Synthesis and antinociceptive activities of some novel benzimidazole-piperidine derivatives

Article abstract of DOI:10.3906/kim-1612-76

In this study, a series of benzimidazole-piperidine derivatives were synthesized with the objective of developing potent antinociceptive agents. Some 2-(4-substituted-phenyl)-1-[2-(piperidin-1-yl)ethyl]-¹H-benzimidazole derivatives were obtaine

Full text of DOI:10.3906/kim-1612-76

Turk J Chem
Turkish Journal of Chemistry
(2017) 41: 672 – 684
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1612-76
Research Article
Synthesis and antinociceptive activities of some novel benzimidazole-piperidine
derivatives
1
1,∗
1
2
¨
˙
¨
¨
˘
Umide DEMIR OZKAY , Ozgu¨r Devrim CAN , Nazlı TURAN , Betu¨l KAYA C¸ AVUS¸OGLU
1Department of Pharmacology, Faculty of Pharmacy, Anadolu University, Eski¸sehir, Turkey
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eski¸sehir, Turkey
Received: 27.12.2016
•
Accepted/Published Online: 29.03.2017
•
Final Version: 10.11.2017
Abstract: In this study, a series of benzimidazole-piperidine derivatives were synthesized with the objective of developing
potent antinociceptive agents. Some 2-(4-substituted-phenyl)-1-[2-(piperidin-1-yl)ethyl]-1H -benzimidazole derivatives
were obtained by microwave-supported reaction of an appropriate 2-(4-substituted-phenyl)-1H -benzimidazole with 2-
(piperidine-1-yl)ethyl chloride. The chemical structures of the compounds were elucidated by FT-IR, 1 H NMR, 13 C
NMR, and HRMS spectral data. Antinociceptive activity was assessed by conducting hot-plate, paw-pressure, and
formalin tests. Morphine (5 mg/kg, i.p) was used as a reference drug. Among the tested compounds 2a–2d and 2f–2h
(10 mg/kg) increased the maximum possible effect (MPE)% values calculated for the hot-plate and paw-pressure tests and
decreased the paw licking time of rats in the early phase of the formalin test, indicating centrally mediated antinociceptive
activities of these derivatives. In the late-phase 2g and 2h were the only compounds reducing the paw licking duration.
These data show additional peripherally mediated antinociceptive activities for these two derivatives. Falling latencies of
animals in the rotarod test did not change upon the administration of test compounds; thus, the observed antinociceptive
effects were specific. Predictions obtained by theoretical calculations of ADME (absorption, distribution, metabolism,
excretion) properties supported the antinociceptive potential of the tested benzimidazole-piperidine derivatives.
Key words: Benzimidazole, formalin test, hot-plate test, paw-pressure test, piperidine, rotarod
1. Introduction
Heterocyclic systems are frequently preferred structures for the synthesis of novel molecules with pharmaco-
logical activity potential. Among them, N -based heterocycles are especially important since many of the
biologically active compounds such as alkaloids, glycosides, and hormones as well as some of the clinically used
drugs carry N -containing heterocycles in their chemical structures.1,2 As one of the N -based heterocycles,
the piperidine ring system has been shown to possess various pharmacological effects such as antibacterial,
antifungal,3−5 anti-HIV,6 antileishmanial,7 anticancer,8,9 renin inhibitory,10 diuretic, and natriuretic11 ef-
fects. Another N -containing heterocyclic structure, benzimidazole, is also known to have the ability to inter-
act with biomolecules of living systems.12 There are many benzimidazole derivative drugs with a wide range
of biologically activities,13−16 such as omeprazole (proton pump inhibitor),17 albendazole (anthelminthic),18
domperidone (antiemetic, gastroprokinetic),19 and pimozide (antipsychotic).20
Recent studies screening the pharmacological activity capacity of various compounds bearing piperidine
rings in their structure have pointed out a notable therapeutic potential of these derivatives on the central ner-
∗Correspondence: ozgurdt@anadolu.edu.tr
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vous system. Cognitive enhancer,21,22 anti-Alzheimer,23 neuroprotective,24 antidepressant-like,25 anxiolytic-
like,26 antipsychotic,27 anticonvulsant,28,29 antiobesity,30 antipyretic,31,32 and wake-promotion activities21 of
various piperidine derivatives have been demonstrated, so far. Antinociception is another pharmacological ac-
tivity induced by compounds carrying piperidine moiety.33 Piperidine derivatives have been shown to possess
antinociceptive effects against acute nociceptive stimuli.31,33−35 Furthermore, they have also been reported
for their efficacy on chronic neuropathic and inflammatory pain.36−38 Central mechanisms of pain seem to
be involved in the antinociceptive effect of various piperidine-derivative compounds.33−36 On the other hand,
peripheral mechanisms of nociception should not be ruled out since numerous piperidine derivatives have been
shown to suppress peripheral inflammation and pain processes.32,36
In this study, based on this current literature indicating the therapeutic potential of piperidine derivatives
for pain disorders as well as the pharmacological activity potential of the benzimidazole core ring, we designed
and synthesized some benzimidazole-piperidine compounds. Then, with the aim of discovering and developing
new analgesic drug candidates, we screened the antinociceptive activities of these novel benzimidazole-piperidine
derivatives by using some well-known in vivo nociceptive tests.
2. Results and discussion
The synthetic route of title compounds (2a–2h) is presented in Figure 1. Condensation of 1,2-phenylenediamine
with diverse sodium bisulfide adducts of benzaldehydes under microwave conditions gave the 2-(4-substituted-
phenyl)-1H -benzimidazole derivatives (1a–1h). In the next step, compounds 1a–1h were reacted with 2-
(piperidine-1-yl)ethyl chloride in the presence of NaH under microwave conditions. Some characteristic proper-
ties of the intermediate and final compounds are given in Table 1. The synthesized compounds (2a–2h) were
characterized by FT-IR, HRMS, 1 H NMR, and 13 C NMR spectroscopic methods. In the IR spectra of the
compounds, stretching bands belonging to C = N and C = C were observed between 1620 and 1442 cm−1 . The
out-of-plane deformation bands belonging 1,4-disubstituted benzene were recorded at 861–819 cm−1 . HRMS
results agreed well with the calculated molecular formula of compounds 2a–2h.
Figure 1. Synthesis of the compounds 2a–2h.
In the 1 H NMR spectra of compound 2a, the 3rd, 4th, and 5th position protons of piperidine were
observed as a broad singlet peak at 1.30 ppm while the 2nd and 6th position protons close to nitrogen were
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Table 1. The percentage of yields and melting points of the compounds (1a–1h, 2a–2h).
Mp (◦C)
Compound
Yield (%) Compound Mp (◦C) Yield (%)
Literature
270–272
273–275
290–292
250–251
261–262
280–281
222–225
149–151
Found
39
40
39
41
39
42
39
43
1a
1b
1c
1d
1e
1f
274–277
275–278
287–288
253–255
261–263
281–284
223–226
152–155
75
69
78
73
80
83
85
70
2a
2b
2c
2d
2e
2f
72–74 69
128–133 74
88–91
68–72
80
76
138–140 72
102–104 66
100–103 65
1g
1h
2g
2h
63–66
70
detected at 2.20 ppm as a broad singlet peak. Methyl group protons attached to the phenyl ring were assigned
as a singlet at 2.40 ppm. A triplet peak due to the 1st CH2 protons belonging to the ethyl moiety between
piperidine and the benzimidazole ring was observed at 2.56 ppm and the 2nd CH2 protons of this moiety were
detected at 4.35 as a triplet. In the aromatic region the 5th and 6th position protons of the benzimidazole
ring were assigned at 7.19–7.29 ppm as a multiplet whereas the 4th and 7th position protons of this ring were
assigned at 7.60–7.67 as a multiplet. Two doublets belonging to the phenyl ring were detected at 7.37 ppm
due to 3rd and 5th position protons and at 7.72 ppm due to 2nd and 6th position protons. In the 13 C NMR
spectra of compound 2a, a signal due to methyl carbon was observed at 21.43 ppm. The other aliphatic carbons
owing to the piperidine ring and ethyl moiety were detected at 24.23, 25.87, 42.63, 54.67, and 57.78 ppm. The
aromatic carbons belonging to benzimidazole and phenyl rings were observed at 111.33, 119.48, 122.27, 122.65,
128.27, 129.61, 129.66, 136.17, 139.67, 143.13, and 153.96 ppm. In the aromatic region, it is difficult to assign
a signal to each carbon because of the similar ppm values of carbons.
In the 1 H NMR and 13 C NMR spectra of the other compounds, similar to the above elucidation, 2nd
and 6th position protons of piperidine were observed as a broad singlet between 2.13 and 2.20 ppm, whereas 3rd,
4th, and 5th position protons of piperidine were recorded between 1.24 and 1.35 ppm as a broad singlet. The
protons of ethylene between piperidine and benzimidazole were assigned as triplet peaks at 2.53–2.60 ppm and
4.35–4.42 ppm, respectively. The other aromatic and aliphatic protons were observed in the expected regions. In
the 13 C NMR spectra, the carbon belonging to the 4th position of piperidine was assigned at 24.17–24.28 ppm.
The carbons at the 3rd and 5th positions of piperidine were observed between 25.76 and 25.94 ppm, whereas the
2nd and 6th position carbons of piperidine were observed at 54.67–57.72 ppm. The peaks belonging to carbons
between piperidine and benzimidazole were recorded at 42.61–57.87 ppm. Aromatic carbons were generally
observed at 110.99–164.91 ppm. In the spectra, splitting associated with neighboring atoms was confirmed
owing to the presence of fluoro in compound 2d.
Following the synthesis and structure elucidation, possible antinociceptive activities of the benzimidazole-
piperidine derivatives were evaluated by using several well-established nociception assays. In experimental
animals, sensation of pain is generally evaluated by monitoring motor responses ranging from spinal reflexes to
complex behaviors. In different pain models mechanical, thermal, chemical, or electrical nociceptive stimuli can
be used as “noxious stimuli”.44 Based on this knowledge, we examined the antinociceptive activity potential of
our novel benzimidazole-piperidine derivatives by using hot-plate, Randall–Selitto paw-pressure, and formalin-
induced paw licking tests.
The hot-plate method is used for assessing the response of animals to acute thermal noxious stimuli. Ob-
tained data showed that benzimidazole-piperidine derivative test compounds 2a–2d and 2f–2h, administrated
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at 10 mg/kg doses, induced significant augmentations in the reaction time (maximum possible effect (MPE)%)
of rats (Figure 2). Enhancements of the MPE% values demonstrated the antinociceptive effects of these com-
pounds on nociceptive pathways carrying thermal noxious stimuli. Findings of the hot-plate test also suggested
a centrally mediated antinociceptive activity profile for these compounds, since this test predominantly measures
supraspinally organized nociceptive signaling.45,46
40
30
***
**
20
10
0
**
*
*
*
*
*
-10
f
l
c
e
r
a
2
g
b
d
h
2
2
2
o
2
2
2
2
ro
t
M
n
o
C
Figure 2. Effects of test compounds (10 mg/kg) and morphine (5 mg/kg) on MPE% values of rats in the hot-plate test.
Significance against control group: *P < 0.05, **P < 0.01, ***P < 0.001. Values are given as mean ꢀ SEM. One-way
ANOVA, post hoc Tukey’s test, n = 7.
In this study, the reaction of the animals to acute mechanical noxious stimuli was evaluated using the
Randall–Selitto paw-pressure test. Obtained results demonstrated that administration of compounds 2a–2d
and 2f–2h induced significant increases in the calculated MPE% values of rats (Figure 3), indicating the
antinociceptive effects of these compounds on nociceptive pathways carrying mechanical noxious stimuli. Similar
to hot-plate tests, results of the paw-pressure tests also suggested centrally mediated antinociceptive activity
profiles for the test compounds, since, in the paw-pressure test, pain caused by the compression of the hind paw
is centrally mediated and is attributed to the direct stimulation of nociceptor afferent fibers.44,47
Antinociceptive efficacy of the compounds against acute chemical noxious stimuli was evaluated using the
formalin-induced paw licking test, which is also a well-established method in the elucidation of a compound’s
mechanism of action at both the peripheral and central levels.48 The obtained findings showed that compounds
2a–2d and 2f–2h significantly shortened the paw licking time of animals measured in the early phase compared
to the control group (Figure 4; Table 2). This finding supported the experimental results of the hot-plate and
the paw-pressure tests since the early phase is characterized by “neurogenic pain”, which is mediated by direct
stimulation of nociceptors in the paw and reflects centrally mediated pain.48,49
Among the tested compounds, 2g and 2h also decreased the paw licking time of rats measured in the
late phase (Figure 5; Table 2). Since the late phase of the formalin test is characterized by “inflammatory
pain”, which is caused by the release of algogenic substances from damaged local tissues, this second phase
reflects peripherally mediated pain.48,49 Therefore, different from compounds 2a, 2b, 2c, 2d, and 2f, the
antinociceptive activities of compounds 2g and 2h are related to the participation of peripheral mechanisms as
well as central ones.
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40
30
20
10
0
100
***
75
**
***
**
**
*
*
*
*
**
50
25
0
***
***
***
***
***
***
-10
f
l
c
e
r
a
g
b
2
d
2
h
2
2
2
o
2
2
f
2
l
c
e
r
a
2
g
2
b
2
d
2
h
2
ro
2
2
2
o
t
ro
M
t
M
n
n
o
o
C
C
Figure 3. Effects of test compounds (10 mg/kg) and
Figure 4. Effects of test compounds (10 mg/kg) and
morphine (5 mg/kg) on paw licking time of rats in the early
phase of the formalin test. Significance against control
Group: *P < 0.05, **P < 0.01, ***P < 0.001. Values
are given as mean ꢀ SEM. One-way ANOVA, post hoc
Tukey’s test, n = 7.
morphine (5 mg/kg) on MPE% values of rats in the paw-
pressure test. Significance against control group: *P <
0.05, **P < 0.01, ***P < 0.001. Values are given as
mean ꢀ SEM. One-way ANOVA, post hoc Tukey’s test,
n = 7.
Table 2. Inhibition % values of experimental groups in the early and late phases of the formalin test.
Early phase Late phase
Treatment
inhibition % inhibition %
Morphine
76.04
60.52
60.81
47.81
76.81
8.50
69.12
49.44
74.53
80.98
26.45
35.60
35.64
9.53
14.52
41.14
46.49
56.28
2a
2b
2c
2d
2e
2f
2g
2h
In all of the nociceptive tests, reference drug morphine sulfate exhibited its antinociceptive efficacy as
expected (Figures 2–5 and Table 2).
Data obtained from the rotarod test did not suggest any alteration in the motor coordination of rats,
indicating that results of the nociceptive tests are specific. In other words, the observed antinociceptive activities
were not affected by any nonspecific sedative or neuromuscular blocker effects caused by the tested compounds.
Theoretically predicted ADME properties of the tested benzimidazole-piperidine derivatives (2a–2h),
namely molecular weight, log P, topological polar surface area (tPSA), number of hydrogen donors and acceptors,
volume, and number of rotatable bonds, are presented in Table 3 along with violations of Lipinski’s rule.50,51
This rule suggests that an orally active drug should not possess more than one violation. Hence, according to
the data presented in Table 3, all compounds 2a–2h are compatible with Lipinski’s rule.
Moreover, it has been determined that the test compounds have ideal lipophilic characters suitable for
crossing to the central nervous system. The tPSA values, described to be a predictive indicator of membrane
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300
250
200
150
100
50
*
**
***
0
f
l
c
e
r
a
g
b
d
h
2
2
o
2
2
o
M
2
2
2
2
r
t
n
Co
Figure 5. Effects of test compounds (10 mg/kg) and morphine (5 mg/kg) on paw licking time of rats in the late phase
of the formalin test. Significance against control group: *P < 0.05, **P < 0.01, ***P < 0.001. Values are given as
mean ꢀ SEM. One-way ANOVA, post hoc Tukey’s test, n = 7.
Table 3. In silico physicochemical parameters of compounds 2a–2h.
Com
2a
2b
2c
2d
2e
2f
2g
R
-CH3
-N(CH3)2 4.65
-Cl
-F
Log P TPSA MW
nON nOHNH nrotb Volume Vio
5
21.06
24.30
21.06
21.06
44.86
21.06
30.30
30.30
319.45
348.49
339.87
323.42
330.44
373.42
335.45
349.48
3
4
3
3
4
3
4
4
0
0
0
0
0
0
0
0
4
5
4
4
4
5
5
6
316.77
346.12
313.75
305.14
317.07
331.51
325.75
342.56
0
0
1
0
0
1
0
0
5.23
4.71
4.31
5.45
4.61
4.98
≤ 5
-CN
-CF3
-OCH3
-OC2H5
2h
Ideal range
≤ 140 ≤ 500 ≤ 10 ≤ 5
≤ 10
≤ 1
log P: log octanol/water partition coefficient; TPSA: total polar surface area; MW: molecular weight; nON: no. of
hydrogen acceptors; nOHNH: no. of hydrogen donors; nrotb: no. of rotatable bonds; Vio: violations were calculated
using the Molinspiration Calculation of Molecular Properties toolkit.
penetration, are positive (21.06–30.30) and suggest that synthesized compounds 2a–2h have abilities to pass
different membranes and reach the central nervous system. These findings supported the efficacy of these
compounds as central antinociceptive agents. On the other hand, participation of peripheral mechanisms in
the antinociceptive activities of compounds 2g and 2h may be related to alkyloxy substituents (methoxy and
ethoxy), which may provide higher ability to these compounds for modifying peripheral nociception mechanisms.
In summary, data acquired from the performed nociceptive tests pointed out centrally mediated antinoci-
ceptive actions induced by the compounds 2a, 2b, 2c, 2d, 2f, 2g, and 2h on nociceptive neuronal pathways
carrying mechanical, thermal, and chemical stimuli. Moreover, peripheral mechanisms also seem to contribute to
the antinociceptive activities of compounds 2g and 2h, as well as central ones. This present study supports the
previous literature reporting on the antinociceptive activities of benzimidazole-piperidine derivatives.31,33−38
Nevertheless, the exact mechanisms of the observed antinociceptive activities need to be clarified with further
detailed investigations.
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3. Experimental
3.1. Drugs
All of the used chemicals were purchased from Sigma-Aldrich Chemicals (Sigma-Aldrich Corp., St. Louis, MO,
USA).
3.2. Chemistry
3.2.1. General
Melting points of the synthesized compounds were determined by an MP90 digital melting point apparatus
(Mettler Toledo, Columbus, OH, USA) and were uncorrected. All reactions were monitored by thin-layer
chromatography (TLC) using silica gel 60 F254 TLC plates (Merck KGaA, Darmstadt, Germany). 1 H NMR
and 13 C NMR spectra were recorded on a Bruker DPX 300 NMR spectrometer (Bruker Bioscience, Billerica,
MA, USA) and on a Bruker DPX 75 MHz spectrometer (Bruker Bioscience) in DMSO-d6 , respectively. In
the NMR spectra splitting patterns were designated as follows: s: singlet; d: doublet; t: triplet; m: multiplet.
Coupling constants (J) were reported as Hz. The IR spectra were obtained on a Shimadzu IR Affinity-1S
(Shimadzu, Tokyo, Japan). HRMS studies were performed on a Shimadzu LCMS-IT-TOF system.
3.2.2. Microwave-assisted synthesis of 2-(4-substituted-phenyl)-1H -benzimidazole derivatives
(1a–1h)
A mixture of suitable benzaldehyde derivative (0.02 mol), sodium disulfite (3.8 g, 0.02 mol), and DMF (10
mL) was added to a vial (30 mL) of microwave synthesis reactor (Anton-Paar, Monowave 300, Austria). The
resultant mixture was heated under conditions of 240 ◦ C and 10 bar for 5 min. After this period, the vial was
cooled down, 1,2-phenylenediamine (2,16 g, 0.02 mol) was added, and then the reaction mixture was kept under
the same reaction conditions in the microwave reactor. After TLC screening, the mixture was poured into ice
water and the solid was washed with water and dried. The products were crystallized from ethanol.52−54
3.2.3. Microwave-assisted synthesis of 2-(4-substituted-phenyl)-1-[2-(piperidin-1-yl)ethyl]-1H -
benzimidazole derivatives (2a–2h)
In a vial (30 mL) of microwave synthesis reactor (Anton-Paar Monowave 300), the corresponding 2-(4-
substituted-phenyl)-1H -benzimidazole derivative (1a–1h) (2.5 mmol) was dissolved in THF (10 mL) and NaH
(0.072 g, 3 mmol) was added. After the addition of 2-(piperidine-1-yl)ethyl chloride (1 mL), the mixture was
heated under conditions of 170 ◦ C and 10 bar for 30 min. After cooling, the mixture was poured into ice water.
The resulting precipitate was washed with water and dried. Crystallization of crude product from ethanol gave
final compounds 2a–2h.
3.2.4. 2-(4-Methylphenyl)-1-[2-(piperidin-1-yl)ethyl]-1H -benzimidazole (2a)
IR (KBr, cm−1): υmax 3049 (aromatic C-H stretching), 2972 (aliphatic C-H stretching), 1610–1448 (C = N
and C = C stretching), 1155 (C-N stretching) 827 (parasubstituted benzene). 1 H NMR (300 MHz, DMSO-d6 ,
ppm) δ: 1.30 (6H, br s, piperidine -CH2 -), 2.20 (4H, br s, piperidine -CH2 -), 2.40 (3H, s, CH3), 2.56 (2H, t,
J = 6.5 Hz, -CH2 -), 4.35 (2H, t, J = 6.5 Hz, -CH2 -), 7.19–7.29 (2H, m, benzimidazole H5 , H6), 7.37 (2H,
d, J = 7.9 Hz, phenyl H3 , H5), 7.60-7.67 (2H, m, benzimidazole H4 , H7), 7.72 (2H, d, J = 8.1 Hz, phenyl
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H2 , H6). 13 C NMR (75 MHz, DMSO-d6 , ppm) δ: 21.43 (CH3), 24.23 (CH2), 25.87 (2CH2), 42.63 (CH2),
54.67 (2CH2), 57.78 (CH2), 111.33 (CH), 119.48 (CH), 122.27 (CH), 122.65 (CH), 128.27 (C), 129.61 (2CH),
129.66 (2CH), 136.17 (C), 139.67 (C), 143.13 (C), 153.96 (C). HRMS (m/z): [M + H]+ calcd for C21 H25 N3 :
320.2121; found: 320.2113.
3.2.5. 2-(4-Dimethylaminophenyl)-1-[2-(piperidin-1-yl)ethyl]-1H -benzimidazole (2b)
IR (KBr, cm−1): υmax 3082 (aromatic C-H stretching), 2933 (aliphatic C-H stretching), 1606–1442 (C = N
and C = C stretching), 1195 (C-N stretching) 819 (parasubstituted benzene). 1 H NMR (300 MHz, DMSO-d6 ,
ppm) δ: 1.35 (6H, br s, piperidine -CH2 -), 2.26 (4H, br s, piperidine -CH2 -), 2.60 (2H, t, J = 6.7 Hz, -CH2 -),
2.99 (6H, s, -N(CH3)2), 4.35 (2H, t, J = 6.7 Hz, -CH2 -), 6.84 (2H, d, J = 8.9 Hz, phenyl H2 , H6), 7.15–7.24
(2H, m, benzimidazole H5 , H6), 7.54–7.62 (2H, m, benzimidazole H4 , H7), 7.67 (2H, d, J = 8.9 Hz, phenyl
H3 , H5). 13 C NMR (75 MHz, DMSO-d6 , ppm) δ: 24.28 (CH2), 25.94 (2CH2), 40.28 (2CH3), 42.77 (CH2),
54.72 (2CH2), 57.78 (CH2), 110.99 (CH), 112.07 (2CH), 117.86 (C), 119.03 (CH), 122.01 (CH), 122.10 (CH),
130.53 (2CH), 136.34 (C), 143.29 (C), 151.36 (C), 154.53 (C). HRMS (m/z): [M + H]+ calcd for C22 H28 N4 :
349.2387; found: 349.2373.
3.2.6. 2-(4-Chlorophenyl)-1-[2-(piperidin-1-yl)ethyl]-1H -benzimidazole (2c)
IR (KBr, cm−1): υmax 3053 (aromatic C-H stretching), 2929 (aliphatic C-H stretching), 1598–1450 (C = N
and C = C stretching), 1157 (C-N stretching) 839 (parasubstituted benzene). 1 H NMR (300 MHz, DMSO-d6 ,
ppm) δ: 1.28 (6H, br s, piperidine -CH2 -), 2.17 (4H, br s, piperidine -CH2 -), 2.55 (2H, t, J = 6.3 Hz, -CH2 -),
4.38 (2H, t, J = 6.3 Hz, -CH2), 7.22-7.32 (2H, m, benzimidazole H5 , H6), 7.62-7.70 (4H, m, phenyl H2 , H6 ,
benzimidazole H4 , H7), 7.89 (2H, d, J = 8.6 Hz, phenyl H3 , H5). 13 C NMR (75 MHz, DMSO-d6 , ppm) δ:
24.21 (CH2), 25.83 (2CH2), 42.70 (CH2), 54.69 (2CH2), 57.80 (CH2), 111.50 (CH), 119.66 (CH), 122.49 (CH),
122.98 (CH), 129.14 (2CH), 130.08 (C), 131.60 (2CH), 134.90 (C), 136.18 (C), 143.06 (C), 152.80 (C). HRMS
(m/z): [M + H]+ calcd for C20 H22 ClN3 : 340.1575; found: 340.1568.
3.2.7. 2-(4-Fluorophenyl)-1-[2-(piperidin-1-yl)ethyl]-1H -benzimidazole (2d)
IR (KBr, cm−1): υmax 3089 (aromatic C-H stretching), 2972 (aliphatic C-H stretching), 1606–1454 (C = N and
C = C stretching), 1224 (C-N stretching) 840 (parasubstituted benzene). 1 H NMR (300 MHz, DMSO-d6 , ppm)
δ: 1.29 (6H, br s, piperidine -CH2 -), 2.17 (4H, br s, piperidine -CH2 -), 2.55 (2H, t, J = 6.4 Hz,-CH2 -), 4.36
(2H, t, J =6.4 Hz,-CH2), 7.24–7.31 (2H, m, benzimidazole H5 , H6), 7.38–7.44 (2H, m, benzimidazole H4 , H7),
7.63–7.69 (2H, m, phenyl H2 , H6), 7.88–7.93 (2H, m, phenyl H3 , H5). 13 C NMR (75 MHz, DMSO-d6 , ppm)
δ: 24.21 (CH2), 25.84 (2CH2), 42.61 (CH2), 54.67 (2CH2), 57.76 (CH2), 111.43 (CH), 116.10 (phenyl C3,3’
,
d, J = 21.8 Hz), 119.58 (CH), 122.40 (CH), 122.85 (CH), 127.71 (phenyl C1 , d, J = 3.8 Hz), 132.16 (phenyl
C2,2’ , d, J = 9.0 Hz), 136.09 (C), 143.03 (C), 153.03 (C), 163.28 (phenyl C4 , d, J = 245.3 Hz). HRMS (m/z):
[M + H]+ calcd for C20 H22 FN3 : 324.1871; found: 324.1860.
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3.2.8. 2-(4-Cyanophenyl)-1-[2-(piperidin-1-yl)ethyl]-1H -benzimidazole (2e)
IR (KBr, cm−1): υmax 3043 (aromatic C-H stretching), 2931 (aliphatic C-H stretching), 2217 (C≡N), 1614–
1446 (C = N and C = C stretching), 1122 (C-N stretching) 848 (parasubstituted benzene). 1 H NMR (300 MHz,
DMSO-d6 , ppm) δ: 1.24 (6H, br s, piperidine -CH2 -), 2.13 (4H, br s, piperidine -CH2 -), 2.53 (2H, t, J = 6.1
Hz, -CH2 -), 4.41 (2H, t, J = 6.1 Hz, -CH2 -), 7.24–7.35 (2H, m, benzimidazole H5 , H6), 7.68–7.72 (2H, m,
benzimidazole H4 , H7), 8.02–8.10 (4H, m, phenyl H2 , H6 , phenyl H3 , H5). 13 C NMR (75 MHz, DMSO-d6 ,
ppm) δ: 24.17 (CH2), 25.78 (2CH2), 42.83 (CH2), 54.71 (2CH2), 57.83 (CH2), 111.71 (CH), 112.45 (C),
118.99 (CN), 119.93 (CH), 122.75 (CH), 123.40 (CH), 130.64 (2CH), 133.00 (2CH), 135.79 (C), 136.30 (C),
143.11 (C), 152.25 (C). HRMS (m/z): [M + H]+ calcd for C21 H22 N4 : 331.1917; found: 331.1902.
3.2.9. 2-[4-(Trifluoromethyl)phenyl]-1-[2-(piperidin-1-yl)ethyl]-1H -benzimidazole (2f)
IR (KBr, cm−1): υmax 3080 (aromatic C-H stretching), 2972 (aliphatic C-H stretching), 1620–1442 (C = N
and C = C stretching), 1126 (C-N stretching) 861 (parasubstituted benzene). 1 H NMR (300 MHz, DMSO-d6 ,
ppm) δ: 1.24 (6H, br s, piperidine -CH2 -), 2.13 (4H, br s, piperidine -CH2 -), 2.54 (2H, t, J = 6.1 Hz, -CH2 -),
4.42 (2H, t, J = 6.2 Hz, -CH2), 7.24–7.34 (2H, m, benzimidazole H5 , H6), 7.67–7.72 (2H, m, benzimidazole
H4 , H7), 7.93 (2H, d, J = 8.3 Hz, phenyl H2 , H6), 8.10 (2H, d, J = 8 Hz, phenyl H3 , H5). 13 C-NMR
(75 MHz, DMSO-d6 , ppm) δ: 24.17 (CH2), 25.76 (2CH2), 42.76 (CH2), 54.70 (2CH2), 57.87 (CH2), 111.65
(CH), 119.86 (CH), 122.66 (CH), 123.26 (CH), 125.93 (phenyl C2,2’ , q, J = 3.7 Hz), 128.12 (CF3 , q, J =261.3
Hz), 130.13 (phenyl C1 , q, J = 22.6 Hz), 130.67 (2CH), 135.33 (C), 136.23 (C), 143.09 (C), 152.48 (C). HRMS
(m/z): [M + H]+ calcd for C21 H22 F3 N3 : 374.1839; found: 374.1833.
3.2.10. 2-(4-Methoxyphenyl)-1-[2-(piperidin-1-yl)ethyl]-1H -benzimidazole (2g)
IR (KBr, cm−1): υmax 3070 (aromatic C-H stretching), 2935 (aliphatic C-H stretching), 1612-1450 (C = N
and C = C stretching), 1307-1029 (C-N and C-O stretching) 837 (parasubstituted benzene). 1 H NMR (300
MHz, DMSO-d6 , ppm) δ: 1.33 (6H, br s, piperidine -CH2 -), 2.21 (4H, br s, piperidine -CH2 -), 2.56 (2H, t,
J = 6.5 Hz, -CH2 -), 3.84 (3H, s, OCH3), 4.35 (2H, t, J =6.6 Hz, -CH2 -), 7.11 (2H, d, J = 8.9 Hz, phenyl
H3 , H5), 7.19–7.28 (2H, m, benzimidazole H5 , H6), 7.59–7.65 (2H, m, benzimidazole H4 , H7), 7.78 (2H, d,
J = 8.8 Hz, phenyl H2 , H6). 13 C NMR (75 MHz, DMSO-d6 , ppm) δ: 24.24 (CH2), 25.89 (2CH2), 42.65
(CH2), 54.69 (2CH2), 55.78 (OCH3), 57.75 (CH2), 111.25 (CH), 114.49 (2CH), 119.35 (CH), 122.21 (CH),
122.51 (CH), 123.32 (C), 131.21 (2CH), 136.17 (C), 143.13 (C), 153.83 (C), 160.09 (C). HRMS (m/z): [M +
H]+ calcd for C21 H25 N3 O: 336.2070; found: 336.2061.
3.2.11. 2-(4-Ethoxyphenyl)-1-[2-(piperidin-1-yl)ethyl]-1H -benzimidazole (2h)
IR (KBr, cm−1): υmax 3053 (aromatic C-H stretching), 2970 (aliphatic C-H stretching), 1612–1452 (C = N and
C = C stretching), 1246–1041 (C-N and C-O stretching) 850 (parasubstituted benzene). 1 H NMR (300 MHz,
DMSO-d6 , ppm) δ: 1.31-1.39 (9H, m, piperidine -CH2 -, -OCH2 CH3), 2.21 (4H, br s, piperidine -CH2 -), 2.57
(2H, t, J = 6.5 Hz, -CH2 -), 4.12 (2H, q, J = 7 Hz, -OCH2 CH3), 4.35 (2H, t, J = 6.5 Hz, -CH2 -), 7.09 (2H, d,
J = 8.8 Hz, phenyl H2 , H6), 7.19–7.28 (2H, m, benzimidazole H5 , H6), 7.59–7.65 (2H, m, benzimidazole H4 ,
H7), 7.76 (2H, d, J = 8.8 Hz phenyl H3 , H5). 13 C NMR (75 MHz, DMSO-d6 , ppm) δ: 15.06 (OCH2 CH3),
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24.24 (CH2), 25.89 (2CH2), 42.62 (CH2), 54.67 (2CH2), 57.75 (CH2), 63.73 (OCH2 CH3), 111.24 (CH), 114.90
(2CH), 119.35 (CH), 122.20 (CH), 122.50 (CH), 123.18 (C), 131.21 (2CH), 136.16 (C), 143.13 (C), 153.86 (C),
159.96 (C). HRMS (m/z): [M + H]+ calcd for C22 H27 N3 O: 350.2227; found: 350.2215.
3.3. Pharmacology
3.3.1. Animals
Experiments were carried out with adult Wistar rats (body weight: 250–350 g) that were housed in well-
ventilated and thermoregulated rooms in a cycle of dark (12 h) and light (12 h) at a temperature of 24 ꢀ 1 ◦ C.
Twelve hours before the experimental session began, animals received only drinking water in order to prevent
food interference with the absorption of the compounds. Animals were brought to the laboratory at least 48 h
before the experiments to ensure that they acclimatized to the environment. The experimental protocol of this
study was approved by the Local Ethics Committee on Animal Experimentation of Anadolu University, Turkey.
3.3.2. Administration of drugs and chemical compounds
Animals were randomly divided into ten groups: control group, reference group (morphine sulfate), and test
groups 2a–2h. Each of the experimental groups consisted of seven rats.
Test compounds were dissolved in sunflower oil and administered orally (p.o.) at a dose of 10 mg/kg.28
The control solution was sunflower oil since test compounds were dissolved in it. The reference drug (morphine
sulfate, 5 mg/kg) was intraperitoneally injected at a volume of 0.1 mL.55
Experiments were performed 30 min after the administration of morphine and 60 min after the adminis-
tration of the control solution and the test compounds.
3.3.3. Nociceptive tests
3.3.3.1. Hot-plate test
The hot-plate test was performed using a hot/cold plate device (Ugo-Basile, 37100, Verase, Italy) that consisted
of an aluminum plate and a Plexiglas compartment (20 × 25 cm) settled on it. Reactions of animals against
thermal stimuli were evaluated by recording the time between placement of rats with all four paws on the plate
maintained at a constant temperature of 55 ꢀ 1 ◦ C and their first hind paw licking and/or jumping behavior.
After each session, the plate was cleaned with ethanol to eliminate the odor of the previous rat. A cut-off time
of 40 s was set as the maximum stimuli period in order to avoid possible tissue damage.56,57
3.3.3.2. Paw-pressure test
The paw-pressure test was performed using a Randall–Selitto analgesy-meter (Ugo-Basile, 37215), as previously
described by Bujalska-Zadroz˙ny et al.58 This device is used to apply incremental pressure at a constant rate (32
g/s) to the dorsal surface of the rat’s paw. The nociceptive threshold was defined as the force (g) at the time
the rat attempted to withdraw its hind paw. A cut-off limit of 480 g was determined to avoid tissue damage.
The data obtained from the hot plate and Randall–Selitto tests were expressed as a percentage of the
maximum possible effect (MPE) using the following equation:59
MPE% = (postdrug value − predrug value) / (cut-off value − predrug value) × 100.
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3.3.3.3. Formalin test
The formalin test was performed by subcutaneous administration of 5% formalin solution (in a volume of 100
µL) into the plantar region of the right hind paw of the animal. The duration of time spent licking or biting
the injected paw was measured every 5 min. Following the formalin injection, the first 0–10 min was accepted
as the “early phase” or “acute phase” and the next 10–45 min was accepted as the “late phase” or “prolonged
tonic response”.60
Inhibition of nociceptive response was calculated by the following equation:
Inhibition% = [(control group − treated group) / control group] × 100.
3.3.4. Motor coordination tests
Motor deficits of the animals were evaluated with a rotarod test device (Ugo Basile, 7560) having five disks
forming four equal sections between them. The rotating mill was adjusted at 16 rpm.
Rats were subjected to pretraining for 3 consecutive days to be acclimatized. On the day of the
experiment, animals were placed on a rotating mill 60 min after the drug administrations and falling latencies
from the mill were recorded automatically by the device.61,62 Endurance time on the treadmill was accepted as
a parameter for motor coordination. Task time-out was chosen as 300 s.
3.4. Statistical evaluation
GraphPad Prism for Windows version 6.01 (GraphPad Software, San Diego, CA, USA) was used for the
statistical evaluation. Analysis of the experimental data was performed using one-way ANOVA with Tukey’s
post hoc test. The results were presented as mean ꢀ standard error of the mean (SEM). P < 0.05 was
considered as statistically significant.
3.5. Theoretical calculation of ADME parameters
In order to evaluate ADME profiles of the synthesized compounds, some physicochemical parameters were calcu-
lated using the Molinspiration property calculation program (http://www.molinspiration.com/services/properties.html).
Acknowledgments
¨
The corresponding author thanks Prof Dr Zafer Asım Kaplancıklı and Assoc Prof Dr Yusuf Ozkay for their
support in discussing structure activity relationships related to the tested compounds.
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