Some Novel Mannich Bases of 5-(3,4-Dichlorophenyl)-1,3,4-oxadiazole-2(3H)-one and Their Anti-Inflammatory Activity

Article abstract of DOI:10.1002/ardp.201700153

Non-steroidal anti-inflammatory drugs (NSAIDs), which are widely used for the treatment of rheumatic arthritis, pain, and many different types of inflammatory disorders, cause serious gastrointestinal (GI) side effects. The free carboxylic acid group exis

Full text of DOI:10.1002/ardp.201700153

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Arch. Pharm. Chem. Life Sci. 2017, 350, e1700153
Full Paper
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Some Novel Mannich Bases of
5
-(3,4-Dichlorophenyl)-1,3,4-oxadiazole-2(3H)-one
and Their Anti-Inflammatory Activity
1
1,2
1
3
4
Meric Koksal
, Irem Ozkan-Dagliyan , Tugce Ozyazici , Beril Kadioglu , Hande Sipahi ,
5
6
Ayhan Bozkurt , and Suleyman S. Bilge
1
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Yeditepe University, Istanbul, Turkey
Faculty of Medicine, Department of Biochemistry and Biophysics, University of North Carolina, Chapel
2
Hill, USA
3
Faculty of Pharmacy, Department of Pharmacology, Yeditepe University, Istanbul, Turkey
Faculty of Pharmacy, Department of Toxicology, Yeditepe University, Istanbul, Turkey
Faculty of Medicine, Department of Physiology, Ondokuz Mayis University, Samsun, Turkey
Faculty of Medicine, Department of Pharmacology, Ondokuz Mayis University, Samsun, Turkey
4
5
6
Non-steroidal anti-inflammatory drugs (NSAIDs), which are widely used for the treatment of
rheumatic arthritis, pain, and many different types of inflammatory disorders, cause serious
gastrointestinal (GI) side effects. The free carboxylic acid group existing on their chemical
structure is correlated with GI toxicity related with all routine NSAIDs. Replacing this functional
group with the 1,3,4-oxadiazole bioisostere is a generally used strategy to obtain an anti-
inflammatory agent devoid of GI side effects. In the present work, a novel group of 5-(3,4-
dichlorophenyl)-1,3,4-oxadiazole-2(3H)-one Mannich bases were synthesized and characterized on
1
the basis of IR, H NMR, and elemental analysis results. The target compounds were first tested
for cytotoxicity to determine a non-toxic concentration for anti-inflammatory screening. Anti-
inflammatory effects of the compounds were evaluated by in vitro lipopolysaccharide (LPS)-
induced NO production and in vivo carrageenan footpad edema with ulcerogenic profile. In
LPS-induced RAW 264.7 macrophages, most of the compounds showed inhibitory activity on
nitrite production while compounds 5a, 5h, and 5j exhibited the best profiles by suppressing the
NO production. To evaluate the in vivo anti-inflammatory potency of the compounds, the
inflammatory response was quantified by increment in paw size in the carrageenan footpad
edema assay. The anti-inflammatory data scoring showed that compounds 5a–d, 5g, and 5j, at the
dose of 100 mg/kg, exhibited anti-inflammatory activity, which for compound 5g was comparable
to that of the reference drug indomethacin with 53.9% and 55.5% inhibition in 60 and 120 min,
respectively.
Keywords: 1,3,4-Oxadiazole-2(3H)-one / Anti-inflammatory activity / Mannich reaction / Piperidine
Received: May 15, 2017; Revised: July 3, 2017; Accepted: July 4, 2017
DOI 10.1002/ardp.201700153
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
Correspondence: Dr. Meric Koksal, Faculty of Pharmacy,
Department of Pharmaceutical Chemistry, Yeditepe University,
Nitrogen and oxygen containing heterocyclic compounds are
well known as important building blocks in organic and
medicinal chemistry [1]. Especially, 1,3,4-oxadiazole is widely
3
4755 Atasehir, Istanbul, Turkey.
E-mail: merickoksal@yeditepe.edu.tr
Fax: þ90 216 5780068
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studied by the chemists due to its pharmacophore feature
for ligand binding [2, 3]. A great number of pharmacological
properties have been associated with this small but
important core structure such as anti-inflammatory
diseases such as asthma and arthritis. Therefore, inhibition of
NO production is important strategy for the screening of anti-
inflammatory agents [30–32].
In view of these observations and in continuation of our
research studies on the synthesis of 5-aryl-1,3,4-oxadiazole-
2(3H)-one Mannich bases [33, 34], we report herein the
synthesis of some novel 3,5-disubstituted-1,3,4-oxadiazole
derivatives which have been found to posses promising anti-
inflammatory activity with significantly reduced ulcerogenic
effect.
[
4–7], antimicrobial [8–11], antitubercular [12–14], and
anticancer [15–19]. Non-steroidal anti-inflammatory drugs
NSAIDs) are a good choice to use for the treatment of pain,
(
fever, and inflammation, particularly arthritis [20, 21].
Principal symptoms of inflammation such as erythema, raised
temperature, hyperalgesia, and pain which are due to
reaction of tissue harm, are caused by aggregation of
leucocytes and serum ingredients [22]. The pharmacological
activity of NSAIDs is related to the suppression of prostaglan-
din (PG) biosynthesis from arachidonic acid by inhibiting the
cyclooxygenase (COX) enzymes. Prostaglandins that cause
fever, pain, and inflammation are formed by cyclooxygenase-
Results and discussion
Chemistry
The preparation of target compounds (5a–j) is described in
Scheme 1. The key intermediate 5-(3,4-dichlorophenyl)-1,3,4-
oxadiazol-2(3H)-one (4) was synthesized in three steps.
Esterification of the 3,4-dichlorobenzoic acid (1) with ethanol
and concentrated sulfuric acid afforded the corresponding
ester(2).Thearoylhydrazide(3)wasobtainedbythereactionof
ethyl 3,4-dichlorobenzoate (2) with hydrazine hydrate mono-
hydrate (85%) in ethanol. Then the treatment of hydrazide (3)
with 1,1-carbonyldiimidazole (CDI) in the presence of triethyl-
amine (TEA) and tetrahydrofuran (THF) by stirring at room
temperature gave the key intermediate (4). The 3,5-disubsti-
tuted-1,3,4-oxadiazole-2(3H)-one derivatives (5a–j) were
prepared via Mannich reaction of 5-(3,4-dichlorophenyl)-
1,3,4-oxadiazol-2(3H)-one (4), substituted piperidines and
formaldehyde in ethanol [33].
2
(COX-2) isoenzyme whereas others regulate beneficial
gastrointestinal cytoprotection due to cyclooxygenase-1
COX-1) mechanism [23]. A prominent side effect on chronic
(
use of NSAIDs is generally attributed to two main factors:
decreased tissue prostaglandin production and local irritation
by the direct contact of carboxylic acid (–COOH) moiety of
NSAIDs with GI mucosal cells [24]. To overcome this problem,
synthetic approaches based upon chemical modifications of
free carboxylic acid group existing on NSAIDs is used to
improve their safety profile. The literature survey revealed
that replacing the free carboxylic functional group with 1,3,4-
oxadiazole enhances the anti-inflammatory activity and
decreases the gastric upset [25–27]. Furthermore, oxadiazole
nucleus enhances the interaction by formation of numerous
hydrogen bonds with cyclooxygenase receptor when it is
compared with carboxylic acid moiety [28, 29].
All of the compounds gave satisfactory analytical and
spectroscopic data, which were in full accordance with their
depicted structures. IR spectra of the synthesized compounds
are similar to the IR values of which were stated in the
literature [35, 36]. For the compounds 5a–j, no absorption
Nitric oxide (NO) is an important regulator in inflammatory
diseases. It gives an anti-inflammatory effect under normal
conditions whereas it causes tissue destruction and cellular
death in high amounts. NO increases in several inflammatory
ꢀ
1
band was detected at 3100–3400 cm , indicating the absence
Scheme 1. Synthesis of 5-(3,4-dichlorophenyl)-1,3,4-oxadiazole-2(3H)-one Mannich bases.
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of an NH group as an evidence for the substitution reaction to
-(3,4-dichlorophenyl)-1,3,4-oxadiazol-2(3H)-one with substi-
tuted piperidine. In the H NMR spectra of all compounds, the
methylene protons representing the Mannich base formation
were seen at about 4.70–4.80 ppm as a singlet. The protons of
the 3,4-dichlorophenyl group were seen approximately at
compounds, 5a–c, 5g–j significantly decreased nitrite
production while the strongest inhibition with 5a, 5h,
and 5j was almost the same as with the reference drug
indomethacin.
The phenyl substituted and carbonyl carrying compounds
(5a, 5h–j) were more active derivatives than the other bulky
piperidine analogs as NO production inhibitors. Furthermore,
benzyl and morpholine containing compounds were inactive
both in in vivo and in vitro anti-inflammatory activity
screening. This type of substitution in the core structure
might not provide the proper orientation at the receptor site
and lead to loss of potency.
5
1
2
5
7
7
.95 (d, J ¼ 2 Hz, 1H, H ), 7.65 (dd, J ¼ 8.2, 2 Hz, 1H, H ), and
0 0
6 3 5
.55 (d, J ¼ 8.8 Hz, 1H, H ) ppm, respectively. As H and H
protons of the piperidine ring are overlapped and seen as a
0
0
6
2
triplet peak at 1.23–1.86 ppm, H and H protons are seen at
.46–3.47 ppm likewise.
2
In vitro anti-inflammatory activity
Cell cytotoxicity
In vivo anti-inflammatory activity
Before the screening studies for inhibition of NO production,
it was essential to carry out cytotoxicity testing in order to
determine safe and non-toxic concentrations of every
compound. Therefore, compounds were analyzed for their
cytotoxic effects on RAW 264.7 macrophages by using the
MTT assay. Indomethacin, which is one of the most effective
anti-inflammatory agents, was used as the reference drug in
all biological studies. It was shown that IC50 values of the
tested compounds were higher than 100 mM (Table 1).
According to the results, in the series of ten compounds,
screening at 100 mM dose level is determined to be safe for NO
production inhibition and data recorded for this dose will not
be due to cytotoxicity against RAW 264.7 cells.
Carrageenan footpad edema
The anti-inflammatory activity of the synthesized compounds
was evaluated using carrageenan-induced rat paw edema
assay. Also, ulcerogenic effect on acute administration
was studied to assess the safety of the compounds.
The inhibition of edema was measured after 60, 120, 180,
and 240 min from the administration of the compounds. The
anti-inflammatory and ulcerogenic results are given in Table 2.
Results revealed that compounds 5a–d, 5g, and 5j
showed anti-inflammatory profile with meaningful statis-
tical results. It was also seen that substitution on fourth
position of piperidine moiety could change the anti-
inflammatory activity with variable edema inhibition
percentages (32–55.5%). In general, phenyl-substituted
analogs were more potent than the corresponding
carbonyl-substituted ones. Although these carboxylic acid
and ester derivatives were found active in in vitro anti-
inflammatory NO assay, absence of activity in carrageenan
footpad edema model except compound 5j might be
because of rapid metabolism of carboxylic acid and ester
derivatives. The compound 5g having piperidine moiety at
fourth position of piperidine ring showed the most
promising inhibition values, 53.9, 55.5, 41.2, and 40.9%,
respectively, in 60–240 min examination period of swelling
Nitrite assay
Anti-inflammatory activity of the tested compounds (5a–j)
were evaluated by measuring NO levels in LPS-stimulated
macrophage cells [37]. Every compound was tested at
1
00 mM concentration which would not cause any cytotoxic
effect at this dose concentration (Fig. 1). Among the tested
Table 1. Structures of the target compounds and
their IC50 values against RAW 264.7 macrophage cell
viability.
Compound
R
IC50 (mM)
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
4-Phenyl
4-Hydroxy-4-phenyl
4-Acetyl-4-phenyl
4-Cyano-4-phenyl
4-Benzyl
4-(Morpholin-4-yl)
4-(Piperidin-4-yl)
3-Carboxylic acid
4-Ethyloxycarbonyl
2-Ethyloxycarbonyl
–
146.35 ꢁ 3.94
150.66 ꢁ 7.56
140.64 ꢁ 5.45
151.07 ꢁ 4.64
147.60 ꢁ 4.91
146.38 ꢁ 3.45
152.17 ꢁ 9.62
127.16 ꢁ 4.89
141.33 ꢁ 4.47
146.05 ꢁ 0.45
>200
Figure 1. The amount of nitrite in LPS-induced RAW 264.7
macrophages. Results are expressed as the mean ꢁ SD (n ¼ 3).
j
ꢂ
ꢂꢂ
ꢂꢂꢂ
p < 0.05, p < 0.01, and
p < 0.001 versus the LPS-stimulated
Indomethacin
group.
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Table 2. Results of the carrageenan-induced paw edema test and incidence of gastrointestinal lesions in rats.
Anti-inflammatory activity swelling in thickness (mm) ꢁ SEM (inhibition %)
Ratio of
ulceration
a)
Compound
60 min
120 min
180 min
240 min
Control
2.01 ꢁ 0.15
2.53 ꢁ 0.19
2.90 ꢁ 0.19
3.02 ꢁ0.22
0/6
0/6
0/6
0/6
0/6
0/6
0/6
1/6
0/6
0/6
0/6
2/6
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
1.39 ꢁ 0.24
1.49 ꢁ 0.19 (41.1)
1.47 ꢁ 0.28 (42.1)
1.58 ꢁ 0.25 (37.5)
1.41 ꢁ 0.32 (44.2)
2.16 ꢁ 0.21
1.96 ꢁ 0.14 (32.6)
1.67 ꢁ 0.25 (42.6)
1.93 ꢁ 0.36
2.05 ꢁ 0.14 (32.0)
1.74 ꢁ 0.37 (42.3)
2.05 ꢁ 0.36
1.22 ꢁ 0.24
ꢂ
ꢂ
1.07 ꢁ 0.12 (46.9)
0.99 ꢁ 0.33 (51.1)
1.50 ꢁ 0.26
2.28 ꢁ 0.36
2.33 ꢁ 0.27
2.74 ꢁ 0.25
2.78 ꢁ 0.19
1.81 ꢁ 0.17
2.36 ꢁ 0.15
2.77 ꢁ 0.19
2.70 ꢁ 0.30
ꢂꢂ
ꢂꢂ
ꢂ
ꢂꢂ
0.93 ꢁ 0.21 (53.9)
1.77 ꢁ 0.30
1.13 ꢁ 0.18 (55.5)
2.45 ꢁ 0.26
1.71 ꢁ 0.20 (41.2)
2.82 ꢁ 0.27
1.78 ꢁ 0.15 (40.9)
2.83 ꢁ 0.29
1.83 ꢁ 0.26
2.09 ꢁ 0.30
2.85 ꢁ 0.38
2.76 ꢁ 0.36
ꢂ
ꢂꢂ
ꢂ
ꢂꢂ
ꢂ
ꢂꢂ
j
1.76 ꢁ 0.33
1.72 ꢁ 0.22 (32.3)
1.16 ꢁ 0.28 (54.3)
1.84 ꢁ 0.16 (36.7)
1.40 ꢁ 0.26 (51.8)
1.94 ꢁ 0.16 (35.5)
1.46 ꢁ 0.27 (51.7)
ꢂꢂ
INDO
1.01 ꢁ 0.15 (49.8)
a)
ꢂ
ꢂꢂ
Number of rats showing ulcer/total number of rats. p < 0.05, p < 0.01 compared to control group.
in thickness of foot paw edema. On the other hand,
replacement of this ring by morpholine resulted in loss of
activity. The compounds which were screened for their
in vivo anti-inflammatory activity were further tested for
their ulcerogenic liability. Results showed that all the
compounds (5a–j) exhibited a safe profile compared to
that of indomethacin.
a rational quantitative structure–activity relationship (QSAR)
mapping, future synthesis of similar derivatives will take place
to create a larger set of compounds.
Experimental
Chemistry
General
Conclusion
All the chemicals used for the synthesis of the compounds
were purchased from Sigma–Aldrich (Germany). Melting
points (°C) were determined by using a Mettler Toledo
FP62 capillary melting point apparatus (Mettler-Toledo,
Greifensee, Switzerland) and are uncorrected. Infrared
spectra were recorded on a Perkin-Elmer Spectrum One series
FTIR apparatus (Version 5.0.1) (Perkin-Elmer, Norwalk, CT,
USA), using potassium bromide pellets, the frequencies were
In this study, a new series of Mannich bases of 5-(3,4-
dichlorophenyl)-1,3,4-oxadiazole-2(3H)-one was synthesized
(
5a–j), tested for their biological effects as inhibitors of LPS-
induced NO production in RAW 264.7 macrophages and
evaluated for their anti-inflammatory profile in carra-
geenan-induced rat paw edema assay. In LPS-induced
macrophages, most of the compounds suppressed the
production of NO which can be useful for targeting the
treatment of anti-inflammatory diseases. Related to these
results, compounds were tested in a simple and routine
animal model for evaluation of activity at the site of
inflammation. The improvement of edema in the rat hind
paw and suppression of the release of NO following the
injection of carrageenan was observed with decrease of
swelling in thickness. Calculated inhibition % values showed
that anti-inflammatory activity profile was generally
parallel to in vivo NO assay except for the carboxylic acid
and ester carrying derivative which can rapidly undergo
biotransformation.
ꢀ
1
1
expressed in cm . The H NMR spectra were recorded with a
Varian Mercury-400 FT-NMR spectrometer (Varian Inc., Palo
Alto, CA, USA), using tetramethylsilane as the internal
3
reference, CDCl as solvent, the chemical shifts were reported
in parts per million (ppm). Coupling constants (J) were given in
hertz (Hz). Elemental analyses were performed on LECO 932
CHNS (Leco-932, St. Joseph, MI, USA) instrument. All the
compounds gave C, H, and N analysis within ꢁ0.4% of the
theoretical values.
The InChI codes of the investigated compounds together
with some biological activity data are provided as Supporting
Information.
As a conclusion, the current study showed that the
synthesized compounds could be used as a part of template
for future development through modification and derivati-
zation to design more potent anti-inflammatory compounds
that carry 1,3,4-oxadiazole core structure. In order to produce
Synthesis of 5-(3,4-dichlorophenyl)-1,3,4-oxadiazol-2(3H)-
one (4)
To a 0°C solution of 3,4-dichlorobenzohydrazide (2.2 mmol,
0.451 g) and triethylamine (TEA) (2.2 mmol) in THF (10 mL),
1,1-carbonyldiimidazole (CDI) was added. The resulting
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cm ): 3083–2827 (C–H), 2238 (C
ꢀN), 1774 (C –– O), 1609 (C –– N),
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Novel Mannich Bases of 5-(3,4-Dichlorophenyl)-1,3,4-oxadiazole-2(3H)-one
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mixture was stirred for 20 h at room temperature and
concentrated in vacuo. The residue was dissolved in diethyl
ether (15 mL), washed with 2 M hydrochloric acid (5 mL) and
saturated aqueous sodium bicarbonate, and then dried with
sodium sulfate. Filtration and concentration in vacuo gave
compound 4, which was recrystallized from ethanol/
water [33].
H
3
, H
J ¼ 8.4 Hz, dichlorophenyl H
dichlorophenyl H
), 7.93 (1H, d, J ¼ 2 Hz, dichlorophenyl H
Anal. calcd. for C22 : C, 59.20; H 4.74; N, 9.41. Found:
C, 59.45; H 4.86; N, 9.55.
4
, H
5
), 7.32–7.36 (2H, m, phenyl H
), 7.66 (1H, dd, J ¼ 8.4, J’ ¼ 2 Hz,
).
2 6
, H ), 7.56 (1H, d,
6
5
2
H
2 3 3
21Cl N O
5-(3,4-Dichlorophenyl)-3-[(4-cyano-4-phenylpiperidin-1-
yl)methyl]-1,3,4-oxadiazol-2(3H)-one (5d)
Synthesis of 5-(3,4-dichlorophenyl)-3-[(4-substituted-
piperidine-1-yl)methyl]-1,3,4-oxadiazol-2(3H)-ones (5a–j)
To a solution of 5-(3,4-dichlorophenyl)-1,3,4-oxadiazol-2(3H)-
one 4 (1 mmol, 0.231 g) in ethanol (10 mL), a mixture of
formaldehyde (1.5 mmol) and piperidine derivative (1 mmol)
in ethanol was added by stirring. After complete addition, the
mixture was refluxed for 4 h. The solution was precipitated by
cooling, the formed compound was filtered and crystallized
with ethanol/water.
White solid; yield: 61%; m.p. 128.9°C; IR (nmax, KBr pellets,
ꢀ
1
ꢀ
ꢀ
1
1254 (C–O). H NMR (CDCl
, H
(2H, d, J ¼ 12.4 Hz, piperidine H
methylene), 7.34–7.36 (3H, m, phenyl H
J ¼ 8.4 Hz, phenyl H , H ), 7.58 (1H, d, J ¼ 8 Hz, dichlorophenyl
), 7.70 (1H, dd, J ¼ 8, J’ ¼ 2 Hz, dichlorophenyl H ), 7.97
(1H, d, J ¼ 2 Hz, dichlorophenyl ). Anal. calcd. for
: C, 58.75; H 4.23; N, 13.05. Found: C, 57.99;
3
) d
H
: 2.12–2.15 (4H, m, piperidine
H
3
5
), 2.98 (2H, t, J ¼ 10 Hz, piperidine H
, H equatorial), 4.77 (2H, s,
, H , H ), 7.50 (2H, d,
2 6
, H axial), 3.18
2
6
3
4
5
2
6
H
6
5
H
2
22 2 3 3
C H21Cl N O
5
-(3,4-Dichlorophenyl)-3-[(4-phenylpiperidin-1-yl)-
H 4.07; N, 12.96.
methyl]-1,3,4-oxadiazol-2(3H)-one (5a)
White solid; yield: 78%; m.p. 153.4°C; IR (nmax, KBr pellets,
cm ): 3059–2806 (C–H), 1773 (C–O), 1604 (C–N), 1245 (C–O);
5-(3,4-Dichlorophenyl)-3-[(4-benzylpiperidin-1-yl)methyl]-
1,3,4-oxadiazol-2(3H)-one (5e)
ꢀ
1
1
H NMR (CDCl
3
) d
H
: 1.81 (2H, t, J ¼ 12.6 Hz, piperidine H
, H axial),
), 2.59 (2H, t, J ¼ 11.6 Hz,
equatorial), 3.17 (2H, d, J ¼ 11.6 Hz,
axial), 4.77 (2H, s, methylene), 7.18–7.12
, H , H ), 7.28–7.31 (2H, m, phenyl H , H ),
.57 (1H, d, J ¼ 8.4 Hz, dichlorophenyl H ), 7.70 (1H, dd, J ¼ 8,
), 7.97 (1H, d, J ¼ 2 Hz, dichlor-
). Anal. calcd. for C20 : C, 59.42; H, 4.74;
N, 10.39. Found: C, 59.36; H, 4.56; N, 10.56.
3
, H
5
White solid; yield: 75%; m.p. 134.6°C; IR (nmax, KBr pellets,
ꢀ
1
equatorial), 1.89 (2H, d, J ¼ 10.4 Hz, piperidine H
3
5
cm ): 3092–2806 (C–H), 1777 (C–O), 1607 (C–N), 1248 (C–O).
1
2
.42–2.48 (1H, m, piperidine H
4
H NMR (CDCl
3
) d
H
: 1.28 (2H, qd, J ¼ 12 Hz, piperidine H
), 1.67 (2H, d, J ¼ 13.2 Hz,
equatorial), 2.39 (2H, t, J ¼ 12.0 Hz, piperi-
axial), 2.52 (2H, d, J ¼ 7.2 Hz, benzyl –CH ), 3.01
(2H, d, J ¼ 11.6 Hz, piperidine H , H equatorial), 4.70 (2H, s,
methylene), 7.11 (2H, d, J ¼ 6.8 Hz, benzyl H , H ), 7.15–7.27
(3H, m, benzyl H , H , H
), 7.55 (1H, d, J ¼ 8.8 Hz, dichlor-
ophenyl H ),
), 7.66 (1H, dd, J ¼ 8, J’ ¼ 2 Hz, dichlorophenyl H
.93 (1H, d, J ¼ 2 Hz, dichlorophenyl H ). Anal. calcd. for
: C, 60.30; H, 5.06; N, 10.05. Found: C, 60.22; H,
5.43; N, 10.17.
3 5
, H
piperidine
piperidine H
H
2
,
H
6
axial), 1.45–1.56 (1H, m, piperidine H
piperidine H , H
dine H , H
4
2
, H
6
3
5
(
3H, m, phenyl H
2
4
6
3
5
2
6
2
7
6
2
6
J’ ¼ 2 Hz, dichlorophenyl H
5
2
6
ophenyl H
2
H19Cl
2
N
3
O
2
3
4
5
6
5
7
2
5
-(3,4-Dichlorophenyl)-3-[(4-hydroxy-4-phenylpiperidin-1-
21 2 3 2
C H21Cl N O
yl)methyl]-1,3,4-oxadiazol-2(3H)-one (5b)
White solid; yield: 75%; m.p. 194.6°C; IR (nmax, KBr pellets,
ꢀ
1
cm ): 3540 (O–H), 3094–2836 (C–H), 1768 (C–O), 1604 (C–N),
5-(3,4-Dichlorophenyl)-3-{[4-(morpholin-4-yl)piperidin-1-
yl]methyl}-1,3,4-oxadiazol-2(3H)-one (5f)
1
1
247 (C–O). H NMR (CDCl
3
) d
axial), 2.12–2.20 (2H, m, piperidine H
equatorial), 2.96 (4H, d, J ¼ 8.4 Hz, piperidine H , H ), 4.78
2H, s, methylene), 7.28 (1H, d, J ¼ 7.2 Hz, phenyl H ), 7.37 (2H,
t, J ¼ 7.6 Hz, phenyl H , H ), 7.50 (2H, d, J ¼ 8 Hz, phenyl H , H ),
.57 (1H, d, J ¼ 8.4 Hz, dichlorophenyl H ), 7.69 (1H, dd, J ¼ 8.4,
J’ ¼ 2 Hz, dichlorophenyl H ), 7.97 (1H, d, J ¼ 2 Hz, dichlor-
ophenyl H ). Anal. calcd. for C20 : C, 57.15; H, 4.56;
N, 10.00. Found: C, 56.77; H, 4.61; N, 10.07.
H
: 1.81 (2H, d, J ¼ 14 Hz,
piperidine H
3
, H
5
3
, H
5
White solid; yield: 59%; m.p. 134.6°C; IR (nmax, KBr pellets,
ꢀ1
cm ): 3097–2821 (C–H), 1786 (C –– O), 1609 (C –– N), 1258
2
6
1
(
4
(C–O). H NMR (CDCl
3
) d
H
: 1.52–1.59 (2H, m, piperidine H
3
,
3
5
2
6
H
5
axial), 1.86 (2H, d, J ¼ 12.8 Hz, piperidine
equatorial), 2.11–2.16 (1H, m, piperidine H ), 2.46 (2H, t,
J ¼ 11.6 Hz, piperidine H , H axial), 2.53 (4H, t, J ¼ 4.8 Hz,
morpholine H , H
), 3.10 (2H, d, J ¼ 11.6 Hz, piperidine H
equatorial), 3.71 (4H, t, J ¼ 4.4 Hz, morpholine H , H
.71 (2H, s, methylene), 7.56 (1H, d, J ¼ 8.4 Hz, dichlor-
ophenyl H
), 7.68 (1H, dd, J ¼ 8.8, J’ ¼ 2 Hz, dichlorophenyl
), 7.95 (1H, d, J ¼ 2 Hz, dichlorophenyl H ). Anal. calcd. for
: C, 52.31; H, 5.37; N, 13.56. Found: 52.26; H,
5.52; N, 13.92.
3 5
H , H
7
6
4
5
2
6
2
H19Cl
N
2 3
O
3
3
5
2
,
H
4
6
3
5
),
5
-(3,4-Dichlorophenyl)-3-[(4-acetyl-4-phenylpiperidin-1-
yl)methyl]-1,3,4-oxadiazol-2(3H)-one (5c)
6
H
5
2
White solid; yield: 52%; m.p. 167.5°C; IR (nmax, KBr pellets,
18 2 4 3
C H22Cl N O
ꢀ
1
cm ): 3078–2819 (C–H), 1776 (C–O), 1698 (C–O, acetyl), 1606
1
(
(
C–N), 1252 (C–O). H NMR (CDCl
3
) d
, H
equatorial), 2.68 (2H, t, J ¼ 12 Hz,
axial), 2.92–2.96 (2H, m, piperidine H , H
H
: 1.88 (3H, s, –CH
3
), 2.06
2H, t, J ¼ 14.8 Hz, piperidine H
, H
3
5
axial), 2.50 (2H, d,
5-(3,4-Dichlorophenyl)-3-{[4-(piperidin-4-yl)piperidin-1-yl]-
methyl}-1,3,4-oxadiazol-2(3H)-one (5g)
J ¼ 12.4 Hz, piperidine H
3
5
piperidine H , H
2
6
2
6
White solid; yield: 63%; m.p. 165.6°C; IR (nmax, KBr pellets,
ꢀ
1
equatorial), 4.68 (2H, s, methylene), 7.27–7.29 (3H, m, phenyl
cm ): 3091–2816 (C–H), 1781 (C–O), 1611 (C–N), 1253 (C–O).
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1
H NMR (CDCl
3
) d
H
: 1.22–1.25 (2H, m, piperidinyl H
, H axial), 1.54–1.64 (4H, m, piperidinyl
), 1.84 (2H, d, J ¼ 12 Hz, piperidinyl H , H equatorial),
.19–2.25 (1H, m, piperidine H
), 2.41 (2H, t, J ¼ 12 Hz,
piperidine H , H
axial), 2.50 (4H, t, J ¼ 4.8 Hz, piperidine
, H , H equatorial),
), 3.09 (2H, d, J ¼ 11.6 Hz, piperidine H
.70 (1H, s, methylene), 5.30 (1H, s, methylene), 7.56 (1H,
dd, J ¼ 8, J’ ¼ 2.4 Hz, dichlorophenyl H ), 7.67–7.69 (1H, m,
dichlorophenyl H ).
), 7.94 (1H, d, J ¼ 2 Hz, dichlorophenyl H
Anal. calcd. for C19 : C, 55.48; H, 5.88; N, 13.62.
Found: C, 54.07; H, 6.03; N, 13.65.
4
), 1.42–1.46
calcd. for C17
51.02; H, 4.92; N, 10.68.
2 3 4
H19Cl N O : C, 51.01; H, 4.78; N, 10.50. Found: C,
(
H
2H, m, piperidinyl H
, H
2
6
3
5
2
6
2
4
Biological evaluation
Cell culture
RAW 264.7 macrophages were obtained from American Type
Culture Collection (ATCC). The cells were cultured in DMEM,
supplemented with 10% FBS and 1% streptomycin and
2
6
H
4
3
5
2
6
6
5
2
2
penicillin at 37°C in 5% CO .
H
2 4 2
24Cl N O
Cell cytotoxicity
4
RAW 264.7 cells at the density of 2 ꢃ 10 cells per well with
5
00 mL of culture medium were incubated for 24 h. The cells
1
-{[5-(3,4-Dichlorophenyl)-2-oxo-1,3,4-oxadiazol-3(2H)-yl]-
were treated with compounds for 24 h and then added 100 mL
of 0.5 mg/mL MTT (AppliChem, Germany) for 2 h. The MTT
solution was removed and 100 mL of isopropanol (Sigma–
Aldrich, Germany) were added in each well and optical
absorbance was measured at 570 nm.
methyl}piperidine-3-carboxylic acid (5h)
White solid; yield: 58%; m.p. 171.3°C. IR (nmax, KBr pellets,
ꢀ
1
cm ): 3100 (OH), 2950–2837 (C–H), 1796 (C–O, RCOOH), 1713
C–O), 1614 (C–N), 1244, 1199 (C–O). 1H NMR (CDCl
) d
axial), 1.75–1.79 (1H, m,
equatorial), 2.42 (2H, t, J ¼ 12.4 Hz, piperidine
axial, H ), 2.81 (1H, d,
axial), 3.02 (1H, dd, J ¼ 11.2, J’ ¼ 3.2
equatorial), 3.42–3.47 (1H, m, piperidine H
equatorial), 4.66 (2H, s, methylene), 7.77 (1H, dd, J ¼ 8.4,
J’ ¼ 2 Hz, dichlorophenyl H ), 7.83 (1H, d, J ¼ 8.8 Hz, dichlor-
ophenyl H ). Anal.
), 7.95 (1H, d, J ¼ 2 Hz, dichlorophenyl H
calcd. for C15 : C, 48.40; H, 4.06; N, 11.29. Found: C,
7.43; H, 4.30; N, 10.84.
(
3
H
:
1
.63–1.68 (1H, m, piperidine H
5
piperidine H
5
Nitrite assay
H
4
), 2.50–2.56 (2H, m, piperidine H
J ¼ 11.2 Hz, piperidine H
Hz, piperidine H
6
3
RAW 264.7 cells were seeded into a 48-well culture plate at
5
2
the density of 1 ꢃ 10 cells per well with 500 mL of culture
6
2
medium and incubated for 24 h. The cells were then
pretreated with compounds (5a–j) and reference molecule,
indomethacin, at 100 mM for 2 h before stimulation with LPS
(1 mg/mL) for 22 h. The nitrite concentration in the medium
was measured by adding 50 mL Griess reagent [1% sulfanil-
amide (Sigma–Aldrich, USA) and 0.1% N-(1-naphthyl)ethyl-
enediamine dihydrochloride (Sigma–Aldrich, USA) in 5%
phosphoric acid (Mettler, Switzerland)] to 50 mL of medium
for 10 min. The absorbance at 570 nm was then measured
using a microplate reader (Microplate photometer, Multiskan
Ascent, Finland). The amount of nitrite in the test samples was
calculated from a sodium nitrite (Fluka Chemika, Germany)
standard curve.
6
5
2
2 3 4
H15Cl N O
4
Ethyl 1-{[5-(3,4-dichlorophenyl)-2-oxo-1,3,4-oxadiazol-
(2H)-yl]methyl}piperidine-4-carboxylate (5i)
White solid; yield: 54%; m.p. 96.9°C; IR (nmax, KBr pellets,
3
ꢀ
1
cm ): 3068–2772 (C–H), 1760 (C–O, RCOOR’), 1732 (C–O),
1
1
613 (C–N), 1213 (C–O). H NMR (CDCl
3
) d
H
: 1.24 (3H, t,
, H axial),
equatorial),
J ¼ 10.8 Hz, –CH
3
), 1.73–1.80 (2H, m, piperidine H
3
5
1
2
.94 (2H, dd, J ¼ 8.8, J’ ¼ 3.2 Hz, piperidine H
.20–2.26 (1H, m, piperidine H
, H
axial), 3.04 (2H, d, J ¼ 12 Hz, piperidine H
equatorial), 4.12 (2H, q, COO–CH –), 4.71 (2H, s, methylene),
), 7.68 (1H, dd, J ¼ 8.4,
), 7.95 (1H, d, J ¼ 2 Hz, dichlor-
: C, 51.01; H, 4.78;
3
, H
5
All results were expressed as the mean ꢁ SD of experiments.
Statistical significance was determined by one-way ANOVA
followed by Tukey’s test using a computerized statistical
program. The data were considered statistically significant if
p < 0.05.
4
), 2.49 (2H, t, J ¼ 11.2 Hz,
, H
piperidine H
2
6
2
6
2
7
.56 (1H, d, J ¼ 8 Hz, dichlorophenyl H
6
J’ ¼ 2 Hz, dichlorophenyl H
5
ophenyl H
2
). Anal. calcd. for C17H19Cl
N
2 3
O
4
In vivo anti-inflammatory activity assays
Animals
N, 10.50. Found: C, 50.88; H, 4.76; N, 10.67.
Sexually mature, 10- to 14-week-old male Sprague-Dawley rats
(weighing 250–300 g) were obtained from Ondokuz Mayis
University (Samsun, Turkey) vivarium sources. Animals were
housed per group (n¼ 6, total 90 rats) in a quiet, temperature-
and humidity-controlled room (22°C and 60 ꢁ 5%, respectively)
in a 12-h light/dark cycle, receiving food and water ad libitum.
All procedures and protocols were conducted in accordance
with the Guide forthe Care and Useof LaboratoryAnimals (NIH
publication 865-23, Bethesda, MD, USA).
Ethyl 1-{[5-(3,4-dichlorophenyl)-2-oxo-1,3,4-oxadiazol-
(2H)-yl]-methyl}piperidine-2-carboxylate (5j)
White solid; yield: 59%; m.p. 80.7°C; IR (nmax, KBr pellets,
3
ꢀ
1
cm ): 3087–2860 (C–H), 1788 (C –– O, RCOOR’), 1732 (C –– O),
1
1
614 (C –– N), 1215, 1187 (C–O). H NMR (CDCl
3
) d
H
: 1.31 (3H, t,
, H , H ),
axial), 3.19–3.24 (1H, m,
equatorial), 3.45 (1H, q, piperidine H ), 4.17–
–), 4.73 (1H, d, J ¼ 14.4 Hz, methylene),
.96 (1H, d, J ¼ 14 Hz, methylene), 7.56 (1H, d, J ¼ 8.4 Hz,
), 7.66 (1H, dd, J ¼ 8, J’ ¼ 2 Hz, dichlor-
), 7.93 (1H, d, J ¼ 2 Hz, dichlorophenyl H ). Anal.
J ¼ 7.2 Hz, –CH
3
), 1.56–1.80 (6H, m, piperidine H
3
4
5
2
.66–2.72 (1H, m, piperidine H
6
piperidine H
6
2
4
.27 (2H, m, COO–CH
2
4
Carrageenan footpad edema
The rats were deprived of food for 12 h prior to the beginning
of experiments. Paw edema was induced by subplantar
dichlorophenyl H
ophenyl H
6
5
2
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injection of 100 mL of 1% sterile carrageenan lambda (Sigma,
St. Louis, MO, USA) in saline into the right hind paw [24]. The
edema component of inflammation was quantified by
measuring the difference in hind footpad thickness before
carrageenan injection and at 60, 120, 180, and 240 min after
carrageenan injection with a micrometer caliper. Vehicle
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0.5% carboxymethyl cellulose, 1 mL/rat) or drugs (100 mg/kg)
were administered orally by gastric intubation to rats
0 min before carrageenan administration. Indomethacin
CAS 53-86-1) was used as a standard at the dose of 10 mg/
3
(
kg by the same application route.
2
010, 45, 2063–2074.
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Statistical analysis
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(
GraphPad software, USA). Following the assurance of
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The authors are grateful to Assoc. Prof. Dilek Telci for proving
RAW 264.7 macrophage cells.
[
The authors have declared no conflicts of interest.
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