C2 + C6), 128.00 (2‐hydroxyphenyl C3), 128.93 (phenyl
C3 + C5), 129.07 (2‐hydroxyphenyl C3), 133.74 (2‐hydroxyphenyl C1),
139.92 (phenyl, C1), 156.46 (2‐hydroxyphenyl C2), 157.98 (oxadiazole
C5), and 176.99 (oxadiazole C═S). Analytical calculated for
C20H20N4O2S.HCl; Cal. C, 64.27; H, 5.14; N, 14.28; S, 8.17. Found: C,
63.86; H, 4.86; N, 14.37; S, 8.28.
5‐(2‐Hydroxyphenyl)‐3‐[(4‐hydroxy‐4‐phenylpiperidin‐1‐yl)methyl]‐
1,3,4‐oxadiazole‐2(3H)‐thione (5d)
5‐(2‐Hydroxyphenyl)‐3‐[(4‐phenylpiperazin‐1‐yl)methyl]‐1,3,4‐
oxadiazole‐2(3H)‐thione (5g)[21]
FT‐IR (KBr, cm−1); 3335 (OH), 2954 (CH aromatic), 2829 (CH aliphatic),
1625 (C═N), 1591 (C═C), 1489 (C═S), and 1252 (C–N). 1H‐NMR
(DMSO, ppm); 1.74 (d, 2H, piperidine H6, J = 6.4 Hz), 2.32–2.20 (m, 1H,
piperidine H4), 2.50 (t, 2H, piperidine H3, J = 3.8 Hz), 3.22 (d, 2H, pi-
peridine H2, J = 7.6 Hz), 5.45 (s, 2H, N–CH2–N), 7.48–7.24 (m, 5H,
phenyl), 7.48–7.46 (m, 1H, 2‐hydroxyphenyl H4), 7.46 (d, 1H,
2‐hydroxyphenyl H3, J = 2 Hz), and 9.08 (bs, 2H, OH). Analytical
calculated for C20H21N3O3S; Cal. C, 62.64; H, 5.52; N, 10.96; S, 8.36.
Found: C, 62.71; H, 5.78; N, 10.45; S, 8.41.
FT‐IR (KBr, cm−1); 3332 (OH), 2886 (CH aromatic), 2830 (CH ali-
phatic), 1626 (C═N), 1598 (C═C), 1489 (C═S), and 1223 (C–N).
1H‐NMR (DMSO, ppm); 2.90 (t, 4H, piperazine H2 + H6, J = 4.8 Hz),
3.13 (t, 4H, piperazine H3 + H5, J = 4.8 Hz), 5.1 (s, 2H, N–CH2–N),
6.76 (t, 1H, phenyl H4, J = 7.2 Hz), 6.90 (bd, 2H, phenyl H2 + H6,
J = 8.0 Hz), 6.95–6.99 (m, 1H, 2‐hydroxyphenyl H5), 7.05 (bd, 1H,
2‐hydroxyphenyl H6, J = 8.4 Hz), 7.18 (t, 2H, phenyl H3 + H5,
J = 7.8 Hz), 7.42–7.46 (m, 1H, 2‐hydroxyphenyl H4), 7.66 (dd, 1H,
2‐hydroxyphenyl H3, J = 8 Hz, Jʹ = 1.6 Hz), and 10.51 (bs, 1H, OH).
5‐(2‐Hydroxyphenyl)‐3‐[(4‐acetyl‐4‐phenylpiperidin‐1‐yl)methyl]‐
1,3,4‐oxadiazole‐2(3H)‐thione (5e)
5‐(2‐Hydroxyphenyl)‐3‐[(4‐(2‐pyridyl)phenylpiperazin‐1‐yl)methyl]‐
1,3,4‐oxadiazole‐2(3H)‐thione (5h)[21]
FT‐IR (KBr, cm−1); 3372 (OH), 2963 (CH aromatic), 2830 (CH ali-
phatic), 1799 (C═O), 1630 (C═N), 1596 (C═C), 1490 (C═S), and
1259 (C–N). 1H‐NMR (DMSO, ppm); 1.84 (s, 3H, CH3), 1.99–1.93 (m,
2H, piperidine H5), 2.43 (d, 2H, piperidine H6, J = 14 Hz), 2.64 (t, 2H,
piperidine H3, J = 9.6 Hz), 2.94–2.91 (m, 2H, piperidine H2), 5.00
(s, 2H, N–CH2–N), 6,99–6.96 (m, 1H, 2‐hydroxyphenyl H5), 7.06
(d, 1H, 2‐hydroxyphenyl H6, J = 8 Hz), 7.56–7.43 (m, 1H,
2‐hydroxyphenyl H4), 7.47–7.24 (m, 5H, phenyl), 7.61 (dd, 1H,
2‐hydroxyphenyl H3, J = 8 Hz, Jʹ = 1 Hz), and 10.56 (bs, 1H, OH).
FT‐IR (KBr, cm‐1); 3435 (OH), 3012 (CH aromatic), 1612 (C═N),
1603, 1560 (C═C), 1484 (C═S), and 1264 (C–N). 1H‐NMR (DMSO,
ppm); 2.48 (t, 4H, piperazine H2 + H6, J = 5 Hz), 3.48 (t, 4H, piperazine
H3 + H5, J = 5 Hz), 5.08 (s, 2H, N–CH2–N), 6.60–6.57 (m, 1H, pyridyl
H5), 6.79 (t, 1H, pyridyl H6, J = 8.8 Hz), 6.93–6.79 (m, 1H, phenyl H5),
7.01 (d, 1H, phenyl H6, J = 8 Hz), 7.44–7.40 (m, 1H, phenyl H4),
7.50–7.46 (m, 1H, 2‐hydroxyphenyl H4), 7.63 (dd, 1H,
2‐hydroxyphenyl H6, J = 8 Hz, Jʹ = 1.6 Hz), 8.05 (dd, 1H,
2‐hydroxyphenyl H3, J = 4Hz, Jʹ = 2 Hz), and 10.51 (bs, 1H, OH).

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KOKSAL ET AL.
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13C‐NMR (DMSO, ppm); 44.64 (piperazine, C2 + C6), 49.38 (piperazine,
C3 + C5), 69.64 (N–CH2–N), 107.06 (pyridyl C5), 108.92 (pyridyl C4),
112.92 (2‐hydroxyphenyl C5), 117.00 (2‐hydroxyphenyl C4), 119.46 (2‐
hydroxyphenyl C6), 129.11 (pyridyl C6), 133.61 (2‐hydroxyphenyl C3),
137.38 (2‐hydroxyphenyl C1), 147.41 (pyridyl C3), 156.36 (2‐
hydroxyphenyl C2), 157.84 (pyridyl C1), 158.77 (C═N), and 176.98
(oxadiazole C═S). Analytical calculated for C18H19N4O3S; Cal. C, 58.52;
H, 5.18; N, 18.96; S, 8.68. Found: C, 58.80; H, 5.34; N, 18.86; S, 8.72.
4.2.3
In vivo anti‐inflammatory activity assay
|
Animals
Adult male BALBc mice (25–35 g) were kept in standard animal
housing conditions at the temperature of 23 1°C and suitable hu-
midity (55 5%) with dark–light cycles (12/12 h). The animals were
kept in home cages prepared with plexiglass (65 × 25 × 15 cm), re-
ceiving food and water ad libitum. The mice were acclimatized to la-
boratory conditions for 1 day before the experiments were carried out.
The experimental protocol was approved by the Ethical Committee of
Yeditepe University Experimental Medicine Research Institute (Deci-
sion number: 2019/10‐12). The animals were fasted (food was with-
drawn, not water) for 3 h before use and during the experiment. Six
mice in one group were used in all sets of the study. The experiments
were performed between 8:00 a.m. and 10:00 a.m. in a soundproof
laboratory. As doses, 10 and 40 mg/kg of compounds 5a, 5g, 5h, and
10 mg/kg of indomethacin were selected and the suspension was made
in normal saline by 0.25% CMC. Control group was used as a standard
drug and normal saline (0.5 ml) was used as a vehicle.
|
4.2
Pharmacological assays
|
4.2.1
MTT assay
Cell viability was examined using the MTT assay. Plated RAW 264.7
cells were exposed to 25, 50, and 100 µM of compounds dissolved in
DMSO. Only compound 5d was applied as 25 and 50 µM due to lack
of solubility in DMSO. After 24 h of incubation, MTT was added to all
wells at 0.5 mg/ml of concentration and incubated for an additional
2 h at 37°C. After discarding the medium from plates, 100 μl of
isopropanol was added to the wells. Absorbance of the MTT for-
mazan was determined at 570 nm by a UV‐spectrophotometric plate
reader (Thermo Multiscan Spectrum). Viability was defined as the
ratio (expressed as a percentage) of absorbance of the cells exposed
to compounds to the cells treated with 0.5% DMSO (as control). All
measurements were done in triplicates.
Carrageenan‐induced mice paw thickness assay
Indomethacin and other compounds were orally given to the animals
30 min before carrageenan injection. Acute inflammation was pro-
duced via injection of 20 µl of 2% carrageenan into the subplantar
region of the right hind paw of the mice.[32] The measurement of
thickness was immediately done before subplantar injection, and 1,
3, 4 h thereafter, using a micrometer that was particularly modified
for the measurement of thickness. The increase in paw thickness was
calculated according to the formula[33]
:
|
4.2.2
Nitrite and PGE2 assays
Percentage of inhibition = [(V − V0)control − (V − V0)treated
]
t
t
RAW 264.7 macrophages cells (ATCC) were maintained in Dulbec-
co's modified Eagle's medium, supplemented with 10% fetal bovine
serum and 1% streptomycin and penicillin at 37°C in 5% CO2. Anti‐
inflammatory activity of the compounds was evaluated by measuring
the stable NO metabolite, nitrite, with Griess assay.[31] Briefly, RAW‐
264.7 cells were plated in a 48‐well plate and incubated for 24 h at 37°C
in 5% CO2. Plated cells were pretreated with the same concentrations in
cell viability assay for 2 h and then stimulated with 1 µg/ml of LPS for
additional 22 h. The culture supernatant (50 µl) was mixed with Griess
reagent (1% sulfanilamide and 0.1% N‐(1‐naphthyl)ethylenediamine di-
hydrochloride in 5% phosphoric acid Mettler]) and incubated at room
temperature for 10 min. The absorbance of the mixture was determined
at 540 nm using a microplate reader (BioTek). The amount of nitrite in
the test samples was calculated using sodium nitrite (Fluka Chemika)
standard curve. As a positive control, 100 µM of indomethacin (Fluka
Chemika) was used. Analgesic and anti‐inflammatory activities were de-
termined with Prostaglandin E2 ELISA Kit (Abcam) according to the
manufacturer's instruction by using cell supernatants of anti‐
inflammatory activity assay. Only doses that showed the highest nitrite
inhibition activity (100 µM) for each molecule were used. SPSS was used
for all statistical analyses. Data related to cell viability, anti‐inflammatory
activity, PGE2 levels were analyzed by using t test. Differences were
considered as significant at p < .05.
/(V − V0)control] × 100,
t
where (Vt – V0)control is the difference in the size of paw in control
mice and (Vt – V0)treated is the difference in the size of paw in mice
treated with the treatments.
|
4.3
Molecular docking
Docking studies were performed on iNOS (PDB ID: 1R35[27]) enzyme
by GLIDE,[34] as a standard docking program. At least one H‐bond
with Glu371 residue was defined as a constraint during the pre-
paration of the grid file. To test if the docking program correctly
reproduces the binding mode and to evaluate the docking program,
redocking experiments were carried out using the cocrystallized in-
hibitors and the crystal structures. Glidescore (SP) was selected as
fitness function after the analysis of the proper pose investigated
according to the root mean square deviation (RMSD) of predicted
conformations versus the related native one, considering the prin-
ciple that docking poses with RMSD of less than 2.0 Å are in
agreement with the X‐ray structure. Therefore, this docking program
and setup were used in further studies. Then, a dataset of eight
samples was generated. For this dataset, 64 conformers for each

KOKSAL ET AL.
9 of 9
|
molecule were produced by ConfGen.[35] In the case of piperidine
derivatives, both equatorial and axial conformations and all stereo-
isomers of 5a were included in the conformer generation. The figures
were rendered by using PyMOL (The PyMOL Molecular Graphics
System, Version 1.8.4, Schrödinger, LLC).
[20] M. Koksal, M. Yarim, A. Erdal, A. Bozkurt, Drug. Res. (Stuttg) 2014,
64, 66. https://doi.org/10.1055/s-0033-1353184
[21] T. Ozyazici, E. E. Gurdal, D. Orak, H. Sipahi, T. Ercetin, H. O. Gulcan,
M. Koksal, Arch. Pharm. 2020, 353, e2000061. https://doi.org/10.
1002/ardp.202000061
[22] M. Koksal, I. Ozkan‐Dagliyan, T. Ozyazici, B. Kadioglu, H. Sipahi,
A. Bozkurt, S. S. Bilge, Arch. Pharm. 2017, 350, 1700153. https://doi.
org/10.1002/ardp.201700153
CONFLICTS OF INTEREST
[23] J. An, H. H. Lee, J. S. Shin, H. S. Yoo, J. S. Park, S. H. Son, S. W. Kim,
J. Yu, J. Lee, K. T. Lee, N. J. Kim, Bioorg. Med. Chem. Lett. 2017, 27,
2613. https://doi.org/10.1016/j.bmcl.2017.03.057
The authors declare that there are no conflicts of interests.
[24] M. M. G. El‐Din, M. I. El‐Gamal, M. S. Abdel‐Maksoud, H. Lee, J. Choi,
T. W. Kim, J. S. Shin, H. H. Lee, H. K. Kim, K. T. Lee, D. Baek, Bioorg.
Med. Chem. Lett. 2020, 30, 126884. https://doi.org/10.1016/j.bmcl.
2019.126884
ORCID
Meric Koksal
http://orcid.org/0000-0001-7662-9364
http://orcid.org/0000-0001-5936-5328
Ayca Dedeoglu‐Erdogan
Enise E. Gurdal
Wolfgang Sippl
Rengin Reis
http://orcid.org/0000-0003-1064-8639
http://orcid.org/0000-0002-5985-9261
http://orcid.org/0000-0002-3484-2201
http://orcid.org/0000-0002-8899-0816
http://orcid.org/0000-0001-6482-3143
http://orcid.org/0000-0003-2787-2485
[25] L. Ma, C. Xie, Y. Ma, J. Liu, M. Xiang, X. Ye, H. Zheng, Z. Chen, Q. Xu,
T. Chen, J. Chen, J. Yang, N. Qiu, G. Wang, X. Liang, A. Peng, S. Yang,
Y. Wei, L. Chen, J. Med. Chem. 2011, 54, 2060. https://doi.org/10.
1021/jm1011534
Melda Ozgurbuz
Hande Sipahi
Turgay Celik
[26] M. F. A. Mohamed, A. A. Marzouk, A. Nafady, D. A. El‐Gamal,
R. M. Allam, G. E. A. Abuo‐Rahma, H. I. El Subbagh, A. H. Moustafa,
Bioorg. Chem. 2020, 105, 104439. https://doi.org/10.1016/j.bioorg.2020.
104439
[27] E. A. Hallinan, S. W. Kramer, S. C. Houdek, W. M. Moore,
G. M. Jerome, D. P. Spangler, A. M. Stevens, H. S. Shieh,
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SUPPORTING INFORMATION
Additional Supporting Information may be found online in the
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How to cite this article: M. Koksal, A. Dedeoglu‐Erdogan, M.
Bader, E. E. Gurdal, W. Sippl, R. Reis, M. Ozgurbuz,
H. Sipahi, T. Celik. Design, synthesis, and molecular docking of
novel 3,5‐disubstituted‐1,3,4‐oxadiazole derivatives as iNOS
inhibitors. Arch. Pharm. 2021, e2000469.
[18] E. Durgashivaprasad, G. Mathew, S. Sebastian, S. M. Reddy,
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