Synthesis, anti-inflammatory activity, and molecular docking studies of some novel Mannich bases of the 1,3,4-oxadiazole-2(3H)-thione scaffold

Article abstract of DOI:10.1002/ardp.202000061

A series of novel ibuprofen and salicylic acid-based 3,5-disubstituted-1,3,4-oxadiazole-2(3H)-thione derivatives was synthesized, and they were evaluated as potential anti-inflammatory agents. Following the structure identification studies employing IR, ¹H nuclear magnetic resonance (NMR), ¹³C NMR, and elemental analysis, the title compounds were tested by cyclooxygenase (COX)-1 and COX-2 inhibition assays concomitant to lipopolysaccharide (LPS)-induced nitric oxide and prostaglandin production prevention experiments. The results indicated that the majority of the compounds displayed either a superior or comparable activity in preventing both LPS-induced NO production and COX-1 activity in comparison to the activities of the reference molecules. Furthermore, docking studies were also performed to reveal possible interactions with the COX enzymes.

Full text of DOI:10.1002/ardp.202000061

Received: 27 February 2020
Revised: 31 March 2020
Accepted: 2 April 2020
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DOI: 10.1002/ardp.202000061
F U L L P A P E R
Synthesis, anti‐inflammatory activity, and molecular docking
studies of some novel Mannich bases of the 1,3,4‐oxadiazole‐
2(3H)‐thione scaffold
Tugce Ozyazici1 | Enise E. Gurdal1
Tuba Ercetin4 | Hayrettin O. Gulcan4
| Duygu Orak2 | Hande Sipahi3
| Meric Koksal1
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1Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Yeditepe University,
Abstract
Istanbul, Turkey
A
series of novel ibuprofen and salicylic acid‐based 3,5‐disubstituted‐1,3,
2Drug, Cosmetic and Medical Device
Research‐Development and Analysis
Laboratory, Faculty of Pharmacy,
Yeditepe University, Istanbul, Turkey
4‐oxadiazole‐2(3H)‐thione derivatives was synthesized, and they were evaluated
as potential anti‐inflammatory agents. Following the structure identification studies
employing IR, 1H nuclear magnetic resonance (NMR), 13C NMR, and elemental
analysis, the title compounds were tested by cyclooxygenase (COX)‐1 and COX‐2
inhibition assays concomitant to lipopolysaccharide (LPS)‐induced nitric oxide and
prostaglandin production prevention experiments. The results indicated that the
majority of the compounds displayed either a superior or comparable activity in
preventing both LPS‐induced NO production and COX‐1 activity in comparison to
the activities of the reference molecules. Furthermore, docking studies were also
performed to reveal possible interactions with the COX enzymes.
3Department of Pharmaceutical Toxicology,
Faculty of Pharmacy, Yeditepe University,
Istanbul, Turkey
4Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Eastern Mediterranean
University, Famagusta, North Cyprus, Turkey
Correspondence
Meric Koksal, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Yeditepe
University, 34755 Atasehir, Istanbul, Turkey.
Email: merickoksal@yeditepe.edu.tr
K E Y W O R D S
1,3,4‐oxadiazole, anti‐inflammatory activity, COX‐1/COX‐2, LPS‐induced NO production,
molecular docking study, PGE2
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1
INTRODUCTION
responses back basal homeostatic set points. Otherwise, sustained
inflammatory states shift through different physiological setpoints, as
Inflammation occurs as an immediate body response to tissue and
cellular damage caused by pathogens, chemicals, or physical injury.
According to causative agents, primary mediators, onset or duration
of actions, and severity of outcomes, inflammation is divided into two
main groups. Acute inflammation is a short‐term action that usually
results in healing, in which the infiltration of leukocytes into the
damaged region ends up with the removal of the stimulus. However,
chronic inflammation is a prolonged, dysregulated and maladaptive
action that includes tissue damages. Such long‐term inflammation is
related to many chronic conditions and diseases, including allergy,
atherosclerosis, cancer, arthritis, and autoimmune diseases.[1–3] Un-
related to the origin of its response, the “purpose” of inflammation is
to eliminate the source of the disturbance. If abnormal conditions are
temporary, the inflamed host is adapted to restore functionality and
homeostasis of the tissue. Therefore, these acute inflammatory
they occur during chronic inflammation.[4]
Steroidal and nonsteroidal anti‐inflammatory drugs are used to
relieve inflammation, pain, and edema through the inhibition of phos-
pholipase A2, cyclooxygenases (COXs), and lipoxygenases, respectively.
The COX family is responsible for the biosynthesis of prostaglandin H2,
which is the precursor for some of the inflammatory mediators, pros-
taglandins, prostacyclins, and thromboxanes. Their biological missions
are coordinated by two major enzyme isoforms, COX‐1 and COX‐2.
COX‐1 is constitutively secreted and located in most tissues to maintain
homeostasis, whereas COX‐2 is synthesized upon an inflammatory re-
sponse and evaluated at the site of inflammation. In many cases of anti‐
inflammatory drug therapy, due to nonselective inhibition of both en-
zymes, there was the emergence of systemic and local gastrointestinal
side effects, thus leading to the requirement of new pharmaceutical
approaches to overcome these problems.[5–8]
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Arch Pharm. 2020;e2000061.
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OZYAZICI ET AL.
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Nitric oxide (NO), which is secreted by inducible nitric oxide
synthase from activated macrophages during the inflammation re-
sponse, plays an important role in cartilage catabolism through the
inflammation. It interacts with inducible COX in pathologic condi-
tions and enhances the formation of prostaglandin E2 (PGE2) pros-
tanoid.[9] PGE2 is involved in many processes leading to the general
symptoms of inflammation: redness, swelling, and pain. Redness and
edema emerge from the increased blood flow into the inflamed tissue
dependent on PGE2‐mediated arterial dilatation and increased mi-
crovascular permeability. Such events mean that PGE2 inhibition also
plays a crucial role in the removal of inflammation, thus, eventually
contributing to tissue remodeling.[10,11]
primary aim was to design new 3,5‐disubstituted‐1,3,4‐oxadiazole‐
2(3H)‐thione compounds and clarify their potent responses for fun-
damental inflammatory enzymes (COX‐1, COX‐2) and mediators
(PGE2, NO) by using in vitro methods. In addition, a molecular
docking analysis was performed to disclose the binding interaction
template of the most active inhibitor with COX‐1 and COX‐2
enzymes.
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2
RESULTS AND DISCUSSION
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2.1
Synthesis
The molecular and conceptual model of inflammation has been
continuously evolving to provide new insights to generate novel
therapeutic approaches for inflammatory diseases. Particularly,
recent studies have aimed to replace the carboxylate functionality
of nonsteroidal anti‐inflammatory drugs with many types of less
acidic heterocyclic bioisosteres, such as 1,3,4‐oxadiazole,[12] 1,2,
4‐triazole,[13,14] and 1,3,4‐thiadiazole,[15] to protect the gastric
mucosa from free carboxylate moiety.[16] On the basis of this
principle, selective COX‐2 inhibitors were developed, which com-
monly have a heterocyclic core and their action on cytoprotective
prostaglandins contribute to physiological homeostasis besides
their anti‐inflammatory actions. However, valdecoxib (Bextra®)
and rofecoxib (Vioxx®) presented severe adverse effects on the
cardiovascular system, and they have been withdrawn from the
market.[17–19]
In this study, derivatives of 5‐(2‐hydroxyphenyl)‐3‐[(substitute-
dpiperazine)methyl]‐1,3,4‐oxadiazole‐2(3H)‐thiones (compounds 5a–o)
and 5‐[1‐(4‐isobutylphenyl)ethyl]‐3‐[(4‐substitutedpiperazine)methyl]‐1,3,
4‐oxadiazole‐2(3H)‐thiones (compounds 10a–p) were synthesized ac-
cording to the reaction pathway depicted in Scheme 1. In the first step,
carboxylic acid groups of salicylic acid (compound 1) or ibuprofen (com-
pound 6) were converted into methyl ester groups (compounds 2, 7) in
the presence of sulfuric acid and methanol. Subsequently, these esters
were reacted with hydrazine hydrate to produce hydrazide derivatives
(compounds 3 and 8). The ring closure of 1,3,4‐oxadiazole‐2(3H)‐thione
(compounds 4 and 9) was accomplished via the reaction of hydrazides
with carbon disulfide (CS2) and potassium hydroxide (KOH) in hot alco-
holic media.[20,21] Finally, compounds 4 and 9 were treated with different
piperazines via Mannich reaction procedure to afford target compounds
5a–o and 10a–p. Novel 3,5‐disubstituted‐1,3,4‐oxadiazole‐2(3H)‐thione
derivatives were yielded in the range of 32–84% for the salicylic acid
Motivated by these approaches and based on the crucial het-
erocyclic skeleton structure of anti‐inflammatory compounds, our
SCHEME 1 The chemical synthesis of compounds 5a–o and 10a–p

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TABLE 1 Yields of compounds 5a–o and 10a–p
Salicylic acid derivatives
Ibuprofen derivatives
R1 = 2‐hydroxyphenyl
R1 = 1‐(4‐isobutylphenyl)ethyl
Compound
R2
Yield (%)
84
Compound
R2
Yield (%)
72
5a
5b
5c
5d
5e
5f
Phenyl
10a
Phenyl
4‐fluorophenyl
4‐trifluoromethylphenyl
3‐trifluoromethylphenyl
4‐chlorophenyl
2‐chlorophenyl
3,4‐dichlorophenyl
2,3‐dichlorophenyl
4‐methylphenyl
2,3‐dimethylphenyl
4‐methoxyphenyl
4‐cyanophenyl
2‐cyanophenyl
2‐pyridyl
76
10b
10c
4‐fluorophenyl
2‐fluorophenyl
3‐trifluorophenyl
4‐chlorophenyl
2‐chlorophenyl
3,4‐dichlorophenyl
2,3‐dichlorophenyl
4‐methylphenyl
2,3‐dimethylphenyl
4‐methoxyphenyl
3‐methoxyphenyl
2‐methoxyphenyl
4‐cyanophenyl
2‐cyanophenyl
2‐pyridyl
69
61
40
30
10d
10e
10f
56
56
54
32
39
5g
5h
5i
75
10g
63
40
10h
10i
52
51
29
5j
66
10j
45
5k
5l
32
10k
10l
47
55
32
5m
5n
5o
56
10m
10n
10o
10p
41
59
50
2‐pyrimidinyl
60
52
55
series (5a–o) and 29–72% for the ibuprofen series (10a–p; Table 1).
Particularly, para‐substituted compounds showed higher yields, compared
with ortho‐ and meta‐substituted ones.
were observed at 2.96–3.28 ppm as triplet signals due to the
substituent effect on the phenyl ring. The other protons belonging
to substituents of piperazine, aromatic ring protons, and ibuprofen
alkyl groups attached to oxadiazole ring were observed at the
expected values.
Structures of the synthesized target compounds were con-
firmed by spectroscopic methods. Basically, the ring closure of
1,3,4‐oxadiazole‐2(3H)‐thione was characterized by using IR,
with the disappearance of N–H and the emergence of C═N and
C═S stretching signals nearly at 3,300, 1,600–1,650, and
1,100–1,250 cm−1, respectively. The signals for C–O stretchings in
the range of 1,200–1,350 cm−1 were also observed to confirm the
ring closure. Additionally, thione and thiol tautomers of 1,3,
4‐oxadiazole were indicated by weak signals around 1,300 and
2,600 cm−1, respectively. Aromatic C–H stretchings of 5a–o and
10a–p were detected between 2,800 and 2,900 cm−1, whereas
aromatic C═C stretchings appeared around 1,450 cm−1 in both
series. Identically, compounds 5a–o presented a broad signal in the
range of 3,304–3,436 cm−1 due to the hydroxy group of the salicylic
acid moiety. IR data also indicated the loss of N–H stretchings at
Molecular structures of the final compounds (5a–o, 10a–p)
were also supported with 13C NMR spectroscopy. On the basis of
carbon nucleus under the influence of a high electronegative en-
vironment, aromatic carbon atoms linked with the fifth position of
1,3,4‐oxadiazole‐2(3H)‐thione ring appeared at a more downfield
region in both compound series. A similar reasoning also explains
the peak values of thione (C═S) and imine (C═N) groups, which
appeared at nearly 177 and 155 ppm. In salicylic acid derivatives
(5a–o), chemical shift values of C2 carbon directly linked to hy-
droxyl group on phenyl ring appeared in the range of
155–153 ppm. Other carbon atoms of two phenyl rings were se-
quenced in a decreasing order of their values, based on their
electronic environments.
3,300 cm−1
, proposing the formation of all newly synthesized
Mannich bases (5a–o and 10a–p).
Besides Fourier‐transform infrared spectroscopy (FT‐IR), 1H
nuclear magnetic resonance (NMR) was another indicator for
methylene hydrogens between oxadiazole and piperazine moieties.
Particularly descriptive singlet signal of methylene protons, found
in the range of 4.99–5.10 ppm, represented Mannich bases of
target compounds. H2 and H6 protons of the piperazine ring were
generally observed at about 2.88 ppm, and H3 and H5 protons
2.2
Biological evaluation
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2.2.1
Cell viability
To determine safe and non‐toxic concentrations of each com-
pound, cytotoxicity testing was carried out before in vitro anti‐
inflammatory activity screening. Safe doses of compounds were

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TABLE 2 Cell viability and nitric oxide (NO) production inhibitory activities of compounds 5a–5o and 10a–10p
Compounda
Cell viability (%)b
NO inhibition (%)b
Compound
Indomethacin
10a
Cell viability (%)b
92.22 6.081
86.64 7.434
92.47 8.097
84.1 4.562
NO inhibition (%)b
40.19 8.097
33.69 6.87
35.00 8.37
35.92 6.49
74.02 5.35
49.72 6.51
24.79 8.52
44.07 0.76
64,61 2.43
45,05 10.43
36.54 1.53
No detection
11.98 5.30
15.54 6.11
14.80 6.25
25.74 1.27
39.03 5.20
Lipopolysaccharide(+)
99.75 0.5
0.00 0.00
5a
5b
5c
5d
5e
5f
84.57 3.10
83.41 7.18
88.25 8.466
86.32 8.968
94.24 8.186
88.31 7.326
83.57 1.226
75.05 8.411
88.26 6.534
81.98 7.92
85.7 2.98
30.07 3.28
36.26 3.74
20.19 4.38
37.59 7.88
38.83 2.92
38.06 5.51
36.79 0.60
55.87 3.053
30.41 3.81
26.79 6.74
26.87 4.42
34.13 4.61
41.45 4.50
32.77 4.98
17.97 5.50
10b
10c
10d
87.34 1.176
87.65 2.245
93.2 7.168
10e
10f
5gc
5h
5i
10gc
10h
77.18 3.467
91.83 4.244
89.89 5.464
89.83 0.663
94.67 3.455
88.26 7.168
81.15 4.714
78.22 7.202
89.11 7.555
85.53 2.204
10i
5j
10j
5k
5l
10k
87.67 4.322
83.13 6.392
87.88 6.911
92.04 7.308
10l
5m
5n
5o
10m
10n
10o
10p
a100‐µM treatment.
bData are expressed as mean standard deviation (n = 3).
c50‐µM treatment.
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determined via cell viabilities that were above 70% in comparison
to the lipopolysaccharide(+) (LPS(+); Table 2). According to the
results, tested compounds were found safe at 100‐μM dose level,
except compounds 5g and 10g that were safely used at 50 μM
concentration.
2.2.3
In vitro COX‐1 and COX‐2 inhibition
The synthesized compounds were tested for their ability to
inhibit COX‐1 and COX‐2 enzymes using in vitro fluorometric
assay (Catalog no. 700100; Cayman Chemicals). The anti‐
inflammatory activity of compounds against the enzyme level
was compared with the known reference drug ibuprofen, using
kit‐specific standards. Results are presented in Table 3 as the
percentage of enzyme inhibition.
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2.2.2
In vitro nitric oxide (NO) inhibition
As an initial anti‐inflammatory activity screening, the
LPS‐induced NO inhibition (%) potential of the synthesized
compounds was applied with the Griess assay method. The re-
sults are summarized in Table 2, and it is observed that all tested
compounds were able to cause NO inhibition except 10k.
NO inhibition results also point out that the ibuprofen
derivatives (10a–p) were more active than the salicylic acid
series (5a–o). Particularly, compounds 10d and 10h exhibited an
excellent nitrite‐suppressor activity, compared with in-
domethacin. Also, NO inhibition values of compounds 5h, 10e,
10g, and 10i were higher than the activity of reference in-
domethacin. These results reveal that halogen fragments have a
significant role in NO inhibition. Especially, 3‐trifluoromethyl
in 10d, 2,3‐dichloro in 5h, and 10h derivatives demonstrate
the importance of halogen atom to obtain the desired
response.
The results for COX inhibition assays indicated that the
title compounds inhibited COX‐1 more selectively than the
COX‐2 enzyme. Especially, ibuprofen‐derived compound 10b,
which contains 4‐fluorophenyl moiety, displayed highest activity,
with 76% for COX‐1 inhibition. Moreover, compounds 5a, 5e,
5i, 5k, 5n, 10b, 10e, and 10h showed more than 50% COX‐1 in-
hibitor response.
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2.2.4
In vitro PGE2 inhibition
On the basis of the cooperation between NO and PGE2, the syn-
thesized compounds that have good and potent NO production in-
hibitory activities were chosen and studied further for PGE2
responses. Compounds 5i, 5n, and 10b that showed the highest
COX inhibition were added to this group to evaluate their

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TABLE 3 COX‐1 and COX‐2 inhibition of compounds 5a–o and 10a–p
COX‐1
inhibition (%)
COX‐2
inhibition (%)
COX‐1
inhibition (%)
COX‐2
Compound
Compound
10a
inhibition (%)
10.2 3.4
9.9 1.2
20.7 2.2
13.9 1.0
20.2 2.5
35.3 0.5
39.3 0.3
32.4 0.4
6.7 1.2
23.2 1.4
6.8 1.5
10.3 1.5
20.5 1.1
24 4.8
5a
5b
5c
5d
5e
5f
51.3 1.6
24.1 1.0
24.7 0.8
32.3 1.9
50.3 2.1
47.9 2.4
14.2 2.8
40.4 0.3
56.7 0.8
35.2 3.1
51.8 2.6
17.3 2.2
40.8 2.3
56 4.8
26.9 1.8
10.5 1.4
20.5 0.5
25.6 0.7
39.8 0.6
20.4 4.1
8.6 0.8
19.3 1.0
76.4 2.1
35.6 1.1
31.5 1.4
51.7 2.8
44.2 3.6
47 0.8
10b
10c
10d
10e
10f
5g
5h
5i
10g
18.6 1.9
20.8 1.8
14.8 1.9
19.7 1.2
10h
54.7 2.2
9.8 1.1
28.4 2.3
12.8 1.5
31.2 0.6
48.3 0.3
32.7 1.3
18.2 2.8
26.7 2.0
83.8 1.8
90 2.6
10i
5j
10j
5k
5l
10k
9
2.5
10l
5m
5n
5o
28.7 3.2
33.8 3.3
18.3 4.2
10m
10n
41.8 3.7
10o
12.5 3.7
20.3 4.5
62.7 4.0
NI
10p
Ibuprofen
SC‐560a
DuP‐697b
NI
90 2.6
Abbreviations: COX, cyclooxygenase; NI, no inhibition.
aSC‐560: Selective COX‐1 inhibitor.
bDuP‐697: Selective COX‐2 inhibitor.
anti‐inflammatory activities in detail. This target set of compounds
expressed varying inhibitory activities for the LPS‐induced NO and
PGE2 productions. Results of these additional assays are summarized
in Table 4.
(PDB ID: 5IKT[23]) enzymes. Docking scores and predicted interac-
tions of the data set are given in Table 5.
Binding to COX‐1 enzyme generally occurs by an interaction with
Arg120, Tyr355, and catalytic Tyr385 residues. Docking studies on
COX‐1 enzyme were performed by using the X‐ray data of 5U6X, in
which the cocrystallized ligand and its conformers made a π–π inter-
action with Tyr355 from isoxazole ring and a halogen bond with the
Arg120 residue (docking score: −7.715, the root mean square deviation
(RMSD: 0.210).[22] Similarly, the S enantiomer of compound 10b
(docking score: −8.695) could make two H‐bonds with Arg120 in ad-
dition to the π–π interaction with Tyr355 from oxadiazole ring, which
may explain its high inhibition observed by in vitro studies. The com-
pound could also interact with Trp387 by π–π stacking from the
4‐fluorophenyl group. Docking studies revealed that the R enantiomer
of the compound 10b could make an additional π–cation interaction
with Arg120 (docking score: −8.386; Figures 1a and 2a).
It is noticeable that the target compounds are more active as in-
hibitors of NO production rather than PGE2 production. The results
indicated that compounds 5h, 10d, 10e, 10g, 10h, and 10i showed a
better NO inhibitory profile than the activity of indomethacin, which is
also found statistically significant. Besides, the other test compounds,
5d, 5e, 5f, 5k, 5n, 10b, 10j, and 10p, also decreased the NO production
in moderate activities of LPS‐induced Raw 264.7 cells. These results
implied that halogen substitution is somewhat additionally beneficial for
the activity. However, all of the compounds that displayed a consider-
able activity in NO production did not show a potent inhibitory effect
on the PGE2 production.
Mutation studies proved that the interaction with Arg120
is not as crucial for binding to COX‐2 enzyme as COX‐1.[24]
The conformer of cocrystallized ligand with the lowest docking score and
the most stable binding pose (−10.165, RMSD: 0.738) made H‐bonds with
Tyr385 and Ser530 from free carboxylate group within the docking
studies performed on X‐ray data of 5IKT.[23] However, both enantiomers
of compound 10b (docking score (S): −8.006, docking score (R): −7.837)
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2.3
Docking studies
To reveal the possible interactions of the compounds with COX en-
zymes, compounds that showed an anti‐inflammatory profile in
screening tests and possessed more than 50% inhibition in the en-
zyme assays were docked into COX‐1 (PDB ID: 5U6X[22]) and COX‐2

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EXPERIMENTAL
TABLE 4 Cytotoxities, NO, and prostaglandin E2 (PGE2)
production inhibition of selected compounds
4
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4.1
Chemistry
NO
Compound
LPS (+)
5d
IC50 (µM)
production (µM) PGE2 level (pg/ml)
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4.1.1
General
41.17 2.96
2,396 154.8
2,227 14.41
2,156 55.78*
2,198 42.66
354.2 36.54**
1,521 27.5**
2,392 0.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
27.47 3.99*
27.33 3.51*
27.74 4.02*
20.75 4.40****
31.95 2.29
All reagents, catalysts, and solvents were purchased from com-
mercial suppliers. Thin‐layer chromatography was performed on
precoated silica gel 60 F245 aluminum plates (Merck) with vi-
sualization under ultraviolet light. Melting points were de-
termined on Mettler Toledo FP62 capillary melting point
apparatus with open capillary tubes, and they were uncorrected.
IR spectra were recorded using KBr pellets in PerkinElmer
Spectrum One series FT‐IR apparatus (Version 5.0.1.), and 1H
NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded
with a Varian Mercury‐400 FT‐NMR spectrometer (Varian Inc.,
Palo Alto, CA), using tetramethylsilane as the internal reference
and dimethyl sulfoxide (DMSO‐d6) as solvent; the chemical shifts
were recorded in parts per million (ppm). Coupling constants
were reported in Hertz. Elemental analyses were performed on
LECO 932 (St. Joseph, MI) instrument.
5e
5f
5h
5k
5m
26.13 3.00**
29.29 2.05
5n
1,659 57.5**
2,285 219.5
1,982 38.46**
2,142 13.85*
2,377 76.84
1,026 59.68**
2,300 29.76
2,330 60.29
2,454 31.75
22.94 4.26**
10b
10d
10e
10g
10h
10i
30.52 6.70
13.16 4.84****
23.77 5.55***
68.47 1.80 23.02 1.51****
>100
>100
>100
>100
>100
14.51 0.13****
24.02 3.90***
28.16 2.93*
27.96 4,83*
24.46 1.80***
The InChI codes of the investigated compounds, together
with some biological activity data, are provided as Supporting
Information.
10j
10p
Indome-
thacin
Note: One‐way analysis of variance, followed by Tukey's post‐hoc tests. The
significant differences between groups and control (lipopolysaccharide(+),
LPS) were defined with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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4.1.2
General procedure for the preparation
of methyl esters 2 and 7
lacked the interactions with Tyr385 and Ser530, which may explain their
selective inhibition against COX‐1 enzyme observed by in vitro studies.
Instead, they could both make a π–cation interaction with Arg120 and a
π–π interaction with Tyr355 from oxadiazole ring (Figures 1b and 2b).
A solution of 1‐g salicylic acid (1) (0.007 mol) or ibuprofen (6)
(0.005 mol), 10‐ml methanol, and 0.75‐ml concentrated sulfuric
acid was prepared. The mixture was gently refluxed at 85°C for
about 75 min by mixing. At the end of the reaction, the solution
mixture was cooled and extracted with hexane. The organic
layer was taken and evaporated. Isolated ester compounds were
checked by thin‐layer chromatography with the benzene/methanol
(90:10) mobile phase system.[25]
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3
CONCLUSİON
In this study, two sets of Mannich bases from salicylic acid and ibu-
profen were designed, synthesized, and evaluated for their in vitro
COX‐1/2 inhibitory activities and NO and PGE2 productions. Among the
compounds, 5h, 10d, 10e, 10g, 10h, and 10j were found to have a
significant anti‐inflammatory activity in NO production in comparison to
the reference drug indomethacin. The in silico studies were also utilized
to predict the binding interactions of these compounds with COX‐1 and
COX‐2. The active compound set showed a significant interaction with
Arg120 residue present in both enzymes. However, lack of interactions
with Tyr385 and Ser530 of COX‐2 was predicted as a reason of se-
lective inhibition against COX‐1 enzyme, which leads to the gastro-
intestinal side effects of anti‐inflammatory drugs. These results indicate
that although ibuprofen‐derived compounds might be regarded as
promising anti‐inflammatory agents, further studies should be con-
ducted for the development of safer and more active anti‐inflammatory
agents.
|
4.1.3
General procedure for the preparation
of aroylhydrazides 3 and 8
The mixture of 0.1‐mol methyl esters of salicylic acid (2) or ibu-
profen (7) and 0.2 mol of hydrazine hydrate was refluxed in 20 ml
absolute alcohol for about 10–20 hr. The excess solvent was
evaporated under reduced pressure and the concentrated
solution was quenched into ice‐cold water. The precipitated
white solid was filtered, rewashed with water, and dried for
about 2 days. The crude product was purified by recrystallization
from water to produce pure hydrazide compounds. Isolated
hydrazide compounds were checked by thin‐layer chromato-
graphy with the benzene/methanol (40:60) mobile phase
system.[18]

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FIGURE 1 Docking pose of 10b‐R (purple) and 10b‐S (cyan). (a) COX‐1: PDB ID: 5U6X; (b) COX‐2: PDB ID: 5IKT
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4.1.4
General procedure for the preparation of
sulfide had ceased. The reaction mixture was cooled to room tem-
perature and diluted with water. The product precipitated after the
acidification with 10% hydrochloric acid was filtered and thoroughly
crystallized with hexane.[18] Isolated 5‐substituted‐1,3,4‐oxadiazole‐
2(3H)‐thione compounds were checked by thin‐layer chromatography
with the benzene/methanol (90:10) mobile phase system.
5‐substituted‐1,3,4‐oxadiazole‐2‐(3H)‐thiones (4, 9)
The mixture of aroyl hydrazide (3, 8) (0.1 mol), KOH (0.1 mol) in ab-
solute alcohol (50 ml), and CS2 (0.2 mol) was taken in a round bottom
flask and refluxed for about 20–25 hr until the evolution of hydrogen
FIGURE 2 Predicted interactions of 10b‐R (right) and 10b‐S (left). (a) COX‐1: PDB ID: 5U6X; (b) COX‐2: PDB ID: 5IKT

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4.1.5
General procedure for the preparation of the
phenyl H3′ + H5′ + H6), 7.40–7.44 (1H, phenyl H4), 7.46 (2H, bd,
J = 8.8 Hz, phenyl H2′ + H6′), 7.64 (1H, dd, J = 7.6 Hz, J′ = 1.6 Hz,
phenyl H3), and 10.51 (1H, bs, –OH). 13C NMR (400 MHz, DMSO‐d6)
δ (ppm): 46.98 (piperazine C2 + C6), 49.31 (piperazine C3 + C5), 69.49
(–N–CH2–N–), 108.99 (phenyl C1), 114.32 (phenyl C2′ + C6′), 117.04
(phenyl C3), 117.43, 117.76, 118.07, 118.39 (phenyl C4′–CF3), 119.44
(phenyl C3′ + C5′), 123.57 (phenyl C6), 126.08, 126.11 (phenyl
C4′–CF3), 129.12 (phenyl C5), 133.65 (phenyl C4), 153.16 (phenyl
C1′), 156.40 (phenyl C2), 158.03 (C═N), and 177.09 (C═S). Anal.
calcd. for C20H19 F3N4O2S: C, 55.04; H, 4.39; N, 12.84; S, 7.35.
Found: C, 54.86; H, 4.47; N, 12.90; S, 7.46.
Mannich bases (5a–o, 10a–p)
A mixture of 5‐substituted‐1,3,4‐oxadiazole‐2(3H)‐thione (4, 9) (0.01 mol),
formaldehyde (0.015 mol), and piperazine derivative (0.01 mol) in
15 ml ethanol or methanol was continuously reacted at room tempera-
ture overnight. The precipitated solids in reaction media were
filtered and recrystallized from ethanol or methanol to obtain the desired
Mannich bases.[18]
5‐(2‐Hydroxyphenyl)‐3‐[(4‐phenylpiperazin‐1‐yl)methyl]‐1,3,4‐
oxadiazole‐2(3H)‐thione (5a)[19]
Compound 5a was obtained as a white powder (84%). Mp: 170°C. IR
νmax (cm−1): 3,332 (O–H), 2,886 (aromatic C–H), 1,626 (C═N), 1,598,
1,573, 1,502, 1,489 (aromatic C═C), 1,326 (C–O), 1,223 (C═S), 766
(phenyl C–H), and 746 (2‐hydroxyphenyl C–H). 1H NMR (400 MHz,
DMSO‐d6) δ (ppm): 2.90 (4H, t, J = 4.8 Hz, piperazin H2 + H6), 3.13 (4H,
t, J = 4.8 Hz, piperazin H3 + H5), 5.1 (2H, s, –N–CH2–N–), 6.76 (1H, t,
J = 7.2 Hz, phenyl H4′), 6.90 (2H, bd, J = 8 Hz, phenyl H2′ + H6′),
6.95–6.99 (1H, m, phenyl H5), 7.05 (1H, bd, J = 8.4 Hz, phenyl H6), 7.18
(2H, t, J = 7.8 Hz, phenyl H3′ + H5′), 7.42–7.46 (1H, m, phenyl H4), 7.66
(1H, dd, J = 8 Hz, J’ = 1.6 Hz, phenyl H3), and 10.51 (1H, bs, –OH).
5‐(2‐Hydroxyphenyl)‐3‐({4‐[3‐(trifluoromethyl)phenyl]piperazin‐1‐
yl}methyl)‐1,3,4‐oxadiazole‐2(3H)‐thione (5d)
Compound 5d was obtained as a white powder (30%). Mp: 173°C. IR
ν
max (cm−1): 3,348 (O–H), 2,961 (aromatic C–H), 1,627 (C═N), 1,598,
1,571, 1,491 (aromatic C═C), 1,331 (C–O), 1,250 (C═S), 849 (3‐
(trifluoromethyl)phenyl C–H), and 743 (2‐hydroxyphenyl C–H). 1H
NMR (400 MHz, DMSO‐d6) δ (ppm): 2.89 (4H, t, J = 4.8 Hz, piperazine
H2 + H6), 3.22 (4H, t, J = 4.8 Hz, piperazine H3 + H5), 5.09 (2H, s, –N–
CH2–N–), 6.95 (1H, t, J = 7.8 Hz, phenyl H5′), 7.03 (2H, bd, J = 8.4 Hz,
phenyl H4′ + H6′), 7.14 (1H, bs, phenyl H2′), 7.18–7.23 (1H, m, phenyl
H5), 7.36 (1H, bd, J = 8 Hz, phenyl H6), 7.40–7.45 (1H, m, phenyl H4),
7.64 (1H, dd, J = 7.6 Hz, J′ = 1.6 Hz, phenyl H3), and 10.53 (1H,
bs, –OH). 13C NMR (400 MHz, DMSO‐d6) δ (ppm): 47.60 (piperazine
C2 + C6), 49.36 (piperazine C3 + C5), 69.44 (–N–CH2–N–), 108.93
(phenyl C5), 111.03, 111.07 (phenyl C2′), 114.65, 114.69 (phenyl C4′),
117 (phenyl C4), 118.91 (phenyl C6′), 119.39 (phenyl C6), 122.97,
125.68 (phenyl C3′–CF3), 129.11 (phenyl C3), 129.84 (phenyl C5′),
129.31, 129.61, 129.92, 130.22 (phenyl C3′–CF3), 133.64 (phenyl C1),
151.13 (phenyl C1′), 156.37 (phenyl C2), 157.92 (C═N), and 177.02
(C═S). Anal. calcd. for C20H19 F3N4O2S: C, 55.04; H, 4.39; N, 12.84; S,
7.35. Found: C, 54.91; H, 4.29; N, 12.79; S, 7.42.
5‐(2‐Hydroxyphenyl)‐3‐{[4‐(4‐fluorophenyl)piperazin‐1‐yl]methyl}‐
1,3,4‐oxadiazole‐2(3H)‐thione (5b)
Compound 5b was obtained as a white powder (76%). Mp: 160°C. IR
νmax (cm−1): 3,425 (O–H), 2,956 (aromatic C–H), 1,613 (C═N); 1,598,
1,499, 1,490 (aromatic C═C), 1,404 (C–O), 1,259 (C═ S), 809 (4‐
fluorophenyl C–H), and 746 (2‐hydroxyphenyl C–H). 1H NMR
(400 MHz, DMSO‐d6) δ (ppm): 2.86 (4H, t, J = 5 Hz, piperazin H2 + H6),
3.06 (4H, t, J = 5 Hz, piperazin H3 + H5), 5.09 (2H, s, –N–CH2–N–), 6.90
(1H, d, J = 4.8 Hz, phenyl H6′), 6.92 (1H, d, J = 4.8 Hz, phenyl H2′),
6.94–6.98 (1H, phenyl H5), 7.00 (1H, d, J = 8.8 Hz, phenyl H5′), 7.02 (1H,
d, J = 8.8 Hz, phenyl H3′), 7.03 (1H, d, J = 8.4 Hz, phenyl H6), 7.40–7.44
(1H, m, phenyl H4), 7.64 (1H, dd, J = 7.6 Hz, J′ = 1.6 Hz, phenyl H3), and
10.51 (1H, bs, –OH). 13C NMR (400 MHz, DMSO‐d6) δ (ppm): 49.08
(piperazine C2 + C6), 49.57 (piperazine C3 + C5), 69.53 (–N–CH2–N–),
109.00 (phenyl C1), 115.09, 115.31 (phenyl C2′ + C6′), 117.04 (phenyl
C3) 117.39, 117.47 (phenyl C3′ + C5′), 119.46 (phenyl C5), 129.09
(phenyl C6), 133.63 (phenyl C4), 147.87, 147.89 (phenyl C1′), 154.90
(phenyl C2), 156.40 (C═N), 157.25, 158.02 (phenyl C4′–F), and 177.11
(C═S). Anal. calcd. for C19H19FN4O2S: C, 59.05; H, 4.96; N, 14.50; S,
8.30. Found: C, 58.56; H, 4.99; N, 14.52; S, 8.52.
5‐(2‐Hydroxyphenyl)‐3‐{[4‐(4‐chlorophenyl)piperazin‐1‐yl]methyl}‐
1,3,4‐oxadiazole‐2(3H)‐thione (5e)
Compound 5e was obtained as a white powder (56%). Mp: 150°C. IR
ν
max (cm−1): 3,348 (O–H), 2,948 (aromatic C–H), 1,622 (C═N), 1,596,
1,569, 1,496, 1,489 (aromatic C═C), 1,323 (C–O), 1,235 (C═S), 815
(4‐chlorophenyl C–H), and 742 (2‐hydroxyphenyl C–H). 1H NMR
(400 MHz, DMSO‐d6) δ (ppm): 2.87 (4H, t, J = 4.8 Hz, piperazine
H2 + H6), 3.12 (4H, t, J = 4.8 Hz, piperazine H3 + H5), 5.08 (2H, s, –N–
CH2–N–), 6.91 (2H, bd, J = 9 Hz, phenyl H2′ + H6′), 6.94–6.98 (1H, m,
phenyl H5), 7.03 (1H, bd, J = 8 Hz, phenyl H6), 7.19 (2H, bd, J = 9 Hz,
phenyl H3′ + H5′), 7.41–7.45 (1H, m, phenyl H4), 7.64 (1H, dd,
J = 7.6 Hz, J′ = 1.6 Hz, phenyl H3), and 10.52 (1H, s, –OH). 13C NMR
(400 MHz, DMSO‐d6) δ (ppm): 48.06 (piperazine C2 + C6), 49.42 (pi-
perazine C3 + C5), 69.51 (–N–CH2–N–), 108.99 (phenyl C5), 117.52
(phenyl C2′ + C6′ + C4), 119.44 (phenyl C6), 122.47 (phenyl C4′),
128.55 (phenyl C3′ + C5′), 129.15 (phenyl C3), 129.15 (phenyl C3),
133.67 (phenyl C1), 149.75 (phenyl C1′), 156.41 (phenyl C2), 157.97
(C═N), and 177.07 (C═S). Anal. calcd. for C19H19ClN4O2S: C, 56.64;
H, 4.75; N, 13.91; S, 7.96. Found: C, 56.49; H, 4.88; N, 13.84; S, 7.98.
5‐(2‐Hydroxyphenyl)‐3‐({4‐[4‐(trifluoromethyl)phenyl]piperazin‐1‐
yl}methyl)‐1,3,4‐oxadiazole‐2(3H)‐thione (5c)
Compound 5c was obtained as a white powder (60%). Mp: 161°C. IR
νmax (cm−1): 3,336 (O–H), 2,837 (aromatic C–H), 1,618 (C═N), 1,598,
1,577, 1,522, 1,490 (aromatic C═C), 1,334 (C–O), 1,256 (C═S), 823
(4‐trifluoromethylphenyl C–H), and 746 (2‐hydroxyphenyl C–H). 1H
NMR (400 MHz, DMSO‐d6) δ (ppm), 2.88 (4H, t, J = 4.8 Hz, piperazine
H2 + H6), 3.28 (4H, t, J = 4.8 Hz, piperazine H3 + H5), 5.09 (2H, s, –N–
CH2–N–), 6.93–6.97 (1H, m, phenyl H5), 7.03 (3H, bd, J = 8.8 Hz,

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5‐(2‐Hydroxyphenyl)‐3‐{[4‐(2‐chlorophenyl)piperazin‐1‐yl]methyl}‐
1,3,4‐oxadiazole‐2(3H)‐thione (5f)
H3), and 10.54 (1H, bs, –OH). 13C NMR (400 MHz, DMSO‐d6) δ (ppm):
49.77 (piperazine C2 + C6), 50.87 (piperazine C3 + C5), 69.65
(–N–CH2–N–), 108.99 (phenyl C1), 117.07 (phenyl C3), 119.49 (phenyl
C5), 119.67 (phenyl C2′), 124.49 (phenyl C6′), 126.06 (phenyl C6), 128.40
(phenyl C4′), 129.09 (phenyl C5′), 132.56 (phenyl C4), 133.65 (phenyl C3′),
151.03 (phenyl C1′), 156.41 (phenyl C2), 158.06 (C═N), and 177.11 (C═S).
Anal. calcd. for C19H18Cl2N4O2S: C, 52.18; H, 4.15; N, 12.81; S, 7.33.
Found: C, 51.75; H, 4.17; N, 13.52; S, 7.07.
Compound 5f was obtained as a white powder (32%). Mp: 180°C.
IR νmax (cm−1): 3,435 (O–H), 3,012 (aromatic C–H), 1,615 (C═N),
1,603, 1,560, 1,484 (aromatic C═C), 1,311 (C–O), 1,264 (C═S), 772
(2‐chlorophenyl C–H s), and 743 (2‐hydroxyphenyl C–H). 1H NMR
(400 MHz, DMSO‐d6) δ (ppm): 2.93 (4H, d, J = 4.8 Hz, piperazine
H2 + H6), 2.97 (4H, d, J = 4.8 Hz, piperazine H3 + H5), 5.1 (2H,
s, –N–CH2–N–), 6.98 (1H, t, J = 7.6 Hz, phenyl H5′), 7.00–7.04 (1H,
m, phenyl H5), 7.05 (1H, bd, J = 8 Hz, phenyl H6′), 7.14 (1H, dd,
J = 7.8 Hz, J′ = 1.4 Hz, phenyl H6), 7.25–7.30 (1H, m, phenyl H4′),
7.37 (1H, dd, J = 8 Hz, J′ = 1.2 Hz, phenyl H3′), 7.43–7.47 (1H, m,
phenyl H4), 7.68 (1H, dd, J = 7.6 Hz, J′ = 1.6 Hz, phenyl H3), and 10.54 (1H,
bs, –OH). 13C NMR (400 MHz, DMSO‐d6) δ (ppm): 49.82 (piperazine
C2 + C6), 50.78 (piperazine C3 + C5), 69.69 (–N–CH2–N–), 108.98
(phenyl C5), 117.07 (phenyl C4), 119.48 (phenyl C6), 120.90 (phenyl C6′),
123.95 (phenyl C4′), 127.61 (phenyl C2′), 128.01 (phenyl C5′), 129.11
(phenyl C3), 130.27 (phenyl C3′), 133.67 (phenyl C1), 148.86 (phenyl
C1′), 156.42 (phenyl C2), 157.99 (C═N), and 177.09 (C═S). Anal. calcd. for
C19H19ClN4O2S: C, 56.64; H, 4.75; N, 13.91; S, 7.96. Found: C, 56.71; H,
4.88; N, 13.88; S, 8.05.
5‐(2‐Hydroxyphenyl)‐3‐[4‐(4‐methylphenyl)piperazin‐1‐yl]methyl‐
1,3,4‐oxadiazole‐2(3H)‐thione (5i)
Compound 5i was obtained as a white powder (51%). Mp: 165°C. IR
ν
max (cm−1): 3,342 (O–H), 3,028 (aromatic C–H), 1,624 (C═N), 1,597,
1,514, 1,498 (aromatic C═C), 1,324 (C–O), 1,260 (C═S), 767
(4‐methylphenyl C–H), and 745 (2‐hydroxyphenyl). 1H NMR (400 MHz,
DMSO‐d6) δ (ppm): 2.16 (phenyl –CH3), 2.87 (4H, d, J = 4.8 Hz, piper-
azine H2 + H6), 3.05 (4H, d, J = 4.8 Hz, piperazine H3 + H5), 5.08 (2H,
s, –N–CH2–N–), 6.79 (2H, d, J = 8.6 Hz, phenyl H3′ + H5′), 6.94–6.97 (1H,
m, phenyl H5), 6.98 (1H, bd, J = 8 Hz, phenyl H6), 7.03 (2H, d, J = 8.6 Hz,
phenyl H2′ + H6′), 7.39–7.44 (1H, m, phenyl H4), 7.34 (1H, dd, J = 7.6 Hz,
J′ = 1.6 Hz, phenyl H3), and 10.53 (1H, bs, –OH). 13C NMR (400 MHz,
DMSO‐d6) δ (ppm): 19.94 (phenyl –CH3), 48.71 (piperazine C2 + C6),
49.53 (piperazine C3 + C5), 69.52 (–N–CH2–N–), 108.93 (phenyl C5),
115.85 (phenyl C2′ + C6′), 116.99 (phenyl C4), 119.39 (phenyl C6),
127.75 (phenyl C4′), 129.08 (phenyl C3), 129.23 (phenyl C3′ + C5′),
133.60 (phenyl C1), 148.84 (phenyl C1′), 156.35 (phenyl C2), 157.89
(C═N), and 177.01 (C═S). Anal. calcd. for C20H22N4O2S: C, 62.80; H,
5.80; N, 14.65; S, 8.38. Found: C, 62.87; H, 5.96; N, 14.56; S, 8.41.
5‐(2‐Hydroxyphenyl)‐3‐{[4‐(3,4‐dichlorophenyl)piperazin‐1‐yl]‐
methyl}‐1,3,4‐oxadiazole‐2(3H)‐thione (5g)
Compound 5g was obtained as a white powder (75%). Mp: 172°C. IR
νmax (cm−1): 3,336 (O–H), 2,837 (aromatic C–H), 1,618 (C═N), 1,598,
1,577, 1,522, 1,490 (aromatic C═C), 1,334 (C–N), 1,256 (C═S), 823
(3,4‐dichlorophenyl C–H), and 746 (2‐hydroxyphenyl C–H). 1H NMR
(400 MHz, DMSO‐d6) δ (ppm): 2.88 (4H, t, J = 5 Hz, piperazine
H2 + H6), 3.39 (4H, t, J = 5 Hz, piperazine H3 + H5), 5.1 (2H,
s, –N–CH2–N–); 6.92 (1H, dd, J = 9 Hz, J′ = 3 Hz, phenyl H6′), 6.95–6.99
(2H, m, phenyl H4 + H5), 7.03 (1H, bd, J = 8 Hz, phenyl H5′); 7.13 (1H, d,
J = 2.4 Hz, phenyl H2′), 7.37 (1H, d, J = 8.8 Hz, phenyl H6), 7.46 (1H, d,
J = 9.2 Hz, phenyl H3), and 10.61 (1H, bs, –OH). 13C NMR (400 MHz,
DMSO‐d6) δ (ppm): 42.43 (piperazine C2 + C6), 44.85 (piperazine C3 + C5),
69.40 (–N–CH2–N–), 109.31 (phenyl C5), 115.45 (phenyl C4), 115.84
(phenyl C6), 116.33 (phenyl C6′), 116.62 (phenyl C5′), 116.98 (phenyl C2′),
119.49 (phenyl C3), 120.78 (phenyl C4′), 130.34 (phenyl C3′), 130.56
(phenyl C1), 131.57 (phenyl C1′), 149.61 (phenyl C2), 150.61 (C═N), and
155.91 (C═S). Anal. calcd. for C19H18Cl2N4O2S: C, 52.18; H, 4.15; N,
12.81; S, 7.33. Found: C, 51.98; H, 4.02; N, 12.83; S, 7.39.
5‐(2‐Hydroxyphenyl)‐3‐{[4‐(2,3‐dimethylphenyl)piperazin‐1‐yl]‐
methyl}‐1,3,4‐oxadiazole‐2(3H)‐thione (5j)
Compound 5j was obtained as a white powder (66%). Mp: 133°C. IR
ν
max (cm−1): 3,364 (O–H), 2,948 (aromatic C–H), 1,630 (C═N), 1,598,
1,492 (aromatic C═C), 1,323 (C–O), 1,255 (C═S), and 746 (2‐
hydroxyphenyl C–H). 1H NMR (400 MHz, DMSO‐d6)
(ppm):
δ
2.09 + 2.17 (6H, s, phenyl –CH3), 2.79–2.91 (8H, m, piperazine H2 +
H3 + H5 + H6), 5.1 (2H, s, –N–CH2–N–), 6.86 (1H, t, J = 8.4 Hz, phenyl H5′),
6.99 (1H, d, J = 4.8 Hz, phenyl H4′), 7.00–7.09 (1H, m, phenyl H5), 7.03
(1H, d, J = 10.8 Hz, phenyl H6′), 7.05 (1H, d, J = 4.8 Hz, phenyl H6),
7.39–7.48 (1H, m, phenyl H4), 7.67 (1H, dd, J = 8 Hz, J′ = 1.6 Hz, phenyl
H3), and 10.58 (1H, bs, –OH). 13C NMR (400 MHz, DMSO‐d6) δ (ppm):
13.66, 20.21 (–CH3), 50.14 (piperazine C2 + C6), 51.72 (piperazine
C3 + C5), 69.81 (methylene –CH2–), 108.03 (phenyl C1), 116.44 (phenyl
C3), 117.05 (phenyl C6′), 119.49 (phenyl C5′), 124.62 (phenyl Cv′), 125.65
(phenyl C4), 129.00 (phenyl C5), 130.44 (phenyl C6), 130.59 (phenyl C4′),
137.22 (phenyl C3′), 151.21 (phenyl C1′), 156.40 (phenyl C2), 158.05
(C═N), and 177.14 (C═S). Anal. calcd. for C21H24N4O2S: C, 63.61; H, 6.10;
N, 14.13; S, 8.09. Found: C, 62.98; H, 6.03; N, 14.37; S, 8.27.
5‐(2‐Hydroxyphenyl)‐3‐{[4‐(2,3‐dichlorophenyl)piperazin‐1‐yl]‐
methyl}‐1,3,4‐oxadiazole‐2(3H)‐thione (5h)
Compound 5h was obtained as a white powder (40%). Mp: 165°C.
IR νmax (cm−1): 3,348 (O–H), 2,961 (aromatic C–H), 1,627 (C═N),
1,610, 1,598, 1,571, 1,491 (aromatic C═C), 1,308 (C–O), 1,265
(C═S), 849 (2,3‐dichlorophenyl C–H), and 743 (2‐hydroxyphenyl
C–H). 1H NMR (400 MHz, DMSO‐d6) δ (ppm): 2.93–2.99 (8H, m,
piperazine H2 + H3 + H5 + H6), 5.1 (2H, s, –N–CH2–N–), 6.98 (1H, t,
J = 7.4 Hz, phenyl H5′), 7.05 (1H, bd, J = 8 Hz, phenyl H4′),
7.12–7.16 (1H, m, phenyl H5), 7.27–7.32 (2H, m, phenyl H6 + H6′),
7.43–7.47 (1H, m, phenyl H4), 7.67 (1H, dd, J = 7.6 Hz, J′ = 1.6 Hz, phenyl
5‐(2‐Hydroxyphenyl)‐3‐{[4‐(4‐methoxyphenyl)piperazin‐1‐yl]methyl}‐
1,3,4‐oxadiazole‐2(3H)‐thione (5k)
Compound 5k was obtained as a white powder (32%). Mp: 148°C. IR
νmax (cm−1): 3,362 (O–H), 2,947 (aromatic C–H), 1,624 (C═N), 1,596,

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1,512 (aromatic C═C), 1,322 (C–O), 1,249 (C═S), 835 (4‐methoxyphenyl
C–H), and 744 (2‐hydroxyphenyl C–H). 1H NMR (400 MHz, DMSO‐d6) δ
(ppm): 2.88 (4H, bt, J = 4.5 Hz, piperazine H2 + H6), 2.99 (4H, bt,
J = 4.5 Hz, piperazine H3 + H5), 3.65 (3H, s, phenyl –OCH3), 5.08 (2H,
s, –N–CH2–N–), 6.77 (2H, bd, J = 9 Hz, phenyl H2′ + H6′), 6.85 (2H, bd,
J = 9 Hz, phenyl H3′ + H5′), 6.96 (1H, t, J = 7.6 Hz, phenyl H5), 7.03 (1H,
bd, J = 8 Hz, phenyl H6), 7.40–7.45 (1H, m, phenyl H4), 7.65 (1H, dd,
J = 7.8 Hz, J′ = 1.4 Hz, phenyl H3), and 10.52 (1H, bs, –OH). 13C NMR
(400 MHz, DMSO‐d6) δ (ppm): 49.69 (piperazine C2 + C3 + C5 + C6),
55.12 (phenyl –OCH3), 69.58 (–N–CH2–N–), 108.99 (phenyl C5), 114.17
(phenyl C2′ + C6′), 117.05 (phenyl C4), 117.66 (phenyl C3′ + C5′), 119.44
(phenyl C6), 129.15 (phenyl C3), 133.66 (phenyl C1), 145.34 (phenyl C1′),
153.12 (phenyl C4′), 156.42 (phenyl C2), 157.94 (C═N), and 177.07
(C═S). Anal. calcd. for C20H22N4O3S: C, 60.28; H, 5.56; N, 14.06; S, 8.05.
Found: C, 60.13; H, 5.76; N, 14.11; S, 8.17.
calcd. for C20H19N5O2S: C, 61.05; H, 4.87; N, 17.80; S, 8.15. Found: C,
60.42; H, 4.87; N, 17.65; S, 8.27.
5‐(2‐Hydroxyphenyl)‐3‐{[4‐(2‐pyridyl)piperazin‐1‐yl]methyl}‐1,3,4‐
oxadiazole‐2‐(3H)‐thione (5n)
Compound 5n was obtained as a white powder (59%). Mp: 148°C. IR νmax
(cm−1): 3,435 (O–H), 3,012 (aromatic C–H), 1,615 (C═N), 1,603, 1,560,
1,484 (aromatic C═C), 1,311 (C–O), 1,264 (C═S), 772 (2‐pyridyl C–H),
and 743 (2‐hydroxyphenyl C–H). 1H NMR (400 MHz, DMSO‐d6) δ (ppm):
22.48 (4H, t, J = 5 Hz, piperazine H2 + H6), 3.48 (4H, t, J = 5 Hz, piperazine
H3 + H5), 5.08 (2H, s, –N–CH2–N–), 6.57–6.60 (1H, m, pyridyl H5), 6.79
(1H, t, J = 8.8 Hz, pyridyl H6), 6.93–6.79 (1H, m, phenyl H5), 7.01 (1H, d,
J = 8 Hz, phenyl H6), 7.40–7.44 (1H, m, phenyl H4), 7.46–7.50 (1H, m,
pyridyl H4), 7.63 (1H, dd, J = 7.8 Hz, J′ = 1.8 Hz, phenyl H3), 8.06 (1H, dd,
J = 4.6 Hz J′ = 1.8 Hz, pyridyl H3), and 10.51 (1H, bs, –OH). 13C NMR
(400 MHz, DMSO‐d6) δ (ppm): 44.64 (piperazine C2 + C6), 49.38 (piper-
azine C3 + C5), 69.64 (–N–CH2–N–), 107.06 (pyridyl C5), 108.92 (pyridyl
C4), 112.92 (phenyl C5), 117.01 (phenyl C4), 119.38 (phenyl C6), 129.11
(pyridyl C6), 133.61 (phenyl C3), 137.38 (phenyl C1), 147.41 (pyridyl C3),
156.36 (phenyl C2), 157.84 (pyridyl C1), 158.77 (C═N), and 176.98 (C═S).
Anal. calcd. for C18H19N5O2S: 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.
5‐(2‐Hydroxyphenyl)‐3‐{[4‐(4‐cyanophenyl)piperazin‐1‐yl]methyl}‐
1,3,4‐oxadiazole‐2(3H)‐thione (5l)
Compound 5l was obtained as a white powder (55%). Mp: 124°C. IR νmax
(cm−1): 3,304 (O–H), 2,943 (aromatic C–H), 2,214 (C≡N), 1,626 (C═N),
1,605, 1,566, 1,515, 1,488 (aromatic C═C), 1,329 (C–O), 1,249 (C═S), 819
(4‐cyanophenyl C–H), and 741 (2‐hydroxyphenyl C–H). 1H NMR
(400 MHz, DMSO‐d6) δ (ppm): 2.86 (8H, t, J = 5 Hz, piperazine H2 +
H3 + H5 + H6), 5.08 (2H, s, –N–CH2–N–), 6.93–6.97 (1H, m, phenyl H5),
6.98 (2H, bd, J = 9.2 Hz, phenyl H2′ + H6′), 7.02 (1H, bd, J = 8 Hz, phenyl
H6), 7.39–7.44 (1H, m, phenyl H4), (2H, m, phenyl H3′ + H5′), 7.63 (1H, dd,
J = 8 J′= 1.6 Hz, phenyl H3), and 10.52 (1H, bs, –OH). 13C NMR (400 MHz,
DMSO‐d6) δ (ppm): 46.26 (piperazine C2 + C6), 49.16 (piperazine C3 + C5),
69.38 (–N–CH2–N–), 98.23 (phenyl C4′), 108.91 (phenyl C5), 114.07
(phenyl C2′ + C6′), 116.98 (phenyl C4), 119.36 (phenyl C6), 119.87 (C≡N),
129.10 (phenyl C3), 133.20 (phenyl C3′ + C5′), 133.62 (phenyl C1), 152.98
(phenyl C1′), 156.98 (phenyl C2), 157.89 (C═N), and 176.99 (C═S). Anal.
calcd. for C20H19N5O2S: C, 61.05; H, 4.87; N, 17.80; S, 8.15. Found: C,
60.47; H, 4.88; N, 17.66; S, 8.25.
5‐(2‐Hydroxyphenyl)‐3‐{[4‐(2‐pyrimidinyl)piperazin‐1‐yl]methyl}‐
1,3,4‐oxadiazole‐2(3H)‐thione (5o)
Compound 5o was obtained as a white powder (60%). Mp: 149°C. IR
ν
max (cm−1): 3,336 (O–H), 3,021 (aromatic C–H), 1,624 (C═N), 1,585,
1,547, 1,489 (aromatic C═C), 1,324 (C–O), 1,262 (C═S), 798 (2‐
pyrimidinyl C–H), and 742 (2‐hydroxyphenyl C–H). 1H NMR
(400 MHz, DMSO‐d6) δ (ppm): 2.80 (4H, t, J = 5 Hz, piperazine
H2 + H6), 3.74 (4H, t, J = 5 Hz, piperazine H3 + H5), 5.09 (2H,
s, –N–CH2–N–), 6.59 (1H, t, J = 4.8 Hz, pyrimidine H4), 6.95 (1H, m,
phenyl H5), 7.02 (1H, bd, J = 7.6 Hz, phenyl H6), 7.40–7.44 (1H, m,
phenyl H4), 7.63 (1H, dd, J = 7.6 Hz, J′ = 1.6 Hz, phenyl H3), 8.32 (2H,
d, J = 4.8 Hz, pyrimidine H3 + H5), and 10.48 (1H, bs, –OH). 13C NMR
(400 MHz, DMSO‐d6) δ (ppm): 43.13 (piperazine C2 + C6), 49.44 (pi-
perazine C3 + C5), 69.73 (–N–CH2–N–), 108.99 (phenyl C5), 110.08
(phenyl C4), 117.04 (pyrimidine C4), 119.43 (phenyl C6), 129.13
(phenyl C3), 133.62 (phenyl C1), 156.38 (phenyl C2), 157.86 (pyr-
imidine C3 + C5, C═N), 161.04 (pyrimidine C1), and 177.01 (C═S).
Anal. calcd. for C17H18N6O2S: C, 55.12; H, 4.90; N, 22.69; S, 8.66.
Found: C, 54.65; H, 4.93; N, 22.68; S, 8.84.
5‐(2‐Hydroxyphenyl)‐3‐{[4‐(2‐cyanophenyl)piperazin‐1‐yl]methyl}‐
1,3,4‐oxadiazole‐2(3H)‐thione (5m)
Compound 5m was obtained as a white powder (56%). Mp: 120°C. IR
νmax (cm−1): 3,336 (O–H), 2,964 (aromatic C–H), 2,220 (C≡N), 1,625
(C═N), 1,595, 1,573, 1,487 (aromatic C═C), 1,322 (C‐O), 1,250 (C═S),
764 (2‐cyanophenyl C–H), and 741 (2‐hydroxyphenyl C–H). 1H NMR
(400 MHz, DMSO‐d6) δ (ppm): 2.94 (4H, t, J = 4.8 Hz, piperazine
H2 + H6), 3.14 (4H, t, J = 4.8 Hz, piperazine H3 + H5), 5.10 (2H,
s, –N–CH2–N–), 6.96 (1H, t, J = 8 Hz, phenyl H5′), 7.04 (1H, t, J = 8 Hz,
phenyl H6′), 7.08 (1H, bd, J = 7.6 Hz, phenyl H6), 7.14 (1H, bd, J = 8 Hz,
phenyl H3′), 7.41–7.45 (1H, m, phenyl H4′), 7.55–7.60 (1H, m, phenyl
5‐[1‐(4‐Isobutylphenyl)ethyl]‐3‐[(4‐phenylpiperazin‐1‐yl)methyl]‐
1,3,4‐oxadiazole‐2(3H)‐thione (10a)
Compound 10a was obtained as a white powder (72%). Mp: 105°C. IR
νmax (cm−1): 2,952 (aromatic C–H), 2,831 (aliphatic C–H), 1,620 (C═N),
1,599, 1,506 (aromatic C═C), 1,441 (C–N), 1,325 (C–O), 1,258 (C═S),
849, and 756 (phenyl C–H). 1H NMR (400 MHz, DMSO‐d6) δ (ppm): 0.84
(6H, d, J = 6.8 Hz, isobutyl –CH3), 1.56 (3H, d, J = 6.8 Hz, –CH–CH3),
1.78–1.81 (1H, m, isobutyl –CH–), 2.41 (2H, d, J = 7.6 Hz, isobutyl –CH2–),
2.84 (4H, t, J = 5 Hz, piperazine H2 + H6), 3.13 (4H, t, J = 5 Hz, piperazine
H3 + H5), 4.33 (1H, q, J = 6.8 Hz, –CH–CH3), 5.00 (2H, s, –N–CH2–N–),
H5), 7.64–7.68 (2H, m, phenyl H3 + H4), and 10.52 (1H, bs, –OH). 13
C
NMR (400 MHz, DMSO‐d6) δ (ppm): 49.67 (piperazine C2 + C6), 51.11
(piperazine C3 + C5), 69.55 (–N–CH2–N–), 104.78 (phenyl C2′),
108.98 (phenyl C1), 117.08 (phenyl C3), 118.14 (phenyl C6′), 119.16
(phenyl C5), 119.47 (phenyl C4′), 122.10 (phenyl C6), 129.07 (phenyl
C5′), 133.67 (phenyl C4), 134.19 (phenyl C3′ + C≡N), 134.31 (phenyl
C1′), 155.13 (phenyl C2), 156.43 (C═N), and 177.10 (C═S). Anal.

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6.77 (1H, t, J = 7.2 Hz, phenyl H4′), 6.91 (2H, d, J = 7.8 Hz, phenyl H3 + H5),
7.13 (2H, d, J = 7.8 Hz, phenyl H2 + H6), and 7.17–7.21 (4H, m, phenyl
H3 + H5), and 7.22 (2H, bd, J = 8.2 Hz, phenyl H2 + H6). Anal. calcd. for
C25H31FN4OS: C, 66.05; H, 6.87; N, 12.32; S, 7.05. Found: C, 66.07; H,
7.12; N, 12.35; S, 7.15.
H2′ + H3′ + H5′ + H6′). 13C NMR (400 MHz, DMSO‐d6)
δ (ppm):
22.13 + 22.14 (isobutyl –CH3), 29.50 (–CH–CH3), 36.09 (isobutyl –CH2–),
44.13 (–CH–CH3), 48.19 (piperazine C2 + C6), 49.52 (piperazine C3 + C5),
69.43 (–N–CH2–N–), 115.58 (phenyl C2′ + C6′), 118.94 (phenyl C4′),
126.99 (phenyl C3 + C5), 128.87 (phenyl C3′ + C5′), 129.44 (phenyl
C2 + C6), 136.77 (phenyl C4), 140.51 (phenyl C1), 157.88 (phenyl C1′),
164.10 (C═N), and 177.76 (C═S). Anal. calcd. for C25H32N4OS: C, 68.77;
H, 7.39; N, 12.83; S, 7.34. Found: C, 68.73; H, 7.59; N, 12.88; S, 7.43.
5‐[1‐(4‐Isobutylphenyl)ethyl]‐3‐{[4‐(3‐(trifluoromethyl)phenyl]
piperazin‐1‐yl}‐methyl}‐1,3,4‐oxadiazole‐2(3H)‐thione (10d)
Compound 10d was obtained as a white powder (56%). Mp: 77°C. IR νmax
(cm−1): 2,954 (aromatic C–H), 2,886 (aliphatic C–H), 1,610 (C═N), 1,512,
1,497 (aromatic C═C), 1,447 (C–N), 1,329 (C–O), 1,242 (C═S), 1,169
(C–F), 851 (1,4‐dialkylphenyl C–H), and 781 (3‐trifluoromethylphenyl
C–H). 1H NMR (400 MHz, DMSO‐d6) δ (ppm): 0.83 (6H, d, J = 6.4 Hz,
isobutyl –CH3), 1.55 (3H, d, J = 7.2 Hz, –CH–CH3), 1.77–1.81 (1H, m,
isobutyl –CH–), 2.40 (2H, d, J = 7.2 Hz, isobutyl –CH2–), 2.84 (4H, t,
J = 4.8 Hz, piperazine H2 + H6), 3.23 (4H, t, J = 4.8 Hz, piperazine H3 + H5),
4.33 (1H, q, J = 7 Hz, –CH–CH3), 5.01 (2H, s, –N–CH2–N–), 7.05 (1H, d,
J = 7.6 Hz, phenyl H6′), 7.12 (2H, d, J = 8 Hz, phenyl H3 + H5), 7.15 (1H, bs,
phenyl H2′), 7.19 (2H, d, J = 8 Hz, phenyl H2 + H6), 7.18–7.22 (1H, m,
phenyl H4′), and 7.40 (1H, t, J = 8 Hz, phenyl H5′). 13C NMR (400 MHz,
DMSO‐d6) δ (ppm): 18.49 (isobutyl –CH–), 22.13 (isobutyl –CH3), 29.50
(–CH–CH3), 36.09 (isobutyl –CH2–), 44.14 (–CH–CH3), 49.83 (piperazine
C2 + C6), 50.72 (piperazine C3 + C5), 69.55 (–N–CH2–N–), 120.94 (phenyl
C6′), 122.97 + 125.68 (–CF3), 123.96 (phenyl C4′), 126.99 (phenyl
C3 + C5), 127.58 (phenyl C2′), 128.02 (phenyl C5′), 129.43 (phenyl
C2 + C6), 130.27 (phenyl C3′–CF3), 136.82 (phenyl C4), 140.52 (phenyl
C1), 148.83 (phenyl C1′), 164.12 (C═N), and 177.76 (C═S). Anal. calcd. for
C26H31F3N4OS: C, 61.88; H, 6.19; N, 11.10; S, 6.35. Found: C, 61.64; H,
6.49; N, 11.14; S, 6.39.
5‐[1‐(4‐Isobutylphenyl)ethyl]‐3‐{[4‐(4‐fluorophenyl)piperazin‐1‐
yl]methyl}‐1,3,4‐oxadiazole‐2(3H)‐thione (10b)[15]
Compound 10b was obtained as a white powder (69%). Mp: 121°C.
IR νmax (cm−1): 2,950 (aromatic C–H), 2,833 (aliphatic C–H), 1,614
(C═N), 1,508 (aromatic C═C), 1,447 (C–N), 1,330 (C–O), 1,254 (C═S),
826 (1,4‐dialkylphenyl C–H), and 780 (4‐fluorophenyl C–H). 1H NMR
(400 MHz, DMSO‐d6) δ (ppm): 0.84 (6H, d, J = 6.8 Hz, isobutyl –CH3),
1.55 (3H, d, J = 6.8 Hz, –CH–CH3), 1.77–1.81 (1H, m, isobutyl –CH–),
2.41 (2H, d, J = 7.2 Hz, isobutyl –CH2–), 2.83 (4H, t, J = 5 Hz, piper-
azine H2 + H6), 3.07 (4H, t, J = 5 Hz, piperazine H3 + H5), 4.33 (1H, q,
J = 6.4 Hz, –CH–CH3), 5.00 (2H, s, –N–CH2–N–), 6.93 (2H, dd,
J = 9.2 Hz, J’ = 4.6 Hz, phenyl H2′ + H6′), 7.03 (2H, t, J = 8.8 Hz, phenyl
H3′ + H5′), 7.13 (2H, bd, J = 8 Hz, phenyl H3 + H5), and 7.19 (2H, bd,
J = 8 Hz, phenyl H2 + H6). 13C NMR (400 MHz, DMSO‐d6) δ (ppm):
18.48 (isobutyl –CH–), 22.11 + 22.13 (isobutyl –CH3), 29.50
(–CH–CH3), 36.09 (isobutyl –CH2–), 44.13 (–CH–CH3), 48.94 (pi-
perazine C2 + C6), 49.48 (piperazine C3 + C5), 69.39 (–N–CH2–N–),
115.09 (phenyl C6′), 115.31 (phenyl C2′), 117.29 (phenyl C5′), 117.36
(phenyl C3′), 126.99 (phenyl C3 + C5), 129.43 (phenyl C2 + C6), 136.75
(phenyl C4), 140.51 (phenyl C1), 147.74 + 147.76 (phenyl C1′),
154.86 + 157.20 (phenyl C4′–F), 164.07 (C═N), and 177.75 (C═S).
5‐[1‐(4‐Isobutylphenyl)ethyl]‐3‐{[4‐(4‐chlorophenyl)piperazin‐1‐
yl]methyl}‐1,3,4‐oxadiazole‐2(3H)‐thione (10e)[18]
Compound 10e was obtained as a white powder (54%). Mp: 99°C. IR νmax
(cm−1): 2,949 (aromatic C–H), 2,827 (aliphatic C–H), 1,617 (C═N), 1,596,
1,496 (aromatic C═C), 1,444 (C–N), 1,329 (C–O), 1,242 (C═S), 812 (1,4‐
dialkylphenyl C–H), and 790 (4‐chlorophenyl C–H). 1H NMR (400 MHz,
DMSO‐d6) δ (ppm): 0.83 (6H, d, J = 6.4 Hz, isobutyl –CH3), 1.55 (3H, d,
J = 7.2 Hz, –CH–CH3), 1.77–1.81 (1H, m, isobutyl –CH–), 2.40 (2H, d,
J = 6.8 Hz, isobutyl –CH2–), 2.87 (4H, t, J = 5 Hz, piperazine H2 + H6), 3.13
(4H, t, J = 5 Hz, piperazine H3 + H5), 4.32 (1H, q, J = 7.2 Hz, –CH–CH3),
4.99 (2H, s, –N–CH2–N–), 6.92 (2H, bd, J = 9.4 Hz, phenyl H2′ + H6′), 7.12
(2H, bd, J = 8.6 Hz, phenyl H3 + H5), 7.18 (2H, bd, J = 8.6 Hz, phenyl
H2 + H6), and 7.21 (2H, bd, J = 9.4 Hz, phenyl H3′ + H5′). 13C NMR
(400 MHz, DMSO‐d6) δ (ppm): 18.48 (isobutyl –CH–), 22.11 + 22.13
(isobutyl –CH3), 29.50 (–CH–CH3), 36.07 (isobutyl –CH2–), 44.13
(–CH–CH3), 47.95 (piperazine C2 + C6), 49.27 (piperazine C3 + C5), 69.36
(–N–CH2–N–), 116.97 (phenyl C2′ + C6′), 122.36 (phenyl C4′), 126.94
(phenyl C3 + C5), 128.56 (phenyl C3′ + C5′), 129.43 (phenyl C2 + C6),
136.75 (phenyl C4), 140.51 (phenyl C1), 149.59 (phenyl C1′), 164.07
(C═N), and 177.73 (C═S).
5‐[1‐(4‐Isobutylphenyl)ethyl]‐3‐{[4‐(2‐fluorophenyl)piperazin‐1‐
yl]methyl}‐1,3,4‐oxadiazole‐2(3H)‐thione (10c)
Compound 10c was obtained as a white powder (40%). Mp: 108°C. IR
νmax (cm−1): 2,950 (aromatic C–H), 2,844 (aliphatic C–H), 1,603 (C═N),
1,578, 1,500 (aromatic C═C), 1,443 (C–N), 1,352 (C–O), 1,242 (C═S), 852
(1,4‐dialkylphenyl C–H), and 751 (2‐fluorophenyl C–H). 1H NMR
(400 MHz, DMSO‐d6) δ (ppm): 0.84 (6H, d, J = 6.8 Hz, isobutyl –CH3), 1.55
(3H, d, J = 6.8 Hz, –CH–CH3), 1.77–1.81 (1H, m, isobutyl –CH–), 2.41 (2H,
d, J = 7.2 Hz, isobutyl –CH2–), 2.83 (4H, t, J = 5 Hz, piperazine H2 + H6),
3.07 (4H, t, J = 5 Hz, piperazine H3 + H5), 4.33 (1H, q, J = 6.4 Hz,
–CH–CH3), 5.00 (2H, s, –N–CH2–N–), 6.93 (2H, dd, J = 9.2 Hz, J′ = 4.6 Hz,
phenyl H2′ + H6′), 7.03 (2H, t, J = 8.8 Hz, phenyl H3′ + H5′), 7.13 (2H, bd,
J = 8 Hz, phenyl H3 + H5), and 7.19 (2H, bd, J = 8 Hz, phenyl H2 + H6). 13
C
NMR (400 MHz, DMSO‐d6) δ (ppm): 18.48 (isobutyl –CH–), 0.85 (6H, d,
J = 6.8 Hz, isobutyl –CH3), 1.57 (3H, d, J = 7.2 Hz, –CH–CH3), 1.77–1.84
(1H, m, isobutyl –CH–), 2.42 (2H, d, J = 7.2 Hz, isobutyl –CH2–), 2.86 (4H,
t, J = 4.8 Hz, piperazine H2 + H6), 3.01 (4H, t, J = 4.8 Hz, piperazine
H3 + H5), 4.35 (1H, q, J = 7 Hz, –CH–CH3), 5.00 (2H, s, –N–CH2–N–),
6.94–7.00 (1H, m, phenyl H6′), 7.03 (1H, bd, J = 9.2 Hz, phenyl H3′),
7.08–7.12 (2H, m, phenyl H4′ + H5′), 7.15 (2H, bd, J = 8.2 Hz, phenyl
5‐[1‐(4‐Isobutylphenyl)ethyl]‐3‐{[4‐(2‐chlorophenyl)piperazin‐1‐
yl]methyl}‐1,3,4‐oxadiazole‐2(3H)‐thione (10f)
Compound 10f was obtained as a white powder (39%). Mp: 94°C. IR νmax
(cm−1): 3,053 (aromatic C–H), 2,819 (aliphatic C–H), 1,618 (C═N), 1,586,

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1,479 (aromatic C═C), 1,443 (C–N), 1,330 (C–O), 1,250 (C═S), 845 (1,4‐
dialkylphenyl C–H), and 741 (2‐chlorophenyl C–H). 1H NMR (400 MHz,
DMSO‐d6) δ (ppm): 0.85 (6H, d, J = 6.4 Hz, isobutyl –CH3), 1.58 (3H, d,
J = 7.2 Hz, –CH–CH3), 1.79–1.83 (1H, m, isobutyl –CH–), 2.42 (2H, d,
J = 7.2 Hz, isobutyl –CH2–), 2.87 (4H, d, J = 4.4 Hz, piperazine H2 + H6),
2.96 (4H, d, J = 4.4 Hz, piperazine H3 + H5), 4.37 (1H, q, J = 7.2 Hz, –CH–
CH3,), 5.01 (2H, s, –N–CH2–N–), 7.02–7.06 (1H, m, phenyl H5′), 7.15 (1H,
dd, J = 8.2 Hz, J′ = 1.8 Hz, phenyl H6′), 7.16 (2H, bd, J = 8 Hz, phenyl
H3 + H5), 7.23 (2H, bd, J = 8 Hz, phenyl H2 + H6), 7.27–7.31 (1H, m,
phenyl H4′), and 7.40 (2H, dd, J = 8.2 Hz, J′ = 1.4 Hz, phenyl H3′).
13C NMR (400 MHz, DMSO‐d6) δ (ppm): 18.50 (isobutyl –CH–), 22.13
(isobutyl –CH3), 29.51 (–CH–CH3), 36.09 (isobutyl –CH2–), 44.14
(–CH–CH3), 49.83 (piperazine C2 + C6), 50.72 (piperazine C3 + C5), 69.55
(–N–CH2–N–), 120.93 (phenyl C4′), 123.96 (phenyl C3′), 126.99 (phenyl
C3 + C5), 127.58 (phenyl C6′), 128.03 (phenyl C5′), 129.43 (phenyl
C2 + C6), 130.28 (phenyl C1), 136.83 (phenyl C4), 140.52 (phenyl C1′),
148.83 (C2′), 164.13 (C═N), and 177.78 (C═S). Anal. calcd. for
C25H31ClN4OS: C, 63.74; H, 6.63; N, 11.89; S, 6.81. Found: C, 63.05; H,
6.83; N, 12.12; S, 6.97.
d, J = 4.2 Hz, piperazine H2 + H6), 2.97 (4H, d, J = 4.2 Hz, piperazine
H3 + H5), 4.36 (1H, q, J = 6.8 Hz, –CH–CH3), 5.00 (2H, s, –N–CH2–N–),
7.14 (1H, t, J = 8 Hz, phenyl H5′), 7.15 (2H, bd, J = 7.4 Hz, phenyl H3 +
H5), 7.22 (2H, bd, J = 7.4 Hz, phenyl H2 + H6), 7.28–7.31 (2H, m,
phenyl H4′ + H6′). 13C NMR (400 MHz, DMSO‐d6) δ (ppm): 18.48
(isobutyl –CH–), 22.12 + 22.13 (isobutyl –CH3), 29.50 (–CH–CH3), 36.08
(isobutyl –CH2–), 44.14 (–CH–CH3), 49.77 (piperazine C2 + C6), 50.79
(piperazine C3 + C5), 69.51 (–N–CH2–N–), 119.68 (phenyl C6′), 124.47
(phenyl C4′), 126.02 (phenyl C2′), 126.99 (phenyl C3 + C5), 128.41 (phenyl
C5′), 129.43 (phenyl C2 + C6), 132.57 (phenyl C3′), 136.81 (phenyl C4),
140.52 (phenyl C1), 150.98 (phenyl C1′), 164.14 (C═N), 177.76 (C═S).
Anal. calcd. for C25H30Cl2N4OS: C, 59.40; H, 5.98; N, 11.08; S, 6.34.
Found: C, 58.70; H, 6.11; N, 11.09; S, 6.36.
5‐[1‐(4‐Isobutylphenyl)ethyl]‐3‐{[4‐(4‐methylphenyl)piperazin‐1‐
yl]methyl}‐1,3,4‐oxadiazole‐2(3H)‐thione (10i)
Compound 10i was obtained as a white powder (70%). Mp: 99°C. IR
νmax (cm−1): 2,948 (aromatic C–H), 2,824 (aliphatic C–H), 1,620
(C═N), 1,574, 1,519 (aromatic C═C), 1,441 (C–N), 1,326 (C–O), 1,245
(C═S), 848 (1,4‐dialkylphenyl C–H), and 807 (4‐methylphenyl C–H).
1H NMR (400 MHz, DMSO‐d6) δ (ppm): 0.83 (6H, d, J = 6.8 Hz, iso-
butyl –CH3), 1.55 (3H, d, J = 7.2 Hz, –CH–CH3), 1.77–1.81 (1H, m,
isobutyl –CH–), 2.18 (3H, s, phenyl CH3), 2.40 (2H, d, J = 7.6 Hz,
isobutyl –CH2–), 2.83 (4H, t, J = 5 Hz, piperazine H2 + H6), 3.06 (4H, t,
J = 5 Hz, piperazine H3 + H5), 4.33 (1H, q, J = 7.2 Hz, –CH–CH3), 4.99
(2H, s, –N–CH2–N–), 6.81 (2H, d, J = 8.8 Hz, phenyl H3′ + H5′), 7.01
(2H, d, J = 8.8 Hz, phenyl H2′ + H6′), 7.13 (2H, d, J = 8.2 Hz, phenyl
H3 + H5), and 7.19 (2H, d, J = 8.2 Hz, phenyl H2 + H6). 13C NMR
5‐[1‐(4‐Isobutylphenyl)ethyl]‐3‐{[4‐(3,4‐dichlorophenyl)piperazin‐1‐
yl]methyl}‐1,3,4‐oxadiazole‐2(3H)‐thione (10g)
Compound 10g was obtained as a white powder (63%). Mp: 116°C. IR
νmax (cm−1): 2,946 (aliphatic C–H), 2,846 (aliphatic C–H), 1,619
(C═N), 1,593, 1,551, 1,512, 1,486 (aromatic C═C), 1,442 (C–N),
1,330 (C–O), 1,258 (C═S), 835 (1,4‐dialkylphenyl C–H), and 802 (3,4‐
dichlorophenyl C–H). 1H NMR (400 MHz, DMSO‐d6) δ (ppm): 0.83
(6H, d, J = 6.4 Hz, isobutyl –CH3), 1.54 (3H, d, J = 6.8 Hz, –CH–
CH3), 1.77–1.81 (1H, m, isobutyl –CH–), 2.40 (2H, d, J = 7.2 Hz, iso-
butyl –CH2–), 2.78 (4H, t, J = 4.8 Hz, piperazine H2 + H6), 3.19 (4H, t,
J = 4.8 Hz, piperazine H3 + H5), 4.32 (1H, q, J = 7.2 Hz, –CH–CH3),
4.99 (2H, s, –N–CH2–N–), 6.91 (1H, dd, J = 9.2 Hz, J′ = 2.8 Hz, phenyl
H6′), 7.11 (1H, d, J = 3.2 Hz, phenyl H2′), 7.12 (2H, d, J = 8 Hz, phenyl
H3 + H5), 7.17 (2H, d, J = 8 Hz, phenyl H2 + H6), and 7.40 (1H, d,
J = 9.2 Hz, phenyl H5′). 13C NMR (400 MHz, DMSO‐d6) δ (ppm): 18.47
(isobutyl –CH–), 22.11 + 22.12 (isobutyl –CH3), 29.50 (–CH–CH3), 36.06
(isobutyl –CH2–), 44.13 (–CH–CH3), 47.44 (piperazine C2 + C6), 49.10
(piperazine C3 + C5), 69.30 (–N–CH2–N–), 115.39 (phenyl C6′), 116.30
(phenyl C5′), 119.54 (phenyl C2′), 126.94 (phenyl C3 + C5), 129.41
(phenyl C2 + C6), 130.40 (phenyl C4′), 131.45 (phenyl C3′), 136.74
(phenyl C4), 140.50 (phenyl C1), 150.45 (phenyl C1′), 164.07 (C═N), and
177.71 (C═S). Anal. calcd. for C25H30Cl2N4OS: C, 59.40; H, 5.98; N,
11.08; S, 6.34. Found: C, 58.78; H, 6.12; N, 11.15; S, 6.40.
(400 MHz, DMSO‐d6)
δ (ppm): 18.49 (isobutyl –CH–), 20.01
(phenyl –CH3), 22.13 + 22.14 (isobutyl –CH3), 29.50 (–CH–CH3),
36.09 (isobutyl –CH2–), 44.14 (–CH–CH3), 48.65 (piperazine C2 + C6),
49.52 (piperazine C3 + C5), 69.44 (–N–CH2–N–), 115.85 (phenyl
C2′ + C6′), 126.99 (phenyl C4′), 127.73 (phenyl C3 + C5), 129.31
(phenyl C3′ + C5v), 129.44 (phenyl C2 + C6), 136.78 (phenyl C4),
140.51 (phenyl C1), 148.79 (phenyl C1′), 164.07 (C═N), and 177.75
(C═S). Anal. calcd. for C26H34N4OS: C, 69.30; H, 7.60; N, 12.43; S,
7.12. Found: C, 69.08; H, 7.67; N, 12.52; S, 7.21.
5‐[1‐(4‐Isobutylphenyl)ethyl]‐3‐{[4‐(2,3‐xylylphenyl)piperazin‐1‐
yl]methyl}‐1,3,4‐oxadiazole‐2(3H)‐thione (10j)
Compound 10j was obtained as a white powder (45%). Mp: 93°C. IR νmax
(cm−1): 2,950 (aromatic C–H), 2,845 (aliphatic C–H), 1,618 (C═N), 1,581,
1,511, 1,470 (aromatic C═C), 1,454 (C–N), 1,326 (C–O), 1,239 (C═S), 842
(1,4‐dialkylphenyl C–H), and 776 (2,3‐dimethylphenyl C–H). 1H NMR
(400 MHz, DMSO‐d6) δ (ppm): 0.84 (6H, d, J = 6.4 Hz, isobutyl –CH3), 1.57
(3H, d, J = 7.2 Hz, –CH–CH3), 1.78–1.82 (1H, m, isobutyl –CH–), 2.11 +
2.19 (6H, s, phenyl –CH3), 2.42 (2H, d, J = 7.2 Hz, isobutyl –CH2–), 2.77
(4H, bd, J = 3.6 Hz, piperazine H2 + H6), 2.85 (4H, bs, piperazine H3 + H5),
4.36 (1H, q, J = 7.2 Hz, –CH–CH3), 5.00 (2H, s, –N–CH2–N–), 6.87 (2H, bd,
J = 8 Hz, phenyl H4′+ H6′), 7.02 (2H, t, J = 7.8 Hz, phenyl H5′), 7.15 (2H, d,
J = 8 Hz, phenyl H3 + H5), and 7.22 (2H, d, J = 8 Hz, phenyl H2 + H6). 13C
NMR (400 MHz, DMSO‐d6) δ (ppm): 13.57 (phenyl C4′–CH3), 18.50
(isobutyl –CH–), 20.01 (phenyl C3′–CH3), 22.12 + 22.13 (isobutyl –CH3),
5‐[1‐(4‐Isobutylphenyl)ethyl]‐3‐{[4‐(2,3‐dichlorophenyl)piperazin‐1‐
yl]methyl}‐1,3,4‐oxadiazole‐2(3H)‐thione (10h)
Compound 10h was obtained as a white powder (52%). Mp: 99°C. IR
νmax (cm−1): 2,946 (aromatic C–H), 2,845 (aliphatic C–H), 1,619
(C═N), 1,593, 1,552, 1,511, 1,486 (aromatic C═C), 1,442 (C–N),
1,326 (C–O), 1,258 (C═S), 835 (1,4‐dialkylphenyl C–H), and 802 (2,3‐
dichlorophenyl C–H). 1H NMR (400 MHz, DMSO‐d6) δ (ppm): 0.84 (6H, d,
J = 6.4 Hz, isobutyl –CH3), 1.57 (3H, d, J = 7.6 Hz, –CH–CH3), 1.77–1.83
(1H, m, isobutyl –CH–), 2.41 (2H, d, J = 6.8 Hz, isobutyl –CH2–), 2.87 (4H,

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29.52 (–CH–CH3), 36.08 (isobutyl –CH2–), 44.14 (–CH–CH3), 50.15
(piperazine C2 + C6), 51.65 (piperazine C3 + C5), 60.61 (–N–CH2–N–),
116.46 (phenyl C6′), 124.62 (phenyl C4′), 125.66 (phenyl C2′), 126.98
(phenyl C3 + C5), 129.43 (phenyl C2 + C6), 130.40 (phenyl C5′), 136.85
(phenyl C4), 137.22 (phenyl C3′), 140.53 (phenyl C1), 148.79 (phenyl C1′),
164.07 (C═N), and 177.75 (C═S). Anal. calcd. for C27H36N4OS: C, 69.79,
H, 7.81, N, 12.06, S, 6.90. Found: C, 69.01, H, 7.61, N, 12.09, S, 6.85.
(C═S), 850 (1,4‐dialkylphenyl C–H), and 737 (2‐methoxyphenyl C–H).
1H NMR (400 MHz, DMSO‐d6) δ (ppm): 0.85 (6H, d, J = 6.8 Hz, iso-
butyl CH3), 1.58 (3H, d, J = 7.6 Hz, –CH–CH3), 1.76–1.86 (1H, m,
isobutyl –CH–), 2.42 (2H, d, J = 7.2 Hz, isobutyl –CH2–), 2.85 (4H, bs,
piperazine H2 + H6), 2.95 (4H, bs, piperazine H3 + H5), 3.76 (3H, s,
phenyl C2′–OCH3), 4.37 (1H, q, J = 7 Hz, –CH–CH3), 4.99 (2H,
s, –N–CH2–N–), 6.86–6.95 (4H, m, phenyl H3′ + H4′ + H5′ + H6′), 7.16
(2H, bd, J = 8 Hz, phenyl H3 + H5), and 7.23 (2H, bd, J = 8 Hz, phenyl
H2 + H6). 13C NMR (400 MHz, DMSO‐d6) δ (ppm): 18.49 (–CH–CH3),
22.12 + 22.13 (isobutyl –CH3), 29.50 (isobutyl –CH–), 36.09
(isobutyl –CH2–), 44.14 (–CH–CH3), 49.86 (piperazine C2 + C6), 49.93
(piperazine C3 + C5), 55.21 (phenyl C2′–OCH3), 69.66 (–N–CH2–N–),
111.78 (phenyl C6′), 118.02 (phenyl C3′), 120.75 (phenyl C4′), 122.51
(phenyl C5′), 127.03 (phenyl C3 + C5), 129.43 (phenyl C2 + C6), 136.79
(phenyl C4), 140.52 (phenyl C1), 140.99 (phenyl C1′), 151.88 (phenyl
C2′), 164.11 (C═N), and 177.75 (C═S). Anal. calcd. for C26H34N4O2S:
C, 66.92, H, 7.34, N, 12.01, S, 6.87. Found: C, 65.99, H, 7.61, N, 11.92,
S, 7.01.
5‐[1‐(4‐Isobutylphenyl)ethyl]‐3‐{[4‐(4‐methoxyphenyl)piperazin‐1‐yl]
methyl}‐1,3,4‐oxadiazole‐2(3H)‐thione (10k)[15]
Compound 10k was obtained as a white powder (47%). Mp: 97°C. IR νmax
(cm−1): 2,950 (aromatic C–H), 2,845 (aliphatic C–H), 1,618 (C═N), 1,581,
1,511, 1,470 (aromatic C═C), 1,454 (C–N), 1,326 (C–O), 1,239 (C═S), 842
(1,4‐dialkylphenyl C–H), and 776 (2,3‐dimethylphenyl C–H). 1H NMR
(400 MHz, DMSO‐d6) δ (ppm): 0.82 (6H, d, J = 6.8 Hz, isobutyl –CH3), 1.54
(3H, d, J = 6.8 Hz, –CH–CH3), 1.77–1.80 (1H, m, isobutyl –CH–), 2.40 (2H,
d, J = 7.2 Hz, isobutyl –CH2–), 2.82 (4H, t, J = 4.6 Hz, piperazine H2 + H6),
2.99 (4H, t, J = 4.6 Hz, piperazine H3 + H5), 3.65 (3H, s, phenyl –OCH3),
4.32 (1H, q, J = 6.8 Hz, –CH–CH3), 4.98 (2H, s, –N–CH2–N–), 6.78 (2H, bd,
J = 9 Hz, phenyl H2′ + H6′), 6.85 (2H, bd, J = 9 Hz, phenyl H3′ + H5′), 7.13
(2H, bd, J = 8.4 Hz, phenyl H3 + H5), and 7.19 (2H, d, J = 8.4 Hz, phe-
nyl H2 + H6).
5‐[1‐(4‐Isobutylphenyl)ethyl]‐3‐{[4‐(4‐cyanophenyl)piperazin‐1‐yl]
methyl}‐1,3,4‐oxadiazole‐2(3H)‐thione (10n)
Compound 10n was obtained as a white powder (50%). Mp: 50°C. IR
νmax (cm−1): 2,946 (aromatic C–H), 2,840 (aliphatic C–H), 2,211
(C≡N), 1,604 (C═N), 1,513 (aromatic C═C), 1,445 (C–N), 1,329 (C–O),
1,248 (C═S), 818 (1,4‐dialkylphenyl C–H), and 789 (4‐cyanophenyl
C–H). 1H NMR (400 MHz, DMSO‐d6) δ (ppm): 0.83 (6H, d, J = 6.4 Hz,
isobutyl –CH3), 1.53 (3H, d, J = 6.8 Hz, –CH–CH3), 1.76–1.80 (1H, m,
isobutyl –CH–), 2.39 (2H, d, J = 7.2 Hz, isobutyl –CH2–), 2.80 (4H, bs,
piperazine H2 + H6), 3.53 (4H, t, J = 5 Hz, piperazine H3 + H5), 4.31 (3H,
q, J = 7.2 Hz, –CH–CH3), 5.00 (2H, s, –N–CH2–N–), 7.01 (2H, bd,
J = 8.8 Hz phenyl H2′ + H6′), 7.09 (2H, bd, J = 8 Hz phenyl H3 + H5), 7.16
(2H, bd, J = 8 Hz, phenyl H2 + H6), and 7.56 (2H, bd, J = 8.8 Hz,
phenyl H3′ + H5′). 13C NMR (400 MHz, DMSO‐d6) δ (ppm): 18.47
(isobutyl –CH–), 20.09 + 20.13 (isobutyl –CH3), 29.50 (–CH–CH3), 36.06
(isobutyl –CH2–), 44.11 (–CH–CH3), 46.32 (piperazine C2 + C6), 49.07
(piperazine C3 + C5), 69.28 (–N–CH2–N–), 98.26 (phenyl C4′–C≡N),
114.12 (phenyl C2′ + C6′), 119.94 (phenyl C4′), 126.93 (phenyl C3 + C5),
129.40 (phenyl C2 + C6), 133.29 (phenyl C3′ + C5′), 136.73 (phenyl C4),
140.51 (phenyl C1), 152.90 (phenyl C1′), 164.07 (C═N), and 177.71
(C═S). Anal. calcd. for C26H31N5OS: C, 67.65, H, 6.77, N, 15.17, S, 6.95.
Found: C, 67.70, H, 6.98, N, 15.20, S, 7.04.
5‐[1‐(4‐Isobutylphenyl)ethyl]‐3‐{[4‐(3‐methoxyphenyl)piperazin‐1‐yl]
methyl}‐1,3,4‐oxadiazole‐2(3H)‐thione (10l)
Compound 10l was obtained as a white powder (45%). Mp: 93°C. IR νmax
(cm−1): 2,950 (aromatic C–H), 2,845 (aliphatic C–), 1,618 (C═N), 1,581,
1,511, 1,470 (aromatic C═C), 1,454 (C–N), 1,326 (C–O), 1,239 (C═S), 842
(1,4‐dialkylphenyl C–H), and 776 (2,3‐dimethylphenyl C–H). 1H NMR
(400 MHz, DMSO‐d6) δ (ppm): 0.84 (6H, d, J = 6.4 Hz, isobutyl CH3), 1.56
(3H, d, J = 6.8 Hz, –CH–CH3), 1.77–1.84 (1H, m, isobutyl –CH–),
2.41 (2H, d, J = 7.2 Hz, isobutyl –CH2–), 2.86 (4H, t, J = 5 Hz,
piperazine H2 + H6), 3.13 (4H, t, J = 5 Hz, piperazine H3 + H5), 4.34 (1H,
q, J = 7.2 Hz, –CH–CH3), 5.00 (2H, s, –N–CH2–N–), 6.36 (1H, dd,
J = 8.2 Hz, J′ = 2.2 Hz, phenyl H6′), 6.44 (1H, t, J = 2.2 Hz, phenyl H2′),
6.51 (1H, dd, J = 8.2 Hz, J′ = 2.2 Hz, phenyl H4′), 7.09 (1H, t, J = 8.4 Hz,
phenyl H5′), 7.14 (2H, d, J = 8.2 Hz, phenyl H3 + H5), and 7.20 (2H, d,
J = 8.2 Hz, phenyl H2 + H6). 13C NMR (400 MHz, DMSO‐d6) δ (ppm):
18.49 (–CH–CH3), 22.13 + 22.14 (isobutyl –CH3), 29.50 (isobutyl –CH–),
36.09 (isobutyl –CH2–), 44.13 (–CH–CH3), 48.13 (piperazine C2 + C6),
49.49 (piperazine C3 + C5), 54.82 (phenyl C3′–OCH3), 69.42
(–N–CH2–N–), 101.69 (phenyl C2′), 104.26 (phenyl C6′), 108.18 (phenyl
C4′), 126.98 (phenyl C2 + C6), 129.44 (phenyl C3 + C5), 129.56 (phenyl
C5′), 136.76 (phenyl C4), 140.50 (phenyl C1), 152.23 (phenyl C1′), 160.14
(phenyl C3′), 164.10 (C═N), and 177.76 (C═S). Anal. calcd. for
C26H34N4O2S: C, 66.92, H, 7.34, N, 12.01, S, 6.87. Found: C, 65.91, H,
7.57, N, 12.43, S, 7.01.
5‐[1‐(4‐Isobutylphenyl)ethyl]‐3‐{[4‐(2‐cyanophenyl)piperazin‐1‐
yl]methyl}‐1,3,4‐oxadiazole‐2(3H)‐thione (10o)
Compound 10o was obtained as a white powder (52%). Mp: 80°C.
IR νmax (cm−1): 2,945 (aromatic C–H), 2,837 (aliphatic C–H), 2,218
(C≡N), 1,617 (C═N), 1,594, 1,569, 1,510, 1,488 (aromatic C═C),
1,445 (C–N), 1,329 (C–O), 1,250 (C═S), 847 (1,4‐dialkylphenyl
C–H), and 765 (2‐cyanophenyl C–H). 1H NMR (400 MHz, DMSO‐
d6) δ (ppm): 0.84 (6H, d, J = 6.8 Hz, isobutyl –CH3), 1.57 (3H, d,
J = 7.2 Hz, –CH–CH3), 1.78–1.82 (1H, m, isobutyl –CH–), 2.41 (2H,
d, J = 7.6 Hz, isobutyl –CH2–), 2.90 (4H, t, J = 3.5 Hz, piperazine
H2 + H6), 3.14 (4H, t, J = 3.5 Hz, piperazine H3 + H5), 4.37 (3H, q,
5‐[1‐(4‐Isobutylphenyl)ethyl]‐3‐{[4‐(2‐methoxyphenyl)piperazin‐1‐yl]
methyl}‐1,3,4‐oxadiazole‐2(3H)‐thione (10m)
Compound 10m was obtained as a white powder (41%). Mp: 98°C. IR
νmax (cm−1): 3,049 (aromatic C–H), 2,909 (aliphatic C–H), 1,614
(C═N), 1,594, 1,498 (aromatic C═C), 1,444 (C–N), 1,324 (C–O), 1,242

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J = 7 Hz, –CH–CH3), 5.03 (2H, s, –N–CH2–N–), 7.10 (2H, t,
J = 7.8 Hz phenyl H5′), 7.15 (3H, d, J = 8 Hz phenyl H3 + H5 + H6′),
7.22 (2H, d, J = 8 Hz, phenyl H2 + H6), 7.58–7.62 (1H, m, phenyl
H4′), and 7.70 (1H, dd, J = 7.6 Hz, J′ = 1.6 Hz, phenyl H3′). 13C
to wells for additional 2 hr at 37°C. After incubation, the cell culture
medium was removed and 100 μl of isopropanol was used to dissolve
formazan. The absorbance was determined at 570 nm wavelength by
a microplate reader (Thermo Multiskan Spectrum, Finland). The ab-
sorbance of the control group was considered as 100%. The per-
centage of cell viability was calculated as follows:
NMR (400 MHz, DMSO‐d6)
δ (ppm): 18.51 (isobutyl –CH–),
22.11 + 22.13 (isobutyl –CH3), 29.49 (–CH–CH3), 36.08
(isobutyl –CH2–), 44.14 (–CH–CH3), 49.66 (piperazine C2 + C6),
51.07 (piperazine C3 + C5), 69.38 (–N–CH2–N–), 104.88 (phenyl
C2′–C≡N), 118.11 (phenyl C6′), 119.19 (phenyl C4′), 122.12
(phenyl C2′), 126.96 (phenyl C3 + C5), 129.46 (phenyl C2 + C6), 134.18
(phenyl C5′), 136.78 (phenyl C4), 140.50 (phenyl C1), 155.11 (phenyl C1′),
164.15 (C═N), and 177.75 (C═S). Anal. calcd. for C26H31N5OS: C, 67.65,
H, 6.77, N, 15.17, S, 6.95. Found: C, 66.91, H, 6.79, N, 15.31, S, 7.09.
%Viability
(Absorbance of treatment group − Absorbance of background)
=
(Absorbance of the control group − Absorbance of background)
× 100%.
|
4.2.2
Anti‐inflammatory activity
COX‐1/COX‐2 enzyme ınhibition assay
5‐[1‐(4‐Isobutylphenyl)ethyl]‐3‐{[4‐(2‐pyridyl)piperazin‐1‐yl]methyl}‐
1,3,4‐oxadiazole‐2(3H)‐thione (10p)
The inhibitory COX activity of the synthesized compounds was as-
sayed using the Cayman COX fluorescent assay kit (Cayman No:
700100; Cayman Chemicals, Ann Arbor, MI) according to manu-
facturer's instructions. Each test compound was analyzed in duplicate
applications. The enzyme‐free and inhibitor‐free assay systems were
used as control experiments. The percentage of enzyme inhibition
was calculated as follows:
Compound 10p was obtained as a white powder (55%). Mp: 85°C. IR
νmax (cm−1): 2,951 (aromatic C–H), 2,844 (aliphatic C–H), 1,618
(C═N), 1,591, 1,563, 1,512, 1,478 (aromatic C═C), 1,445 (C–N),
1,326 (C–O), 1,274 (C═S), 843 (1,4‐dialkylphenyl C–H), and 774
(2‐pyridyl C–H). 1H NMR (400 MHz, DMSO‐d6) δ (ppm): 0.83 (6H, d,
J = 6.4 Hz, isobutyl –CH3), 1.54 (3H, d, J = 6.8 Hz, –CH–CH3), 1.74–1.82
(1H, m, isobutyl –CH–), 2.42 (2H, d, J = 7.6 Hz, isobutyl –CH2–), 2.78
(4H, t, J = 4.8 Hz, piperazine H2 + H6), 3.49 (4H, t, J = 4.8 Hz, piperazine
H3 + H5), 4.32 (1H, q, J = 7.2 Hz, –CH–CH3), 5.00 (2H, s, –N–CH2–N–),
6.61–6.64 (1H, m, pyridyl H5), 6.81 (1H, d, J = 8.4 Hz, pyridyl H6), 7.11
(2H, d, J = 8 Hz, phenyl H3 + H5), 7.17 (2H, d, J = 8 Hz, phenyl H2 + H6),
7.49–7.53 (1H, m, pyridyl H4), and 8.09 (1H, dd, J = 4.6 Hz, J′ = 1.4 Hz,
(Initial activity − Sample activity)
%Inhibition =
× 100%.
(Initial activity)
NO inhibition assay
The anti‐inflammatory activity of compounds was evaluated by
measuring the stable NO metabolite, nitrite, levels in cell culture
media, with Griess reagent. RAW264.7 cells were plated, with a
density of 1 × 106/ml, into a 48‐well plate and incubated for 24 hr at
37°C in 5% CO2. After cell culture medium was aspirated, cells were
pretreated with various concentrations of compounds (50–100 µM)
for 2 hr and then stimulated with 1 µg/ml of LPS (lipopolysaccharide
from Escherichia coli 0111:B4; Sigma‐Aldrich) for additional 22 hr. The
collected culture supernatant was mixed with an equal volume of
Griess reagent (1% sulfanilamide and 0.1% N‐(1‐naphthyl) ethylene-
diamine dihydrochloride in 5% phosphoric acid) in a 96‐well plate and
incubated at room temperature for 10 min in the dark. The absor-
bance was determined using a microplate reader (Multiskan Ascent,
Finland) at 540 nm wavelength. The concentration of nitrite in sam-
ples was calculated by using sodium nitrite standard curve. In-
domethacin (100 µM) was used as a positive control.[25,26]
pyridyl H3). 13C NMR (400 MHz, DMSO‐d6)
δ (ppm): 18.45
(isobutyl –CH–), 22.11 + 22.13 (isobutyl –CH3), 29.49 (–CH–CH3), 36.06
(isobutyl –CH2–), 44.12 (–CH–CH3), 44.51 (piperazine C2 + C6), 49.34
(piperazine C3 + C5), 69.52 (–N–CH2–N–), 107.17 (pyridyl C6), 112.99
(pyridyl C4), 126.95 (phenyl C3 + C5), 129.41 (phenyl C2 + C6), 136.74
(phenyl C4), 137.45 (pyridyl C5), 140.49 (phenyl C1), 147.49 (pyridyl C3),
159.32 (pyridyl C1), 164.58 (C═N), and 178.22 (C═S). Anal. calcd. for
C24H31N5OS: C, 65.87, H, 7.14, N, 16.00, S, 7.33. Found: C, 65.32, H,
7.10, N, 16.12, S, 7.48.
|
4.2
Biological evaluation
|
4.2.1
Cell viability
PGE2 ınhibition assay
The murine macrophage RAW264.7 cell line (American Type Culture
Collection) was maintained in Dulbecco's modified Eagle's medium
High Glucose supplemented with 10% fetal bovine serum and 1%
penicillin (10,000 units/ml) and streptomycin (10,000 µg/ml) at 37°C
under a 5% CO2 atmosphere. Cell viability was measured by using
3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT)
colorimetric assay, which depends on the mitochondria‐based re-
duction of MTT formazan. Plated RAW 264.7 cells were treated with
various concentrations of compounds (50–100 µM). After 24 hr, the
cell medium was discarded and MTT solution (0.5 mg/ml) was added
PGE2 concentrations in cell culture supernatants of the compounds
that have shown to reduce inflammation in NO assay were measured
by using a commercially available quantitative enzyme‐linked im-
munosorbent assay (ELISA) kit (Abcam PGE2 ELISA Kit, UK) ac-
cording to manufacturer's instructions.
|
4.2.3
Statistical analysis
All repeated experiments were conducted in triplicate. Statistical analysis
was performed by using GraphPad Prism 6 (Version 6.01; GraphPad

16 of 16
OZYAZICI ET AL.
|
Software, Inc., San Diego, CA). Differences between groups were ana-
lyzed by using one‐way analysis of variance, followed by Tukey's post‐hoc
tests. The statistical correlation was measured by Pearson's coefficient.
Group differences were considered to be significant at p < 0.05.
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4.3
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ORCID
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Hayrettin O. Gulcan
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Meric Koksal
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